Publications by authors named "Marta Szabat"

16 Publications

  • Page 1 of 1

RNA Secondary Structure as a First Step for Rational Design of the Oligonucleotides towards Inhibition of Influenza A Virus Replication.

Pathogens 2020 Nov 7;9(11). Epub 2020 Nov 7.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.

Influenza is an important research subject around the world because of its threat to humanity. Influenza A virus (IAV) causes seasonal epidemics and sporadic, but dangerous pandemics. A rapid antigen changes and recombination of the viral RNA genome contribute to the reduced effectiveness of vaccination and anti-influenza drugs. Hence, there is a necessity to develop new antiviral drugs and strategies to limit the influenza spread. IAV is a single-stranded negative sense RNA virus with a genome (viral RNA-vRNA) consisting of eight segments. Segments within influenza virion are assembled into viral ribonucleoprotein (vRNP) complexes that are independent transcription-replication units. Each step in the influenza life cycle is regulated by the RNA and is dependent on its interplay and dynamics. Therefore, viral RNA can be a proper target to design novel therapeutics. Here, we briefly described examples of anti-influenza strategies based on the antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA) and catalytic nucleic acids. In particular we focused on the vRNA structure-function relationship as well as presented the advantages of using secondary structure information in predicting therapeutic targets and the potential future of this field.
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http://dx.doi.org/10.3390/pathogens9110925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694947PMC
November 2020

Parental experience of hope in pediatric palliative care: Critical reflections on an exemplar of parents of a child with trisomy 18.

Authors:
Marta Szabat

Nurs Inq 2020 04 3;27(2):e12341. Epub 2020 Jan 3.

Department of Philosophy and Bioethics, Medical College, Faculty of Health Sciences, Jagiellonian University, Krakow, Poland.

The purpose of this study is to analyze the experience of hope that appears in a parent's blog presenting everyday life while caring for a child with Trisomy 18 (Edwards syndrome). The author, Rebekah Peterson, began her blog on 17 March 2011 and continues to post information on her son Aaron's care. The analysis of hope in the blog is carried out using a mixed methodology: initial and focused coding using Charmaz's constructed grounded theory and elements of Colaizzi's method. Each aspect of hope is coded through the blog author's statements, from which three main aspects of hope emerge: hope for the longest possible presence of Aaron with his family, hope for control over situations, pain, and symptoms, and existential facets of hope. These various aspects reveal to what extent the experience of hope is unique. Additionally, analyzing the experience of parental hope uncovers the additional problem of inappropriate communication by health care professionals (HCPs) in intensive care units, particularly when discussing the termination of causal treatment. The problem may be solved through proper education for HCPs and serious consideration of parental involvement in order to properly elaborate guidelines on this issue. The three main aspects of parental hope discussed in this paper might expand knowledge on the issue, helping HCPs to better understand the parents' experience of care and to help sustain parental hope in pediatric palliative care.
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http://dx.doi.org/10.1111/nin.12341DOI Listing
April 2020

Modified RNA triplexes: Thermodynamics, structure and biological potential.

Sci Rep 2018 08 29;8(1):13023. Epub 2018 Aug 29.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland.

The occurrence of triplexes in vivo has been well documented and is determined by the presence of long homopurine-homopyrimidine tracts. The formation of these structures is the result of conformational changes that occur in the duplex, which allow the binding of a third strand within the major groove of the helix. Formation of these noncanonical forms by introducing synthetic triplex-forming oligonucleotides (TFOs) into the cell may have applications in molecular biology, diagnostics and therapy. This study focused on the formation of RNA triplexes as well as their thermal stability and biological potential in the HeLa cell line. Thermodynamics studies revealed that the incorporation of multiple locked nucleic acid (LNA) and 2-thiouridine (2-thioU) residues increased the stability of RNA triplexes. These data suggest that the number and position of the modified nucleotides within TFOs significantly stabilize the formed structures. Moreover, specificity of the interactions between the modified TFOs and the RNA hairpin was characterized using electrophoretic mobility-shift assay (EMSA), and triplex dissociation constants have been also determined. Finally, through quantitative analysis of GFP expression, the triplex structures were shown to regulate GFP gene silencing. Together, our data provide a first glimpse into the thermodynamic, structural and biological properties of LNA- and 2-thioU modified RNA triplexes.
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http://dx.doi.org/10.1038/s41598-018-31387-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115336PMC
August 2018

Reducing insulin via conditional partial gene ablation in adults reverses diet-induced weight gain.

FASEB J 2018 03 3;32(3):1196-1206. Epub 2018 Jan 3.

Department of Cellular and Physiological Sciences, Diabetes Research Group, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada.

