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Hydrogen Internal Combustion Engines (H2-ICEs) are subject to increased attention thanks to their extremely low criteria pollutant emission and near-zero CO2 tailpipe emissions. However, to further minimize exhaust emissions and increase the efficiency of a H2-ICE, it is important to carefully control the relative air-fuel ratio of operation, i.e. Lambda (λ), which will lead in turn to an optimal combustion process. The precise λ control mainly relies upon the methodology to calculate λ on board of the engine, where the availability of reliable sensors specifically-developed for hydrogen combustion is currently limited. In this article, a comparative analysis of different methodologies for the calculation of λ is performed, comparing four methodologies: exhaust gas analysis through a Spindt-Brettschneider approach (λEMI), raw Universal Exhaust Gas Oxygen (λR-UEGO), processed Universal Exhaust Gas Oxygen (λP-UEGO) and speed-density (λSD) outputs. The experimental data used to compare the four methodologies were acquired through detailed and systematic experimentation on a fully-instrumented single-cylinder H2-ICE. Results show that the λP-UEGO is the closest one to the reference Spindt-Brettschneider analysis λEMI and the most robust to ample variations in the nominal λ values. The sensor’s raw UEGO output λR-UEGO is instead affected by the sensor calibration which is usually performed across a range of carbon-based fuels, a procedure that introduces a bias. The results can be used for the selection of the correct methodology to calculate λ in a H2-ICE and to choose optimal sensors for mobile applications.

The propagation of foreign DNA in Escherichia coli is central to molecular biology. Recent advances have dramatically expanded the ability to engineer (bacterial) cells; however, most of these techniques remain time-consuming. The aim of the present work was to explore the possibility to use the cloning-free genome editing (CFGE) approach, proposed by Döhlemann and coworkers (2016), for E. coli genetics, and to deepen the knowledge about the homologous recombination mechanism. The E. coli auxotrophic mutant strains FB182 (hisF892) and FB181 (hisI903) were transformed with the circularized wild-type E. coli (i) hisF gene and hisF gene fragments of decreasing length, and (ii) hisIE gene, respectively. His+ clones were selected based on their ability to grow in the absence of histidine, and their hisF/hisIE gene sequences were characterized. CFGE method allowed the recombination of wild-type his genes (or fragments of them) within the mutated chromosomal copy, with a different recombination frequency based on the fragment length, and the generation of clones with a variable number of in tandem his genes copies. Data obtained pave the way to further evolutionary studies concerning the homologous recombination mechanism and the fate of in tandem duplicated genes.

The urban plan of Palermo (Sicily, Italy) has evolved throughout Punic, Roman, Byzantine, Arab, and Norman ages until it stabilized within the borders that correspond to the current historic center. During the 2012 to 2013 excavation campaign, new remains of the Arab settlement, directly implanted above the structures of the Roman age, were found. The materials investigated in this study derived from the so-called Survey No 3, which consists of a rock cavity of subcylindrical shape covered with calcarenite blocks: it was probably used to dispose of garbage during the Arabic age and its content, derived from daily activities, included grape seeds, scales and bones of fish, small animal bones, and charcoals. Radiocarbon dating confirmed the medieval origin of this site. The composition of the bacterial community was characterized through a culture-dependent and a culture-independent approach. Culturable bacteria were isolated under aerobic and anaerobic conditions and the total bacterial community was characterized through metagenomic sequencing. Bacterial isolates were tested for the production of compounds with antibiotic activity: a Streptomyces strain, whose genome was sequenced, was of particular interest because of its inhibitory activity, which was due to the Type I polyketide aureothin. Moreover, all strains were tested for the production of secreted proteases, with those belonging to the genus Nocardioides having the most active enzymes. Finally, protocols commonly used for ancient DNA studies were applied to evaluate the antiquity of isolated bacterial strains. Altogether these results show how paleomicrobiology might represent an innovative and unexplored source of novel biodiversity and new biotechnological tools. IMPORTANCE One of the goals of paleomicrobiology is the characterization of the microbial community present in archaeological sites. These analyses can usually provide valuable information about past events, such as occurrence of human and animal infectious diseases, ancient human activities, and environmental changes. However, in this work, investigations about the composition of the bacterial community of an ancient soil sample (harvested in Palermo, Italy) were carried out aiming to screen ancient culturable strains with biotechnological potential, such as the ability to produce bioactive molecules and secreted hydrolytic enzymes. Besides showing the biotechnological relevance of paleomicrobiology, this work reports a case of germination of putatively ancient bacterial spores recovered from soil rather than extreme environments. Moreover, in the case of spore-forming species, these results raise questions about the accuracy of techniques usually applied to estimate antiquity of DNA, as they could lead to its underestimation.

Membrane vesicles (MVs) are spherical particles with nanoscale dimensions and characterized by the presence of diverse cargos, such as nucleic acids, proteins, lipids, and cellular metabolites. Many examples of (micro)organisms producing MVs are reported in literature. Among them, bacterial MVs are of particular interest because they are now considered as the fourth mechanism of horizontal gene transfer. Streptomyces bacteria are well-known for their ecological roles and ability to synthesize bioactive compounds, with Streptomyces coelicolor being the model organism. It was previously demonstrated that it can produce distinct populations of MVs characterized by different protein and metabolite cargos. In this work we demonstrated for the first time that MVs of S. coelicolor carry both DNA and RNA and that their DNA content represents the entire chromosome of the bacterium. These findings suggest that MV DNA could have a role in the evolution of Streptomyces genomes and that MVs could be exploited in new strain engineering strategies.

