Engineering Lactococcus lactis for the scaffold protein-mediated surface display of recombinant enzymes

Multi-enzyme complexes are responsible for the synthesis of a number of biochemical compounds and the degradation of complex polymers. An example of the latter is the degradation of cellulose by enzyme complexes termed “cellulosomes” which are produced by several bacteria of the class Clostridia. The basic structure of a cellulosome comprises a central scaffold protein, which associates with a multitude of cellulases via cohesin-dockerin interactions. The advent of cellulose utilization as feedstock for producing biofuels has garnered much interest towards designing custom-tailored recombinant cellulosomes and expressing them in microbes of interest. The metabolic diversity among bacteria also make this approach an appealing strategy for bestowing cellulolytic capabilities upon organisms which produce non-biofuel commodity chemicals such as lactic acid, succinic acid, acetone, amino acids, food additives and carotenoids. In addition, the display of recombinant multi-enzyme complexes in bacteria can yield novel insights into the mechanisms and parameters affecting their secretion, assembly and function. The industrially relevant lactic acid bacterium, Lactococcus lactis, is a model organism for the secretion and display of recombinant proteins, and the numerous biological techniques available for its manipulation make this organism particularly appealing for such a task. In this thesis, I present my work describing the incremental steps taken towards the surface display of custom-tailored multi-enzyme complexes on the surface of L. lactis. Chapter 1 describes the proof of concept for this project, including the choice of promoters, secretion signal peptide, and reporter enzymes. It also discusses the major bottlenecks observed based on the organism’s physiology. Chapter 2 describes the engineering of scaffold chimeras with cohesins of different specificity and the display of two enzymes on such scaffolds. I also investigated the catalytic profiles of the resulting complexes when enzymes were simultaneously or sequentially bound to the displayed scaffold. Finally, chapter 3 describes the optimization of the type 2 dockerin-cohesin interaction by the inclusion of the CipA “X” module, as well as the engineering of enzyme complexes with novel architectures by use of secondary “adapter” scaffolds and the subsequent assembly of multi-scaffold complexes. Also investigated is the potential of using a dual-plasmid system for the full in vivo assembly of such complexes without the exogenous addition of components

A comparative morphological revision of the aphid genus Myzaphis van der Goot, 1913 (Insecta: Hemiptera: Aphididae) revealed a new genus and three new species

The aphid genus Myzaphis van der Goot, 1913 from the tribe Macrosiphini is revised to include eight species. Apterous and alate viviparous females, known fundatrices and known sexual morphs (oviparous females and males) of Myzaphis bucktoni, M. juchnevitschae, M. rosarum, M. tianshanica and M. turanica are re-described and illustrated. Lectotype and paralectotypes of Myzaphis bucktoni and M. turanica are designated. The status of M. komatsubarae nomen dubium is discussed. Myzaphis avariolosa is regarded as a species belonging to the genus Ericaphis. Three new species: M. oezdemirae Kanturski & Barjadze sp. nov., M. tuatayae Kanturski & Barjadze sp. nov. from Turkey and M. rezwanii Kanturski & Barjadze sp. nov. from Iran are described and illustrated. Myzaphis bucktoni is recorded from Portugal for the first time. Diagnosis of the genus Myzaphis van der Goot, 1913 is redefined and a new genus Richardsaphis Kanturski & Barjadze gen. nov. is erected with the type species R. canadensis (Richards) comb. nov. Richardsaphis is for the first time recorded from the USA and hitherto unknown oviparous female and alate male are described and illustrated. Original keys to species of the genus Myzaphis and aphid genera of the tribe Macrosiphini with 2-2-2 first tarsal chaetotaxy are also provided

The Effect of Caffeic Acid Phenethyl Ester (CAPE) on H2O2-Induced Oxidative Stress in Cultured H9c2 Cells Compared to Common Antioxidants

