BAL9141, a novel extended-spectrum cephalosporin active against methicillin-resistant Staphylococcus aureus in treatment of experimental endocarditis.

The therapeutic efficacy of BAL9141 (formerly Ro 63-9141), a novel cephalosporin with broad in vitro activity that also has activity against methicillin-resistant Staphylococcus aureus (MRSA), was investigated in rats with experimental endocarditis. The test organisms were homogeneously methicillin-resistant S. aureus strain COL transformed with the penicillinase-encoding plasmid pI524 (COL Bla+) and homogeneously methicillin-resistant, penicillinase-producing isolate P8-Hom, selected by serial exposure of parent strain P8 to methicillin. The MICs of BAL9141 for these organisms (2 mg/liter) were low, and BAL9141was bactericidal in time-kill curve studies after 24 h of exposure to either two, four, or eight times the MIC. Rats with experimental endocarditis were treated in a three-arm study with a continuous infusion of BAL5788 (formerly Ro 65-5788), a carbamate prodrug of BAL9141, or with amoxicillin-clavulanate or vancomycin. The rats were administered BAL9141 to obtain steady-state target levels of 20, 10, and 5 mg of per liter or were administered either 1.2 g of amoxicillin-clavulanate (ratio 5:1) every 6 h or 1 g of vancomycin every 12 h at changing flow rates to simulate the pharmacokinetics produced in humans by intermittent intravenous treatment. Treatment was started 12 h after bacterial challenge and lasted for 3 days. BAL9141 was successful in the treatment of experimental endocarditis due to either MRSA isolate COL Bla+ or MRSA isolate P8-Hom at the three targeted steady-state concentrations and sterilized >90% of cardiac vegetations (P < 0.005 versus controls; P < 0.05 versus amoxicillin-clavulanate and vancomycin treatment groups). These promising in vivo results with BAL9141 correlated with the high affinity of the drug for PBP 2a and its stability to penicillinase hydrolysis observed in vitro.

Analysis of LEDGF/p75 expression regulation

Background To replicate, retroviruses must insert DNA copies of their RNA genomes into the host genome. This integration process is catalyzed by the viral integrase protein. The site of viral integration has been shown to be non-random and retrovirus-specific. LEDGF/p75, a splice variant encoded by PSIP1 gene and described as a general transcription coactivator, was identified as a tethering factor binding both to chromatin and to lentiviral integrases, thereby affecting integration efficiency as well as integration site selection. LEDGF/p75 is still a poorly characterized protein, and its cellular endogenous function has yet to be fully determined. In order to start unveiling the roles of LEDGF/p75 in the cell, we started to investigate the mechanisms involved in the regulation of LEDGF/p75. Materials and methods To identify PSIP1 minimal promoter and associated regulatory elements, we cloned a region starting 5 kb upstream the transcription start site (TSS, +1 reference position) to the ATG start codon (+816), as well as systematic truncations, in a plasmid containing the firefly luciferase reporter gene. These constructs were co-transfected into HEK293 cells with a plasmid encoding the Renilla luciferase under the pTK promoter as an internal control for transfection efficiency. Both luciferase activities were assessed by luminescence as an indicator of promoter activity. Results Luciferase assays identified regions -76 to +1 and +1 to +94 as two independent minimal promoters showing respectively a 3.7x and 2.3x increase in luciferase activity. These two independent minimal promoters worked synergistically increasing luciferase activity up to 16.3x as compared to background. Moreover, we identified five regulatory blocks which modulated luciferase activity depending on the DNA region tested, three enhancers (- 2007 to -1159, -284 to -171 and +94 to +644) and two silencers (-171 to -76 and +796 to +816). However, the silencing effect of the region -171 to -76 is dependent on the presence of the +94 to +644 region, ruling out the enhancer activity of the latter. Computational analysis of PSIP1 promoter revealed the absence of TATA box and initiator (INR) sequences, classifying this promoter as nonconventional. TATA-less and INR-less promoters are characterized by multiple Sp1 binding sites, involved in the recruitment of the RNA pol II complex. Consistent with this, PSIP1 promoter contains multiple putative Sp1 binding sequences in regions -76 to +1 and +1 to +94.

Increased therapeutic window for the R91W mutant form of Rpe65 compared to Rpe65 null backgroud

