Riboswitches as targets for metabolic engineering in Bacillus subtilis

Many metabolic pathways in bacteria are modulated by metabolite-sensing riboswitches, which regulate gene expression at the level of transcription elongation or translation initiation. Riboswitches represent promising targets to modulate expression of genes and operons relevant for the biotechnological production of commercially relevant compounds. In Firmicutes, approximately 70% of all putative and validated riboswitches (are predicted to) act exclusively at the transcriptional level using a termination-antitermination mechanism. In a first attempt to interfere with purine-sensing riboswitches and deregulate purine metabolism in Bacillus subtilis, a set of synthetic small RNAs (sRNAs) targeting the purine-sensing aptamers were designed to impair ligand binding using rational design combined with in silico evolution. However, the designed sRNAs did not show any activity in vivo on the riboswitch controlling purine biosynthesis (pur operon riboswitch). The effect of the antisense RNA (asRNA) perfectly complementary to the aptamer of the pur operon riboswitch was also tested; The asRNA did not affect negatively expression of a riboswitch-regulated lacZ gene, yet similarly to the partially complementary sRNAs, the asRNA did not impair the downregulation exerted by the riboswitch in the presence of ligand. Finally, expression of the small RNAs in B. subtiliswas quantified, and the kinetic limitations for their hybridization with the aptamer and their competition with the ligand are discussed. A second metabolic engineering strategy based on editing the genome of B. subtilis with regard to transcriptional riboswitches was investigated. Removal of the riboswitches that control purine biosynthesis and riboflavin biosynthesis in B. subtilis led to auxotrophic strains. As an alternative, a rational approach was developed for engineering transcriptional riboswitches independently from the availability of their 3D structures. This approach consists in the identification and deletion of a key nucleotide sequence exclusively involved in transcription termination without affecting formation of other secondary and tertiary structures potentially involved in other roles. To demonstrate the efficacy of the approach, it was applied to derepress the purine and the riboflavin biosynthetic pathways in B. subtilis. Following the proof of concept using specialized reporter strains, the approach was implemented into a B. subtilis wild-type strain employing CRISPR-Cas genome editing. The CRISPRCas9 system displayed an efficiency of 61% in editing the genome, and the resulting purine and riboflavin production strains were characterized at the level of gene expression, metabolite synthesis, and growth. With a substantial enhancement observed at each level, the strategy established here represents a powerful tool for deregulating pathways modulated by transcriptional riboswitches. Finally, applying this strategy to derepress the purine pathway of an industrial riboflavin overproducing strain, with impaired growth, led to an increase in biomass by 53% and resulted in an enhanced total production of riboflavin in the culture

An Investigation into the Physical-Mechanical Indices of Igneous Rocks and Their Aggregates

The Present investigation is a study of the physical and mechanical properties of a suite of igneous rocks and aggregates derived from them, with special emphasis on their geological nature and the post emplacement processes which affects them. The study concentrated on: i- Petrographical characteristics. ii- Weathering effects and characterisation. iii- Index properties for intact and aggregate rock strength. iv- Los Angeles Abrasion Value, methodological and geological factors affecting values. v- Correlation between index properties for intact rock and aggregate. The rock suite ranges from volcanic to plutonic, basic to acid in composition and fresh to completely weathered. This provided an opportunity for testing and evaluating various textural and weathering variables. Weathering which systematically affects the engineering properties of rocks, can be quantified by secondary mineral content or alternatively the well established micropetrographic index (Ip). It can also be quantified by physical indices such as specific gravity, porosity and water absorption or mechanical indices such as Rebound Number and Point Load Strength. Weathering and other geological variables such as grain size and texture are prominent factors affecting the strength of both intact and aggregate strength values. An investigation into the Los Angeles Abrasion Value (LAAV) and the factors which affect it established that test results are as systematic and rational as the other recognised strength tests. Aggregate Impact (AIV) and Crushing Values (ACV). In the Los Angeles Abrasion Test It was demonstrated that the mechanism of comminution is dominantly one of impact loading (80 %) with a minor component of attrition (20%). The LAAV is consistently affected by geological variables such as grain size, texture, clast shape and degree of weathering in a manner similar to that established for AIV and ACV. Intact and aggregate strength indices are related in a simple manner and provided geological and methodological variables are known and evaluated, aggregate strength indices can be predictable from intact rock properties

