The glutaminase-dependent acid resistance system. Qualitative and quantitative assays and analysis of its distribution in enteric bacteria

Neutralophilic bacteria have developed several strategies to overcome the deleterious effects of acid stress. In particular, the amino acid-dependent systems are widespread, with their activities overlapping, covering a rather large pH range, from 6 to <2. Recent reports showed that an acid resistance (AR) system relying on the amino acid glutamine (AR2_Q), the most readily available amino acid in the free form, is operative in Escherichia coli, Lactobacillus reuteri and some Brucella species. This system requires a glutaminase active at acidic pH and the antiporter GadC to import L-glutamine and export either glutamate (the glutamine deamination product) or GABA. The latter occurs when the deamination of glutamine to glutamate, via acid-glutaminase (YbaS/GlsA), is coupled to the decarboxylation of glutamate to GABA, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR (AR2) system, together with GadC. Taking into account that AR2_Q could be widespread in bacteria and that until now assays based on ammonium ion detection were typically employed, this work was undertaken with the aim to develop assays that allow a straightforward identification of the acid-glutaminase activity in permeabilised bacterial cells (qualitative assay) as well as a sensitive method (quantitative assay) to monitor in the pH range 2.5-4.0 the transport of the relevant amino acids in vivo. The qualitative assay is colorimetric, rapid and reliable and provides several additional information, such as co-occurrence of AR2 and AR2_Q in the same bacterial species and assessment of the growth conditions that support maximal expression of glutaminase at acidic pH. The quantitative assay is HPLC-based and allows to concomitantly measure the uptake of glutamine and the export of glutamate and/or GABA via GadC in vivo and depending on the external pH. Finally, an extensive bioinformatic genome analysis shows that the gene encoding the glutaminase involved in AR2_Q is often nearby or in operon arrangement with the genes coding for GadC and GadB. Overall, our results indicate that AR2_Q is likely to be of prominent importance in the AR of enteric bacteria and that it modulates the enzymatic as well as antiport activities depending on the imposed acidic stress

Assessment of retrofit measures to prevent progressive collapse in steel structures

Man-made hazards, such as fires, explosions, or impacts, may have severe social and economic consequences and, therefore, should be carefully considered during the design of new, as well as, during the retrofitting of existing structures. Among others, these events could induce the progressive collapse of structures, in which the localised failure spreads from the single affected structural component to other parts of the structure. It is important to highlight that most existing structures worldwide have been designed before the introduction of design rules against progressive collapse. Therefore, it is nowadays of paramount importance to identify effective retrofit measures to renovate existing structures and return safer buildings to the community, including explicit design considerations against progressive collapse. The present paper investigates the effectiveness of three different retrofit measures, namely roof-truss, bracing, and cable systems, conceived to increase the structural robustness and hence mitigate the progressive collapse risk in steel structures. A case study steel moment resisting frame (MRF) was studied by performing non-linear static analyses in OpenSees and investigating its response before and after retrofitting. The progressive collapse was simulated by considering central column loss scenarios, and the ability to prevent the spread of failures of the original and retrofitted structures was examined. The present study sheds some light on the effectiveness and limitations of the considered retrofit measures in improving the overall robustness of the frame. The results show that, after the column removal, the original configuration of the selected MRF fails due to column buckling. Therefore, only the roof-truss and bracings strategies effectively improve the frame’s robustness and allow the creation of alternative load paths. Additionally, some critical aspects to be carefully considered in the design of the retrofit measures are indicated

Dynamic increase factors for progressive collapse analysis of steel structures considering column buckling

Progressive or disproportionate collapse of structures may have severe socio-economic consequences. Aiming at buildings that can withstand such events, one solution is to prevent or minimize the propagation of damage that may lead to progressive collapse by means of robust design strategies. As a typical approach to model progressive collapse and assess robustness, the Alternate Path Method (APM) allows for static analyses, in which the dynamic effects induced by a sudden column loss are taken into account by amplifying loads through a Dynamic Increase Factor. Current recommendations for Dynamic Increase Factors to be used within non-linear static analyses have mainly considered beam-type collapse, overlooking other failure mechanisms, e.g., column buckling. The present paper investigates the dynamic effects of steel structures subjected to progressive collapse when buckling of columns is relevant. Five low- to high-rise case study building structures are considered together with three different column loss scenarios. A numerical procedure is introduced to evaluate the Dynamic Increase Factors considering two different Engineering Demand Parameters (EDPs), suited for describing beam- and column-type mechanisms, respectively. As Dynamic Increase Factors are typically assessed by increasing the loads on all the spans (DIF), a procedure was proposed for deriving factors that apply only on the spans above the removal (DIF*), consistently with UFC guidelines. The obtained DIF and DIF* are compared with the current literature and with values recommended in the UFC guidelines, highlighting the limits of current recommendations. Relevant considerations on the derived Dynamic Increase Factors and failure mechanisms involved are provided

Dynamic Increase Factors for progressive collapse anaylsis of steel structures accounting for column buckling

Man-made hazards, such as fire, explosions, or impacts, may induce the progressive collapse of structures, in which the localised failure spreads from the single affected structural component to other parts of the structure. A typical approach to model progressive collapse consists in performing static column removal analyses considering a Dynamic Increase Factor (DIF), whose determination becomes paramount to account for the dynamic effects related to a sudden column loss scenario. Current recommendations on the definition of such factor mainly consider a beam-type collapse in non-linear analyses, though different mechanisms, e.g., column buckling, may govern progressive collapse events. This paper presents the determination of the DIFs through a numerical procedure for five steel structures with an increasing number of storeys. Both global and local imperfections are modeled to account for the geometric non-linearities of the structure and column buckling. DIF values are obtained considering two different Engineering Demand Parameters (EDPs), suited for describing beam-type and column-type mechanisms respectively. The evaluated DIFs are compared with the values recommended in the current UFC design prescriptions for progressive collapse, and considerations on the choice of the appropriate DIF values are provided

Residually stressed beams

In this paper we derive a theory for a linearly elastic residually stressed rod through an asymptotic analysis based on Γ-convergence

The Glutaminase-dependent system confers extreme acid resistance to new species and atypical strains of Brucella

Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad)-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB) and of the glutamate (Glu)/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the "classical" species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q), relying on the deamination of glutamine (Gln) into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA)-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH =2.5), in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC-glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis

Digital volume correlation can be used to estimate local strains in natural and augmented vertebrae: An organ-level study

Digital Volume Correlation (DVC) has become popular for measuring the strain distribution inside bone structures. A number of methodological questions are still open: the reliability of DVC to investigate augmented bone tissue, the variability of the errors between different specimens of the same type, the distribution of measurement errors inside a bone, and the possible presence of preferential directions. To address these issues, five augmented and five natural porcine vertebrae were subjected to repeated zero-strain micro-CT scan (39 μm voxel size). The acquired images were processed with two independent DVC approaches (a local and a global one), considering different computation sub-volume sizes, in order to assess the strain measurement uncertainties. The systematic errors generally ranged within ±100 microstrain and did not depend on the computational sub-volume. The random error was higher than 1000 microstrain for the smallest sub-volume and rapidly decreased: with a sub-volume of 48 voxels the random errors were typically within 200 microstrain for both DVC approaches. While these trends were rather consistent within the sample, two individual specimens had unpredictably larger errors. For this reason, a zero-strain check on each specimen should always be performed before any in-situ micro-CT testing campaign. This study clearly shows that, when sufficient care is dedicated to preliminary methodological work, different DVC computation approaches allow measuring the strain with a reduced overall error (approximately 200 microstrain). Therefore, DVC is a viable technique to investigate strain in the elastic regime in natural and augmented bones

Mécanismes d’acido-résistance extrême chez les souches nouvelles et atypiques de Brucella

Brucella est l'agent causal de la brucellose, une zoonose bactérienne répandue à l'échelle mondiale. Durant les dernières années, de nouvelles souches et espèces de Brucella (dont Brucella microti) ont été isolées de l’environnement et d’animaux sauvages. Ces souches, phylogénétiquement anciennes, sont plus acido-résistantes que les espèces classiques, plus récentes et inféodées aux animaux domestiques et à l’homme. Chez Escherichia coli, le système glutamate décarboxylase (GAD) et le système glutaminase (AR2_Q), basés respectivement sur la décarboxylation du glutamate et la déamination de la glutamine, sont les systèmes d’acido-résistance (AR) les plus efficaces. Notre équipe a démontré que le système GAD (GadB et GadC) est fonctionnel seulement dans les nouvelles souches et espèces de Brucella, et participe à la réussite de l’infection des souris par voie orale. Dans cette thèse, le rôle de nouveaux facteurs et les mécanismes moléculaires impliqués dans l’acido-résistance ont été explorés. Premièrement, nous avons montré que GlsA et GadC sont les deux protéines structurales du système AR2_Q qui, avec le système GAD, joue un rôle essentiel dans l’AR de ces nouvelles souches. De plus, chez ces mêmes souches, le système uréase intervient également dans la survie en milieu acide.Nos résultats suggèrent que les systèmes GAD, AR2_Q et uréase, en fonction de la disponibilité des substrats, pourraient contribuer à améliorer l’adaptation des nouvelles espèces dans les environnements acides naturels et/ou dans le tractus gastro-intestinal de leurs hôtes.Brucella is the etiological agent of brucellosis, a worldwide bacterial zoonosis. In the last ten years, new and atypical strains of Brucella (among which Brucella microti) were isolated from the environment and wild hosts. These strains, of ancient origin, are considered more environmental and acid resistant than classical Brucella species, which are mostly pathogenic for livestock and humans. In Escherichia coli, the glutamate decarboxylase (GAD)-dependent system and the glutaminase (AR2_Q) system, based on the decarboxylation of glutamate and on the deamination of glutamine, respectively, are most efficient in conferring acid resistance (AR). Our team has previously demonstrated that in Brucella the GAD system (GadB and GadC) is functional only in new/atypical strains and contributes to murine infection by oral route. In this thesis, novel molecular mechanisms and factors involved in specific AR of new/atypical Brucella species were explored. Firstly, we have shown that in these strains, GlsA and GadC are the two structural proteins of the AR2_Q system, which, in concert with the GAD system, plays an essential role in AR. In addition, the functionality and role of the urease system in AR was also demonstrated in these strains.Our results suggest that the GAD, AR2_Q and urease systems may participate in a better adaptation of new Brucella species to certain natural acidic habitats and/or to the gastrointestinal tract of their hosts, depending on substrate availability

rapros: A ROS Package for Rapid Prototyping

ROS framework lacks of an internal tool to design or test control algorithms and therefore developers have to test their algorithms on-line, directly on the robotic platform they are working with. This is not always safe and possible, and a rapid prototyping tool can help during the design phase. Users can develop their algorithms directly on the controller board and safely test them in a simulated scenario. Although some rapid prototyping tools exist in the ROS community, none of them take Simulink® into consideration. In this work the authors provide an open source Rapid Prototyping tool which integrates ROS and Simulink. The proposed package is useful for control designers, who are frequently used to exploit Simulink features for control deployment. The tool can be downloaded from https://​github.​com/​gionatacimini/​rapros