Prototyping a Tool for Processing Genetic Meta-Data in Microbiological Laboratories

Next generation sequencing (NGS) technologies allow improved understanding of pathogens. In the upstream processing of generating genomic data, there is still a lack of process-oriented tools for managing corresponding meta data. In this paper, we provide a description of how a process-oriented software prototype was developed that allowed the capture and collation of metadata involved when doing NGS. Our question was: How to develop an interactive web application that supports the process-oriented management of genetic data independent of any sequencing technique

Exploiting 3D printing and digital imaging technology to develop affordable phenotypic assays for rapid and portable detection of bacterial antibiotic resistance

Rising antimicrobial resistance (AMR) is a challenge to public health, animal health, and the environment. Early detection of phenotypic-based antimicrobial resistance remains an essential step to effectively optimizing antibiotic treatments. By using 3D printed technology and Raspberry Pi - computers, two novel assays were developed for early detection of antimicrobial resistance based on motility and the bacterial response to antibiotics on agar media. Integrating culture steps and determination of minimum inhibitory concentration (MIC) into one single test, this thesis elucidated the identification of bacterial species and their MIC within hours using a frame dip slide, as well as within just 5 minutes of testing to show the results of bacterial susceptibility based on their movement or stop swimming. In addition, the temperature remains a crucial factor for bacterial growth. By designing a mobile incubator, all microbiology tests can be carried out in a variety of settings including next to the bed of patients or in the field, instead of in the laboratory. Automation of image analysis contributes another tool to better understand physiological bacteria when living in vitro conditions. These successful tools contributed alternative approaches to surveying at real-time detection of antimicrobial resistance in the field. Beyond the laboratory, these tools brought the concept to combine all-in-one. Using a frame dip slide combined with a mobile incubator as well as combining Microcapillary Film (MCF) with 3D printed microscopy created a portable kit for affordable phenotypic assays for rapid detection of bacterial antibiotic resistance anywhere, not just for performing in the laboratory

Flow cytometry for microbial sensing in environmental sustainability applications: current status and future prospects

Practical and accurate microbial assessment of environmental systems is predicated on the detection and quantification of various microbial parameters in complex matrices. Traditional growth-based assays, considered to be both slow and biased, are increasingly being replaced by optical detection methods such as flow cytometry. Flow cytometry (FCM) offers high-speed multi-parametric data acquisition, compatibility with current molecular-based microbial detection technologies, and is a proven technology platform. The unique technical properties of flow cytometry have allowed the discrimination of bacteria based on nucleic acid staining, microbial identification based on genomic and immunologic characteristics, and determination of cell viability. For this technology to achieve the ultimate goal of monitoring the microbial ecology of distributed systems, it will be necessary to develop a fully functional, low cost, and networkable microsystem platform capable of rapid detection of multiple species of microorganisms simultaneously under realistic environmental conditions. One such microsystem, miniaturized and integrated in accordance with recent advances in micro-electro-mechanical systems technology, is named the Micro Integrated Flow Cytometer. This manuscript is a minireview of the current status and future prospects for environmental application of flow cytometry in general, and micro-flow cytometry in particular.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75610/1/j.femsec.2004.01.014.pd

How underground systems can contribute to meet the challenges of energy transition

The paper provides an overview of the several scientific and technical issues and challenges to be addressed for underground storage of carbon dioxide, hydrogen and mixtures of hydrogen and natural gas. The experience gained on underground energy systems and materials is complemented by new competences to adequately respond to the new needs raised by transition from fossil fuels to renewables. The experimental characterization and modeling of geological formations (including geochemical and microbiological issues), fluids and fluid-flow behavior and mutual interactions of all the systems components at the thermodynamic conditions typical of underground systems as well as the assessment and monitoring of safety conditions of surface facilities and infrastructures require a deeply integrated teamwork and fit-for-purpose laboratories to support theoretical research. The group dealing with large-scale underground energy storage systems of Politecnico di Torino has joined forces with the researchers of the Center for Sustainable Future Technologies of the Italian Institute of Technology, also based in Torino, to meet these new challenges of the energy transition era, and evidence of the ongoing investigations is provided in this paper

A survey on pre-processing techniques: relevant issues in the context of environmental data mining

