Optimization of Pyrazole Compounds as Antibiotic Adjuvants Active against Colistin- and Carbapenem-Resistant Acinetobacter baumannii.

The diffusion of antibiotic-resistant, Gram-negative, opportunistic pathogens, an increasingly important global public health issue, causes a significant socioeconomic burden. Acinetobacter baumannii isolates, despite causing a lower number of infections than Enterobacterales, often show multidrug-resistant phenotypes. Carbapenem resistance is also rather common, prompting the WHO to include carbapenem-resistant A. baumannii as a "critical priority" for the discovery and development of new antibacterial agents. In a previous work, we identified several series of compounds showing either direct-acting or synergistic activity against relevant Gram-negative species, including A. baumannii. Among these, two pyrazole compounds, despite being devoid of any direct-acting activity, showed remarkable synergistic activity in the presence of a subinhibitory concentration of colistin on K. pneumoniae and A. baumannii and served as a starting point for the synthesis of new analogues. In this work, a new series of 47 pyrazole compounds was synthesized. Some compounds showed significant direct-acting antibacterial activity on Gram-positive organisms. Furthermore, an evaluation of their activity as potential antibiotic adjuvants allowed for the identification of two highly active compounds on MDR Acinetobacter baumannii, including colistin-resistant isolates. This work confirms the interest in pyrazole amides as a starting point for the optimization of synergistic antibacterial compounds active on antibiotic-resistant, Gram-negative pathogens.

Screen of Unfocused Libraries Identified Compounds with Direct or Synergistic Antibacterial Activity.

Antibiotic resistance is an increasingly important global public health issue, as major opportunistic pathogens are evolving toward multidrug- and pan-drug resistance phenotypes. New antibiotics are thus needed to maintain our ability to treat bacterial infections. According to the WHO, carbapenem-resistant Acinetobacter, Enterobactericaeae, and Pseudomonas are the most critical targets for the development of new antibacterial drugs. An automated phenotypic screen was implemented to screen 634 synthetic compounds obtained in-house for both their direct-acting and synergistic activity. Fourteen percent and 10% of the compounds showed growth inhibition against tested Gram-positive and Gram-negative bacteria, respectively. The most active direct-acting compounds showed a broad-spectrum antibacterial activity, including on some multidrug-resistant clinical isolates. In addition, 47 compounds were identified for their ability to potentiate the activity of other antibiotics. Compounds of three different scaffolds (2-quinolones, phenols, and pyrazoles) showed a strong potentiation of colistin, some being able to revert colistin resistance in Acinetobacter baumannii.

The plakin domain of C. elegans VAB-10/plectin acts as a hub in a mechanotransduction pathway to promote morphogenesis.

Mechanical forces can elicit a mechanotransduction response through junction-associated proteins. In contrast to the wealth of knowledge available for focal adhesions and adherens junctions, much less is known about mechanotransduction at hemidesmosomes. Here, we focus on the C. elegans plectin homolog VAB-10A, the only evolutionary conserved hemidesmosome component. In C. elegans, muscle contractions induce a mechanotransduction pathway in the epidermis through hemidesmosomes. We used CRISPR to precisely remove spectrin repeats (SRs) or a partially hidden Src homology 3 (SH3) domain within the VAB-10 plakin domain. Deleting the SH3 or SR8 domains in combination with mutations affecting mechanotransduction, or just the part of SR5 shielding the SH3 domain, induced embryonic elongation arrest because hemidesmosomes collapse. Notably, recruitment of GIT-1, the first mechanotransduction player, requires the SR5 domain and the hemidesmosome transmembrane receptor LET-805. Furthermore, molecular dynamics simulations confirmed that forces acting on VAB-10 could make the central SH3 domain, otherwise in contact with SR4, available for interaction. Collectively, our data strongly indicate that the plakin domain plays a central role in mechanotransduction and raise the possibility that VAB-10/plectin might act as a mechanosensor.

