Secretion of Malaria Transmission-Blocking Proteins from Paratransgenic Bacteria

Malaria is a debilitating and deadly disease that afflicts over 200 million people and kills over 600 thousand each year. Due to quickly evolving drug resistance and lack of an affordable vaccine, novel interventions are needed to fight the Plasmodium parasites that cause malaria. Targeting Plasmodium inside their mosquito hosts is one approach that could complement other preventative and medicinal interventions by reducing the ability of the mosquitoes to transmit the disease to humans. The research presented here uses paratransgenesis, the genetic modification of symbiotic bacteria within the mosquito midgut, to provide antimalarial protein to the mosquito and to interfere with the life cycle of Plasmodium within the insect host. This research has produced three new antimalarial paratransgenic tools. The first tool is a set of new antimalarial effector proteins that were constructed by converting anti-Plasmodium mouse antibodies into single-chain variable fragment (scFv) versions for expression by bacteria. These antibodies bind to Plasmodium surface proteins and interfere with critical steps in the parasite life cycle. The second tool is a modified bacterial species, Pantoea agglomerans , which was engineered to secrete diverse antimalarial proteins via the hemolysin secretion pathway. Modified P. agglomerans were fed to mosquitoes and were capable of inhibiting the invasion of Plasmodium within the midgut. The third tool is another modified bacterial species, Asaia sp. SF2.1. Native Type II secretion signals were discovered that enable the creation of paratransgenic strains of these bacteria. Modified strains of Asaia sp. SF2.1 were also demonstrated to interfere with the invasion of Plasmodium within the mosquito. These tools have laid the groundwork for the future use of paratransgenic bacteria to combat malaria in the wild. Asaia sp. SF2.1 bacteria, in particular, are capable of spreading throughout mosquito populations, so they provide their own drive mechanism to establish themselves within the mosquito vectors of malaria. While further modifications will be required to make these bacteria ready for field use, the findings of this research provide proof of concept that the bacteria are suitable for eventual use in malaria transmission-blocking interventions

Numerical Computation of approximate Generalized Polarization Tensors

In this paper we describe a method to compute Generalized Polarization Tensors. These tensors are the coefficients appearing in the multipolar expansion of the steady state voltage perturbation caused by an inhomogeneity of constant conductivity. As an alternative to the integral equation approach, we propose an approximate semi-algebraic method which is easy to implement. This method has been integrated in a Myriapole, a matlab routine with a graphical interface which makes such computations available to non-numerical analysts

Self-Assembled Lipid Nanoparticles for Oral Delivery of Heparin-Coated Iron Oxide Nanoparticles for Theranostic Purposes

Recently, solid lipid nanoparticles (SLNs) have attracted increasing attention owing to their potential as an oral delivery system, promoting intestinal absorption in the lymphatic circulation which plays a role in disseminating metastatic cancer cells and infectious agents throughout the body. SLN features can be exploited for the oral delivery of theranostics. Therefore, the aim of this work was to design and characterise self-assembled lipid nanoparticles (SALNs) to encapsulate and stabilise iron oxide nanoparticles non-covalently coated with heparin (Fe@hepa) as a model of a theranostic tool. SALNs were characterised for physico-chemical properties (particle size, surface charge, encapsulation efficiency, in vitro stability, and heparin leakage), as well as in vitro cytotoxicity by methyl thiazole tetrazolium (MTT) assay and cell internalisation in CaCo-2, a cell line model used as an indirect indication of intestinal lymphatic absorption. SALNs of about 180 nm, which are stable in suspension and have a high encapsulation efficiency (>90%) were obtained. SALNs were able to stabilise the heparin coating of Fe@hepa, which are typically unstable in physiological environments. Moreover, SALNs-Fe@hepa showed no cytotoxicity, although their ability to be internalised into CaCo-2 cells was highlighted by confocal microscopy analysis. Therefore, the results indicated that SALNs can be considered as a promising tool to orally deliver theranostic Fe@hepa into the lymphatic circulation, although further in vivo studies are needed to comprehend further potential applications

A comparative study of diagnostic and imaging techniques for osteoarthritis of the trapezium

Objectives. The aims of this study were to determine whether micro-CT is a reliable investigation method to evaluate the severity of OA in the trapezium and to develop a novel micro-CT scoring system based on a quantitative assessment of the subchondral bone thickness in order to better assess OA through an objective parameter.Methods. We compared different diagnostic and imaging techniques performed consecutively on each sample: X-ray, visual analysis, micro-CT and histology. OA and healthy trapezia were subjected to semi-quantitative and quantitative analyses to be classified in four degrees of severity in OA (control, OA-2, OA-3 and OA-4). Specifically, samples were analysed using Dell's score for X-ray, Brown's score for visual analysis and Mankin's score for histology. Micro-CT was scored using a novel quantitative scoring system based on subchondral bone thickness measurements. Results obtained with each technique were then compared and correlated.Results. X-ray analysis showed a higher frequency of OA-2 (27%) and OA-3 (32%) compared with OA-4 (5%), whereas visual analysis, micro-CT and histology showed a lower percentage for OA-2 (18%, 18% and 14%) and OA-3 (23%) and increased frequency for OA-4 (45%, 32% and 40%). Only the micro-CT score of subchondral bone thickness correlated significantly with all the other techniques (P < 0.05).Conclusion. This is the first comparison of techniques proposing a novel scoring system based on objective and quantitative micro-CT data that can be applied as a useful diagnostic tool for OA, providing a deeper comprehension of the pathophysiology of OA in trapezium

