Self-organized partitioning of dynamically localized proteins in bacterial cell division

The Min proteins are equally partitioned between daughter cells at division.The mechanism allowing this accurate distribution is intrinsic to the Min system.Individual oscillations appear in each daughter cell before cytokinesis is completed.Diffusion through the gradually constricting septum is key to this process

Go in! Go out! Inducible control of nuclear localization

Cells have evolved a variety of mechanisms to regulate the enormous complexity of processes taking place inside them. One mechanism consists in tightly controlling the localization of macromolecules, keeping them away from their place of action until needed. Since a large fraction of the cellular response to external stimuli is mediated by gene expression, it is not surprising that transcriptional regulators are often subject to stimulus-induced nuclear import or export. Here we review recent methods in chemical biology and optogenetics for controlling the nuclear localization of proteins of interest inside living cells. These methods allow researchers to regulate protein activity with exquisite spatiotemporal control, and open up new possibilities for studying the roles of proteins in a broad array of cellular processes and biological functions

Engineering a synthetic p53-Mdm2 network in budding yeast

p53 is among the most thoroughly studied proteins to date. Its tumour suppressor activity and the consideration that inactivation of the p53 pathway is a common, if not universal, feature of all human cancers, have gained it the interest of a multitude of researches seeking new therapies against this disease. As a consequence, p53 is at the centre of a feverish research and nowadays is reported to be involved in most cellular processes. Despite the vast amount of data published, much is yet to be unravelled about the mechanisms regulating the p53 signalling network. Indeed, the p53 network is extremely intricate and complex, and from a system biology point of view it can be seen as a series of interconnected negative and positive feedback loops, which can give rise to complex dynamics such as oscillations. In the combined effort to understand more of the biological meaning of these oscillations and to study the properties of this network motif from an engineering perspective, a synthetic p53 network has been built in budding yeast with the aim of studying the network in isolation while being embedded in living cells. p53 and most proteins in the network are absent from the budding yeast genome. This diminishes the likelihood of interferences on the engineered module from the cellular environment. Surprisingly, despite the evolutionary conservation of the ubiquitin pathway from yeast to humans, p53 ubiquitylation by the E3 ubiquitin ligase Mdm2 - an event central to the oscillatory dynamics of the system - does not appear to take place in budding yeast, even when the human E2 enzyme UbcH5B is exogenously expressed. p53 is instead sumoylated by Mdm2 and sumoylation dictates the co-localization of p53 and Mdm2 to a nuclear body reminiscent of human PML bodies. In conclusion, attempting to rebuild from scratch a simplified version of the intricate p53 network, isolating it from its natural context, has proven to be a very powerful means leading to unexpected findings, testifying the usefulness of the synthetic biology approach

Ultrafast processes triggered by one- and two-photon excitation of a photochromic and luminescent hydrazone

In this work we apply a combination of steady state and time resolved luminescence and absorption spectroscopies to investigate the excited-state dynamics of a recently developed molecular photoswitch, belonging to the hydrazone family. The outstanding properties of this molecule, involving fluorescence toggling, bistability, high isomerization quantum yield and non-negligible two-photon absorption cross section, make it very promising for numerous applications. Here we show that the light induced Z/E isomerization occurs on a fast <1 ps timescale in both toluene and acetonitrile, while the excited state lifetime of the Z-form depends on solvent polarity, suggesting a partial charge transfer nature of its low lying excited state. Time-resolved luminescence measurements evidence the presence of a main emission component in the 500\u2013520 nm spectral range, attributed to the Z-isomer, and a very short living blue-shifted emission, attributed to the E-isomer. Finally, transient absorption measurements performed upon far-red excitation are employed as an alternative method to determine the two-photon absorption cross-section of the molecule

Brucella abortus Strain RB51 Vaccine: Immune Response after Calfhood Vaccination and Field Investigation in Italian Cattle Population

Immune response to Brucella abortus strain RB51 vaccine was measured in cattle vaccinated at calfhood. After an increase at day 6 post-vaccination (pv), the antibody level recorded in the 10 vaccinated animals remained constant for two months, and then progressively decreased. All vaccinated animals remained negative from day 162 pv to the end of the study (day 300 pv). Only at days 13 and 14 pv the RB51-CFT showed 100% sensitivity (credibility interval (CI) 76.2%–100%). The results indicate that the possibility to use RB51-CFT for the identification of cattle vaccinated at calfhood with RB51 is limited in time. A field investigation was carried out on 26,975 sera collected on regional basis from the Italian cattle population. The study outcomes indicate that in case of RB51-CFT positive results observed in officially Brucellosis-free (OBF) areas and, in any case, when an illegal use of RB51 vaccine is suspected, the use of the RB51-CFT alone is not sufficient to identify all the vaccinated animals. The design of a more sophisticated diagnostic protocol including an epidemiological investigation, the use of RB51-CFT, and the use of the skin test with RB51 as antigen is deemed more appropriate for the identification of RB51 vaccinated animals

Exact simulation of hybrid stochastic and deterministic models for biochemical systems