Purpose:Given the advances of gene therapy studies to cure RPE65-derived Leber Congenital Amaurosis (LCA) (clinical trials phase I) and the heterogeneity of the targeted patients both genetically and phenotypically, it is of prime importance to examine the rescue efficiency of gene transfer in different mutant contexts. Indeed, half of these mutations are missense mutations, leading to potential residual RPE65 activity. Consequently, we wanted to evaluate the effect on retinal activity and cone survival of lentivirus-mediated gene therapy in the R91W knock-in mouse model expressing the mutant Rpe65R91W gene (Samardzija et al. 2008), a mutation found in LCA patients. Notably we investigated whether if the therapeutic window is prolonged in comparison to null mutations. Methods:An HIV-1-derived lentiviral vector (LV) expressing either the GFP or the mouse Rpe65 cDNA under the control of a 0.8 kb fragment of the human Rpe65 promoter (R0.8) was produced by transient transfection of 293T cells. LV-R0.8-RPE65 or GFP was injected into 5-days-old (P5) or 1 month-old R91W mice. Functional rescue was assessed by ERG (1 and 4 months post-injection) and pupillary light response (PLR) recordings and cone survival by histological analysis. Results:Increased light sensitivity was detected by scotopic ERG in animals injected with LV-R0.8-RPE65 at both P5 and 1 month compared to GFP-treated animals or untreated mice. PLR was also improved in some eyes and histological analysis of cone markers showed that the density of cones reached the wild type level in the region of wt RPE65 delivery after treatment at P5. However, the rescue effect of the injection at 1 month was limited and attained 60% of the wild type level, but still more cones were observed in the treated area than in 1 month-old untreated Rpe65R91W mice. Conclusions:We were able to show that lentivirus-mediated Rpe65 gene transfer not only increases retinal activity of the Rpe65R91W mouse and survival of cones after treatment at P5 but also after treatment at 1 month. However even if the treatment at 1 month is more limited (60% of the wild type level) than treatment at P5, the amount of cone markers is increased compared to the proportion found at 1 month of age in untreated animals. This results contrast with the lack of cone rescue by treatment at 1 month of age in Rpe65-/- (Bemelmans et al, 2006). Thus patient suffering from R91W mutation might benefit from a prolonged therapeutic window.

Silencing mutant ataxin-3 rescues motor deficits and neuropathology in machado-joseph disease transgenic mice.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein - ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.

Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism.

Abstract The plasmid pME6863, carrying the aiiA gene from the soil bacterium Bacillus sp. A24 that encodes a lactonase enzyme able to degrade N-acyl-homoserine lactones (AHLs), was introduced into the rhizosphere isolate Pseudomonas fluorescens P3. This strain is not an effective biological control agent against plant pathogens. The transformant P. fluorescens P3/pME6863 acquired the ability to degrade AHLs. In planta, P. fluorescens P3/pME6863 significantly reduced potato soft rot caused by Erwinia carotovora and crown gall of tomato caused by Agrobacterium tumefaciens to a similar level as Bacillus sp. A24. Little or no disease reduction was observed for the wild-type strain P3 carrying the vector plasmid without aiiA. Suppression of potato soft rot was observed even when the AHL-degrading P. fluorescens P3/pME6863 was applied to tubers 2 days after the pathogen, indicating that biocontrol was not only preventive but also curative. When antagonists were applied individually with the bacterial plant pathogens, biocontrol activity of the AHL degraders was greater than that observed with several Pseudomonas 2,4-diacetylphloroglucinol-producing strains and with Pseudomonas chlororaphis PCL1391, which relies on production of phenazine antibiotic for disease suppression. Phenazine production by this well characterized biological control strain P. chlororaphis PCL1391 is regulated by AHL-mediated quorum sensing. When P. chlororaphis PCL1391 was co-inoculated with P. fluorescens P3/pME6863 in a strain mixture, the AHL degrader interfered with the normally excellent ability of the antibiotic producer to suppress tomato vascular wilt caused by Fusarium oxysporum f. sp. lycopersici. Our results demonstrate AHL degradation as a novel biocontrol mechanism, but also demonstrate the potential for non-target interactions that can interfere with the biocontrol efficacy of other strains.

Supercoiling, knotting and replication fork reversal in partially replicated plasmids.

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.

ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.

BACKGROUND: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA). RESULTS: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NPâeuro0/00>âeuro0/00chitosan DNA NPâeuro0/00=âeuro0/00DNA unloaded chitosan NPâeuro0/00>âeuro0/00control (culture medium). CONCLUSIONS: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

Management of major accidents with on-site 144 dispatcher

Introduction: An excellent coordination between firefighters, policemen and medical rescue is the key to success in the management of major accidents. In order to improve and assist the medical teams engaged on site, the Swiss "medical command and control system" for rescue operations is based on a binomial set up involving one head emergency doctor and one head rescue paramedic, both trained in disaster medicine. We have recently experimented an innovative on-site "medical command and control system", based on the binomial team, supported by a dedicated 144 dispatcher. Methods: A major road traffic accident took place on the highway between Lausanne and Vevey on April 9th 2008. We have retrospectively collected all data concerning the victims as well as the logistics and dedicated structures, reported by the 144, the Hospitals, the Authority of the State and the Police and Fire Departments. Results: The 72-car pileup caused one death and 26 slightly injured patients. The management on the accident site was organized around a tripartite system, gathering together the medical command and control team with the police and fire departments. On the medical side, 16 ambulances, 2 medical response teams (SMUR), the Rega crew and the medical command and control team were dispatched by the 144. On that occasion an advanced medical command car equipped with communication devices and staffed with a 144 dispatcher was also engaged, allowing efficient medical regulation directly from the site. Discussion: The specific skills of one doctor and one paramedic both trained for disaster's management proved to be perfectly complementary. The presence of a dispatcher on site with a medical command car also proved to be useful, improving orders transmission from the medical command team to all other on- and off-site partners. It relieved the need of repeated back-and-forth communication with the 144, allowing both paramedic and doctor to focus on strategy and tactics rather than communication and logistics.

