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A large body of data both in animals and humans demonstrates that the gut microbiome plays a fundamental role in cancer immunity and in determining the efficacy of cancer immunotherapy. In this work, we have investigated whether and to what extent the gut microbiome can influence the antitumor activity of neo-epitope-based cancer vaccines in a BALB/c-CT26 cancer mouse model. Similarly to that observed in the C57BL/6-B16 model, Bifidobacterium administration per se has a beneficial effect on CT26 tumor inhibition. Furthermore, the combination of Bifidobacterium administration and vaccination resulted in a protection which was superior to vaccination alone and to Bifidobacterium administration alone, and correlated with an increase in the frequency of vaccine-specific T cells. The gut microbiome analysis by 16S rRNA gene sequencing and shotgun metagenomics showed that tumor challenge rapidly altered the microbiome population, with Muribaculaceae being enriched and Lachnospiraceae being reduced. Over time, the population of Muribaculaceae progressively reduced while the Lachnospiraceae population increased—a trend that appeared to be retarded by the oral administration of Bifidobacterium. Interestingly, in some Bacteroidales, Prevotella and Muribaculacee species we identified sequences highly homologous to immunogenic neo-epitopes of CT26 cells, supporting the possible role of “molecular mimicry” in anticancer immunity. Our data strengthen the importance of the microbiome in cancer immunity and suggests a microbiome-based strategy to potentiate neo-epitope-based cancer vaccines.

Significance Thanks to their potent built-in adjuvanticity, bacterial outer membrane vesicles (OMVs) represent an attractive vaccine platform. However, their full-blown exploitation relies on the availability of efficient engineering strategies. This work provides strong experimental evidence that OMVs can be successfully decorated with heterologous antigens by channeling them to the lipoprotein transport machinery. Not only did lipidated antigens accumulate in the vesicular compartment at high levels but they also interfered with the acylation process of lipid A, thus substantially reducing the lipopolysaccharide-mediated reactogenicity, a major hurdle for vaccine applications. The approach was validated with 5 Staphylococcus aureus antigens, and mice immunized with engineered OMVs were completely protected against S. aureus challenge, paving the way for development of vaccines against this important pathogen., Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigen-specific antibody titers when administered to mice in quantities as low as 0.2 μg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo-OMVs a promising vaccine candidate to be tested in clinics.

Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines.

Because of their potent adjuvanticity, ease of manipulation and simplicity of production Gram‐negative Outer Membrane Vesicles OMVs have the potential to become a highly effective vaccine platform. However, some optimization is required, including the reduction of the number of endogenous proteins, the increase of the loading capacity with respect to heterologous antigens, the enhancement of productivity in terms of number of vesicles per culture volume. In this work we describe the use of Synthetic Biology to create Escherichia coli BL21(DE3)Δ60, a strain releasing OMVs (OMVsΔ60) deprived of 59 endogenous proteins. The strain produces large quantities of vesicles (> 40 mg/L under laboratory conditions), which can accommodate recombinant proteins to a level ranging from 5% to 30% of total OMV proteins. Moreover, also thanks to the absence of immune responses toward the inactivated endogenous proteins, OMVsΔ60 decorated with heterologous antigens/epitopes elicit elevated antigens/epitopes‐specific antibody titers and high frequencies of epitope‐specific IFN‐γ‐producing CD8+ T cells. Altogether, we believe that E. coli BL21(DE3)Δ60 have the potential to become a workhorse factory for novel OMV‐based vaccines.

Prejudices on psychiatric disorders frequently turn into stigmatizating attitudes, also among health care providers. The Opening Minds Stigma Scale for Health Care Providers (OMS-HC) was introduced in 2012 to measure stigma. No Italian versions of this tool exist so far. We wanted to investigate stigma among healthcare students in Italy, and to prepare an Italian version of the scale. A multicentric, cross-sectional study was conducted on a sample of Nursing, Physiotherapy, Occupational therapy, and Dietistics students. The Italian version of the scale was obtained through back-translation. 561 students were enrolled, median age 21 years, IQR [20;23], 62.22% females (n = 349). 262 students declared having met subjetcs affected by psychiatric disorders during their training; 50 had one or more psychiatric disorders in their lives. The Italian version proved valid and reliable. Older students had lower stigma scores. No differences existed between stigma scores according to gender and personal experience of mental illness.