One of the important issues related with all types of data analysis, either statistical data analysis, machine learning, data mining, data science or whatever form of data-driven modeling, is data quality. The more complex the reality to be analyzed is, the higher the risk of getting low quality data. Unfortunately real data often contain noise, uncertainty, errors, redundancies or even irrelevant information. Useless models will be obtained when built over incorrect or incomplete data. As a consequence, the quality of decisions made over these models, also depends on data quality. This is why pre-processing is one of the most critical steps of data analysis in any of its forms. However, pre-processing has not been properly systematized yet, and little research is focused on this. In this paper a survey on most popular pre-processing steps required in environmental data analysis is presented, together with a proposal to systematize it. Rather than providing technical details on specific pre-processing techniques, the paper focus on providing general ideas to a non-expert user, who, after reading them, can decide which one is the more suitable technique required to solve his/her problem.Peer ReviewedPostprint (author's final draft

Going synthetic: how scientists and engineers imagine and build a new biology

Synthetic biology practitioners look through an engineer's lens at the incredibly complex, sensitive and seemingly endless resource of living reproductive material and contemplate turning biology into a substrate – composed of modular, wellcharacterised parts – that can be used to design and build new functional devices and systems. It is often explained that this vision for engineering biology may deliver future forms of efficient drug production, renewable sources of biofuel, methods to sense and remediate toxins and numerous other applications. Yet, synthetic biology remains a field in its infancy, facing a barrage of interconnected challenges across technical, social, ethical, legal and political realms. This multifaceted dynamic makes it a timely and important locus for sociophilosophical investigation. This thesis provides a highly empirical ethnographic account of two research groups as they were challenged to design and build a microbiological machine for the International Genetically Engineered Machine competition (iGEM) in 2009. The work examines forms of knowledge and material production in synthetic biology and, in focusing on iGEM, argues that this field is not only a feat of technical engineering, but also one of social engineering as it educates and indoctrinates a next generation of researchers through this unique contest. In this narrative, one discovers a microsocial sphere in which new ideas and biological entities at the intersection of natural and synthetic kingdoms of life are being constructed. Forms of teaching, tools, practices and processes that make imagining, designing and building new living systems possible are illustrated. The reader is also introduced to some international stakeholders and dynamics at play. With gathering media interest, attention from art and design perspectives, as well as publications across social, philosophical, political and legal studies of this ‘new’ biotechnology, there is a great need for the kind of detailed, insider view that this thesis provides – it contributes to an informed space through which constructive questions may be asked as the debate around engineering synthetic life continues to unfold. As such, this work helps to enable a reflection on the kinds of intervention possible in the process of dreaming up ideas of potential future living machines. Involved collaborators, as well as the resistance of life itself, will ultimately govern the limits of synthetic biology

Dual-function nanoparticles enzymatically conjugated with a custom-made polyurethane hydrogel for chronic wound treatment

Hydrogels are attractive drug delivery systems with the potential to protect their cargo and control its release. In particular, hydrogels based on synthetic polymers are gaining increasing interest by virtue of their controllable chemistry, ease of modification, and reproducibility. Moreover, the presence of specific side chains and pending functional groups in the polymer structure allows for the conjugation of drugs and other compounds resulting in improved control over drug release. Enzymes that catalyse reactions in a very specific way could also be used to control the conjugation of compounds to the polymeric chains to improve reproducibility and biocompatibility of the conjugation process. This contribution describes an innovative system for drug delivery comprising a bioartificial supramolecular hydrogel based on a customised polyurethane and α- cyclodextrins, and nanoparticles, for application in the treatment of chronic wounds. The system has the potential to reduce inflammation and eradicate infection by virtue of dual-function nanoparticles which incorporate cobalt as antimicrobial agent, and phenolated lignin as antioxidant. The nanoparticles are enzymatically conjugated to the hydrogel by means of the amine side groups exposed along the backbone of the ad-hoc synthesised polyurethane. The oxidase enzyme laccase is exploited to oxidize the phenol groups of lignin, to allow their interaction with the amines on the hydrogel. The effects of nanoparticles conjugation to the hydrogel are studied through gelification tests, stability tests, and rheology. Moreover, the release of nanoparticles from the hydrogel and their effects on patients’ wound fluids and against relevant bacterial strains are analysed in vitro

Solvent-free nanoparticles synthesis for encapsulation of water-soluble compounds

