Moderate Neonatal Stress Decreases Within-Group Variation in Behavioral, Immune and HPA Responses in Adult Mice

BACKGROUND: The significance of behavioral neuroscience and the validity of its animal models of human pathology largely depend on the possibility to replicate a given finding across different laboratories. Under the present test and housing conditions, this axiom fails to resist the challenge of experimental validation. When several mouse strains are tested on highly standardized behavioral test batteries in different laboratories, significant strain x lab interactions are often detected. This limitation, predominantly due to elevated within-group variability observed in control subjects, increases the number of animals needed to address fine experimental questions. Laboratory rodents display abnormal stress and fear reactions to experimental testing, which might depend on the discrepancy between the stability of the neonatal environment and the challenging nature of the adult test and housing conditions. METHODOLOGY/PRINCIPAL FINDINGS: Stimulating neonatal environments (e.g. brief maternal separations, increased foraging demands or maternal corticosterone supplementation) reduce stress and fear responses in adulthood. Here we tested whether reduced fearfulness associated with experimental testing would also reduce inter-individual variation. In line with our predictions, we show that a moderate elevation in neonatal corticosterone through maternal milk significantly reduces fear responses and inter-individual variability (average 44%) in adult mouse offspring. CONCLUSIONS/SIGNIFICANCE: We observed reduced variation in pain perception, novelty preference, hormonal stress response and resistance to pathogen infection. This suggests that the results of this study may apply to a relatively broad spectrum of neuro-behavioral domains. Present findings encourage a reconsideration of the basic principles of neonatal housing systems to improve the validity of experimental models and reduce the number of animals used

A Web-based Software System for Behavior Analysis of Laboratory Animals

The analysis of locomotion in laboratory animals plays a crucial role in many scientific research areas. In fact, important information on animals’ behavior and their reaction to a particular stimulus is deduced from a careful analysis of their movements. The techniques commonly adopted to support such analysis have many limitations, which make the related systems particularly ineffective. On the one hand, the human observation and annotation process is strongly observer-dependent and expensive in terms of time and efforts. On the other hand, the use of more sophisticated systems based on video recordings and recognition algorithms is very expensive and complex. In order to face this challenge, this paper presents a tracking solution based on passive Radio Frequency Identification (RFID) technology in Ultra High Frequency (UHF) band, allowing the tracking of laboratory animals with a high accuracy. The overall solution consists of a hybrid system including hardware and software components. In particular, in this paper, the attention is focused on the software component as the hardware has already been described in previous works. The software component is a Web-oriented solution that offers a complete 2D and 3D information tool including reports, dashboards, and tracking graphs. The proposed solution was widely tested using twelve laboratory mice and compared with an automated video-tracking software (i.e., EthoVision) in order to demonstrate its effectiveness and reliability. The obtained results have demonstrated that the proposed solution is able to correctly detect and reconstruct the events occurring in the animals’ cage, and to offer a complete and user-friendly tool to support researchers in behavioral analysis of small laboratory animals

Evaluation of the analgesic effect of 4-anilidopiperidine scaffold containing ureas and carbamates

Fentanyl is a powerful opiate analgesic typically used for the treatment of severe and chronic pain, but its prescription is strongly limited by the well-documented side-effects. Different approaches have been applied to develop strong analgesic drugs with reduced pharmacologic side-effects. One of the most promising is the design of multitarget drugs. In this paper we report the synthesis, characterization and biological evaluation of twelve new 4-anilidopiperidine (fentanyl analogues). In vivo hot-Plate test, shows a moderate antinociceptive activity for compounds OMDM585 and OMDM586, despite the weak binding affinity on both μ and δ-opioid receptors. A strong inverse agonist activity in the GTP-binding assay was revealed suggesting the involvement of alternative systems in the brain. Fatty acid amide hydrolase inhibition was evaluated, together with binding assays of cannabinoid receptors. We can conclude that compounds OMDM585 and 586 are capable to elicit antinociception due to their multitarget activity on different systems involved in pain modulation. © 2016 Informa UK Limited, trading as Taylor & Francis Group

Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands

ToxT promoter recognition by ToxR transcription factor, a co-activator within the Vibrio cholerae virulence cascade