Motivation : Over the last years it has become evident that stochastic effects play an important role in biological processes leading to an increase in stochastic modelling attempts. Despite the availability of exact algorithms to numerically solve the chemical master equation that entirely describes a stochastic system, stochastic simulations are most of the times very computationally expensive. Hybrid methods that treat some processes as in the deterministic framework and others as stochastic are a promising way to speed up simulations for those cases involving different time scales, e.g., systems integrating metabolic pathways and gene regulatory networks

Antioxidant and Anti-Inflammaging Ability of Prune (Prunus Spinosa L.) Extract Result in Improved Wound Healing Efficacy

none12sìPrunus spinosa L. fruit (PSF) ethanol extract, showing a peculiar content of biologically active molecules (polyphenols), was investigated for its wound healing capacity, a typical feature that declines during aging and is negatively affected by the persistence of inflammation and oxidative stress. To this aim, first, PSF anti-inflammatory properties were tested on young and senescent LPS-treated human umbilical vein endothelial cells (HUVECs). As a result, PSF treatment increased miR-146a and decreased IRAK-1 and IL-6 expression levels. In addition, the PSF antioxidant effect was validated in vitro with DPPH assay and confirmed by in vivo treatments in C. elegans. Our findings showed beneficial effects on worms’ lifespan and healthspan with positive outcomes on longevity markers (i.e., miR-124 upregulation and miR-39 downregulation) as well. The PSF effect on wound healing was tested using the same cells and experimental conditions employed to investigate PSF antioxidant and anti-inflammaging ability. PSF treatment resulted in a significant improvement of wound healing closure (ca. 70%), through cell migration, both in young and older cells, associated to a downregulation of inflammation markers. In conclusion, PSF extract antioxidant and antiinflammaging abilities result in improved wound healing capacity, thus suggesting that PSF might be helpful to improve the quality of life for its beneficial health effects.openSofia Coppari, Mariastella Colomba, Daniele Fraternale, Vanessa Brinkmann, Margherita Romeo, Marco Bruno Luigi Rocchi, Barbara Di Giacomo, Michele Mari, Loretta Guidi, Seeram Ramakrishna, Natascia Ventura, Maria Cristina AlbertiniCoppari, Sofia; Colomba, Mariastella; Fraternale, Daniele; Brinkmann, Vanessa; Romeo, Margherita; Rocchi, MARCO BRUNO LUIGI; DI GIACOMO, Barbara; Mari, Michele; Guidi, Loretta; Ramakrishna, Seeram; Ventura, Natascia; Albertini, MARIA CRISTIN

Assessment of Plasmodium Falciparum RNA Pseudouridylate Synthase (Putative) as Novel Drug Target

Malaria is a major public health problem associated with high mortality, morbidity rates and undue economic burden in sub-Saharan countries. Presently, every year, 300 to 500 million people suffer clinically from malaria and 90% of them in sub-Saharan Africa. About 1.5 to 3 million people die of malaria every year and 85% of these occur in Africa. One child dies of malaria somewhere in Africa every 20 second, and there is one malarial death every 12 sec somewhere in the world. This is also a damaging economic burden for these sub-Saharan Africa countries as huge work force time and resources are expended for treatment. Plasmodium falciparum (hence forth Pf) is the most severe of all the human malaria parasites. This organism is continuing to develop resistance to all known drugs and therapeutic regime. One of the mechanisms of resistance in Pf is the modification of the drug target. Hence, it is expedient to continuously discover novel drug targets in Pf and to discover or develop new drugs against such targets. Drug-able signaling pathways have been shown to have inherent mechanism capable of deterring drug resistance. Using computational techniques, we have identified some proteins in the signaling pathways of Pf as putative targets for anti-plasmodia drug. RNA pseudouridylate synthase, which also plays a key role in RNA synthesis and ribosomal function, is one of such proteins. Initial virtual screening of this enzyme against drug and chemical databases has been performed to identify compounds that can inhibit this enzyme. This led us to compounds which inhibit nucleotide metabolism. This is a work in progress whose current state is hoped for presentation at this conference. In order to determine the identified compounds IC50, the identified compounds will be screened in vitro against the enzyme. We have currently completed the establishment of the enzyme functionally expression in E. coli and purification. Thereafter, the drugs will be screened for their anti-plasmodia activity using cultured Pf and the IC50 for each drug will be determined. In order to assess their safety, the selectivity index of compounds that showed in vitro anti-plasmodia activity will be determined using human cultured cell lines. The last stage of this study will involve screening the compounds in an in vivo mouse model of malaria. It is hoped that the result of this study will prove this enzyme as a novel target for antimalarial drug. And provide as input, critical drug targets in to our established Structure Based Drug Design (SBDD) pipelin

BBF RFC 105: The Intein standard - a universal way to modify proteins after translation

This Request for Comments (RFC) proposes a new standard that allows for easy and flexible cloning of intein constructs and thus makes this technology accessible to the synthetic biology community

Standard for Synthesis of Customized Peptides by Non-Ribosomal Peptide Synthetases

The purpose of this RFC is to introduce a standardized framework for the engineering of customizable non-ribosomal peptide synthetases (NRPS) and their application for in vivo and in vitro synthesis of short non-ribosomal peptides (NRPs) of user-defined sequence and structure