Other Publications (15)
- Biomedical Materials (Bristol, England)
- Lab on a Chip
- Journal of Neurotrauma
- Molecular BioSystems
- Methods in Molecular Biology (Clifton, N.J.)
- Stem Cell Research
- Sensors (Basel, Switzerland)
- Advanced Healthcare Materials
- BioResearch Open Access
- Stem Cells Translational Medicine
- Current Biology : CB
- Journal of Materials Chemistry. B, Materials for Biology and Medicine
- Scientific Reports
- FEBS Letters
Articles by Salvatore Pernagallo in JoVE
High-throughput Identification of Bacteria Repellent Polymers for Medical Devices Seshasailam Venkateswaran*1, Peter J. Gwynne*2, Mei Wu1, Ailsa Hardman2, Annamaria Lilienkampf1, Salvatore Pernagallo1, Garry Blakely2, David G. Swann3, Mark Bradley1, Maurice P. Gallagher2 1School of Chemistry, EaStCHEM, University of Edinburgh, 2School of Biological Sciences, University of Edinburgh, 3Critical Care, NHS Lothian, Royal Infirmary of Edinburgh A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.
Other articles by Salvatore Pernagallo on PubMed
Deciphering Cellular Morphology and Biocompatibility Using Polymer Microarrays Biomedical Materials (Bristol, England). Sep, 2008 | Pubmed ID: 18708702 A quantitative and qualitative analysis of cellular adhesion, morphology and viability is essential in understanding and designing biomaterials such as those involved in implant surfaces or as tissue-engineering scaffolds. As a means to simultaneously perform these studies in a high-throughput (HT) manner, we report a normalized protocol which allows the rapid analysis of a large number of potential cell binding substrates using polymer microarrays and high-content fluorescence microscopy. The method was successfully applied to the discovery of optimal polymer substrates from a 214-member polyurethane library with mouse fibroblast cells (L929), as well as simultaneous evaluation of cell viability and cellular morphology. Analysis demonstrated high biocompatibility of the binding polymers and permitted the identification of several different cellular morphologies, showing that specific polymer interactions may provoke changes in cell shape. In addition, SAR studies showed a clear correspondence between cellular adhesion and polymer structure. The approach can be utilized to perform multiple experiments (up to 1024 single experiments per slide) in a highly reproducible manner, leading to the generation of vast amounts of data in a short time period (48-72 h) while reducing dramatically the quantities of polymers, reagents and cells used.
A Cooperative Polymer-DNA Microarray Approach to Biomaterial Investigation Lab on a Chip. Feb, 2009 | Pubmed ID: 19156288 In this study, polymer microarrays were used for the rapid identification of polymer substrates upon which a suspension cell line would both adhere and proliferate giving a detailed and rapid understanding of cell-biomaterial interactions. Analysis demonstrated that suspension K562 human erythroleukemic cells, which normally grow in suspension, adhered and proliferated on several different polymers. Phenotypic and transcriptomic analysis techniques allowed examination of the interaction between cells and polymers permitting the elucidation of putative links between phenotypic responses to cell-biomaterial interactions and global gene expression.
Transcriptomics of Traumatic Brain Injury: Gene Expression and Molecular Pathways of Different Grades of Insult in a Rat Organotypic Hippocampal Culture Model Journal of Neurotrauma. Feb, 2010 | Pubmed ID: 19903084 Traumatic brain injury (TBI) is the one of the most common forms of head trauma, and it remains a leading cause of death and disability. It is known that the initial mechanical axonal injury triggers a complex cascade of neuroinflammatory and metabolic events, the understanding of which is essential for clinical, translational, and pharmacological research. These can occur even in mild TBI, and are associated with several post-concussion manifestations, including transiently heightened vulnerability to a second insult. Recent studies have challenged the tenet that ischemia is the ultimate modality of tissue damage following TBI, as metabolic dysfunction can develop in the presence of normal perfusion and before intracranial hypertension. In order to elucidate the cellular and molecular changes occurring in TBI as a direct result of neuronal injury and in the absence of ischemic damage, we performed a microarray analysis of expressed genes and molecular interaction pathways for different levels of severity of trauma using an in-vitro model. A stretch injury, equivalent to human diffuse axonal injury, was delivered to rat organotypic hippocampal slice cultures, and mRNA levels following a 10% (mild) and 50% (severe) stretch were compared with controls at 24 h. More genes were differentially expressed following 10% stretch than 50% stretch, indicating the early activation of complex cellular mechanisms. The data revealed remarkable differential gene expression following mTBI, even in the absence of cell damage. Pathway analysis revealed that molecular interactions in both levels of injury were similar, with IL-1beta playing a central role. Additional pathways of neurodegeneration involving RhoA (ras homolog gene family, member A) were found in 50% stretch.
Investigation of Microsphere-mediated Cellular Delivery by Chemical, Microscopic and Gene Expression Analysis Molecular BioSystems. Feb, 2010 | Pubmed ID: 20094660 Amino functionalized cross-linked polystyrene microspheres of well defined sizes (0.2-2 mum) have been prepared and shown to be efficient and controllable delivery devices, capable of transporting anything from small dye molecules to bulky proteins into cells. However, the specific mechanism of cellular entry is largely unknown and widely variant from study to study. As such, chemical, biological and microscopic methods are used to elucidate the mechanism of cellular uptake for polystyrene microspheres of 0.2, 0.5 and 2 mum in mouse melanoma cells. Uptake is found to be wholly unreliant upon energetic processes, while lysosomal and endosomal tracking agents failed to show co-localisation with lysosomes/endosomes, suggesting a non-endocytic uptake pathway. To further explore the consequences of microsphere uptake, gene expression profiling is used to determine if there is a transcriptional response to "beadfection" in both murine and human cells. None of the common transcriptional responses to enhanced endocytosis are observed in beadfected cells, further supporting a non-endocytic uptake mechanism. Furthermore, the microspheres are noted to have a limited interaction with cells at a transcriptional level, supporting them as a non-toxic delivery vehicle.
Polymer Microarrays for Cellular High-content Screening Methods in Molecular Biology (Clifton, N.J.). 2011 | Pubmed ID: 21104063 Polymer microarrays as platforms for cell-based assays are presented, offering a unique approach to high-throughput cellular analysis. These high-throughput (HT) platforms are used for the screening of new materials with the purpose of first finding substrates upon which a specific cell line would adhere and second to gain a rapid understanding of the interactions between cells and biomaterials. Arrays presented here are fabricated using pre-synthesised polymers by contact printing via a robotic microarrayer. These large arrays of polymers are then incubated with cell cultures and the results obtained are used to significantly help the design of synthetic biomaterials, implant surfaces and tissue-engineering scaffolds by finding correlations between their chemical structure and their biological performance. The flexibility of polymer microarrays analysis not only greatly refines our knowledge of multitude of cell-biomaterial interactions but could also be used in biocompatibility assessments as novel biomarkers.
Unbiased Screening of Polymer Libraries to Define Novel Substrates for Functional Hepatocytes with Inducible Drug Metabolism Stem Cell Research. Mar, 2011 | Pubmed ID: 21277274 Maintaining stable differentiated somatic cell function in culture is essential to a range of biological endeavors. However, current technologies, employing, for example, primary hepatic cell culture (essential to the development of a bio-artificial liver and improved drug and toxicology testing), are limited by supply, expense, and functional instability even on biological cell culture substrata. As such, novel biologically active substrates manufacturable to GMP standards have the potential to improve cell culture-based assay applications. Currently hepatic endoderm (HE) generated from pluripotent stem cells is a genotypically diverse, cheap, and stable source of "hepatocytes"; however, HE routine applications are limited due to phenotypic instability in culture. Therefore a manufacturable subcellular matrix capable of supporting long-term differentiated cell function would represent a step forward in developing scalable and phenotypically stable hESC-derived hepatocytes. Adopting an unbiased approach we screened polymer microarrays and identified a polyurethane matrix which promoted HE viability, hepatocellular gene expression, drug-inducible metabolism, and function. Moreover, the polyurethane supported, when coated on a clinically approved bio-artificial liver matrix, long-term hepatocyte function and growth. In conclusion, our data suggest that an unbiased screening approach can identify cell culture substrate(s) that enhance the phenotypic stability of primary and stem cell-derived cell resources.
Novel Biochip Platform for Nucleic Acid Analysis Sensors (Basel, Switzerland). 2012 | Pubmed ID: 22969389 This manuscript describes the use of a novel biochip platform for the rapid analysis/identification of nucleic acids, including DNA and microRNAs, with very high specificity. This approach combines a unique dynamic chemistry approach for nucleic acid testing and analysis developed by DestiNA Genomics with the STMicroelectronics In-Check platform, which comprises two microfluidic optimized and independent PCR reaction chambers, and a sequential microarray area for nucleic acid capture and identification by fluorescence. With its compact bench-top "footprint" requiring only a single technician to operate, the biochip system promises to transform and expand routine clinical diagnostic testing and screening for genetic diseases, cancers, drug toxicology and heart disease, as well as employment in the emerging companion diagnostics market.
Novel Biopolymers to Enhance Endothelialisation of Intra-vascular Devices Advanced Healthcare Materials. Sep, 2012 | Pubmed ID: 23184801 Rapid endothelisation is of critical importance in the prevention of adverse remodelling after device implantation. Currently, there is a need for alternative strategies to promote re-endothelialisation for intravascular stents and vascular grafts. Using polymer microarray technology 345 polymers are comprehensively assessed and a matrix is identified that specifically supports both progenitor and mature endothelial cell activity in vitro and in vivo while minimising platelet attachment.
Maintaining Hepatic Stem Cell Gene Expression on Biological and Synthetic Substrata BioResearch Open Access. Jan, 2012 | Pubmed ID: 23515003 The liver is a highly resilient organ that possesses enormous regenerative capacity. This is mediated mainly through the most abundant cell type found in the liver, the hepatocyte. When the regenerative capacity of the hepatocyte is compromised, during chronic or acute liver injury, hepatic progenitor cells (HPCs) are activated to replace the damaged tissue. The HPC resides in a laminin-rich environment; as HPCs differentiate toward a hepatic or biliary fate, the extracellular matrix (ECM) composition changes, influencing cell behavior. To assess the impact that the biological ECM and the synthetic ECM have on the maintenance of hepatic stem cell gene expression, a murine hepatic stem cell line was employed. We demonstrate that hepatic stem cell gene expression could be maintained using a biological or synthetic substratum, but not on plastic alone.
Developing High-fidelity Hepatotoxicity Models from Pluripotent Stem Cells Stem Cells Translational Medicine. Jul, 2013 | Pubmed ID: 23757504 Faithfully recapitulating human physiology "in a dish" from a renewable source remains a holy grail for medicine and pharma. Many procedures have been described that, to a limited extent, exhibit human tissue-specific function in vitro. In particular, incomplete cellular differentiation and/or the loss of cell phenotype postdifferentiation play a major part in this void. We have developed an interdisciplinary approach to address this problem, using skill sets in cell biology, materials chemistry, and pharmacology. Pluripotent stem cells were differentiated to hepatocytes before being replated onto a synthetic surface. Our approach yielded metabolically active hepatocyte populations that displayed stable function for more than 2 weeks in vitro. Although metabolic activity was an important indication of cell utility, the accurate prediction of cellular toxicity in response to specific pharmacological compounds represented our goal. Therefore, detailed analysis of hepatocellular toxicity was performed in response to a custom-built and well-defined compound set and compared with primary human hepatocytes. Importantly, stem cell-derived hepatocytes displayed equivalence to primary human material. Moreover, we demonstrated that our approach was capable of modeling metabolic differences observed in the population. In conclusion, we report that pluripotent stem cell-derived hepatocytes will model toxicity predictably and in a manner comparable to current gold standard assays, representing a major advance in the field.
A Conserved Oct4/POUV-dependent Network Links Adhesion and Migration to Progenitor Maintenance Current Biology : CB. Nov, 2013 | Pubmed ID: 24210613 The class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarily conserved regulator of progenitor cell differentiation during embryonic development.
Bacteria Repelling Poly(methylmethacrylate-co-dimethylacrylamide) Coatings for Biomedical Devices†Electronic Supplementary Information (ESI) Available: Polymer Microarray Screening, Including Analysis of Bacterial Adhesion by Fluorescence Microscopy and SEM, and Chemical Composition of Bacteria Repelling Polymers Identified in the Screen; Polymer Synthesis and Characterisation; Preparation of Catheter Pieces and Solvent Studies, and Details for Confocal Imaging/analysis. See DOI: 10.1039/c4tb01129eClick Here for Additional Data File Journal of Materials Chemistry. B, Materials for Biology and Medicine. Sep, 2014 | Pubmed ID: 25580245 Nosocomial infections due to bacteria have serious implications on the health and recovery of patients in a variety of medical scenarios. Since bacterial contamination on medical devices contributes to the majority of nosocomical infections, there is a need for redesigning the surfaces of medical devices, such as catheters and tracheal tubes, to resist the binding of bacteria. In this work, polyurethanes and polyacrylates/acrylamides, which resist binding by the major bacterial pathogens underpinning implant-associated infections, were identified using high-throughput polymer microarrays. Subsequently, two 'hit' polymers, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)) and PA515 (poly(methoxyethylmethacrylate-co-diethylaminoethylacrylate-co-methylmethacrylate)), were used to coat catheters and substantially shown to decrease binding of a variety of bacteria (including isolates from infected endotracheal tubes and heart valves from intensive care unit patients). Catheters coated with polymer PA13 showed up to 96% reduction in bacteria binding in comparison to uncoated catheters.
Number of Nanoparticles Per Cell Through a Spectrophotometric Method - A Key Parameter to Assess Nanoparticle-based Cellular Assays Scientific Reports. May, 2015 | Pubmed ID: 25976173 Engineered nanoparticles (eNPs) for biological and biomedical applications are produced from functionalised nanoparticles (NPs) after undergoing multiple handling steps, giving rise to an inevitable loss of NPs. Herein we present a practical method to quantify nanoparticles (NPs) number per volume in an aqueous suspension using standard spectrophotometers and minute amounts of the suspensions (up to 1 μL). This method allows, for the first time, to analyse cellular uptake by reporting NPs number added per cell, as opposed to current methods which are related to solid content (w/V) of NPs. In analogy to the parameter used in viral infective assays (multiplicity of infection), we propose to name this novel parameter as multiplicity of nanofection.
Identification and Characterization of a Bacterial Hyaluronidase and Its Production in Recombinant Form FEBS Letters. Jul, 2016 | Pubmed ID: 27311405 Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection.
Novel Bead-based Platform for Direct Detection of Unlabelled Nucleic Acids Through Single Nucleobase Labelling Talanta. Dec, 2016 | Pubmed ID: 27769437 Over the last decade, circulating microRNAs have received attention as diagnostic and prognostic biomarkers. In particular, microRNA122 has been demonstrated to be an early and more sensitive indicator of drug-induced liver injury than the widely used biomarkers such as alanine aminotransferase and aspartate aminotransferase. Recently, microRNA122 has been used in vitro to assess the cellular toxicity of new drugs and as a biomarker for the development of a rapid test for drug overdose/liver damage. In this proof-of-concept study, we report a PCR-free and label-free detection method that has a limit of detection (3 standard deviations) of 15 fmoles of microRNA122, by integrating a dynamic chemical approach for "Single Nucleobase Labelling" with a bead-based platform (Luminex(®)) thereby, in principle, demonstrating the exciting prospect of rapid and accurate profiling of any microRNAs related to diseases and toxicology.