Localization of Magic-F1 Transgene, Involved in Muscular Hypertrophy, during Early Myogenesis

We recently showed that Magic-F1 (Met-activating genetically improved chimeric factor 1), a human recombinant protein derived from hepatocyte growth factor/scatter factor (HGF/SF) induces muscle cell hypertrophy but not progenitor cell proliferation, both in vitro and in vivo. Here, we examined the temporal and spatial expression pattern of Magic-F1 in comparison with Pax3 (paired box gene 3) transcription factor during embryogenesis. Ranging from 9.5 to 17.5 dpc (days post coitum) mouse embryos were analyzed by in situ hybridization using whole mounts during early stages of development (9.5–10.5–11.5 dpc) and cryostat sections for later stages (11.5–13.5–15.5–17.5 dpc). We found that Magic-F1 is expressed in developing organs and tissues of mesenchymal origin, where Pax3 signal appears to be downregulated respect to the wt embryos. These data suggest that Magic-F1 could be responsible of muscular hypertrophy, cooperating with Pax3 signal pathway in skeletal muscle precursor cells

Systemic and Local Administration of Antimicrobial and Cell Therapies to Prevent Methicillin-Resistant Staphylococcus epidermidis-Induced Femoral Nonunions in a Rat Model

S. epidermidis is responsible for biofilm-related nonunions. This study compares the response to S. epidermidis-infected fractures in rats systemically or locally injected with vancomycin or bone marrow mesenchymal stem cells (BMSCs) in preventing the nonunion establishment. The 50% of rats receiving BMSCs intravenously (s-rBMSCs) died after treatment. A higher cytokine trend was measured in BMSCs locally injected rats (l-rBMSCs) at day 3 and in vancomycin systemically injected rats (l-VANC) at day 7 compared to the other groups. At day 14, the highest cytokine values were measured in l-VANC and in l-rBMSCs for IL-10. \ub5CT showed a good bony bridging in s-VANC and excellent both in l-VANC and in l-rBMSCs. The bacterial growth was lower in s-VANC and l-VANC than in l-rBMSCs. Histology demonstrated the presence of new woven bone in s-VANC and a more mature bony bridging was found in l-VANC. The l-rBMSCs showed a poor bony bridging of fibrovascular tissue. Our results could suggest the synergic use of systemic and local injection of vancomycin as an effective treatment to prevent septic nonunions. This study cannot sustain the systemic injection of BMSCs due to high risks, while a deeper insight into local BMSCs immunomodulatory effects is mandatory before developing cell therapies in clinics

Localization of Magic-F1 Transgene, Involved in Muscular Hypertrophy, during Early Myogenesis

We recently showed that Magic-F1 (Met-activating genetically improved chimeric factor 1), a human recombinant protein derived from hepatocyte growth factor/scatter factor (HGF/SF) induces muscle cell hypertrophy but not progenitor cell proliferation, both in vitro and in vivo. Here, we examined the temporal and spatial expression pattern of Magic-F1 in comparison with Pax3 (paired box gene 3) transcription factor during embryogenesis. Ranging from 9.5 to 17.5 dpc (days post coitum) mouse embryos were analyzed by in situ hybridization using whole mounts during early stages of development (9.5-10.5-11.5 dpc) and cryostat sections for later stages (11.5-13.5-15.5-17.5 dpc). We found that Magic-F1 is expressed in developing organs and tissues of mesenchymal origin, where Pax3 signal appears to be downregulated respect to the wt embryos. These data suggest that Magic-F1 could be responsible of muscular hypertrophy, cooperating with Pax3 signal pathway in skeletal muscle precursor cells

Novel self-gelling injectable hydrogel/alpha-tricalcium phosphate composites for bone regeneration:Physiochemical and microcomputer tomographical characterization

Mineralized hydrogels are increasingly gaining attention as biomaterials for bone regeneration. The most common mineralization strategy has been addition of preformed inorganic particles during hydrogel formation. This maintains injectability. One common form of bone cement is formed by mixing particles of the highly reactive calcium phosphate alpha-tricalcium phosphate (α-TCP) with water to form hydroxyapatite (HA). The calcium ions released during this reaction can be exploited to crosslink anionic, calcium-binding polymers such as the polysaccharide gellan gum (GG) to induce hydrogel formation. In this study, three different amounts of α-TCP particles were added to GG polymer solution to generate novel, injectable hydrogel-inorganic composites. Distribution of the inorganic phase in the hydrogel was studied by high resolution microcomputer tomography (µCT). Gelation occurred within 30 min. α-TCP converted to HA. µCT revealed inhomogeneous distribution of the inorganic phase in the composites. These results demonstrate the potential of the composites as alternatives to traditional α-TCP bone cement and pave the way for incorporation of biologically active substances and in vitro and in vivo testing