Swelling Clay Parameters Investigation Using Design of Experiments (A Case Study)

The present paper aims to investigate geotechnical parametric effects on the expansion behavior of clayey soils in Tebessa province northeast of Algeria using the Design Of Experiments (DOE) methodology. It has been used as powerful tools based on physical and mechanical properties, data results obtained within laboratory soil mechanics testing. This statistical tool methodology presents the factor screening design to determine the effect of different parameters such as dry unit weight, saturation degree, water content, plasticity index, etc., on the swelling pressure parameter which can be used as expansion behavior of clay indicator. All data previously collected in the studied prone area allows the ability of detailed analysis using design of experiment and parametric optimization process with response surface methodology (RSM). Each variable that present effects on swelling pressure is also discussed. Besides, the obtained models and equations related the factors affecting the expansion process have been determined. At the output process; the response desirability of the screening design methodology can be optimized by maximization or minimization of the optimal values affecting the swelling behavior. This process allows us to find the best describing models, whereas output results may be compared to empirical laboratory tests results to assess the RSM models

Breeding biology of the White-headed Duck Oxyura leucocephala at Lake Tonga (Algeria)

The study of the reproductive phenology of the White-headed Duck was carried out at Lake Tonga, a wetland of international importance for the nesting of this species across the Mediterranean basin. The following breeding parameters have been studied in 2007 and 2008 : dates of laying and hatching, clutch size and breeding success. The breeding population was estimated at 78 males and 26 females in 2007 and at 71 males and 22 females in 2008. Laying is recorded during a period of 9 to 11 weeks from late April to mid-July. A brood size of 3.2 ± 1.6 young (n = 17) was recorded in the first year of study and another of 3.9 ± 1.4 young (n = 37) in the second one. Young fledged between late June and early September. The data obtained in this study suggest that the White-headed Duck may be considered as a bird almost constant in terms of life-history traits in AlgeriaLa phénologie de la reproduction de l'Érismature à tête blanche Oxyura leucocephala a été étudiée au lac Tonga, Parc national d'El Kala (Algérie), une zone humide d'importance internationale pour la nidification de l'espèce à l'échelle du bassin Méditerranéen. Les paramètres de la reproduction étudiés durant les années 2007 et 2008 furent les dates et périodes de ponte, la grandeur des nichées, le succès de reproduction et l'envol des jeunes. La population reproductrice a été estimée à 78 mâles et 26 femelles en 2007 et à 71 mâles et 22 femelles en 2008. La ponte s'étale de fin avril à la mi-juillet, sur une période allant de 9 à 11 semaines. La taille des nichées enregistrées fut de 3,2 ± 1,6 canetons (n = l7) pour la première année d'étude et de 3,9 ± 1,4 (n = 37) pour la seconde. L'envol des jeunes a lieu entre fin juin et début septembre. Les données obtenues au cours de cette étude suggèrent que l'Érismature à tête blanche est un oiseau relativement constant en termes de caractéristiques liées à la reproduction en Algérie

Changes of the breeding population of White-headed Duck Oxyura leucocephala at Lake Tonga (Algeria)

The White-headed Duck Oxyura leucocephala is a species listed in the IUCN Red List as threatened (Endangered), it is also protected by the Algerian law as at risk of extinction. Based on weekly surveys of adults, males and females, and nests (or families) on the Ramsar site of Lake Tonga, the most important nesting site of the species in southern Mediterranean, our work aims at checking the present status of this population in order to develop a national action plan for its preservation. The observations of adults and families during pre-breeding and breeding periods, conducted each year in 2006, 2007 and 2008, show a clear increase of the population relative to existing data on this site obtained in 1991-1992L'Érismature à tête blanche Oxyura leucocephala est une espèce classée dans la liste rouge de l'UICN comme menacée, elle est également protégée par la législation algérienne en tant qu'espèce menacée de disparition. Basé sur les recensements hebdomadaires des adultes, mâles et femelles, et des nichées (ou familles) sur le site Ramsar du Lac Tonga, site de nidification le plus important de l'espèce sur la rive sud de Méditerranée, notre travail cherche à actualiser l'état de cette population afin d'élaborer un plan d'action national pour sa sauvegarde. Les observations sur les adultes et les nichées (ou familles) en période de pré-nidification et de nidification, effectuées chaque année en 2006, 2007 et 2008, mettent en évidence une nette augmentation des effectifs par rapport aux données déjà obtenues sur ce site en 1991-199