Phytodepuration occurs in the plant-mediated remediation processes exploited to remove pollutants from wastewater, and

Context Several case reports of Graves’ disease (GD) occurrence after COVID-19 vaccination that are possibly related to the autoimmune syndrome induced by adjuvants (ASIA) were published recently. Objective The aim of our study was to evaluate possible distinctive features in the presentation and clinical course of patients with GD occurring early (within 4 weeks) after COVID-19 vaccination who attended our Endocrine Unit in 2021. Methods Patients with a first episode of GD attending a tertiary endocrine center between January 1, 2021, and December 31, 2021, were included. Results Sixty-four patients with a first episode of GD were seen in 2021: 20 (31.2%) of them had onset within 4 weeks following vaccine administration. Compared with the other 44 patients, the 20 patients with postvaccine early-onset (PoVEO) GD were older (median age 51 years vs 35 years, P = .003) and more likely to be male (40.0% vs 13.6%, P = .018). At diagnosis, the biochemical and immune profiles were similar between the 2 groups. However, at 3 months after starting methimazole, patients with PoVEO GD had significantly lower thyrotropin receptor antibody titer and were taking lower doses of methimazole than the other patients with GD. None in the PoVEO group had sustained free triiodothyronine elevation. Conclusion This relatively large series suggests that in 2021 PoVEO GD may be a new nosologic entity representing one-third of patients evaluated for new-onset GD in our center. Distinctive features included older age at onset, higher male prevalence, and a better initial biochemical and immunologic response to treatment. Further studies are warranted to clinically and biochemically differentiate these cases from sporadically occurring GD.

Medicinal aromatic plants' essential oils (EOs) are mixtures of volatile compounds showing antimicrobial activity, which could be exploited to face the emerging problem of multi-drug resistance. Their chemical composition can depend on the interactions between the plant and its endophytic microbiota, which is known to synthesize volatile organic compounds (VOCs). However, it is still not clear whether those volatile metabolites can contribute to the composition of the aroma profile of plants' EOs. The aims of this study were to characterize medicinal plant

Food contamination can be a serious concern for public health because it can be related to the severe spreading of pathogens. This is a main issue, especially in the case of fresh fruits and vegetables; indeed, they have often been associated with gastrointestinal outbreak events, due to contamination with pathogenic bacteria. However, little is known about the physiological adaptation and bacterial response to stresses encountered in the host plant. Thus, this work aimed to investigate the adaptation of a commensal E. coli strain while growing in tomato pericarp. Pre-adapted and non-adapted cells were compared and used to contaminate tomatoes, demonstrating that pre-adaptation boosted cell proliferation. DNA extracted from pre-adapted and non-adapted cells was sequenced, and their methylation profiles were compared. Hence, genes involved in cell adhesion and resistance against toxic compounds were identified as genes involved in adaptation, and their expression was compared in these two experimental conditions. Finally, pre-adapted and non-adapted E. coli were tested for their ability to resist the presence of toxic compounds, demonstrating that adaptation exerted a protective effect. In conclusion, this work provides new information about the physiological adaptation of bacteria colonizing the tomato fruit pericarp.

Medicinal plants play an important role in the discovery of new bioactive compounds with antimicrobial activity, thanks to their pharmacological properties. However, members of their microbiota can also synthesize bioactive molecules. Among these, strains belonging to the genera Arthrobacter are commonly found associated with the plant’s microenvironments, showing plant growth-promoting (PGP) activity and bioremediation properties. However, their role as antimicrobial secondary metabolite producers has not been fully explored. The aim of this work was to characterize the Arthrobacter sp. OVS8 endophytic strain, isolated from the medicinal plant Origanum vulgare L., from molecular and phenotypic viewpoints to evaluate its adaptation and influence on the plant internal microenvironments and its potential as a producer of antibacterial volatile molecules (VOCs). Results obtained from the phenotypic and genomic characterization highlight its ability to produce volatile antimicrobials effective against multidrug-resistant (MDR) human pathogens and its putative PGP role as a producer of siderophores and degrader of organic and inorganic pollutants. The outcomes presented in this work identify Arthrobacter sp. OVS8 as an excellent starting point toward the exploitation of bacterial endophytes as antibiotics sources.

The optimization of the piston bowl design has been shown to have a great potential for air–fuel mixing improvement, leading to significant fuel consumption and pollutant emissions reductions for diesel engines. With this aim, a conventional re-entrant bowl for a 1.6 L light-duty diesel engine was compared with two innovative piston designs: a stepped-lip bowl and a radial-bumps bowl. The potential benefits of these innovative bowls were assessed through 3D-CFD simulations, featuring a calibrated spray model and detailed chemistry. To analyse the impact of these innovative designs, two different engine operating conditions were scrutinized, corresponding to the rated power and a partial load, respectively. Under the rated power engine operating condition, a start of injection sensitivity was then carried out to assess the optimal spray–wall interaction. Results highlighted that, thanks to optimal injection phasing, faster mixing-controlled combustion could be reached with both the innovative designs. Moreover, the requirements in terms of swirl were also investigated, and a higher swirl ratio was found to be necessary to improve the mixing process, especially for the radial-bumps design. Finally, at part-load operating conditions, different exhaust gas recirculation (EGR) rates were analysed for two injection pressure levels. The stepped-lip and radial-bumps bowls highlighted reduced indicated specific fuel consumption (ISFC) and soot emissions values over different rail pressure levels, guaranteeing NOx control thanks to the higher EGR tolerance compared with the re-entrant bowl. The results suggested the great potential of the investigated innovative bowls for improving efficiency and reducing emissions, thus paving the way for further possible optimization through the combination of these designs.