Caffeic Acid Phenethyl Ester (CAPE) is a natural compound that has previously exhibited anti-proliferative, anti-inflammation and antioxidant activities. However, CAPE’s effects have not been fully elucidated in myoblasts under oxidative stress. We compared the effects of 24 hour pretreatment of CAPE to several known antioxidants (caffeic acid, vitamin C, and trolox) in H9c2 cells following oxidative injury by hydrogen peroxide (H2O2). H9c2 cells incubated with H2O2 treatment (100-700 μM, n=4) for 24 hours dose-dependently reduced cell viability (assessed by a cell counting assay). Compared to the reduction in viability from H2O2 500 μM treatment (22 ± 4%), H9c2 cell viability was significantly restored by pretreatment of CAPE (at 10 μM (100 ± 25%); 20 μM (112 ± 15%); 40 μM (109 ± 15%) n=5, p\u3c0.001) and Trolox (at 50 μM (83 ± 10%); 100 μM (89 ± 8%) n=4, p\u3c0.001). In contrast, pretreatment of H9c2 cells with caffeic acid (1-80 μM, n=3) and vitamin C (1000-10,000 μM, n=3) did not restore cell viability following H2O2-induced injury. CAPE’s mechanism was further investigated by measuring reactive oxygen species via a dichlorofluorescin diacetate assay and by evaluating heme oxygenase-1 (HO-1) expression via western blot. Increases in ROS caused by H2O2 500 μM (239 ± 30% of control, n=3) were significantly restored to control by pretreatment of CAPE dose-dependently (n=3, p\u3c0.001). Moreover, CAPE dose-dependently increased HO-1 expression (n=3). These results suggest CAPE can mitigate oxidative stress in H9c2 cells which may involve the induction of HO-1

The Effects of Caffeic Acid Phenethyl Ester (Cape) on Oxidative Stress and Hypoxia

Oxidative stress has been implicated in pathogenesis of hypoxia and ischemia/reperfusion (I/R) injury. In previous studies, we have shown that the antioxidant CAPE exerted cardioprotection in an isolated rat heart I (30 min)/R (60 min) injury model. In this study, we further evaluated the effects of CAPE on oxidative stress and hypoxia-induced cell damage. We evaluated the inhibition of absorbance in the phorbol 12-myristate 13-acetate (30 nM) induced superoxide production spectrophotometrically in isolated rat neutrophils via reduction of exogenous cytochrome C. We found that CAPE (0.5 µM- 40 µM; n=4-13) reduced phorbol 12-myristate 13-acetate induced neutrophil superoxide release dose-dependently from 29±3% to 95±2%. In a rat hind limb I (30 min)/R (60 min) model, blood hydrogen peroxide levels serves as an indicator of blood oxidative stress and was measured in real-time via a hydrogen peroxide microsensor (100 μm) inserted into both femoral veins (one served as sham, the other as I/R). We found that in the control group, I/R significantly increased blood hydrogen peroxide levels to 2.1±0.8 μM relative to the sham limb at 60 minutes reperfusion when saline was given at the beginning of reperfusion (n=5). By contrast, CAPE when given at reperfusion (40 µM, n=5) significantly reduced blood hydrogen peroxide levels from 30 min reperfusion and throughout the rest of experiment (p This study was supported by Division of Research and Department of Bio-Medical Sciences at Philadelphia College of Osteopathic Medicine

Development and Characterization of a Eukaryotic Expression System for Human Type II Procollagen

Background Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. Results Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. Conclusions Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation

Unpacking sustainabilities in diverse transition contexts: solar photovoltaic and urban mobility experiments in India and Thailand

It is generally accepted that the concept of sustainability is not straightforward, but is subject to ongoing ambiguities, uncertainties and contestations. Yet literature on sustainability transitions has so far only engaged in limited ways with the resulting tough questions around what sustainability means, to whom and in which contexts. This paper makes a contribution to this debate by unpacking sustainability in India and Thailand in the context of solar photovoltaic and urban mobility experimentation. Building on a database of sustainability experiments and multicriteria mapping techniques applied in two workshops, the paper concludes that sustainability transition scholarship and associated governance strategies must engage with such questions in at least three important ways. First, there is a need for extreme caution in assuming any objective status for the sustainability of innovations, and for greater reflection on the normative implications of case study choices. Second, sustainability transition scholarship and governance must engage more with the unpacking of uncertainties and diverse possible socio-technical configurations even within (apparently) singular technological fields. Third, sustainability transition scholarship must be more explicit and reflective about the specific geographical contexts within which the sustainability of experimentation is addressed

