EXP-SA: Explosives Tracking: A Microsystem for Detection of Bacterial Endospores as Self-Replicating Nucleic Acid Taggants

This proposal presents an integrated research and educational plan directed toward the production, detection, and identification of bacterial endospore taggants for explosive tracking. While the most immediate application of the research is related to stemming the activities of bioterrorists, the anticipated fundamental advances in bioengineering and sensor science and engineering will have significant societal relevance to other applications, including first-responder activities, healthcare, food safety, and pollution avoidance and mitigation. Intellectual Merit The investigators propose to combine bioengineering of Bacillus stearothermophilus endospores with microdevices for sample processing and taggant identification. A surface acoustic wave (SAW) microdroplet mixing/transport/incubator system will be coupled with molecular padlock probe technology for sensitive identification of bioengineered endospores. The specific research tasks are to: (i) Generate a number of different Bacillus spores, each with a unique DNA sequence or sequences spliced into its genome; (ii) Investigate and identify the optimal SAW device designs needed to germinate spores, lyse vegetative bacteria, transport, mix, and heat microdroplet samples; (iii) Design subsystems for DNA isolation; (iv) Develop a fluorescence-based molecular padlock probe system for DNA identification that can operate effectively in conjunction with the SAW fabrication microsystem platform; (v) Fabricate and test the proposed prototype identification system. Broader Impacts Broader impacts will be achieved through the following programs and activities to: (i) Train and interact with high school audiences through two major ongoing programs at University of Maine (UMaine), NSF Research Experiences for Teachers (RET) and the GK-12 Sensors; (ii) Involve undergraduates from Maine and other institutions directly into the research project under the umbrella of the ongoing NSF Research Experience for Undergraduates (REU) program at the UMaine; (iii) Identify appropriate Capstone projects for undergraduates involving cross-disciplinary research and design projects; (iv) Enhance existing graduate level courses (1) Microscale Bioengineering and (2) Design and Fabrication of Acoustic Wave Devices by incorporating research results into each course; (v) Contribute to the interdisciplinary multi-institutional NSF Integrative Graduate Education and Research Traineeship (IGERT) program in functional genomics, which involves UMaine, the Jackson Laboratory, and the Maine Medical Center Research Institute; (vi) Provide thesis topics for M.S. and Ph.D. students; (vii) Disseminate the research and educational material on a project website, and through conferences and printed literature. Project Outcomes ReportNew investigative tools are desperately needed to determine the origin and transit routes of contraband explosive materials, and the individuals who transport them. A powerful strategy for tracking and identifying specific lots of explosives is the incorporation or labeling with pre-and post-detonation identification tags, or taggants. This project involves the production, detection, and identification of bacterial endospore taggants for explosive tracking. It combines bioengineering of environmentally resistant Geobacillus thermoglucosidasius endospores with development of microdevices for sample processing and taggant identification. A surface acoustic wave (SAW) bacterial lysis system is coupled with on-chip fluorescence-based quantitative polymerase chain reaction (PCR) for identification of bioengineered endospores.Geobacillus spores with a unique DNA sequence encoded in well-retained plasmids have been generated. Optimal SAW device structures have been designed, fabricated and tested for lysis of the vegetative bacteria. A number of on-chip structures for multiplex PCR analysis have been created and tested. DNA release and fluorescence-based PCR analysis for identification of specific genomic DNA sequences can now be interfaced to the SAW microsystem platform to comprise an important part of the overall detection system. We anticipate that aspects of this technology will be useful for tracking contraband materials such as explosives, environmental monitoring, and potentially medical diagnostic applications. This project has fostered the multidisciplinary training of numerous undergraduate and graduate students in molecular biology, microbiology, biochemistry, and bioengineering

