Targeting of Regulators as a Promising Approach in the Search for Novel Antimicrobial Agents

Since the discovery of penicillin in the first half of the last century, antibiotics have become the pillars of modern medicine for fighting bacterial infections. However, pathogens resistant to antibiotic treatment have increased in recent decades, and efforts to discover new antibiotics have decreased. As a result, it is becoming increasingly difficult to treat bacterial infections successfully, and we look forward to more significant efforts from both governments and the scientific community to research new antibacterial drugs. This perspective article highlights the high potential of bacterial transcriptional and posttranscriptional regulators as targets for developing new drugs. We highlight some recent advances in the search for new compounds that inhibit their biological activity and, as such, appear very promising for treating bacterial infections

NGF-response of EGF-dependent progenitor cells obtained from human sympathetic ganglia

SIGNALLING molecules are thought to play a significant role in determining the fate of neural crest progenitor cells. The human sympathetic chain was identified at 6.5, 7.5, 8.2, 10.2 and 11.4 postconception (PC) weeks demonstrating low affinity nerve growth factor (NGF) receptors, and was processed for tissue culture. In the presence of epidermal growth factor (EGF), floating spheres of proliferating progenitor cells were developed in vitro. In the absence of EGF progenitor cells differentiated into tyrosine hydroxylase (TH)-immunoreactive neuronal and TH-negative flat cells. NGF treatment significantly increased neurite outgrowth and survival of TH-immunoreactive cells. The multipotent cells we isolated differ from previously reported sympathoadrenal progenitors in that they give rise to TH immunoreactive neurones precociously sensitive to NGF

Feeling the heat: The campylobacter jejuni HrcA transcriptional repressor is an intrinsic protein thermosensor

The heat-shock response, a universal protective mechanism consisting of a transcriptional reprogramming of the cellular transcriptome, results in the accumulation of proteins which coun-teract the deleterious effects of heat-stress on cellular polypeptides. To quickly respond to thermal stress and trigger the heat-shock response, bacteria rely on different mechanisms to detect temperature variations, which can involve nearly all classes of biological molecules. In Campylobacter jejuni the response to heat-shock is transcriptionally controlled by a regulatory circuit involving two re-pressors, HspR and HrcA. In the present work we show that the heat-shock repressor HrcA acts as an intrinsic protein thermometer. We report that a temperature upshift up to 42°C negatively affects HrcA DNA-binding activity to a target promoter, a condition required for de-repression of regu-lated genes. Furthermore, we show that this impairment of HrcA binding at 42°C is irreversible in vitro, as DNA-binding was still not restored by reversing the incubation temperature to 37°C. On the other hand, we demonstrate that the DNA-binding activity of HspR, which controls, in combi-nation with HrcA, the transcription of chaperones’ genes, is unaffected by heat-stress up to 45°C, portraying this master repressor as a rather stable protein. Additionally, we show that HrcA binding activity is enhanced by the chaperonin GroE, upon direct protein–protein interaction. In conclu-sion, the results presented in this work establish HrcA as a novel example of intrinsic heat-sensing transcriptional regulator, whose DNA-binding activity is positively modulated by the GroE chap-eronin

A new test for equilibrium based on clinopyroxene-melt pairs: Clues on the solidification temperatures of Etnean alkaline melts at post-eruptive conditions

We have performed new global regression analyses to calibrate a model of equilibrium between clinopyroxene and co-existing melt. Then we have applied this model to a restricted but important range of clinopyroxene and melt compositions from Mt. Etna volcano. The degree of disequilibrium is determined through the comparison between components “predicted” for clinopyroxene via regression analyses of clinopyroxene-liquid pairs in equilibrium conditions, with those “measured” in the analyzed crystals. The model is tested using compositions not included into the calibration dataset, i.e., clinopyroxene-melt pairs obtained from equilibrium and cooling rate experiments conducted at ambient pressure on an Etnean trachybasalt. The experiments were duplicated at the NNO+1.5 and QFM oxygen buffering conditions estimated for magmas at Mt. Etna. Both equilibrium and disequilibrium clinopyroxene-melt pairs from the experiments were also used as input data for one of the most recent thermometers based on the Jd-DiHd exchange reaction. Results from calculations indicate that, under rapid cooling rate conditions, clinopyroxenes do not equilibrate with the melt. Consequently, the thermometers predict higher crystallization temperatures compared to the final experimental temperature, prior to rapid quenching of the experiment. The systematic difference between expected and measured compositions and temperatures allows us to calibrate a model that describes undercooling based on disequilibrium exchange reactions. We use this new tool to estimate the thermal history of naturally cooled lava flows and dikes at Mt. Etna volcano