Longitudinal mouse-PET imaging: a reliable method for estimating binding parameters without a reference region or blood sampling

International audienceLongitudinal mouse PET imaging is becoming increasingly popular due to the large number of transgenic and disease models available but faces challenges. These challenges are related to the small size of the mouse brain and the limited spatial resolution of microPET scanners, along with the small blood volume making arterial blood sampling challenging and impossible for longitudinal studies. The ability to extract an input function directly from the image would be useful for quantification in longitudinal small animal studies where there is no true reference region available such as TSPO imaging.METHODS:Using dynamic, whole-body 18F-DPA-714 PET scans (60 min) in a mouse model of hippocampal sclerosis, we applied a factor analysis (FA) approach to extract an image-derived input function (IDIF). This mouse-specific IDIF was then used for 4D-resolution recovery and denoising (4D-RRD) that outputs a dynamic image with better spatial resolution and noise properties, and a map of the total volume of distribution (VT) was obtained using a basis function approach in a total of 9 mice with 4 longitudinal PET scans each. We also calculated percent injected dose (%ID) with and without 4D-RRD. The VT and %ID parameters were compared to quantified ex vivo autoradiography using regional correlations of the specific binding from autoradiography against VT and %ID parameters.RESULTS:The peaks of the IDIFs were strongly correlated with the injected dose (Pearson R = 0.79). The regional correlations between the %ID estimates and autoradiography were R = 0.53 without 4D-RRD and 0.72 with 4D-RRD over all mice and scans. The regional correlations between the VT estimates and autoradiography were R = 0.66 without 4D-RRD and 0.79 with application of 4D-RRD over all mice and scans.CONCLUSION:We present a FA approach for IDIF extraction which is robust, reproducible and can be used in quantification methods for resolution recovery, denoising and parameter estimation. We demonstrated that the proposed quantification method yields parameter estimates closer to ex vivo measurements than semi-quantitative methods such as %ID and is immune to tracer binding in tissue unlike reference tissue methods. This approach allows for accurate quantification in longitudinal PET studies in mice while avoiding repeated blood sampling

The SIGMA rat brain templates and atlases for multimodal MRI data analysis and visualization

Preclinical imaging studies offer a unique access to the rat brain, allowing investigations that go beyond what is possible in human studies. Unfortunately, these techniques still suffer from a lack of dedicated and standardized neuroimaging tools, namely brain templates and descriptive atlases. Here, we present two rat brain MRI templates and their associated gray matter, white matter and cerebrospinal fluid probability maps, generated from ex vivo [Formula: see text]-weighted images (90 µm isotropic resolution) and in vivo T2-weighted images (150 µm isotropic resolution). In association with these templates, we also provide both anatomical and functional 3D brain atlases, respectively derived from the merging of the Waxholm and Tohoku atlases, and analysis of resting-state functional MRI data. Finally, we propose a complete set of preclinical MRI reference resources, compatible with common neuroimaging software, for the investigation of rat brain structures and functions.This work is part of the SIGMA project with the reference FCT-ANR/NEU-OSD/0258/2012, co-financed by the French public funding agency ANR (Agence Nationale pour laRecherche, APP Blanc International II 2012), the Portuguese FCT (Fundação para aCiência e Tecnologia) and the Portuguese North Regional Operational Program (ON.2—O Novo Norte) under the National Strategic Reference Framework (QREN), through theEuropean Regional Development Fund (FEDER) as well as the Projecto Estratégico co-funded by FCT (PEst-C/SAU/LA0026-/2013) and the European Regional DevelopmentFund COMPETE (FCOMP-01-0124-FEDER-037298). D.A.B. and A.N. were funded bygrants from FCT-ANR/NEU-OSD/0258/2012. R.M. was supported by the FCT fellow-ship grant with the reference PDE/BDE/113604/2015 from the PhDiHES program. A.C.was supported by a grant from the foundation NRJ. P.M. was funded by FundaçãoCalouste Gulbenkian (Portugal;‘Better mental health during ageing based on temporalprediction of individual brain ageing trajectories TEMPO’) with Grant Number P-139977. France Life Imaging is acknowledged for its support in funding the NeuroSpinplatform of preclinical MRI scanners. The authors also acknowledge and thank EdwardGanz, MD, for proof reading our work