Excess circulating insulin is associated with obesity in humans and in animal models. However, the physiologic causality of hyperinsulinemia in adult obesity has rightfully been questioned because of the absence of clear evidence that weight loss can be induced by acutely reversing diet-induced hyperinsulinemia. Herein, we describe the consequences of inducible, partial insulin gene deletion in a mouse model in which animals have already been made obese by consuming a high-fat diet. A modest reduction in insulin production/secretion was sufficient to cause significant weight loss within 5 wk, with a specific effect on visceral adipose tissue. This result was associated with a reduction in the protein abundance of the lipodystrophy gene polymerase I and transcript release factor ( Ptrf; Cavin) in gonadal adipose tissue. RNAseq analysis showed that reduced insulin and weight loss also associated with a signature of reduced innate immunity. This study demonstrates that changes in circulating insulin that are too fine to adversely affect glucose homeostasis nonetheless exert control over adiposity.-Page, M. M., Skovsø, S., Cen, H., Chiu, A. P., Dionne, D. A., Hutchinson, D. F., Lim, G. E., Szabat, M., Flibotte, S., Sinha, S., Nislow, C., Rodrigues, B., Johnson, J. D. Reducing insulin via conditional partial gene ablation in adults reverses diet-induced weight gain.
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http://dx.doi.org/10.1096/fj.201700518RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892722PMC
March 2018

Parallel-stranded DNA and RNA duplexes - structural features and potential applications.

FEBS J 2017 12 22;284(23):3986-3998. Epub 2017 Aug 22.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

Nowadays, decades after the discovery of the right-handed B form of DNA, it is well known that nucleic acids have great conformational flexibility, exhibiting a large degree of variation in their structure. In nature, DNA and RNA exist in an antiparallel orientation, stabilized by Watson-Crick base pairs. However, in some cases, nucleic acid fragments with specific nucleotide sequences can adopt a parallel orientation involving non-canonical base pairing. Interestingly, parallel-stranded duplexes have been found in specific chromosome regions. Furthermore, parallel oriented regions have also been found in bacterial (Escherichia coli, Listeria innocua) and insect genomes (Drosophila melanogaster). These unusual structures could have a remarkable evolutionary role, as well as significant impact on biological processes. For example, parallel stretches were shown to be involved in processing the 3' ends of mRNAs and in specific gene silencing. Moreover, certain types of parallel-stranded duplexes may be useful tools, with several practical applications. They can constitute excellent templates for the formation of other structures and for the development of antigene and antisense approaches.
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http://dx.doi.org/10.1111/febs.14187DOI Listing
December 2017

High-content screening identifies a role for Na(+) channels in insulin production.

R Soc Open Sci 2015 Dec 2;2(12):150306. Epub 2015 Dec 2.

Department of Cellular and Physiological Sciences University of British Columbia Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.

Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 β-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of β-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect β-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and β-cell differentiation status. In particular, our unbiased screen identified a novel role for a β-cell sodium channel gene in insulin production.
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http://dx.doi.org/10.1098/rsos.150306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807443PMC
December 2015

Thermodynamic Features of Structural Motifs Formed by β-L-RNA.

PLoS One 2016 23;11(2):e0149478. Epub 2016 Feb 23.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland.

This is the first report to provide comprehensive thermodynamic and structural data concerning duplex, hairpin, quadruplex and i-motif structures in β-L-RNA series. Herein we confirm that, within the limits of experimental error, the thermodynamic stability of enantiomeric structural motifs is the same as that of naturally occurring D-RNA counterparts. In addition, formation of D-RNA/L-RNA heterochiral duplexes is also observed; however, their thermodynamic stability is significantly reduced in reference to homochiral D-RNA duplexes. The presence of three locked nucleic acid (LNA) residues within the D-RNA strand diminishes the negative effect of the enantiomeric, complementary L-RNA strand in the formation of heterochiral RNA duplexes. Similar behavior is also observed for heterochiral LNA-2'-O-methyl-D-RNA/L-RNA duplexes. The formation of heterochiral duplexes was confirmed by 1H NMR spectroscopy. The CD curves of homochiral L-RNA structural motifs are always reversed, whereas CD curves of heterochiral duplexes present individual features dependent on the composition of chiral strands.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149478PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801053PMC
July 2016

Reduced Insulin Production Relieves Endoplasmic Reticulum Stress and Induces β Cell Proliferation.

Cell Metab 2016 Jan 25;23(1):179-93. Epub 2015 Nov 25.