A convenient and robust in vivo reporter system to monitor gene expression in the human pathogen helicobacter pylori

Thirty years of intensive research have significantly contributed to our understanding of Helicobacter pylori biology and pathogenesis. However, the lack of convenient genetic tools, in particular the limited effectiveness of available reporter systems, has notably limited the toolbox for fundamental and applied studies. Here, we report the construction of a bioluminescent H. pylori reporter system based on the Photorhabdus luminescens luxCDABE cassette. The system is constituted of a promoterless lux acceptor strain in which promoters and sequences of interest can be conveniently introduced by double homologous recombination of a suicide transformation vector. We validate the robustness of this new lux reporter system in noninvasive in vivo monitoring of dynamic transcriptional responses of inducible as well as repressible promoters and demonstrate its suitability for the implementation of genetic screens in H. pylori. © 2012, American Society for Microbiology

Carbonate assimilation in magmas: a reappraisal based on experimental petrology

The main effect of magma-carbonate interaction on magma differentiation is the formation of a silica-undersaturated, alkali-rich residual melt. Such a desilication process was explained as the progressive dissolution of CaCO3 in melt by consumption of SiO2 and MgO to form diopside sensu stricto. Magma chambers emplaced in carbonate substrata, however, are generally associated with magmatic skarns containing clinopyroxene with a high Ca-Tschermak activity in their paragenesis. Data are presented from magma-carbonate interaction experiments, demonstrating that carbonate assimilation is a complex process involving more components than so far assumed. Experimental results show that, during carbonate assimilation, a diopside-hedenbergite-Ca-Tschermak clinopyroxene solid solution is formed and that Ca-Tschermak/diopside and hedenbergite/diopside ratios increase as a function of the progressive carbonate assimilation. Accordingly, carbonate assimilation reaction should be written as follows, taking into account all the involved magmatic components: CaCO3solid+SiO2melt+MgOmelt+FeOmelt+Al2O3melt → (Di-Hd-CaTs)sssolid+CO2fluid The texture of experimental products demonstrates that carbonate assimilation produces three-phases (solid, melt, and fluid) whose main products are: i) diopside-hedenbergite-Ca-Tschermak clinopyroxene solid solution; ii) silica-undersaturated CaO-rich melt; and iii) C-O-H fluid phase. The silica undersaturation of the melt and, more importantly, the occurrence of a CO2-rich fluid phase, must be taken into account as they significantly affect partition coefficients and the redox state of carbonated systems, respectively

A general viscosity model of Campi Flegrei (Italy) melts

Viscosities of shoshonitic and latitic melts, relevant to the Campi Flegrei caldera magmas, have been experimentally determined at atmospheric pressure and 0.5 GPa, temperatures between 840 K and 1870 K, and H2O contents from 0.02 to 3.30 wt%. The concentric cylinder technique was employed at atmospheric pressure to determine viscosity of nominally anhydrous melts in the viscosity range of 101.5 - 103 Pa·s. The micropenetration technique was used to determine the viscosity of hydrous and anhydrous melts at atmospheric pressure in the high viscosity range (1010 Pa·s). Falling sphere experiments were performed at 0.5 GPa in the low viscosity range (from 100.35 to 102.79 Pa·s) in order to obtain viscosity data of anhydrous and hydrous melts. The combination of data obtained from the three different techniques adopted permits a general description of viscosity as a function of temperature and water content using the following modified VFT equation: where η is the viscosity in Pa·s, T the temperature in K, w the H2O content in wt%, and a, b, c, d, e, g are the VFT parameters. This model reproduces the experimental data (95 measurements) with a 1σ standard deviation of 0.19 and 0.22 log units for shoshonite and latite, respectively. The proposed model has been applied also to a more evolved composition (trachyte) from the same area in order to create a general model applicable to the whole compositional range of Campi Flegrei products. Moreover, speed data have been used to constrain the ascent velocity of latitic, shoshonitic, and trachytic melts within dikes. Using petrological data and volcanological information (geometrical parameters of the eruptive fissure and depth of magma storage), we estimate a time scale for the ascent of melt from 9 km to 4 km depth (where deep and shallow reservoirs, respectively, are located) in the order of few minutes. Such a rapid ascent should be taken into account for the hazard assessment in the Campi Flegrei area

International cooperation during volcanic crisis: an example from the Italy-El Salvador monitoring system installed at Chaparrastique volcano, El Salvador