Multidrug-resistant pathogens represent a serious threat to human health. The inefficacy of traditional antibiotic drugs could be surmounted through the exploitation of natural bioactive compounds of which medicinal plants are a great reservoir. The finding that bacteria living inside plant tissues, (i.e., the endophytic bacterial microbiome) can influence the synthesis of the aforementioned compounds leads to the necessity of unraveling the mechanisms involved in the determination of this symbiotic relationship. Here, we report the genome sequence of four endophytic bacterial strains isolated from the medicinal plant Origanum vulgare L. and able to antagonize the growth of opportunistic pathogens of cystic fibrosis patients. The in silico analysis revealed the presence of gene clusters involved in the production of antimicrobial compounds, such as paeninodin, paenilarvins, polymyxin, and paenicidin A. Endophytes’ adaptation to the plant microenvironment was evaluated through the analysis of the presence of antibiotic resistance genes in the four genomes. The diesel fuel degrading potential was also tested. Strains grew in minimum media supplemented with diesel fuel, but no n-alkanes degradation genes were found in their genomes, suggesting that diesel fuel degradation might occur through other steps involving enzymes catalyzing the oxidation of aromatic compounds.

Multidrug-resistant pathogens represent a serious threat to human health. The inefficacy of traditional antibiotic drugs could be surmounted through the exploitation of natural bioactive compounds of which medicinal plants are a great reservoir. The finding that bacteria living inside plant tissues, (i.e., the endophytic bacterial microbiome) can influence the synthesis of the aforementioned compounds leads to the necessity of unraveling the mechanisms involved in the determination of this symbiotic relationship. Here, we report the genome sequence of four endophytic bacterial strains isolated from the medicinal plant

Cities that are densely populated are reservoirs of antibiotic resistant genes (ARGs). The overall presence of all resistance genes in a specific environment is defined as a resistome. Spatial proximity of surfaces and different hygienic conditions leads to the transfer of antibiotic resistant bacteria (ARB) within urban environments. Built environments, public transportation, green spaces, and citizens’ behaviors all support persistence and transfer of antimicrobial resistances (AMR). Various unique aspects of urban settings that promote spread and resilience of ARGs/ARB are discussed: (i) the role of hospitals and recreational parks as reservoirs; (ii) private and public transportation as carriers of ARGs/ARB; (iii) the role of built environments as a hub for horizontal gene transfer even though they support lower microbial biodiversity than outdoor environments; (iv) the need to employ ecological and evolutionary concepts, such as modeling the fate of a specific ARG/ARB, to gain enhanced health risk assessments. Our understanding and our ability to control the rise of AMR in an urban setting is linked to our knowledge of the network connecting urban reservoirs and the environment.

The optimization of diesel engine piston bowl geometries has a crucial role in improving the near-wall flame evolution for better air/fuel mixing and soot reduction. With these aims, an innovative piston bowl for a light-duty diesel engine was designed, coupling both a sharp-stepped lip and radial bumps in the inner bowl rim. The impact of the proposed design was investigated through both 3D-CFD and single-cylinder optical engine. The numerical model, featuring a calibrated spray model and a detailed combustion mechanism, was used to analyse the non-reactive air/fuel mixing and the combustion processes. Results highlighted a reduced jet-to-jet interaction and better air/fuel mixing for the innovative bowl with respect to a conventional re-entrant design, thus enabling faster combustion process after the end of main injection. Numerical results in terms of flame’s kinematic and oxidation process were compared with the combustion image velocimetry (CIV) and OH* chemiluminescence from the optical engine, showing higher velocity near the radial bumps, and faster flame recirculation towards the piston center. Moreover, both experiments and simulations showed a more intense OH distribution in the radial-bumps region and above the step during the first stage of the combustion process, thanks to the enhanced air/fuel mixing.

Streptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-positive bacteria that undergoes a complex life cycle and produces a broad repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view; this was achieved by combining microscopic, physical and -omics analyses. Two main populations of MVs, with different sizes and cargos, were isolated and purified. S. coelicolor MV cargo was determined to be complex, containing different kinds of proteins and metabolites. In particular, a total of 166 proteins involved in cell metabolism/differentiation, molecular processing/transport, and stress response were identified in MVs, the latter functional class also being important for bacterial morpho-physiological differentiation. A subset of these proteins was protected from degradation following treatment of MVs with proteinase K, indicating their localization inside the vesicles. Moreover, S. coelicolor MVs contained an array of metabolites, such as antibiotics, vitamins, amino acids, and components of carbon metabolism. In conclusion, this analysis provides detailed information on S. coelicolor MVs under basal conditions and on their corresponding content, which may be useful in the near future to elucidate vesicle biogenesis and functions. IMPORTANCE Streptomycetes are widely distributed in nature and characterized by a complex life cycle that involves morphological differentiation. They are very relevant in industry because they produce about half of all clinically used antibiotics, as well as other important pharmaceutical products of natural origin. Streptomyces coelicolor is a model organism for the study of bacterial differentiation and bioactive molecule production. S. coelicolor produces extracellular vesicles that carry many molecules, such as proteins and metabolites, including antibiotics. The elucidation of S. coelicolor extracellular vesicle cargo will help us to understand different aspects of streptomycete physiology, such as cell communication during differentiation and response to environmental stimuli. Moreover, the capability of these vesicles for carrying different kinds of biomolecules opens up new biotechnological possibilities related to drug delivery. Indeed, decoding the molecular mechanisms involved in cargo selection may lead to the customization of extracellular vesicle content.