In many Countries legislation forces Oil&Gas Companies to implement a Process Safety Management System (PSMS) to prevent and mitigate major accidents associated to the release of energy or hazardous substances. In plants where activities may have lower process risks, such as contaminated groundwater treatment plants, the implementation of a PSMS usually is not mandatory. This paper outlines how also these plants could benefit from a Process SafetySafety framework. A PSMS can be developed in line with the framework of an internationally recognized organization (CPPS, Energy Institute, IOGP, OSHA) to be applied to activities with significant HSE risks while it may be considered as an internal standard for low-risk activities. The effectiveness of the PSMS adopted and its correct and complete implementation in accordance with the selected framework may be evaluated by a dedicated audit protocol. The typical findings from audits carried out on reference upstream and downstream installations as well as on some low-risk plants were collected and performance compared in order to identify peculiarities in terms of good practices and recurrent areas for improvement. The main results of the benchmark are presented in this paper. The results confirm that the effectiveness of a PSMS is strictly linked with the level of risks in the activities and how such risks are managed both in administrative and operating processes. A robust risk-based approach in all processes, fostered by a visible commitments of top management, is the successful key for enhancing the Process Safety culture of personnel and ensuring safe performances and business continuity in operations, irrespective of the risk level in activities. The paper also provides a roadmap for implementing a PSMS in contaminated groundwater treatment plants, tailoring the Process Safety framework to the specific operative needs and risks.

Background The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions. Results We first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 “dispensable” genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions. Conclusions This work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0681-1) contains supplementary material, which is available to authorized users.

Glycine is involved in several physiological functions, e.g. as a neurotransmitter in the central nervous system, and sarcosine has been identified as a differential metabolite greatly enhanced during prostate cancer progression and metastasis. Glycine oxidase from Bacillus subtilis (GO) was engineered with the final aim of producing specific analytical systems to detect these small achiral amino acids. Based on in silico analysis, site-saturation mutagenesis was independently performed at 11 positions: a total of 16 single-point GO variants were analyzed. Significantly improved kinetic parameters were observed on glycine for the A54R, H244K-N-Q-R, Y246W and M261R variants. The introduction of multiple mutations then identified the H244K/M261R variant showing a 5.4-fold increase in maximal activity on glycine. With sarcosine as substrate, a number of single-point variants showed increased maximal activity and/or affinity: the kinetic efficiency was increased 6-fold for the M49L variant. Two GO variants with a high substrate specificity ratio for glycine (versus sarcosine, i.e. H244K GO) or for sarcosine (versus glycine, i.e. M49L GO) combined with high substrate affinity were used to set up a simple fluorescence-based biosensor. This optical sensing assay represents a novel, inexpensive and fast tool to assay glycine or sarcosine concentrations in biological samples (detection limit

In the oil and gas business incidents are still raising. This is mainly due to the increase of men-hours worked by contractors. In order to take an action involving leadership of the top and middle management of contractors, the methodology of Performance Audit and focus groups has been applied in some of our affiliate Companies. The performance audits focus on the weak points of the Company field activities, such as some poor HSE performance in peculiar areas such as Construction. Evidences from metrics such as stop cards show which areas need improvements. The audit programme implies a preliminary phase where a visit is planned together with two "escorts", coached by hazard spotting specialists. The escorts are two middle managers of the Company (mainly from Operations and Logistics). On the basis of the safety interventions made, the two escorts prepared a high impact presentation which was delivered to the very top management of the company (CEO). This methodology was able to make a mutual commitment between the two middle managers (escorts) and the CEO, who took leadership to enhance the HSE performance, after acknowledging it was his own fault (and no other's) if some HSE performances were poor. The exercise was concluded by a focus group in which all company took consciousness of the issues and committed mutually with the CEO to take leadership at all Company's levels. The paper illustrates how this methodology achieved the objectives to: Increase Company's leaders' understanding of the real status of HSE within their operations.Make the leaders owning the HSE issues and feeling compelled to act and change. Help intervening in front of unsafe acts.Improve performance.