Publisher Correction: An actin-based viscoplastic lock ensures progressive body-axis elongation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: An actin-based viscoplastic lock ensures progressive body-axis elongation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

An actin-based viscoplastic lock ensures progressive body-axis elongation.

Body-axis elongation constitutes a key step in animal development, laying out the final form of the entire animal. It relies on the interplay between intrinsic forces generated by molecular motors1-3, extrinsic forces exerted by adjacent cells4-7 and mechanical resistance forces due to tissue elasticity or friction8-10. Understanding how mechanical forces influence morphogenesis at the cellular and molecular level remains a challenge1. Recent work has outlined how small incremental steps power cell-autonomous epithelial shape changes1-3, which suggests the existence of specific mechanisms that stabilize cell shapes and counteract cell elasticity. Beyond the twofold stage, embryonic elongation in Caenorhabditis elegans is dependent on both muscle activity7 and the epidermis; the tension generated by muscle activity triggers a mechanotransduction pathway in the epidermis that promotes axis elongation7. Here we identify a network that stabilizes cell shapes in C. elegans embryos at a stage that involves non-autonomous mechanical interactions between epithelia and contractile cells. We searched for factors genetically or molecularly interacting with the p21-activating kinase homologue PAK-1 and acting in this pathway, thereby identifying the α-spectrin SPC-1. Combined absence of PAK-1 and SPC-1 induced complete axis retraction, owing to defective epidermal actin stress fibre. Modelling predicts that a mechanical viscoplastic deformation process can account for embryo shape stabilization. Molecular analysis suggests that the cellular basis for viscoplasticity originates from progressive shortening of epidermal microfilaments that are induced by muscle contractions relayed by actin-severing proteins and from formin homology 2 domain-containing protein 1 (FHOD-1) formin bundling. Our work thus identifies an essential molecular lock acting in a developmental ratchet-like process.

LiveFly: A Toolbox for the Analysis of Transcription Dynamics in Live Drosophila Embryos.

We present the LiveFly toolbox for quantitative analysis of transcription dynamics in live Drosophila embryos. The toolbox allows users to process two-color 3D confocal movies acquired using nuclei-labeling and the fluorescent RNA-tagging system described in the previous chapter and export the nuclei's position as a function of time, their lineages and the intensity traces of the active loci. The toolbox, which is tailored for the context of Drosophila early development, is semiautomatic, and requires minimal user intervention. It also includes a tool to combine data from multiple movies and visualize several features of the intensity traces and the expression pattern.

Alkyl-guanidine Compounds as Potent Broad-Spectrum Antibacterial Agents: Chemical Library Extension and Biological Characterization.

Nowadays, the increasing of multidrug-resistant pathogenic bacteria represents a serious threat to public health, and the lack of new antibiotics is becoming a global emergency. Therefore, research in antibacterial fields is urgently needed to expand the currently available arsenal of drugs. We have recently reported an alkyl-guanidine derivative (2), characterized by a symmetrical dimeric structure, as a good candidate for further developments, with a high antibacterial activity against both Gram-positive and Gram-negative strains. In this study, starting from its chemical scaffold, we synthesized a small library of analogues. Moreover, biological and in vitro pharmacokinetic characterizations were conducted on some selected derivatives, revealing notable properties: broad-spectrum profile, activity against resistant clinical isolates, and appreciable aqueous solubility. Interestingly, 2 seems neither to select for resistant strains nor to macroscopically alter the membranes, but further studies are required to determine the mode of action.

Precision of Readout at the hunchback Gene: Analyzing Short Transcription Time Traces in Living Fly Embryos.