Antibiotic resistance is one of the biggest threats to global health, food security and development. Urgent action is needed at all levels of society to reduce the impact and spread of antibiotic resistance. For a more sustaining approach, education in children, college students, citizens and caregivers are essential. The One-Heath approach is a collaborative, multisectoral and transdisciplinary strategy in which, no single organizations or sector can address the issue of antimicrobial resistance at the human–environment interface alone. Within this strategy, education plays a central role. In this scoping review, we highlighted a range of learning activities on antibiotic resistance as part of the One-Health approach. In particular, those applications that can be introduced to a wide audience to help arrest the current crisis for the next generation. The review identifies a high number of teaching opportunities: board and role-play games, round tables, musicals, e-learning and environmental experiments to couple with more curricula and formal education to inform a diverse group of audiences.

In actinomycetes, antibiotic production is often associated with a morpho-physiological differentiation program that is regulated by complex molecular and metabolic networks. Many aspects of these regulatory circuits have been already elucidated and many others still deserve further investigations. In this regard, the possible role of many small open reading frames (smORFs) in actinomycete morpho-physiological differentiation is still elusive. In Streptomyces coelicolor, inactivation of the smORF trpM (SCO2038) – whose product modulates L-tryptophan biosynthesis – impairs production of antibiotics and morphological differentiation. Indeed, it was demonstrated that TrpM is able to interact with PepA (SCO2179), a putative cytosol aminopeptidase playing a key role in antibiotic production and sporulation. In this work, a S. coelicolor trpM knock-in (Sco-trpMKI) mutant strain was generated by cloning trpM into overexpressing vector to further investigate the role of trpM in actinomycete growth and morpho-physiological differentiation. Results highlighted that trpM: (i) stimulates growth and actinorhodin (ACT) production; (ii) decreases calcium-dependent antibiotic (CDA) production; (iii) has no effect on undecylprodigiosin production. Metabolic pathways influenced by trpM knock-in were investigated by combining two-difference in gel electrophoresis/nanoliquid chromatography coupled to electrospray linear ion trap tandem mass spectrometry (2D-DIGE/nanoLC-ESI-LIT-MS/MS) and by LC-ESI-MS/MS procedures, respectively. These analyses demonstrated that over-expression of trpM causes an over-representation of factors involved in protein synthesis and nucleotide metabolism as well as a down-representation of proteins involved in central carbon and amino acid metabolism. At the metabolic level, this corresponded to a differential accumulation pattern of different amino acids – including aromatic ones but tryptophan – and central carbon intermediates. PepA was also down-represented in Sco-trpMKI. The latter was produced as recombinant His-tagged protein and was originally proven having the predicted aminopeptidase activity. Altogether, these results highlight the stimulatory effect of trpM in S. coelicolor growth and ACT biosynthesis, which are elicited through the modulation of various metabolic pathways and PepA representation, further confirming the complexity of regulatory networks that control antibiotic production in actinomycetes.

Medicinal aromatic plants’ essential oils (EOs) are mixtures of volatile compounds showing antimicrobial activity, which could be exploited to face the emerging problem of multi-drug resistance. Their chemical composition can depend on the interactions between the plant and its endophytic microbiota, which is known to synthesize volatile organic compounds (VOCs). However, it is still not clear whether those volatile metabolites can contribute to the composition of the aroma profile of plants’ EOs. The aims of this study were to characterize medicinal plant O. vulgare ssp. vulgare bacterial endophyte VOCs, evaluating their ability to antagonize the growth of opportunistic human pathogens belonging to the Burkholderia cepacia complex (Bcc) and compare them with O. vulgare EO composition. Many of the tested endophytic strains showed (i) a bactericidal and/or bacteriostatic activity against most of Bcc strains and (ii) the production of VOCs with widely recognized antimicrobial properties, such as dimethyl disulfide, dimethyl trisulfide, and monoterpenes. Moreover, these monoterpenes were also detected in the EOs extracted from the same O. vulgare plants from which endophytes were isolated. Obtained results suggest that endophytes could also play a role in the antibacterial properties of O. vulgare ssp. vulgare and, potentially, in determining its aromatic composition.

[EN] The development of new piston geometries has shown great potential to achieve the low levels of soot emissions required by regulation. Thus, the present paper aims to characterize the influence of a new piston design over combustion process. It is characterized by the introduction of protrusions around the periphery of the bowl, evenly spaced. The performance of this geometry is compared to other geometries that have been extensively analyzed in literature, under similar operating conditions. To achieve this objective, a single cylinder optical compression ignition engine was used with full-quartz pistons rep resenting three bowl geometries: re-entrant, stepped lip and wave-stepped lip. Two optical techniques (OH* chemiluminescence and Natural Luminosity-NL) were applied for identifying the near stoichiometric zones and the differences in the combustion evolution. The flame movement was analyzed by applying the combustion image velocimetry (CIV) algorithms. In addition, an in-cylinder pressure analysis was performed for each piston at 4.5 bar and 7.5 bar IMEP and the differences in terms of Rate of Heat Release were highlighted. A more intense reverse flow was clearly identified when using wave protrusions inside the bowl. The stepped lip and wave-stepped lip bowl present faster late cycle oxidation with much near-stoichiometric zones than re-entrant piston, The authors gratefully acknowledge General Motors Propulsion Systems-Torino S. r.I. for support the project. Daniel Lerida for his laboratory work on the engine maintenance, operation, and control. This work was partially funded by Generalitat Valenciana through the Programa Santiago Grisolia (GRISOLIAP/2018/142) program.