On December 29th, 2013, after 12 years of inactivity, a new explosive eruption occurred at Chaparrastique volcano (San Miguel, el Salvador) prompting the evacuation of more than 5000 people. The new eruption that occurred at the volcano has so far been an isolated single explosion of vulcanian type, and was the first eruption since 2002, when the volcano produced a small VEI 1 eruption. The explosion produced an ash plume of considerable (5-10 km) height, generating heavy ash fall in nearby areas downwind, such as in the towns of Chinameca and San Jorge. Pyroclastic density currents also affected the flanks, damaging the coffee plantations and small inhabited areas around the volcano. On January 2014, following a request of support by the government of El Salvador, INGV (Istituto Nazionale di Geofisica e Vulcanologia), organized a task force, V-Emer (Volcano Emergency) to improve the existing monitoring network at Chaparrastque volcano. During a 10 days campaign in El Salvador a temporary network was successfully installed, and it is now run by the volcanologists of MARN (Ministerio de Medio Ambiente y Recursos Naturales, El Salvador). The network is composed of five broadband seismic stations, 3 infrasonic microphones, 2 radiometers, 10 GPS stations, 1 thermal camera, 1 DOAS and 1 multi-gas geochemical station for measurement of SO2 and CO2 fluxes, respectively. Since 27 January, significant collaborative efforts are being done between MARN and INGV for the processing and interpretation of the data collected during monitoring. For facilitating communication and exchange between the members of the cooperation, a mailing list has been created, and weekly meetings are attended by the members for the discussion on a number of scientific and technical aspects. This initiative seeks to make significant advance into volcano monitoring network and data analysis, as well as improving international cooperation during volcanic crisis management. V-EMER group: A. Bonforte, G. Giuffrida,A. La Spina, F. Montalvo, M. Liuzzo, S. Rapisarda, G. Salerno, D. Andronico, E. Biale, A. Cannata, T. Caltabiano, E. Del Bello, M. La Rocca, D. Granieri, L. Lodato, G. Giudice, F. Murè, E. Pecora, M. Prestifilippo, L. Scuderi, L. Zuccarello, G. De Natale, R. Favara, E. Privitera. MARN group: M. Diaz, D. Escobar, E. Gutierrez, D. Hernandez, G. Marroquin, C. Bolaños, L. Handal, C. Polío, B. Palacios, N. Galvez, R. Torres, E. Escobar

Multilayer Regulation of Neisseria meningitidis NHBA at Physiologically Relevant Temperatures

Neisseria meningitidis colonizes the nasopharynx of humans, and pathogenic strains can disseminate into the bloodstream, causing septicemia and meningitis. NHBA is a surface-exposed lipoprotein expressed by all N. meningitidis strains in different isoforms. Diverse roles have been reported for NHBA in heparin-mediated serum resistance, biofilm formation, and adherence to host tissues. We determined that temperature controls the expression of NHBA in all strains tested, with increased levels at 30–32◦C compared to 37◦C. Higher NHBA expression at lower temperatures was measurable both at mRNA and protein levels, resulting in higher surface exposure. Detailed molecular analysis indicated that multiple molecular mechanisms are responsible for the thermoregulated NHBA expression. The comparison of mRNA steady-state levels and half-lives at 30◦C and 37◦C demonstrated an increased mRNA stability/translatability at lower temperatures. Protein stability was also impacted, resulting in higher NHBA stability at lower temperatures. Ultimately, increased NHBA expression resulted in higher susceptibility to complement-mediated killing. We propose that NHBA regulation in response to temperature downshift might be physiologically relevant during transmission and the initial step(s) of interaction within the host nasopharynx. Together these data describe the importance of NHBA both as a virulence factor and as a vaccine antigen during neisserial colonization and invasion

Stable hybrid myotubes: a new model for studying re-expression of enzymatic activities in vitro

Heterokaryons represent a stable and reproducible model system for the study of biochemical and molecular aspects responsible for muscle gene activation. Previous experiments have used this fusion system to demonstrate human gene activation in hybrids formed between human and non-human cells. The aim of this research was to apply this experimental model to the correction of a cytoplasmic activity, namely glucose-6-phosphate dehydrogenase (G6PD), in vitro, in hybrid myotubes formed between G6PD-negative and positive myoblasts. Different identification methods were used (Hoechst stain and Fluorescent Latex Microspheres, FLMs) to identify hybrid myotubes formed. We demonstrated the restoration of G6PD activity in all hybrid myotubes formed; we then tried to elucidate the mechanisms underlying the restoration of this specific activity and apply the results obtained to the understanding of more complex mechanisms involved in muscle gene activation