Lorentzian spin foam amplitudes: graphical calculus and asymptotics

The amplitude for the 4-simplex in a spin foam model for quantum gravity is defined using a graphical calculus for the unitary representations of the Lorentz group. The asymptotics of this amplitude are studied in the limit when the representation parameters are large, for various cases of boundary data. It is shown that for boundary data corresponding to a Lorentzian simplex, the asymptotic formula has two terms, with phase plus or minus the Lorentzian signature Regge action for the 4-simplex geometry, multiplied by an Immirzi parameter. Other cases of boundary data are also considered, including a surprising contribution from Euclidean signature metrics.Comment: 30 pages. v2: references now appear. v3: presentation greatly improved (particularly diagrammatic calculus). Definition of "Regge state" now the same as in previous work; signs change in final formula as a result. v4: two references adde

Evaluation of a model photo-caged dehydropeptide as a stimuli-responsive supramolecular hydrogel

Short peptides capped on the N-terminus with aromatic groups are often able to form supramolecular hydrogels, via self-assembly, in aqueous media. The rheological properties of these readily tunable hydrogels resemble those of the extracellular matrix (ECM) and therefore have potential for various biological applications, such as tissue engineering, biosensors, 3D bioprinting, drug delivery systems and wound dressings. We herein report a new photo-responsive supramolecular hydrogel based on a “caged” dehydropeptide (CNB-Phe-ΔPhe-OH 2), containing a photo-cleavable carboxy-2-nitrobenzyl (CNB) group. We have characterized this hydrogel using a range of techniques. Irradiation with UV light cleaves the pendant aromatic capping group, to liberate the corresponding uncaged model dehydropeptide (H-Phe-ΔPhe-OH 3), a process which was investigated by 1H NMR and HPLC studies. Crucially, this cleavage of the capping group is accompanied by dissolution of the hydrogel (studied visually and by fluorescence spectroscopy), as the delicate balance of intramolecular interactions within the hydrogel structure is disrupted. Hydrogels which can be disassembled non-invasively with temporal and spatial control have great potential for specialized on-demand drug release systems, wound dressing materials and various topical treatments. Both 2 and 3 were found to be non-cytotoxic to the human keratinocyte cell line, HaCaT. The UV-responsive hydrogel system reported here is complementary to previously reported related UV-responsive systems, which are generally composed of peptides formed from canonical amino acids, which are susceptible to enzymatic proteolysis in vivo. This system is based on a dehydrodipeptide structure which is known to confer proteolytic resistance. We have investigated the ability of the photo-activated system to accelerate the release of the antibiotic, ciprofloxacin, as well as some other small model drug compounds. We have also conducted some initial studies towards skin-related applications. Moreover, this model system could potentially be adapted for on-demand “self-delivery”, through the uncaging of known biologically active dehydrodipeptides.This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CQUM (UID/QUI/00686/2019) and of IPC (UID/CTM/50025/2019). L.H. acknowledges funding from the FCT Investigator Programme through grant IF/00606/2014. FCT, FEDER, PORTUGAL2020 and COMPETE2020 are also acknowledged for funding under research project PTDC/QUI-QOR/29015/2017 (POCI-01-0145-FEDER-029015)

A comparison of the CARATKids and CARAT10 questionnaires for the evaluation of control of asthma and allergic rhinitis in adolescents

[Excerpt] The Control of Allergic Rhinitis and Asthma Test (CARAT) was introduced to assess control of allergic rhinitis and asthma (ARA) simultaneously. It is the first tool to implement ARIA guidelines in clinical practice [1-5].CARAT10 was developed for adults [5], and CARATKids was designed for children aged 6 to 12 years [6]. There is no validated questionnaire to assess control of ARA in patients between the ages of 12 to 17 years.Financial support for this work was provided by FEDER funds through the Operational Programme Competitiveness Factors—COMPETE and National Funds through FCT— Foundation for Science and Technology under project POCI-01-0145-FEDER--007038 and project NORTE-01-0145-FEDER-000013, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

Dehydropeptide supramolecular hydrogels and nanostructures as potential peptidomimetic biomedical materials