The uncontrolled eruption of a well is one of the most critical accidents that could happen in the oil and gas industry. Thanks to challenging R&D activities and collaborations, eni Upstream and Technical Services has developed and implemented Rainbow, an integrated methodology for blow-out risk analyses. As the Methodology has been applied in a wide range of HSE studies, the database of the analyzed blowout scenarios has been increasing for about 15 years, including for example release rates, total oil spill volumes and impact parameters (e.g. beached volumes for offshore scenarios and rainout area extension for onshore blowouts). Considering, for example, oil rates (BOPD), they could extend in a range as wide as 5 orders of magnitude, therefore a probabilistic analysis has been performed in order to evaluate the stochastic distribution of the above mentioned impact parameters. Applying short-cut models included in the Rainbow Methodology, the database of the analyzed blowout events has been integrated in order to include all potential scenarios. Moreover, in order to take into account both the local parameters affecting the well blowout frequency and the different flow paths (i.e. in-well geometry) distribution, a complete frequency assessment has been carried out. The stochastic distributions of the following parameters have been obtained as results: total spill volume (all the scenarios).extension of impacted areas involved in the oil fallout (for onshore wells).length of impacted shoreline, oil volumes beached and open sea impacted area extension (for offshore wells, considering both underwater and atmospheric releases). The distribution curves have been clustered distinguishing among: HPHT and normal wells.Subsea and atmospheric.Crude oil and gas condensate wells. The above information can be useful for several purposes, e.g.: To categorize wells and potential blowouts in a rational risk ranking.To support the Company clustering procedure focused on the cost evaluation of the blowout events.To compare the blowout impact distribution with other accidents associated to hydrocarbon releases in the environment (e.g. tanker/ship, pipeline, storage, etc.) The comparison between the stochastic distribution curves and some real accidents (including Macondo blowout) occurred in the oil and gas industry is also presented in the Paper.

d-Amino acid oxidase (DAAO; EC1.4.3.3) has been proposed to play a main role in the degradation of d-serine, an allosteric activator of the N-methyl-d-aspartate-type glutamate receptor in the human brain, and to be associated with the onset of schizophrenia. To prevent excessive d-serine degradation, novel drugs for schizophrenia treatment based on DAAO inhibition were designed and tested on rats. However, the properties of rat DAAO are unknown and various in vivo trials have demonstrated the effects of DAAO inhibitors on d-serine concentration in rats. In the present study, rat DAAO was efficiently expressed in Escherichia coli. The recombinant enzyme was purified as an active, 40 kDa monomeric flavoenzyme showing the basic properties of the dehydrogenase-oxidase class of flavoproteins. Rat DAAO differs significantly from the human counterpart because: (a) it possesses a different substrate specificity; (b) it shows a lower kinetic efficiency, mainly as a result of a low substrate affinity; (c) it differs in affinity for the binding of classical inhibitors; (d) it is a stable monomer in the absence of an active site ligand; and (e) it interacts with the mammalian protein modulator pLG72 yielding a ∼ 100 kDa complex in addition to the ∼ 200 kDa one, as formed by the human DAAO. Furthermore, the concentration of endogenous d-serine in U87 glioblastoma cells was not affected by transfection with rat DAAO, whereas it was significantly decreased when expressing the human homologue. These results raise doubt on the use of the rat as a model system for testing new drugs against schizophrenia and indicate a different physiological function of DAAO in rodents and humans. Structured digital abstract • pLG72 binds rDAAO by molecular sieving (View interaction)

Objective/ Scope Despite all the technological advancements and lessons learnt from past incidents, the oil and gas business has still a long way ahead in the journey towards an accident free working environment. In particular process-safety incidents characterized by low-frequency but severe consequences represent a big concern not only for downstream plants and refineries, but also for upstream activities due to their dramatic aftermath: loss of containments and release of toxic substances have in fact the deadly potential to escalate to fires, explosions and unwanted releases into the environment. Therefore e&p operators needs to get a better understanding of how managing major accident hazards through structured and systematic approaches. This issue becomes increasingly important when it comes to deal with mature assets where ageing wells, flowlines and facilities can pose integrity issues and operational risks. In such circumstances the possible presence of H2S can make the picture even more complex as regards health and Safety of people and Emergency Response readiness. This paper provides an overview of the holistic and systematic approach that has been set up in the company and implemented throughout the asset life with the precise aim to ensure integrity of its operation, managing major accident and deliver the fundamentals of process safety consistently across its subsidiaries Method Procedures, process In the Company efforts to enhance process safety have been translated into the identification of focus areas representing the main pillars for Process Safety: Risk identification assessment and management, assurance verification and continuous improvement and last but not least Process Safety Leadership, culture and training. Results, Observations, Conclusions Within each focus area, a number of elements have been singled out to define the key aspects of operations that organizations need to get right in order to assure integrity (e.g. management of change, incident investigation and analysis, emergency planning and response, Safety Critical Elements etc.) Novel Additive Information In the paper all support strategies, methodologies and dedicated tools (e.g. BART BAse line Risk assessment Tool, production checks, Safety Critical Element Management, PS Audits) that constitute the bedrocks of the system and contribute to an effective and thorough implementation of the Process Safety framework are described through its successful roll out on selected pilot cases, two sour mature fields onshore.