Location, location, location: utilizing pipelines and services to more effectively georeference the world's biodiversity data

Abstract Background Increasing the quantity and quality of data is a key goal of biodiversity informatics, leading to increased fitness for use in scientific research and beyond. This goal is impeded by a legacy of geographic locality descriptions associated with biodiversity records that are often heterogeneous and not in a map-ready format. The biodiversity informatics community has developed best practices and tools that provide the means to do retrospective georeferencing (e.g., the BioGeomancer toolkit), a process that converts heterogeneous descriptions into geographic coordinates and a measurement of spatial uncertainty. Even with these methods and tools, data publishers are faced with the immensely time-consuming task of vetting georeferenced localities. Furthermore, it is likely that overlap in georeferencing effort is occurring across data publishers. Solutions are needed that help publishers more effectively georeference their records, verify their quality, and eliminate the duplication of effort across publishers. Results We have developed a tool called BioGeoBIF, which incorporates the high throughput and standardized georeferencing methods of BioGeomancer into a beginning-to-end workflow. Custodians who publish their data to the Global Biodiversity Information Facility (GBIF) can use this system to improve the quantity and quality of their georeferences. BioGeoBIF harvests records directly from the publishers' access points, georeferences the records using the BioGeomancer web-service, and makes results available to data managers for inclusion at the source. Using a web-based, password-protected, group management system for each data publisher, we leave data ownership, management, and vetting responsibilities with the managers and collaborators of each data set. We also minimize the georeferencing task, by combining and storing unique textual localities from all registered data access points, and dynamically linking that information to the password protected record information for each publisher. Conclusion We have developed one of the first examples of services that can help create higher quality data for publishers mediated through the Global Biodiversity Information Facility and its data portal. This service is one step towards solving many problems of data quality in the growing field of biodiversity informatics. We envision future improvements to our service that include faster results returns and inclusion of more georeferencing engines

Model-informed classification of broadband acoustic backscatter from zooplankton in an in situ mesocosm

Funding: The fieldwork was registered in the Research in Svalbard database (RiS ID 11578). Fieldwork and research were financed by Arctic Field Grant Project AZKABAN-light (Norwegian Research Council project no. 322 332), Deep Impact (Norwegian Research Council project no. 300 333), Deeper Impact (Norwegian Research Council project no. 329 305), Marine Alliance for Science and Technology in Scotland (MASTS), the Ocean Frontier Institute (SCORE grant no. HR09011), and Glider Phase II financed by ConocoPhillips Skandinavia AS. Geir Pedersen’s participation was co-funded by CRIMAC (Norwegian Research Council project no. 309 512). Maxime Geoffroy was financially supported by the Ocean Frontier Institute of the Canada First Research Excellence Fund, the Natural Sciences and Engineering Research Council Discovery Grant Programme, the ArcticNet Network of Centres of Excellence Canada, the Research Council of Norway Grant Deep Impact, and the Fisheries and Oceans Canada through the Atlantic Fisheries Fund.Classification of zooplankton to species with broadband echosounder data could increase the taxonomic resolution of acoustic surveys and reduce the dependence on net and trawl samples for ‘ground truthing’. Supervised classification with broadband echosounder data is limited by the acquisition of validated data required to train machine learning algorithms (‘classifiers’). We tested the hypothesis that acoustic scattering models could be used to train classifiers for remote classification of zooplankton. Three classifiers were trained with data from scattering models of four Arctic zooplankton groups (copepods, euphausiids, chaetognaths, and hydrozoans). We evaluated classifier predictions against observations of a mixed zooplankton community in a submerged purpose-built mesocosm (12 m3) insonified with broadband transmissions (185–255 kHz). The mesocosm was deployed from a wharf in Ny-Ålesund, Svalbard, during the Arctic polar night in January 2022. We detected 7722 tracked single targets, which were used to evaluate the classifier predictions of measured zooplankton targets. The classifiers could differentiate copepods from the other groups reasonably well, but they could not differentiate euphausiids, chaetognaths, and hydrozoans reliably due to the similarities in their modelled target spectra. We recommend that model-informed classification of zooplankton from broadband acoustic signals be used with caution until a better understanding of in situ target spectra variability is gained.Publisher PDFPeer reviewe