The simultaneous expression of the hunchback gene in the numerous nuclei of the developing fly embryo gives us a unique opportunity to study how transcription is regulated in living organisms. A recently developed MS2-MCP technique for imaging nascent messenger RNA in living Drosophila embryos allows us to quantify the dynamics of the developmental transcription process. The initial measurement of the morphogens by the hunchback promoter takes place during very short cell cycles, not only giving each nucleus little time for a precise readout, but also resulting in short time traces of transcription. Additionally, the relationship between the measured signal and the promoter state depends on the molecular design of the reporting probe. We develop an analysis approach based on tailor made autocorrelation functions that overcomes the short trace problems and quantifies the dynamics of transcription initiation. Based on live imaging data, we identify signatures of bursty transcription initiation from the hunchback promoter. We show that the precision of the expression of the hunchback gene to measure its position along the anterior-posterior axis is low both at the boundary and in the anterior even at cycle 13, suggesting additional post-transcriptional averaging mechanisms to provide the precision observed in fixed embryos.

New methods to image transcription in living fly embryos: the insights so far, and the prospects.

The regulation of transcription is a fundamental process underlying the determination of cell identity and its maintenance during development. In the last decades, most of the transcription factors, which have to be expressed at the right place and at the right time for the proper development of the fly embryo, have been identified. However, mostly because of the lack of methods to visualize transcription as the embryo develops, their coordinated spatiotemporal dynamics remains largely unexplored. Efforts have been made to decipher the transcription process with single molecule resolution at the single cell level. Recently, the fluorescent labeling of nascent RNA in developing fly embryos allowed the direct visualization of ongoing transcription at single loci within each nucleus. Together with powerful imaging and quantitative data analysis, these new methods provide unprecedented insights into the temporal dynamics of the transcription process and its intrinsic noise. Focusing on the Drosophila embryo, we discuss how the detection of single RNA molecules enhanced our comprehension of the transcription process and we outline the potential next steps made possible by these new imaging tools. In combination with genetics and theoretical analysis, these new imaging methods will aid the search for the mechanisms responsible for the robustness of development. For further resources related to this article, please visit the WIREs website.

Transcriptional Memory in the Drosophila Embryo.

Transmission of active transcriptional states from mother to daughter cells has the potential to foster precision in the gene expression programs underlying development. Such transcriptional memory has been specifically proposed to promote rapid reactivation of complex gene expression profiles after successive mitoses in Drosophila development [1]. By monitoring transcription in living Drosophila embryos, we provide the first evidence for transcriptional memory in animal development. We specifically monitored the activities of stochastically expressed transgenes in order to distinguish active and inactive mother cells and the behaviors of their daughter nuclei after mitosis. Quantitative analyses reveal that there is a 4-fold higher probability for rapid reactivation after mitosis when the mother experienced transcription. Moreover, memory nuclei activate transcription twice as fast as neighboring inactive mothers, thus leading to augmented levels of gene expression. We propose that transcriptional memory is a mechanism of precision, which helps coordinate gene activity during embryogenesis.

Live imaging of bicoid-dependent transcription in Drosophila embryos.

The early Drosophila embryo is an ideal model to understand the transcriptional regulation of well-defined patterns of gene expression in a developing organism. In this system, snapshots of transcription measurements obtained by RNA FISH on fixed samples cannot provide the temporal resolution needed to distinguish spatial heterogeneity from inherent noise. Here, we used the MS2-MCP system to visualize in living embryos nascent transcripts expressed from the canonical hunchback (hb) promoter under the control of Bicoid (Bcd). The hb-MS2 reporter is expressed as synchronously as endogenous hb in the anterior half of the embryo, but unlike hb it is also active in the posterior, though more heterogeneously and more transiently than in the anterior. The length and intensity of active transcription periods in the anterior are strongly reduced in absence of Bcd, whereas posterior ones are mostly Bcd independent. This posterior noisy signal decreases progressively through nuclear divisions, so that the MS2 reporter expression mimics the known anterior hb pattern at cellularization. We propose that the establishment of the hb pattern relies on Bcd-dependent lengthening of transcriptional activity periods in the anterior and may require two distinct repression mechanisms in the posterior.