Lay abstract In the present work, 21 bacteria associated to

Enhancing mixture preparation upstream of the premixed autoignition zone is a solution to reduce soot emission formation in compression ignition engines. With this aim, in recent years, Ducted Fuel Injection (DFI) concept has been developed: DFI is based on the idea of injecting the fuel spray through a small cylindrical pipe within the combustion chamber at a certain distance from the nozzle injector hole. Recent research studies have highlighted the high potential of this innovative concept for soot mitigation in both constant volume vessel and engine-like operating conditions. However, the mechanisms driving the soot reduction have not yet been fully understood. The aim of this research work is to further investigate the DFI concept, evaluating its impact on the spray characteristics, on the air/fuel mixing and, therefore, on the soot formation phenomena. Firstly, an experimental activity was carried out by means of a constant volume vessel test bench with optical accesses to compare the spray evolution and sizing with and without duct adoption, over a wide range of vessel thermodynamic conditions and injection pressures. After that, a simulation setup was defined in the commercially available 3D-CFD software CONVERGE reproducing the experimental test bench, calibrating and validating the spray model. Firstly, the spray model was calibrated and validated considering the same non-reacting conditions exploited in the experimental analysis. Then, the calibrated spray model was used as a virtual tool to investigate the air entrainment process. As a results, the DFI adoption increases the air entrainment in the near-nozzle region caused by the high velocity spray that generates a pumping effect at the duct inlet. Moreover, mixing process is also enhanced by the turbulence distribution at the duct exit resulting in a narrower distribution of equivalence ratio at the ignition. As a consequence of the more effective air entrainment and improved turbulent mixing, DFI remarkably mitigates soot emissions, with a reduction up to 80% with respect to free spray configuration in all tested operating conditions.

The pressing issue of the abundance of antibiotic resistance genes and resistant bacteria in the environment (ARGs and ARB, respectively) requires procedures for assessing the risk to health. The chemo-centric environmental risk assessment models identify hazard(s) in a dose–response manner, obtaining exposure, toxicity, risk, impact and policy. However, this risk assessment approach based on ARGs/ARB evaluation from a quantitative viewpoint shows high unpredictability because ARGs/ARB cannot be considered as standard hazardous molecules: ARB duplicate and ARGs evolve within a biological host. ARGs/ARB are currently listed as Contaminants of Emerging Concern (CEC). In light of such characteristics, we propose to define ARGs/ARB within a new category of evolving CEC (or e-CEC). ARGs/ARB, like any other evolving determinants (e.g., viruses, bacteria, genes), escape environmental controls. When they do so, just one molecule left remaining at a control point can form the origin of a new dangerous and selection-responsive population. As a consequence, perhaps it is time to acknowledge this trait and to include evolutionary concepts within modern risk assessment of e-CEC. In this perspective we analyze the evolutionary responses most likely to influence risk assessment, and we speculate on the means by which current methods could measure evolution. Further work is required to implement and exploit such experimental procedures in future risk assessment protocols.

One of the most studied metabolic routes is the biosynthesis of histidine, especially in enterobacteria where a single compact operon composed of eight adjacent genes encodes the complete set of biosynthetic enzymes. It is still not clear how his genes were organized in the genome of the last universal common ancestor community. The aim of this work was to analyze the structure, organization, phylogenetic distribution, and degree of horizontal gene transfer (HGT) of his genes in the Bacteroidota-Rhodothermota-Balneolota-Chlorobiota superphylum, a group of phylogenetically close bacteria with different surviving strategies. The analysis of the large variety of his gene structures and organizations revealed different scenarios with genes organized in more or less compact—heterogeneous or homogeneous—operons, in suboperons, or in regulons. The organization of his genes in the extant members of the superphylum suggests that in the common ancestor of this group, genes were scattered throughout the chromosome and that different forces have driven the assembly of his genes in compact operons. Gene fusion events and/or paralog formation, HGT of single genes or entire operons between strains of the same or different taxonomic groups, and other molecular rearrangements shaped the his gene structure in this superphylum.

The Sasso Fratino Integral Nature Reserve (Italy) aims to protect nature and territory. Since no anthropic activities are allowed, it represents a good model to study the bacterial community of a wild environment. The aim of this work was to characterise the cultivable and the total bacterial community of soil samples from the reserve in terms of taxonomy, composition, and structure. Seven soil samples were collected at different altitudes, and the chemical composition, the total and the cultivable microbiota, and the antibiotic resistance profiles of isolates were investigated. Total bacterial communities, studied through Next Generation Sequences analysis, included 390 genera. Samples differed in terms of microbial composition basing on the different altitude/vegetation of collection points. Random Amplified Polymorphic DNA Analysis (RAPD) allowed to identify 82 haplotypes out of 158 bacterial isolates. The taxonomic identification through 16S rDNA sequencing revealed that the strains were affiliated to 21 genera. Antibiotic resistance profiles of bacteria were also investigated, highlighting a high resistance against streptomycin and kanamycin. This work represents the first description of the soil bacterial community from the Natural Reserve of Sasso Fratino, and it is the first study considering the soil microbiota of an Italian integral nature reserve.

Medicinal plants (MPs) have been used since antiquity in traditional and popular medicine, and they represent a very important source of bioactive molecules, including antibiotic, antiviral, and antifungal molecules. Such compounds are often of plant origin, but in some cases, an origin or a modification from plant microbiota has been shown. Actually, the research continues to report the production of bioactive molecules by plants, but the role of plant–endophytic interaction is emerging. Classic examples are mainly concerned with fungal endophytes; however, it has been recently shown that bacterial endophytes can also play an important role in influencing the plant metabolism related to the synthesis of bioactive compounds. In spite of this, a deep investigation on the power of MP bacterial endophytes is lacking. Here, an overview of the studies on MP bacterial microbiota and its role in the production of plant antimicrobial compounds contributing to prime host defense system and representing a huge resource for biotech and therapeutic applications is provided.

Background: Rising number of multidrug-resistant human pathogens demands novel antibiotics: to this aim, unexplored natural sources are investigated to find new compounds. In this context, bacteria associated to medicinal plants, including Phragmites australis, might represent an important source of antimicrobial compounds. Materials & methods: In the present work, 21 bacterial endophytes isolated from P. australis roots were tested, by cross-streaking, for their inhibitory activity against 36 multidrug-resistant pathogens isolated from food, clinical patients and hospitals. Results & conclusion: Seven endophytes, belonging to Pseudomonas and Stenotrophomonas, were able to inhibit the growth of most of the target strains. In conclusion, this preliminary work could pave the way for the discovery of new antibiotics against superbugs.

Histidine biosynthesis is one of the most characterized metabolic routes for its antiquity and its central role in cellular metabolism; indeed, it represents a cross-road between nitrogen metabolism and de novo synthesis of purines. This interconnection is due to the activity of imidazole glycerol phosphate synthase, a heterodimeric enzyme constituted by the products of two his genes, hisH and hisF, encoding a glutamine amidotransferase and a cyclase, respectively. Despite their interaction was suggested by several in vitro experiments, their in vivo complex formation has not been demonstrated. On the contrary, the analysis of the entire Escherichia coli interactome performed using the yeast two hybrid system did not suggest the in vivo interaction of the two IGP synthase subunits. The aim of this study was to demonstrate the interaction of the two proteins using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system. Data obtained demonstrated the in vivo interaction occurring between the proteins encoded by the E. coli hisH and hisF genes; this finding might also open the way to pharmaceutical applications through the design of selective drugs toward this enzyme.

Improvement of industrial productions through more environment-friendly processes is a hot topic. In particular, land and marine environment pollution is a main concern, considering that recalcitrant compounds can be spread and persist for a long time. In this context, an efficient and cost-effective treatment of wastewater derived from industrial applications is crucial. Phytodepuration has been considered as a possible solution and it is based on the use of plants and their associated microorganisms to remove and/or transform pollutants. In this work we investigated the culturable microbiota of Phragmites australis roots, sampled from the constructed wetlands (CWs) pilot plant in the G.I.D.A. SpA wastewater treatment plant (WWTP) of Calice (Prato, Tuscany, Italy) before and after the CW activation in order to check how the influx of wastewater might affect the resident bacterial community. P. australis specimens were sampled and a panel of 294 culturable bacteria were isolated and characterized. This allowed to identify the dynamics of the microbiota composition triggered by the presence of wastewater. 27 out of 37 bacterial genera detected were exclusively associated to wastewater, and Pseudomonas was constantly the most represented genus. Moreover, isolates were assayed for their resistance against eight different antibiotics and synthetic wastewater (SWW). Data obtained revealed the presence of resistant phenotypes, including multi-drug resistant bacteria, and a general trend regarding the temporal evolution of resistance patterns: indeed, a direct correlation linking the appearance of antibiotic- and SWW-resistance with the time of exposure to wastewater was observed. In particular, nine isolates showed an interesting behavior since their growth was positively affected by the highest concentrations of SWW. Noteworthy, this study is among the few investigating the P. australis microbiota prior to the plant activation.

Improvement of industrial productions through more environment-friendly processes is a hot topic. In particular, land and marine environment pollution is a main concern, considering that recalcitrant compounds can be spread and persist for a long time. In this context, an efficient and cost-effective treatment of wastewater derived from industrial applications is crucial. Phytodepuration has been considered as a possible solution and it is based on the use of plants and their associated microorganisms to remove and/or transform pollutants. In this work we investigated the culturable microbiota of Phragmites australis roots, sampled from the constructed wetlands (CWs) pilot plant in the G.I.D.A. SpA wastewater treatment plant (WWTP) of Calice (Prato, Tuscany, Italy) before and after the CW activation in order to check how the influx of wastewater might affect the resident bacterial community. P. australis specimens were sampled and a panel of 294 culturable bacteria were isolated and characterized. This allowed to identify the dynamics of the microbiota composition triggered by the presence of wastewater. 27 out of 37 bacterial genera detected were exclusively associated to wastewater, and Pseudomonas was constantly the most represented genus. Moreover, isolates were assayed for their resistance against eight different antibiotics and synthetic wastewater (SWW). Data obtained revealed the presence of resistant phenotypes, including multi-drug resistant bacteria, and a general trend regarding the temporal evolution of resistance patterns: indeed, a direct correlation linking the appearance of antibiotic- and SWW-resistance with the time of exposure to wastewater was observed. In particular, nine isolates showed an interesting behavior since their growth was positively affected by the highest concentrations of SWW. Noteworthy, this study is among the few investigating the P. australis microbiota prior to the plant activation.

