Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature

Background: In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic α-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular proteolytic activity. Results: P. haloplanktis TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of P. haloplanktis TAC125 in which the gspE gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-β-lactamase chimera. Conclusion: Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic α-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the gspE mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium

Cooperation for innovation in liberal market economies

This study focuses on the collaboration patterns that small firms hold with other agents within liberal market economies and identifies the collaborative drivers that in this context deliver a superior impact on innovation output measured by product and process innovations. To explore this research question, the study combines the literature on innovation systems with a growing literature on business innovation modes that studies whether businesses are driven by science and technology factors (STI), or experience-based factors such as learning-by-doing, by-using and by-interacting (DUI). In the UK liberal market economy, universities and research centres are expected to play a critical role for innovation well beyond the typical impact they produce in coordinated market economies. This hypothesis is largely verified through our empirical evidence. Methodologically, this research is developed through the application of propensity score matching in the context of the UK longitudinal small business survey (LSBS) for 2015

Business innovation modes and their impact on innovation outputs: Regional variations and the nature of innovation across EU regions

This work contributes to the literature on innovation systems and, in particular, delivers a thorough analysis on business innovation modes across a range of regional contexts. This analysis refers to the strand of literature on STI (Science and Technology-based Innovation) and DUI innovation modes (Innovation based on learning-by-Doing, learning-by-Using, learning-by-Interacting) that have been intensely debated over the past few years. It is a relevant area of research because it discusses the most effective innovation mode adopted by firms and their regions in the context of increasing global competition. In this scientific area, we inquire whether and how the regional context and its specific technological capabilities produce a differentiated impact of STI and DUI innovation modes on innovation outputs, alongside the nature of innovation outputs. In this respect, this study advances the literature on regional innovation systems that have not been analyzed by other scholarly contributions in this strand who have mostly discussed the differentiated impact of innovation modes across individual countries, industries, and business networks. Based on the large heterogeneity of regions across the European geography, we move beyond the set of individual country studies and develop a thorough analysis based on the Community Innovation Survey (CIS 2014) data from the Eurostat office about EU regions. Empirical evidence based on the application of a multiple treatment model suggests that both regional specificities and the nature of innovation matter. In addition, the DUI innovation mode proves to be often more important than expected for most types of innovation output

Environmental innovation across SMEs in Europe

This paper focuses on the relation between business innovation modes and environmental innovation. Over time firms have recognized the importance of prioritizing innovation to gain competitive advantages in open markets. Yet, in more recent times with the more recent international agreements on environmental sustainability (rounds in Doha in 2004; Copenhagen in 2009; Paris in 2016; and Glasgow in 2021), innovation needs to be guided through new boundaries and requirements that individual businesses and the business system as a whole face. One of these boundaries is nature and its resources which require significant protection as part of the international priority agenda on climate change agreed by most countries with the 2016 Paris Agreement on the Environment and recently confirmed with COP26. As firms are found to adopt alternative archetypical strategies of innovation, some science-driven (STI innovation mode) and others practice-driven (DUI innovation mode), we investigate whether any of these strategic modes is beneficial in relation to the capacity of the firms to produce eco-innovations, and which one is more beneficial in relation to which type of eco-innovation (e.g. technological and non-technological innovation). This analysis is seen in relation to the size of the firms as SMEs typically rely on practice and interactive-based innovation activities (DUI mode). This may help design environment protection-orientated policies that focus on specific drivers, thus making policy action efficient and effective. The analysis is based on the Community Innovation Survey (CIS) database for European countries. Our findings support the view that both STI and DUI drivers support eco-innovation through technological nuances that work also in the specific case of SME environmental innovation

Plant Dynamic Metabolic Response to Bacteriophage Treatment After Xanthomonas campestris pv. campestris Infection