Small-conductance Ca2+-activated K+ channels modulate action potential-induced Ca2+ transients in hippocampal neurons.

In hippocampal pyramidal neurons, voltage-gated Ca(2+) channels open in response to action potentials. This results in elevations in the intracellular concentration of Ca(2+) that are maximal in the proximal apical dendrites and decrease rapidly with distance from the soma. The control of these action potential-evoked Ca(2+) elevations is critical for the regulation of hippocampal neuronal activity. As part of Ca(2+) signaling microdomains, small-conductance Ca(2+)-activated K(+) (SK) channels have been shown to modulate the amplitude and duration of intracellular Ca(2+) signals by feedback regulation of synaptically activated Ca(2+) sources in small distal dendrites and dendritic spines, thus affecting synaptic plasticity in the hippocampus. In this study, we investigated the effect of the activation of SK channels on Ca(2+) transients specifically induced by action potentials in the proximal processes of hippocampal pyramidal neurons. Our results, obtained by using selective SK channel blockers and enhancers, show that SK channels act in a feedback loop, in which their activation by Ca(2+) entering mainly through L-type voltage-gated Ca(2+) channels leads to a reduction in the subsequent dendritic influx of Ca(2+). This underscores a new role of SK channels in the proximal apical dendrite of hippocampal pyramidal neurons.

Promising in Vitro anti-Alzheimer Properties for a Ruthenium(III) Complex.

Metal complexes represent today an attractive class of experimental anti-Alzheimer agents with the potential of blocking ß-amyloid 1-42 aggregation and scavenging its toxicity. Three representative ruthenium(III) complexes, namely NAMI A, KP1019, and PMRU20, were specifically evaluated to this end in an established in vitro model of AD relying on primary cortical neurons. Notably, PMRU20 turned out to be highly effective in protecting cortical neurons against Aß 1-42 toxicity, while the other tested ruthenium compounds were poorly active or even inactive; we also found that PMRU20 is virtually devoid of any significant toxicity in vitro at the applied concentrations. Interestingly, PMRU20 was neuroprotective even against the toxicity induced by Aß 25-35. The direct reaction of PMRU20 with Aß 1-42 was explored through ESI MS analysis and some adduct formation evidenced. In addition, thioflavin T assays revealed that PMRU20 greatly reduces Aß 1-42 aggregation. The implications of these findings are discussed in relation to emerging treatment strategies for the Alzheimer's disease.

Analysis of skeletal status by quantitative ultrasonometry in a cohort of postmenopausal women with high blood cholesterol without documented osteoporosis.

Osteoporosis and atherosclerosis are leading causes of mortality and morbidity in the Western world. A link between osteoporosis and atherosclerosis was proposed by epidemiologic and laboratory data. In the present study, we investigated skeletal status in postmenopausal women with hypercholesterolemia using quantitative ultrasonometry (QUS). Six hundred healthy postmenopausal subjects were enrolled within a 2-mo period by primary care physicians. Information on lifestyle and calcium intake was collected for each enrolled subject. Subjects (n = 256) were divided into two groups according to lipid profile: normal (n = 180) with serum cholesterol <200 mg/dL and hypercholesterolemic (n = 76) with serum cholesterol >or=200 mg/dL. Hypercholesterolemic subjects were further stratified into two groups, one receiving dietary treatment (n = 34) and the other receiving statin treatment (n = 42). We found a statistically significant reduction in amplitude dependent speed of sound (AD-SoS) in hypercholesterolemic subjects compared with subjects with normal cholesterol (p = 0.006). Calcium intake behaved similarly to AD-SoS (p = 0.0001). No statistical significant difference in AD-SoS were observed between the group on diet treatment versus the group on statin (p = 0.52). Calcium intake was lower in patients on statins treatment compared with subjects on diet treatment only (p < 0.0001). Our data suggest that hypercholesterolemia per se is a risk factor for impaired skeletal status. Our data also call attention to the risk of a poor calcium intake in patient receiving diet to lower plasma cholesterol. Moreover, our data suggest that statins per se may exert a protective effect on bone independently from calcium intake.