The insurgence of antibiotic resistance and emergence of multidrug-resistant (MDR) pathogens prioritize research to discover new antimicrobials. In this context, medicinal plants produce bioactive compounds of pharmacological interest: some extracts have antimicrobial properties that can contrast different pathogens. For such a purpose, Origanum vulgare L. (Lamiaceae family) is a medicinal aromatic plant, whose essential oil (EO) is recognized for its antiseptic, antimicrobial and antiviral activities. The cultivable bacteria from different compartments (i.e., flower, leaf, stem and soil) were isolated in order to: (i) characterize the bacterial microbiota associated to the plant, determining the forces responsible for the structuring of its composition (by evaluation of cross inhibition); (ii) investigate if bacterial endophytes demonstrate antimicrobial activities against human pathogens. A pool of plants belonging to O. vulgare species was collected and the specimen chemotype was defined by hydrodistillation of its essential oil. The isolation of plant associated bacteria was performed from the four compartments. Microbiota was further characterized through a culture-independent approach and next-generation sequencing analysis, as well. Isolates were molecularly typed by Random Amplified Polymorphic DNA (RAPD) profiling and taxonomically assigned by 16S rRNA gene sequencing. Antibiotic resistance profiles of isolates and pairwise cross-inhibition of isolates on agar plates (i.e., antagonistic interactions) were also assessed. High level of diversity of bacterial isolates was detected at both genus and strain level in all different compartments. Most strains were tolerant against common antibiotics; moreover, they produced antagonistic patterns of interactions mainly with strains from different compartments with respect to that of original isolation. Strains that exhibited high inhibitory properties were further tested against human pathogens, revealing a strong capacity to inhibit the growth of strains resistant to several antibiotics. In conclusion, this study regarded the characterization of O. vulgare L. chemotype and of the bacterial communities associated to this medicinal plant, also allowing the evaluation of antibiotic resistance and antagonistic interactions. This study provided the bases for further analyses on the possible involvement of endophytic bacteria in the production of antimicrobial molecules that could have an important role in clinical and therapeutic applications.

The introduction of new light-duty vehicle emission limits to comply under real driving conditions (RDE) is pushing the diesel engine manufacturers to identify and improve the technologies and strategies for further emission reduction. The latest technology advancements on the after-treatment systems have permitted to achieve very low emission conformity factors over the RDE, and therefore, the biggest challenge of the diesel engine development is maintaining its competitiveness in the trade-off "CO2-system cost" in comparison to other propulsion systems. In this regard, diesel engines can continue to play an important role, in the short-medium term, to enable cost-effective compliance of CO2-fleet emission targets, either in conventional or hybrid propulsion systems configuration. This is especially true for large-size cars, SUVs and light commercial vehicles. In this framework, a comprehensive approach covering the whole powertrain is of primary importance in order to simultaneously meet the performance, efficiency, noise and emission targets, and therefore, further development of the combustion system design and injection system represent important levers for additional improvements. For this purpose, a dedicated 0.5 dm3 single-cylinder engine has been developed and equipped with, a state-of-the-art Euro 6 combustion system, and an advanced common rail fuel injection system (FIS) offering higher flexibility in terms of injection strategy and higher quantity accuracy. Three injector nozzles with different hydraulic flow rates (HF) have been selected and employed for the overall combustion process optimization. The optimization has been performed by means of an extensive DoE-based test campaign in which the engine and FIS operating parameters have been parametrized with the aim to carry out a proper combination in terms of HF and injection strategy. The results at partial load conditions evidence significant advantages in applying an advanced injection pattern, while the HF reduction can significantly improve the smoke emission and combustion noise without fuel consumption penalties. Therefore, a proper combination and optimization of the HF and injection strategy can provide low noise and engine-out smoke while maintaining the rated power performance targets.

Diesel will continue to be an indispensable energy carrier for the car fleet CO2 emission targets in the short-term. This is particularly relevant for heavy-duty vehicles as for mid-size cars and SUVs. Looking at the latest technology achievements on the after-treatment systems, it can be stated that the concerning about the NOx emission gap between homologation test and real road use is basically solved, while the future challenge for diesel survival is to keep its competitiveness in the CO2 vs cost equation in comparison to other propulsion systems. The development of the combustion system design still represents an important leverage for further efficiency and emissions improvements while keeping the current excellent performance in terms of power density and low-end torque. The paper describes the results achieved in developing a new diesel combustion system for car application that, leveraging on the high flexibility of the latest fuel injection technology, combines outstanding power and fuel efficiency with low pollutant emissions in ultralight engine designed for lower maximum peak cylinder pressure. The study has been carried out on a 0.5l single-cylinder engine on which an advanced and last generation common rail system, capable of very high injection pressure, has been installed. Through an extensive DoE-based test campaign in which all engine operating parameters have been carefully parametrized, the capability to achieve high power density and excellent fuel economy with low engine-out pollutant emissions has been demonstrated and discussed in the paper.