Supramolecular peptide hydrogels are gaining increased attention, owing to their potential in a variety of biomedical applications. Their physical properties are similar to those of the extracellular matrix (ECM), which is key to their applications in the cell culture of specialized cells, tissue engineering, skin regeneration, and wound healing. The structure of these hydrogels usually consists of a di- or tripeptide capped on the N-terminus with a hydrophobic aromatic group, such as Fmoc or naphthalene. Although these peptide conjugates can offer advantages over other types of gelators such as cross-linked polymers, they usually possess the limitation of being particularly sensitive to proteolysis by endogenous proteases. One of the strategies reported that can overcome this barrier is to use a peptidomimetic strategy, in which natural amino acids are switched for non-proteinogenic analogues, such as D-amino acids, β-amino acids, or dehydroamino acids. Such peptides usually possess much greater resistance to enzymatic hydrolysis. Peptides containing dehydroamino acids, i.e., dehydropeptides, are particularly interesting, as the presence of the double bond also introduces a conformational restraint to the peptide backbone, resulting in (often predictable) changes to the secondary structure of the peptide. This review focuses on peptide hydrogels and related nanostructures, where α,β-didehydro-α-amino acids have been successfully incorporated into the structure of peptide hydrogelators, and the resulting properties are discussed in terms of their potential biomedical applications. Where appropriate, their properties are compared with those of the corresponding peptide hydrogelator composed of canonical amino acids. In a wider context, we consider the presence of dehydroamino acids in natural compounds and medicinally important compounds as well as their limitations, and we consider some of the synthetic strategies for obtaining dehydropeptides. Finally, we consider the future direction for this research area.This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CQUM (UID/QUI/00686/2019). FCT, FEDER, PORTUGAL2020 and COMPETE2020 are also acknowledged for funding under research project PTDC/QUI-QOR/29015/2017 (POCI-01-0145-FEDER-029015)

Aryl-capped lysine-dehydroamino acid dipeptide supergelators as potential drug release systems

Employing amino acids and peptides as molecular building blocks provides unique opportunities for generating supramolecular hydrogels, owing to their inherent biological origin, bioactivity, biocompatibility, and biodegradability. However, they can suffer from proteolytic degradation. Short peptides (<8 amino acids) attached to an aromatic capping group are particularly attractive alternatives for minimalistic low molecular weight hydrogelators. Peptides with low critical gelation concentrations (CGCs) are especially desirable, as the low weight percentage required for gelation makes them more cost-effective and reduces toxicity. In this work, three dehydrodipeptides were studied for their self-assembly properties. The results showed that all three dehydrodipeptides can form self-standing hydrogels with very low critical gelation concentrations (0.050.20 wt%) using a pH trigger. Hydrogels of all three dehydrodipeptides were characterised by scanning tunnelling emission microscopy (STEM), rheology, fluorescence spectroscopy, and circular dichroism (CD) spectroscopy. Molecular modelling was performed to probe the structural patterns and interactions. The cytotoxicity of the new compounds was tested using human keratinocytes (HaCaT cell line). In general, the results suggest that all three compounds are non-cytotoxic, although one of the peptides shows a small impact on cell viability. In sustained release assays, the effect of the charge of the model drug compounds on the rate of cargo release from the hydrogel network was evaluated. The hydrogels provide a sustained release of methyl orange (anionic) and ciprofloxacin (neutral), while methylene blue (cationic) was retained by the network.This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CQUM (UID/QUI/00686/2019), IPC (UIDP/CTM/05256/2020 and UIDB/05256/2020) and REQUIMTE/LAQV (UIDB/50006/2020). L.H. acknowledges grant CEECINST/00156/2018. FCT, FEDER, PORTUGAL2020 and COMPETE2020 are also acknowl edged for funding under research project PTDC/QUI-QOR/29015/2017 (POCI-01-0145-FEDER 029015). TGC thanks FCT under the scope of the strategic funding of UIDB/04469/2020 unit, and LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio