Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

Situation Background Assessment Recommendation (SBAR): Undergraduate Perspectives

Situation Background Assessment and Recommendation (SBAR): Undergraduate Perspectives C Morgan, L Adams, J Murray, R Dunlop, IK Walsh. Ian K Walsh, Centre for Medical Education, Queen’s University Belfast, Mulhouse Building, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6DP Background and Purpose: Structured communication tools are used to improve team communication quality.1,2 The Situation Background Assessment and Recommendation (SBAR) tool is widely adopted within patient safety.3 SBAR effectiveness is reportedly equivocal, suggesting use is not sustained beyond initial training.4-6 Understanding perspectives of those using SBAR may further improve clinical communication. We investigated senior medical undergraduate perspectives on SBAR, particularly when communicating with senior colleagues. Methodology: Mixed methods data collection was used. A previously piloted questionnaire with 12 five point Lickert scale questions and 3 open questions was given to all final year medical students. A subgroup also participated in 10 focus groups, deploying strictly structured audio-recorded questions. Selection was by convenience sampling, data gathered by open text questions and comments transcribed verbatim. In-vivo coding (iterative, towards data saturation) preceded thematic analysis. Results: 233 of 255 students (91%) completed the survey. 1. There were clearly contradictory viewpoints on SBAR usage. A recurrent theme was a desire for formal feedback and a relative lack of practice/experience with SBAR. 2. Students reported SBAR as having variable interpretation between individuals; limiting use as a shared mental model. 3. Brief training sessions are insufficient to embed the tool. 4. Most students reported SBAR helping effective communication, especially by providing structure in stressful situations. 5. Only 18.5% of students felt an alternative resource might be needed. Sub analysis of the themes highlighted: A. Lack of clarity regarding what information to include and information placement within the acronym, B. Senior colleague negative response to SBAR C. Lack of conciseness with the tool. Discussion and Conclusions: Despite a wide range of contradictory interpretation of SBAR utility, most students wish to retain the resource. More practice opportunities/feedback may enhance user confidence and understanding. References: (1) Leonard M, Graham S, Bonacum D. The human factor: the critical importance of effective teamwork and communication in providing safe care. Quality & Safety in Health Care 2004 Oct;13(Suppl 1):85-90. (2) d'Agincourt-Canning LG, Kissoon N, Singal M, Pitfield AF. Culture, communication and safety: lessons from the airline industry. Indian J Pediatr 2011 Jun;78(6):703-708. (3) Dunsford J. Structured communication: improving patient safety with SBAR. Nurs Womens Health 2009 Oct;13(5):384-390. (4) Compton J, Copeland K, Flanders S, Cassity C, Spetman M, Xiao Y, et al. Implementing SBAR across a large multihospital health system. Jt Comm J Qual Patient Saf 2012 Jun;38(6):261-268. (5) Ludikhuize J, de Jonge E, Goossens A. Measuring adherence among nurses one year after training in applying the Modified Early Warning Score and Situation-Background-Assessment-Recommendation instruments. Resuscitation 2011 Nov;82(11):1428-1433. (6) Cunningham NJ, Weiland TJ, van Dijk J, Paddle P, Shilkofski N, Cunningham NY. Telephone referrals by junior doctors: a randomised controlled trial assessing the impact of SBAR in a simulated setting. Postgrad Med J 2012 Nov;88(1045):619-626.

Pervasive eHealth services a security and privacy risk awareness survey

The human factor is often recognised as a major aspect of cyber-security research. Risk and situational perception are identified as key factors in the decision making process, often playing a lead role in the adoption of security mechanisms. However, risk awareness and perception have been poorly investigated in the field of eHealth wearables. Whilst end-users often have limited understanding of privacy and security of wearables, assessing the perceived risks and consequences will help shape the usability of future security mechanisms. This paper present a survey of the the risks and situational awareness in eHealth services. An analysis of the lack of security and privacy measures in connected health devices is described with recommendations to circumvent critical situations.

MATCH: a new industry-focused approach to medical device development

MATCH (Multidisciplinary Assessment of Technology Centre for Healthcare) is a new collaboration in the UK that aims to support the healthcare sector by creating methods to assess the value of medical devices from concept through to mature product. A major aim of MATCH is to encourage the inclusion of the user throughout the product lifecycle in order to achieve devices that truly meet the requirements of their users. A review of the published literature indicates that user requirements are mainly collected during the design and evaluation stage of the product lifecycle whilst other areas, including the concept stage, have less user involvement. Complementing the literature review is an in-depth consultation with the medical device industry, which has identified a number of barriers encountered by companies when attempting to capture user requirements. These will be addressed by a number of case study projects, performed in collaboration with our industrial partners, that will examine the application and utility of different approaches to collecting and analysing data on user requirements. MATCH is focused on providing advice to device developers on how to select and apply methods that have maximum theoretical strength, practical application, cost-effectiveness and likelihood of wide sector acceptance. Feedback will be sought in order to ensure that the needs of the diverse medical device sector are met.

The use of digital educational resources : the contribution of research in the supervised teaching practice in basic education

This article aims to reflect on the impact of Information and Communication Technologies (ICT) in the educational context, focusing on the potential contributions of the use of Digital Educational Resources (RED) in the process of teaching and learning. For this purpose, the results of the use of the RED will be presented:. Digital Classroom - The World's 1st Year Carochinha The study was accomplished in a class of the 1st grade of the 1st CEB, composed of 27 students, aged 6-7 years in Castelo Branco City Schools Group within the Supervised Teaching Practice. The results obtained after the analysis and processing of the data showed that when using this RED students show they have acquired the content covered by the fact that they enhanced levels of greater interest, commitment, motivation, commitment and initiative in the course of activities proposals. But, perhaps because they are students of 1st year of the 1st CEB, do not neglect the presence and monitoring of the teacher and the use of paper-based resources. This means that there should be a complementarity that reconciles the human factor (teacher), with the use of digital media resources and paper support resources (Manual).

A study on situational awareness security and privacy of wearable health monitoring devices

Situational Awareness provides a user centric approach to security and privacy. The human factor is often recognised as the weakest link in security, therefore situational perception and risk awareness play a leading role in the adoption and implementation of security mechanisms. In this study we assess the understanding of security and privacy of users in possession of wearable devices. The findings demonstrate privacy complacency, as the majority of users trust the application and the wearable device manufacturer. Moreover the survey findings demonstrate a lack of understanding of security and privacy by the sample population. Finally the theoretical implications of the findings are discussed.

Medición de la cultura de seguridad del paciente en un hospital público de primer nivel en el municipio de Villeta

Esta investigación midió la percepción del personal asistencial sobre la cultura de seguridad de los pacientes en un hospital de primer nivel de complejidad por medio de un estudio descriptivo de corte transversal. Se utilizó como herramienta de medición la encuesta ‘Hospital Survey on Patient Safety Cultura’ (HSOPSC) de la Agency of Healthcare Research and Quality (AHRQ) versión en español, la cual evalúa doce dimensiones. Los resultados mostraron fortalezas como el aprendizaje organizacional, las mejoras continuas y el apoyo de los administradores para la seguridad del paciente. Las dimensiones clasificadas como oportunidades de mejora fueron la cultura no punitiva, el personal, las transferencias y transiciones y el grado en que la comunicación es abierta. Se concluyó que aunque el personal percibía como positivo el proceso de mejoramiento y apoyo de la administración también sentía que era juzgado si reportaba algún evento adverso.

Liderazgo y autoeficacia en el comportamiento organizacional positivo

El objetivo que tiene este proyecto es revisar los conceptos básicos acerca de las relaciones que crean los líderes con sus colaboradores dentro de las organizaciones, dichas relaciones y vínculos pueden afectar positiva o negativamente el desempeño de sus actividades diarias dentro de una organización. Para darle inicio a la investigación se estudió como primer paso el concepto de liderazgo transformacional, capital psicológico y que componentes hacían parte de este factor. El desarrollo de la investigación se enfatizó entre el liderazgo transformacional y la autoeficacia ya que son factores claves dentro del desarrollo de las actividades organizacionales debido a que afectan claramente el capital humano de las compañías y están directamente relacionados con el crecimiento de las mismas, lo que nos llevó a preguntarnos ¿qué relación tendrá el liderazgo transformacional y la autoeficacia en la productividad de las empresas? Es aquí donde radica la importancia de esta investigación ya que el cambio de pensamiento de las organizaciones hacia un liderazgo transformacional podría lograr una maximización del desempeño del personal de trabajo en relación al objetivo de la compañía. Como conclusión llegamos a que efectivamente hay un efecto positivo en los individuos que desarrollan un capital psicológico específicamente en el factor de autoeficacia para lograr un desempeño destacable, productivo y eficiente dentro de las organizaciones.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from 12 age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the Wnt pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

Brivanib alaninate, a dual inhibitor of vascular endothelial growth factor receptor and fibroblast growth factor receptor tyrosine kinases, induces growth inhibition in mouse models of human hepatocellular carcinoma

PURPOSE: Hreceptor (VEGFR) and FGF receptor (FGFR) signaling pathways. EXPERIMENTAL DESIGN: Six different s.c. patient-derived HCC xenografts were implanted into mice. Tumor growth was evaluated in mice treated with brivanib compared with control. The effects of brivanib on apoptosis and cell proliferation were evaluated by immunohistochemistry. The SK-HEP1 and HepG2 cells were used to investigate the effects of brivanib on the VEGFR-2 and FGFR-1 signaling pathways in vitro. Western blotting was used to determine changes in proteins in these xenografts and cell lines. RESULTS: Brivanib significantly suppressed tumor growth in five of six xenograft lines. Furthermore, brivanib-induced growth inhibition was associated with a decrease in phosphorylated VEGFR-2 at Tyr(1054/1059), increased apoptosis, reduced microvessel density, inhibition of cell proliferation, and down-regulation of cell cycle regulators. The levels of FGFR-1 and FGFR-2 expression in these xenograft lines were positively correlated with its sensitivity to brivanib-induced growth inhibition. In VEGF-stimulated and basic FGF stimulated SK-HEP1 cells, brivanib significantly inhibited VEGFR-2, FGFR-1, extracellular signal-regulated kinase 1/2, and Akt phosphorylation. CONCLUSION: This study provides a strong rationale for clinical investigation of brivanib in patients with HCC.

Cloning and characterization of a novel human gene related to vascular endothelial growth factor

This paper describes the cloning and characterization of a new member of the vascular endothelial growth factor (VEGF) gene family, which we have designated VRF for VEGF-related-factor. Sequencing of cDNAs from a human fetal brain library and RT-PCR products from normal and tumor tissue cDNA pools indicate two alternatively spliced messages with open reading frames of 621 and 564 bp, respectively. The predicted proteins differ at their carboxyl ends resulting from a shift in the open reading frame. Both isoforms show strong homology to VEGF at their amino termini, but only the shorter isoform maintains homology to VEGF at its carboxyl terminus and conserves all 16 cysteine residues of VEGF165. Similarity comparisons of this isoform revealed overall protein identity of 48% and conservative substitution of 69% with VEGF189. VRF is predicted to contain a signal peptide, suggesting that it may be a secreted factor. The VRF gene maps to the D11S750 locus at chromosome band 11q13, and the protein coding region, spanning approximately 5 kb, is comprised of 8 exons that range in size from 36 to 431 bp. Exons 6 and 7 are contiguous and the two isoforms of VRF arise through alternate splicing of exon 6. VRF appears to be ubiquitously expressed as two transcripts of 2.0 and 5.5 kb; the level of expression is similar among normal and malignant tissues.