Periodic epidemics of black rot disease occur worldwide causing substantial yield losses. Xanthomonas campestris pv. campestris (Xcc) represents one of the most common bacteria able to cause the above disease in cruciferous plants such as broccoli, cabbage, cauliflower, and Arabidopsis thaliana. In agriculture, several strategies are being developed to contain the Xanthomonas infection. The use of bacteriophages could represent a valid and efficient approach to overcome this widespread phenomenon. Several studies have highlighted the potential usefulness of implementing phage therapy to control plant diseases as well as Xcc infection. In the present study, we characterized the effect of a lytic phage on the plant Brassica oleracea var. gongylodes infected with Xcc and, for the first time, the correlated plant metabolic response. The results highlighted the potential benefits of bacteriophages: reduction of bacterium proliferation, alteration of the biofilm structure and/or modulation of the plant metabolism and defense response

Identification of putative adult stem cells in the rat thyroid and their use in ex situ bioengineering

Adult stem cells have been recently isolated from the human and mouse thyroid. Identification has been possible by their capacity to form floating cell spheroids or thyrospheres when primary cells are cultured in the absence of serum but presence of epidermal growth factor and basic fibroblast growth factor, as well as by the presence of stem cell markers like the breast cancer-resistant protein 1 (Bcrp1)/ATP-binding cassette subfamily G member 2 (ABCG2). Using this strategy, and an innovative in vitro growing system, we have attempted identification of stem / progenitor elements from the adult rat thyroid. Sprague-Dawley male rats (50-75 gr) were used as thyroid donors. After penthobarbital anesthesia rats were thyroidectomised, thyroids surgically excised, and primary cells prepared using enzymatic breaking. After 72 hs in standard monolayer culture, cells were trypsinized and either seeded (20 x103/cm2) and grown for 8 days in a 3D Matrigel (12.5-50%) system using low-glucose DMEM and serum, or immediately cytospinned (1200 RPM x 5 min) for immunocytochemistry, or harvested and frozen with lysis buffer for Western blotting (WB). Bcrp1/ ABCG2-immunoreactivity (IR) was detected using a rabbit anti-human, polyclonal antibody (1:500, Cell Signalling), and visualized either with the ABC technique and DAB as a chromogen, or with a chemiluminescence-based staining. The human plasmocytoma cell line, RPMI 8226 (B lymphocytes) and the acute lymphoblastic leukemia cell line CCRF-CEM (T lymphocytes) were used as positive and negative controls, respectively. Thyrosphere-like aggregates were transiently observed after initial monolayer expansion and, more consistently, at day 3 in 50% Matrigel culture, followed by rapid cell differentiation (days 4-8), including epithelial-mesenchymal transitions, formation of follicles and pavment layering. Similar differentiation changes were also detected after seeding of primary thyroid cells onto decellularized rat thyroid matrixes, as previously reported [1]. Less than 0.4% of cytospinned thyroid cells exhibited cytoplasmic Bcrp1/ABCG2-IR, and a band of around 72kD was detected by WB in cell lysates. We conclude that the thyroid of the adult rat contains a small population of stem / progenitor-like elements, likely contributing to the regenerative processes that occur during ex situ recellularization of acellular thyroid matrixes [1, 2]

A new approach to migration: communities on the move as assets

The aim of this article (opening the special issue) is to examine the impact of migration on a specific set of issues at the regional level: innovation, entrepreneurship and economic performance. In particular, we look at migration through a new lens of analysis, which we have termed the “Communities-on-the-Move (CoM)” approach. In a nutshell, this approach focuses on migrant communities, emerging from the capacity of specific national/regional groups to carry the heritage of their social capital when moving from one place to another. More precisely, the CoM approach focuses on the social capital migrants can rely on to “bond” their in-group relations and to “bridge” with extra-group ones during the migration process. The CoM approach represents a different, though complementary approach to the analysis of diasporas in the migration literature. Indeed, CoM relates to diasporas similarly to how “clustering effects” relate to “networking activities” in the regional economics of innovation literature. CoM approach takes account of the local effects such communities generate in the localities they are embedded in (“clustering effect”), while the related ‘diaspora communities concept’ captures the non-local “networking activities” that connect ethnic communities across the world. As we will also maintain in the following, through this specificity the CoM approach is likely to capture a significant impact on innovation, entrepreneurship and economic performance, which would remain otherwise hidden by using more standard approaches to migration