[EN] This article describes the main results of an investigation about counter-bore injector nozzle impact on the combustion process in a modern Euro 6 diesel engine. First, hydraulic and spray visualization tests have been performed, showing a potential advantage of such nozzle design in fuel-air mixing efficiency. Then, combustion performance has been assessed on a GM-designed 1.6-L four-cylinder engine. The engine has been installed on a dynamometric test bench and instrumented with an AVL cylinder pressure transducer for heat release rate analysis, as well as HORIBA MEXA gas analyzer for exhaust emissions and AVL 415 Smoke Meter. Engine efficiency and emissions have been analyzed on four different part-load steady-state points, representative of New European Driving Cycle and Worldwide harmonized Light duty Test Cycle certification cycles, and covering engine speeds from 1250 to 2000 r/min and brake mean effective pressure between 0.2 and 1.4 MPa. Results of indicated analysis show that counter-bore nozzles have significant differences in terms of pilot injection combustion at low load points, which in turn lead to a better ignition and shorter combustion of the main injection. In addition, an improvement of diffusive combustion is observed as load increases. Because of both, fuel consumption is reduced by approximately 1% with respect to a standard nozzle. Finally, an appreciable decrease in engine exhaust emissions has been recorded, especially in terms of particulate matter and hydrocarbon emissions. This reduction has been linked to the improvement of fuel-air mixing promoted by the counter-bore nozzle previously observed., The authors would like to thank General Motors Global Propulsion Systems-Torino S.r.l. for sponsoring the current work. Part of the equipment was purchased with the help of Generalitat Valenciana in project IDIFEDER2018 with title "Equipamiento de diagnostico optico de alta velocidad para estudiar procesos de inyeccion''.

Histidine biosynthesis is an ancestral pathway that was assembled before the appearance of the Last Universal Common Ancestor; afterwards, it remained unaltered in all the extant histidine-synthesizing (micro)organisms. It is a metabolic cross-road interconnecting histidine biosynthesis to nitrogen metabolism and the de novo synthesis of purines. This interconnection is due to the reaction catalyzed by the products of hisH and hisF genes. The latter gene is an excellent model to study which trajectories have been followed by primordial cells to build the first metabolic routes, since its evolution is the result of different molecular rearrangement events, i.e. gene duplication, gene fusion, gene elongation, and domain shuffling. Additionally, this review summarizes data concerning the involvement of hisF and its product in other different cellular processes, revealing that HisF very likely plays a role also in cell division control and involvement in virulence and nodule development in different bacteria. From the metabolic viewpoint, these results suggest that HisF plays a central role in cellular metabolism, highlighting the interconnections of different metabolic pathways.

Gene elongation is a molecular mechanism consisting of an in-tandem duplication of a gene and divergence and fusion of the two copies, resulting in a gene constituted by two divergent paralogous modules. The aim of this work was to evaluate the importance of gene elongation in the evolution of histidine biosynthetic genes and to propose a possible evolutionary model for some of them. Concerning the genes hisA and hisF, which code for two homologous (&beta, /&alpha, )8-barrels, it has been proposed that the two extant genes could be the result of a cascade of gene elongation/domain shuffling events starting from an ancestor gene coding for just one (&beta, ) module. A gene elongation event has also been proposed for the evolution of hisB and hisD, structural analyses revealed the possibility of an early elongation event, resulting in the repetition of modules. Furthermore, it is quite possible that the gene elongations responsible for the evolution of the four proteins occurred before the earliest phylogenetic divergence. In conclusion, gene elongation events seem to have played a crucial role in the evolution of the histidine biosynthetic pathway, and they may have shaped the structures of many genes during the first steps of their evolution.

The paper describes the results achieved in developing a new diesel combustion system for passenger car application that, while capable of high power density, delivers excellent fuel economy through a combination of mechanical and thermodynamic efficiencies improvement. The project stemmed from the idea that, by leveraging the high fuel injection pressure of last generation common rail systems, it is possible to reduce the engine peak firing pressure (pfp) with great benefits on reciprocating and rotating components light-weighting and friction for high-speed light-duty engines, while keeping the power density at competitive levels. To this aim, an advanced injection system concept capable of injection pressure greater than 2500 bar was coupled to a prototype engine featuring newly developed combustion system. Then, the matching among these features have been thoroughly experimentally examined. The results confirmed the benefits of the employment of high fuel injection pressures as a way to reduce the pfp, combining competitive performance and excellent fuel efficiency with emissions and Noise Vibration Harshness (NVH) requirements of last generation diesel engines for passenger car applications. In particular, the paper discusses the engine power and efficiency sensitivities to the boundary conditions of the charging/exhaust systems, the fuel injection pressure and the mechanical base engine design (with particular reference to the pfp). Eventually, a balanced set of targets for the entire system based on such results are carried out.

Objectives:Health technology financing is often based on randomized controlled trials (RCTs), which are often the same ones used for licensing. Because they are designed to show the best possible results, typically Phase III studies are conducted under ideal and highly controlled conditions. Consequently, it is not surprising that technologies do not always perform in real life in the same way as controlled conditions. Because financing (and price paid) decisions can be made with overestimated results, health authorities need to ask whether health systems achieve the results they expect when they choose to pay for a technology. The optimal way to answer this question is to assess the performance of financed technologies in real-world settings. Health technology performance assessment (HTpA) refers to the systematic evaluation of the properties, effects, and/or impact of a health intervention or health technology in the real world to provide information for investment/disinvestment decisions and clinical guideline updates. The objective is to describe the development and principal aspects of the Guideline for HTpA commissioned by the Brazilian Ministry of Health.Methods:Our methods used include extensive literature review, refinement with experts across countries, and public consultation.Results:A comprehensive guideline was developed, which has been adopted by the Brazilian government.Conclusion:We believe the guideline, with its particular focus on disinvestment, along with the creation of a specific program for HTpA, will allow the institutionalization and continuous improvement of the scientific methods to use real-world evidence to optimize available resources not only in Brazil but across countries.