The increasing concerns on green manufacturing practices with low environmental impact have put pressure on the pharmaceutical industry. Of particular concern is the large number of organic solvents used in a wide range of pharmaceutical products, posing a significant risk on human and environmental health. Recently, nanoparticles(NPs) emerged as advantageous drug-delivery systems with the potential to maximize drug efficacy and minimize side effects. Unfortunately, NPs synthesis processes are still environmentally unsustainable, due to the large amount of organic solvent involved. This contribution describes the synthesis and characterization of NPs encapsulating proteins or nucleic acids for metastatic melanoma treatment using alternative synthesis methods that replace organic solvents with water-based solutions. Two different green synthesis techniques have been investigated; (i)Ionic gelation was used to prepare chitosan(CS) NPs, exploiting the electrostatic interaction between the CS amino groups and tripolyphosphate(TPP), (ii)self-assembly technique to prepare siRNA/phosphate-poly(allylamine-hydrochloride)(PAH) NPs, exploiting the interactions between primary amines in the polymer and siRNAs to form stable complexes. CS and PAH NPs with the appropriate hydrodynamic diameters (~200 nm), polydispersity index, and Z potential for high cell internalization and tissue extravasation were obtained. Preliminary in vitro tests demonstrated that particles are well tolerated by human fibroblast which has shown high viability even when treated with the highest NPs concentration (viability ~85% at 48h from the treatment). Additional tests are currently ongoing to demonstrate the efficacy of the drug- loaded system on human fibroblasts. Carlotta Mattioda acknowledges PON "Ricerca e Innovazione" 2014-2020 Azione IV.R "dottorati su tematiche green" for co-financing her Ph.D scholarship

The Development of a Primer Payload with Microparticles for UTI Pathogen Identification Using Polythymidine- Modified LAMP Primers in Droplet LAMP

Nucleic acid amplification tests (NAATs) are among the diagnostic tests with the highest sensitivity and specificity. However, they are more complex to develop than other diagnostic tests such as biochemical tests and lateral flow immunoassay tests. Polymerase chain reaction (PCR) is the gold standard for NAATs. PCR requires thermal cycling to achieve clonal amplification of the target pathogen DNA for diagnosis. Thermal cycling poses a challenge in the development of PCR diagnostics for point-of-care (POC) settings. Loop-mediated isothermal amplification (LAMP) offers an isothermal method for NAATs diagnostics. The advancement of the microfluidics field significantly enhances the development of LAMP diagnostics devices for POC testing. Another challenge with NAATs, is the limitation in the development of multiplex NAATs. Multiplexing however, occupies an important role in the efforts to address the antimicrobial resistance global crisis. Multiplexing will help to provide more thorough and complete diagnostics of infections, and enable doctors to prescribe the most effective antibiotics to the patients. This will help slow the emergence of antibiotic resistant pathogens. We are currently in a period of discovery void, with regards to antibiotics discovery. At this rate, more pathogens are becoming resistant to the antibiotics that we have, faster than we are developing new classes of antibiotics. According to the World Health Organization (WHO) interagency coordination group on AMR report to the secretary general of the United Nations, by 2050, there will be 10 million annual deaths globally, as a result of AMR-related events. There will also be $55 billion productivity losses globally due to AMR. In addition, there will be a total of$ 1 trillion in healthcare costs, and 28 million people will be living in poverty, as a result of the economic impact of uncontrolled AMR. Another area where multiplex diagnostics play a crucial role is infection control in the era of epidemics and pandemics. The increasing prevailing frequency of global pandemics stresses the need for the development of highly accurate and decentralized POC diagnostics. Over the last ten years, there have been more than 30 epidemics and pandemics around the world, including SARS-CoV-2, Monkey pox, India black fungus, Dengue fever, Measles, Zika, Avian influenza, Influenza A and Ebola. With advancing technology and international commerce and relations, we are now more connected than ever. This means that if there are no developments to make molecular tests more accessible at the POC, the future waves of epidemics and pandemics will have faster spread, further reach and more devastating impacts on the lives of the 8 billion people on our planet. We have developed a diagnostic method for executing droplet microfluidics LAMP via a microparticle primer payload mechanism and have demonstrated it with urinary tract infection (UTI) pathogens. With inspiration from overhang PCR and RNA-Seq, we engineered LAMP primers with 5’ polythymidine (PolyT) oligonucleotide (PolyT is placed in the middle of the Forward inner primers and Backward inner primers). The PolyT sequence is recognized by a biotinylated capture oligonucleotide engineered with a polyadenylated (PolyA) polynucleotide on the 3’ end. The streptavidin-coated microparticles functionalized with the PolyA oligonucleotide and PolyT primers, capture their specific target DNA and deliver the cargo into emulsion droplets of LAMP reagents for amplification. This platform provides the ability to multiplex by coding specific pathogen target DNA with different fluorescent signatures of the microparticles