Ex situ bioengineering of the rat thyroid using as a scaffold the three-dimensional (3D) decellularized matrix of the glandular lobe: clues to the organomorphic principle

Recently, we designed a bioreactor system for bioengineering ex situ (i.e. on the laboratory bench) a bioartificial thyroid gland suitable for transplantation. It is based on the organomorphic principle, i.e. the bioreactor mimics the macro-microscopic architecture of the thyroid stromal-vascular scaffold (SVS). To prove the reliability of this approach, we have initiated a pilot study using as a model the rat thyroid, and its natural decellularized 3D matrix to be eventually recellularized up to formation of a viable 3D thyroid lobe ex situ. Sprague-Dawley male rats (220-240 g) were used as thyroid donors. After penthobarbital anesthesia, rats were thyroidectomised and thyroid matrixes obtained by decellularization of the native SVS. Initially, we applied a sequence of liquid N2 freezing at - 80°C / thawing at 4°C for a total of 72 h, various washings with 0.02% trypsin / 0.05% EDTA for 1 h at 37°C, 3% Triton X-100 for 72 h at 4°C, and 4% deoxycholic acid for 24 h at 4°C, followed by sterilization with 0.1% peracetic acid, and 1% penicillin / streptomycin / fungizone for 24 h. Test matrixes were made electrondense with uranium / bismuth / lead counterstaining, and analyzed by microtomography (microTC). Primary thyroid cultures were prepared using enzymatic breaking of the native thyroid tissue. Cells were seeded at 19.000 / cm 2, and grown 72 h in low-glucose DMEM supplemented with 10% FBS / 5% FCS. Following trypsinization, 450.000 cells were harvested to coat the inner surface of the matrix. After 7 and 14 days, colonized matrixes were fixed in aldheydes and processed for light (LM), transmission (TEM) and scanning electron (SEM) microscopy. Culture supernatants were collected every 48 h, and thyroid hormones assessed with chemiluminescent immunoassays. Complete decellularization and maintenance of the 3D architecture of the thyroid SVS were achieved. Thyroid-derived cells were found to aggregate, link and give rise to intracytoplasmic cavities up to follicular coating, whereas secretory de-differentiation occurred. These results show that the 3D matrix of the rat thyroid can be used as a natural scaffold to recellularize the thyroid lobe with progenitor-like elements, supporting the validity of the organomorphic principle for ex situ bioengineering of a bioartificial thyroid gland. Grants FIL09, PRIN082008ZCCJX4, FIRB2010RBAP10MLK

The present state of research into industrial clusters and districts. Content analysis of material published 1997-2006

[EN] The aim of this work is to help gain a better understanding of the research conducted on territorial agglomerations of firms at a multidisciplinary level. To this end, an analysis was performed of the contents of the most significant scientific literature on economics, management, planning and development, urban studies and geography published over the period 1997-2006. From the results, a database of 142 papers from 43 journals was then elaborated. An analysis of these works reveals the level of development of the main lines of research in this field and, consequently, makes it possible to detect the topics that require greater attention and that can be the object of future research for researchers and academics. The main conclusions include the growing number of studies conducted on the subject in recent years, as well as a greater predominance of empirical research over conceptual work, and the existence of a significant change in the topics or lines of research throughout the period under study. © 2012 Copyright Taylor and Francis Group, LLC.This research was financed by the Spanish Ministry of Education and Science, National R&D & Innovation Plan 2007–2010, under the project entitled “El Distrito Industrial: el impacto del Capital Social sobre la Gestio´n de la Cadena de Suministro” (The Industrial District: the impact of Social Capital on the management of the Supply Chain) (SEJ2007- 62876/ECON).Martinez-Fernandez, M.; Capó Vicedo, J.; Vallet-Bellmunt, T. (2012). The present state of research into industrial clusters and districts. Content analysis of material published 1997-2006. European Planning Studies. 20(2):281-304. doi:10.1080/09654313.2012.650906S28130420