Optimal bail out policy, conditionality and constructive ambiguity

This paper addresses the issue of the optimal behaviour of the Lender of Last Resort (LOLR) in its microeconomic role regarding individual financial institutions in distress. It has been argued that the LOLR should not intervene at the microeconomic level and let any defaulting institution face the market discipline, as it will be confronted with the consequences of the risks it has taken. By considering a simple costbenefit analysis we show that this position may lack a sufficient foundation. We establish that, instead, uder reasonable assumptions, the optimal policy has to be conditional on the amount of uninsured debt issued by the defaulting bank. Yet in equilibrium, because the rescue policy is costly, the LOLR will not rescue all the banks that fulfill the uninsured debt requirement condition, but will follow a mixed strategy. This we interpret as the confirmation of the "creative ambiguity" principle, perfectly in line with the central bankers claim that it is efficient for them to have discretion in lending to individual institutions. Alternatively, in other cases, when the social cost of a bank's bankruptcy is too high, it is optimal for the LOLR to bail out the insititution, and this gives support to the "too big to fail" policy.

Dicarboxylic amino acids and glycine-betaine regulate chaperone-mediated protein-disaggregation under stress.

Active protein-disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress-induced protein aggregates in vitro and in Escherichia coli cells. K-glutamate and glycine-betaine (betaine) naturally accumulate in salt-stressed cells. In addition to providing thermo-protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone-mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K-glutamate or betaine. The combined protective, counter-negative and net activatory effects of K-glutamate and betaine, allowed protein disaggregation and refolding under heat-shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte-activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat-shock conditions.

How well do individuals predict the selling prices of their homes?

Self-reported home values are widely used as a measure of housing wealth by researchers employing a variety of data sets and studying a number of different individual and household level decisions. The accuracy of this measure is an open empirical question, and requires some type of market assessment of the values reported. In this research, we study the predictive power of self-reported housing wealth when estimating sales prices utilizing the Health and Retirement Study. We find that homeowners, on average, overestimate the value of their properties by between 5% and 10%. More importantly, we are the first to document a strong correlation between accuracy and the economic conditions at the time of the purchase of the property (measured by the prevalent interest rate, the growth of household income, and the growth of median housing prices). While most individuals overestimate the value of their properties, those who bought during more difficult economic times tend to be more accurate, and in some cases even underestimate the value of their house. These results establish a surprisingly strong, likely permanent, and in many cases long-lived, effect of the initial conditions surrounding the purchases of properties, on how individuals value them. This cyclicality of the overestimation of house prices can provide some explanations for the difficulties currently faced by many homeowners, who were expecting large appreciations in home value to rescue them in case of increases in interest rates which could jeopardize their ability to live up to their financial commitments.

Complete Nucleotide Sequence of Mouse Mammary Tumor Virus from JYG Chinese Wild Mice: Absence of Bacterial Insertion Sequences in the Cloned Viral gag Gene.

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5' long terminal repeat (LTR)(partial)-gag-pol-env-3' LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor V beta recognition as a superantigen is implicated among these MMTV species. Analysis of the viral gag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing the gag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids of gag, pol, protease and env products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.

The efficiency of a novel delivery system (PEI600-Tat) in development of potent DNA vaccine using HPV16 E7 as a model antigen.

DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. The mode of plasmid DNA delivery is critical to make progress in DNA vaccination. Using human papillomavirus type 16 E7 as a model antigen, this study evaluated the effect of peptide-polymer hybrid including PEI600-Tat conjugate as a novel gene delivery system on the potency of antigen-specific immunity in mice model. At ratio of 10:50 PEI-Tat/E7DNA (w/w), both humoral and cellular immune responses were significantly enhanced as compared with E7DNA construct and induced Th1 response. Therefore, this new delivery system could have promising applications in gene therapy.

A morphological reappraisal of the immature stages and life history of Elachista synethes Meyrick (Lepidoptera, Elachistidae), an Australian leaf miner alien to Chile

ABSTRACT Elachista synethes was recently recognized as an alien species in northern Chile, where its larvae mine the rescue grass Bromus catharticus (Poaceae). In order to provide the necessary information to allow field detection of E. synethes during early ontogeny, we conducted a morphological reappraisal of the immature stages of this leaf-miner moth, based on light and scanning electron microscopy, including the first descriptions of the egg and the first-instar larva. This is the first report of the existence of an apodal early larva for a species of Elachista Treitschke. The legs and prolegs are absent in the first two instars, but are well developed in the last two. Additional observations on the life history are also provided, including a description of the mine.