This paper presents the toolkit developed by Eni E&P to strengthen the manager’s HSE leadership and commitment. The first tool is a self assessment form to measure the leadership strength of each manager on the 5 key elements for accident prevention: - Communication of HSE issues with the workforce. - Reduction of accidents causes. - Promotion of Contractors HSE issues. - Supervision. - Audits and visits. The self-assessment then guides the user to express his/her personal commitment into visible actions like: - Site visits. - Participation to HSE Audits. - Periodical meetings/reports/newsletters. - Pre job meetings. Regular site visits, in particular, help managers understand in depth the company’s activities and where the challenges are, delivering the message that HSE is an essential aspect of everybody’s job and it deserves the highest attention and full commitment. To help managers get the most from site visits, some checklists have been developed guiding them on the task: - An "aide memoire" which condenses the key HSE aspects to observe on a site together with some tips on how to reinforce a safe behaviour or how to manage an observed unsafe act. - A pocket-size checklist covering the main aspects of the HSE Management System which, guiding through common management system faults ad typical rule–breaking mechanisms, provides useful indications on the health condition of the site HSE Management System. - A suite of checklists to analyse in detail the compliance to each of the 10 Eni Safety Golden Rules. These checklists are intended to be used with the assistance of an HSE professional and the involvement of the concerned contractor’s representatives. The leadership and commitment of company managers is essential for the achievement of zero incidents on work activities. This paper aims at disseminating some fundamentals for an effective and visible HSE leadership together with some useful tools which can be deployed to reinforce the HSE leadership and commitment of the managers.

Background Teicoplanin is a glycopeptide antibiotic used clinically in Europe and in Japan for the treatment of multi-resistant Gram-positive infections. It is produced by fermenting Actinoplanes teichomyceticus. The pharmaceutically active principle is teicoplanin A2, a complex of compounds designated T-A2-1-A2-5 differing in the length and branching of the fatty acid moiety linked to the glucosamine residue on the heptapeptide scaffold. According to European and Japanese Pharmacopoeia, components of the drug must be reproduced in fixed amounts to be authorized for clinical use. Results We report our studies on optimizing the fermentation process to produce teicoplanin A2 in A. teichomyceticus ATCC 31121. Robustness of the process was assessed on scales from a miniaturized deep-well microtiter system to flasks and 3-L bioreactor fermenters. The production of individual factors T-A2-1-A2-5 was modulated by adding suitable precursors to the cultivation medium. Specific production of T-A2-1, characterized by a linear C10:1 acyl moiety, is enhanced by adding methyl linoleate, trilinoleate, and crude oils such as corn and cottonseed oils. Accumulation of T-A2-3, characterized by a linear C10:0 acyl chain, is stimulated by adding methyl oleate, trioleate, and oils such as olive and lard oils. Percentages of T-A2-2, T-A2-4, and, T-A2-5 bearing the iso-C10:0, anteiso-C11:0, and iso-C11:0 acyl moieties, respectively, are significantly increased by adding precursor amino acids L-valine, L-isoleucine, and L-leucine. Along with the stimulatory effect on specific complex components, fatty acid esters, oils, and amino acids (with the exception of L-valine) inhibit total antibiotic productivity overall. By adding industrial oils to medium containing L-valine the total production is comparable, giving unusual complex compositions. Conclusions Since the cost and the quality of teicoplanin production depend mainly on the fermentation process, we developed a robust and scalable fermentation process by using an industrial medium in which a complex composition can be modulated by the combined addition of suitable precursors. This work was performed in the wild-type strain ATCC 31121, which has a clear genetic background. This is important for starting a rational improvement program and also helps to better control teicoplanin production during process and strain development.

The aim of our present research is to produce mutant forms of d -amino acid oxidase from Rhodotorula gracilis in order to determine d -amino acid content in different biological samples. During the past few years, our group has produced yeast d -amino acid oxidase variants with altered substrate specificity (e.g., active on acidic, or hydrophobic, or on all d -amino acids) both by rational design and directed evolution methods. Now, the kinetic constants for a number of amino acids (even for unnatural ones) of the most relevant d -amino acid oxidase variants have been investigated. This information constitutes the basis for considering potential analytical applications in this important field.

Background Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO). Results Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from ~500 up to ~25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies. Conclusions Comparison of our results with those published on non-covalent (type I) COs expressed in recombinant form (either in E. coli or Streptomyces spp.), shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.