[eng] Cholera is an acute diarrheal infection caused by the bacterium Vibrio cholerae. An estimated 2.9 million of cases and about 100000 cholera deaths occur annually all over the world. Upon ingestion of the V. cholerae, the bacterium travels to small intestine where it colonizes and produces the cholera toxin. Cholera toxin raises intracellular cyclic AMP and leads to chloride secretion and the subsequent secretory diarrhoea. Cholera toxin production is regulated through the master virulence regulator ToxT. Trascriptional activation of toxT is activated by membrane-localized ToxR, in association with another membrane protein named TcpP. Both ToxR and TcpP work as a two-component regulatory system merged in single proteins: they receive an external signal through its periplasmic C-terminal domain and bind to the toxT promoter by their cytoplasmic N-terminal domains. This project thesis aims at characterizing part of the system by studying ToxR-DNA complexes, since two ToxR molecules are supposed to bind the promoter to recruit TcpP and hence the RNA polymerase for transcription activation. Using X-ray crystallography, we have solved the structure of three complexes of the ToxR DNA binding domain with 20-bp, 40-bp and 25-bp oligonucleotides at 2.0 A, 2.6 A and 3.2 A resolution, respectively. According to the three structures, ToxR is able to bind to an extensive region of the toxT promoter that goes from the position -97 to the position -45. Considering an integrated model of the three structures, there are four ToxR molecules binding the toxT promoter: two molecules bind the DNA in tandem, one molecule binds the ToxR degenerate box and the last one is binding what is supposed to be the TcpP binding site. The structure determination of the three complexes is important to define with more accuracy the ToxR binding site in the toxT promoter. This site is characterized by eleven bases with a high A-T rich region sequence followed by a CATA/CATG/TGTA box, where the last two bases perform direct and specific contacts with the protein. In the three structures, ToxR shows a winged helix-turn-helix (w-HTH) fold. The wing interacts with the minor groove in an A-T rich region sequence while the recognition helix enters in the major groove at the region with a sequence corresponding to a CATA box. We have compared ToxR with the other w-HTH family proteins and we have found a new structural element, the secondary wing, which displays interactions with the DNA. We have analyzed the protein-DNA contacts in the three structures, and also the protein-protein interactions in the ToxR-DNA40 structure, thus validating the data published on ToxR defective mutations. Finally, we put forward a model of toxT promoter activation at molecular level, based on our crystal structures and on what is known in literature and from our collaborators. We propose that ToxR acts as co-activator in the first steps of toxT transcription activation at different levels. First, it would capture the DNA and hold it close to the cytoplasmic membrane, since both ToxR and TcpP are membrane proteins. Second, it would play a key-role in relieving H-NS from the toxT promoter: H-NS binds the DNA and transcription is repressed, but ToxR is able to replace it in a region that goes from the position -97 to the position -45. Third, ToxR would not be recruiting the RNA polymerase directly, but creating the suitable conditions for the action of TcpP. ToxR recruits TcpP probably through a hand-holding mechanism since one of the ToxR binding site is very close to the TcpP's binding site.[spa] El cólera es una infección diarreica aguda causada por la bacteria Vibrio cholerae. La producción de la toxina colérica se controla a través del regulador maestro de virulencia ToxT, cuya activación se lleva a cabo por las proteínas de membrana ToxR y TcpP. Este proyecto de tesis tiene como objetivo el estudio de los complejos formados por ToxR junto con el ADN, dado que se conoce que ToxR se une al promotor toxT para reclutar TcpP y consecuentemente la ARN polimerasa, produciendo la activación de la transcripción. Mediante cristalografía de rayos X hemos resuelto la estructura de tres complejos del dominio de unión a ADN de ToxR con oligonucleótidos de 20, 40 y 25 pares de bases a resoluciones de 2.0 A, 2.6 A y 3.2 A, respectivamente. De acuerdo con las tres estructuras, ToxR es capaz de unirse a una amplia región del promotor toxT que se expande desde la posición -97 hasta la posición -45. Teniendo en cuenta el modelo integrado de las tres estructuras, cuatro moléculas de ToxR se unen al promotor toxT en tándem e invertidas. En las tres estructuras, ToxR muestra un tipo de plegamiento winged helix-turn-helix (w-HTH). El ala (wing) interactúa con el surco menor del ADN, mientras que la hélice de reconocimiento penetra en el surco mayor. Comparando ToxR con el resto de proteínas de la familia w-HTH, hemos encontrado un nuevo elemento estructural, el ala secundaria (secondary wing), que interacciona con el ADN. La determinación de la estructura de los tres complejos es importante para definir con mayor precisión el sitio de unión de ToxR en el promotor toxT. Este sitio se caracteriza por once bases con una secuencia rica en A-T seguida de una caja CATA/CATG/TGTA, donde las dos últimas bases contactan directamente y específicamente con la proteína. Proponemos que ToxR actuaría como co-activador de la transcripción de toxT a diferentes niveles: (i) podría ser responsable de capturar el ADN y mantenerlo cerca de la membrana citoplasmática, (ii) podría jugar un papel clave en el desplazamiento de H-NS, (iii) podría reclutar TcpP y estabilizar su interacción con el promotor

Toxt promoter recognition by toxr transcription factor, a co-activator within the vibrio cholerae virulence cascade

Peer Reviewe

Reporting epidemiology of antibiotic resistance

The aim of antimicrobial resistance surveillance is to monitor temporal trends and provide clinicians with data to define empirical treatment protocols. The surveillance methods adopted in different settings can be significantly different and, therefore, no reference can be made to a single set of standards. This paper outlines the main features of analysis and reporting of antimicrobial resistance data according to the guidelines issued by the US Clinical and Laboratory Standards Institute, and the surveillance systems adopted in Europe. In this article the strengths and weaknesses of the various types of analyses will be discussed highlighting the critical aspects to be taken into account in surveillance data reporting