Molecular cloning and characterization of a novel human variant of RIC-3, a putative chaperone of nicotinic acetylcholine receptors.

Recent reports demonstrate that the RIC-3 (resistant to inhibitors of cholinesterase-3) protein is important for the maturation of nAChRs (nicotinic acetylcholine receptors). In the present study RIC-3e, a novel variant of RIC-3, is described. This variant contains a deletion of exons 4 and 5 of RIC-3, resulting in a protein product lacking a conserved coiled-coil domain. Like RIC-3, the new variant is predominantly, but not exclusively, expressed in the brain. The analysis of expression of variant RIC-3 mRNA and of alpha7-nAChR mRNA in a set of human tissues shows a similar profile. The RIC-3e protein is functionally active and enables surface expression of mature alpha7-nAChRs in cell lines not otherwise permissive for the expression of this receptor.

An amino acid outside the pore region influences apamin sensitivity in small conductance Ca2+-activated K+ channels.

Small conductance calcium-activated potassium channels (SK, K(Ca)) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity.

Impact of occult hepatitis B virus infection in HIV patients naive for antiretroviral therapy.

OBJECTIVE: To study the impact of occult hepatitis B virus (HBV) infection in 115 consecutive anti-HIV-positive, hepatitis B surface antigen-negative patients, naive for antiretroviral treatment. METHODS: Of these 115, 86 patients were followed for at least 6 months (range 6-36) with serial determinations of HIV RNA and HBV DNA by polymerase chain reaction and other laboratory tests. RESULTS: Of the 86 patients having a follow-up, plasma HBV DNA was detected in 17 (19.8%), 13 on admission and four during follow-up. HBV DNA was more frequently found in patients with isolated anti-hepatitis B core (HBc; 35.5% of 31 cases) than in those lacking anti-HBc and anti-hepatitis B surface (8.8% of 41, P < 0.005), or showing both (21.4% of 14). Twenty-eight patients (32.5%) experienced a hepatic flare during the follow-up; this event was more frequent in the 17 HBV-DNA-positive patients than in the 69 negative (64.7% versus 24.6%, P < 0.005). Of the 13 HBV-DNA-positive patients on admission, 11 receiving HAART containing lamivudine became HBV-DNA negative, but two of these again became positive and experienced a hepatic flare during treatment and two both during and after lamivudine treatment. A hepatic flare also occurred under lamivudine treatment in two of the four patients in whom HBV DNA became detectable during follow-up. The role of immune reconstitution inflammatory syndrome and HAART in inducing a hepatic flare was found to be marginal in 49 patients with no HBV or hepatitis C virus marker. CONCLUSION: The study suggests that HBV occult infection, relatively frequent in anti-HIV-positive patients, is associated with hepatic flares.

The management of hepatitis B virus/HIV-1 co-infected patients starting their first HAART regimen. Treating two infections for the price of one drug?

We examined the impact of a lamivudine-containing highly active antiretroviral therapy (HAART) regimen on 164 hepatitis B virus/HIV co-infected individuals starting their first HAART. Lamivudine-treated patients (accounting for 73% of the study population) showed a significantly lower level of alanine aminotransferase over follow-up [-81.1 mU/ml mean difference; 95% confidence intervals (95% CI): -30.3; -131.7, P=0.003] and a significantly reduced risk of liver-related morbidity/mortality [Relative hazard (RH)=0.07; 95% CI: 0.01-0.38, P=0.002] than those starting a lamivudine sparing-regimen.

Functional characterization of WNT7A signaling in PC12 cells: interaction with A FZD5 x LRP6 receptor complex and modulation by Dickkopf proteins.

WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased beta-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5.LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.