Department of Cellular and Physiological Sciences, Diabetes Research Group, Life Sciences Institute, University of British Columbia, BC V6T1Z3, Canada. Electronic address:

Pancreatic β cells are mostly post-mitotic, but it is unclear what locks them in this state. Perturbations including uncontrolled hyperglycemia can drive β cells into more pliable states with reduced cellular insulin levels, increased β cell proliferation, and hormone mis-expression, but it is unknown whether reduced insulin production itself plays a role. Here, we define the effects of ∼50% reduced insulin production in Ins1(-/-):Ins2(f/f):Pdx1Cre(ERT):mTmG mice prior to robust hyperglycemia. Transcriptome, proteome, and network analysis revealed alleviation of chronic endoplasmic reticulum (ER) stress, indicated by reduced Ddit3, Trib3, and Atf4 expression; reduced Xbp1 splicing; and reduced phospho-eIF2α. This state was associated with hyper-phosphorylation of Akt, which is negatively regulated by Trib3, and with cyclinD1 upregulation. Remarkably, β cell proliferation was increased 2-fold after reduced insulin production independently of hyperglycemia. Eventually, recombined cells mis-expressed glucagon in the hyperglycemic state. We conclude that the normally high rate of insulin production suppresses β cell proliferation in a cell-autonomous manner.
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http://dx.doi.org/10.1016/j.cmet.2015.10.016DOI Listing
January 2016

Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2'-O-Methyl RNA and RNA Oligonucleotides.

PLoS One 2015 18;10(11):e0143354. Epub 2015 Nov 18.

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.

This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2'-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2'-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143354PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666348PMC
June 2016

Arabidopsis protein phosphatase 2C ABI1 interacts with type I ACC synthases and is involved in the regulation of ozone-induced ethylene biosynthesis.

Mol Plant 2014 Jun 17;7(6):960-976. Epub 2014 Mar 17.

Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, Poznań 61-614, Poland; Institute of Plant Genetics Polish Academy of Science, Strzeszyńska 34, Poznan 60-479, Poland.

Ethylene plays a crucial role in various biological processes and therefore its biosynthesis is strictly regulated by multiple mechanisms. Posttranslational regulation, which is pivotal in controlling ethylene biosynthesis, impacts 1-aminocyclopropane 1-carboxylate synthase (ACS) protein stability via the complex interplay of specific factors. Here, we show that the Arabidopsis thaliana protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, is involved in the regulation of ethylene biosynthesis under oxidative stress conditions. We found that ABI1 interacts with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6. In addition, ABI1 controls MPK6 activity directly and by this means also affects the ACS6 phosphorylation level. Consistently with this, ozone-induced ethylene production was significantly higher in an ABI1 knockout strain (abi1td) than in wild-type plants. Importantly, an increase in stress-induced ethylene production in the abi1td mutant was compensated by a higher ascorbate redox state and elevated antioxidant activities. Overall, the results of this study provide evidence that ABI1 restricts ethylene synthesis by affecting the activity of ACS6. The ABI1 contribution to stress phenotype underpins its role in the interplay between the abscisic acid (ABA) and ethylene signaling pathways.
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http://dx.doi.org/10.1093/mp/ssu025DOI Listing
June 2014

Modulation of β-cell fate and function by TGFβ ligands: a superfamily with many powers.

Endocrinology 2013 Nov;154(11):3965-9

PhD, Associate Professor, Medicine and Cellular and Physiological Sciences, Surgery, Diabetes Research Group, Cardiovascular Research Group, The University of British Columbia, Point Grey Campus, 5358-2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.

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http://dx.doi.org/10.1210/en.2013-1880DOI Listing
November 2013

Maintenance of β-cell maturity and plasticity in the adult pancreas: developmental biology concepts in adult physiology.

Diabetes 2012 Jun;61(6):1365-71

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

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http://dx.doi.org/10.2337/db11-1361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3357305PMC
June 2012

Kinetics and genomic profiling of adult human and mouse β-cell maturation.

Islets 2011 Jul-Aug;3(4):175-87. Epub 2011 Jul 1.

Laboratory of Molecular Signaling in Diabetes, Diabetes Research Group, Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.

Diabetes is a multifactorial metabolic disorder defined by the loss of functional pancreatic insulin-producing β-cells. The functional maturation and dedifferentiation of adult β-cells is central to diabetes pathogenesis and to β-cell replacement therapy for the treatment of diabetes. Despite its importance, the dynamics and mechanisms of adult β-cell maturation remain poorly understood. Using a novel Pdx1/Ins1 dual fluorescent reporter lentiviral vector, we previously found that individual adult human and mouse β-cells exist in at least two differentiation states distinguishable by the activation of the rat Ins1 promoter and performed the first real-time imaging of the maturation of individual cultured β-cells. Our previous study focused on transformed (MIN6) β-cells as a model to investigatethe kinetics of β-cell maturation. In the present study, we investigated the kinetics of the maturation process in primary human and mouse β-cells and performed gene expression profiling. Gene expression profiling of FACS purified immature Pdx1 (+) /Ins1 (low) cells and mature Pdx1 (high) /Ins1 (high ) cells from cultures of human islets, mouse islets and MIN6 cells revealed that Pdx1 (+) /Ins1 (low) cells are enriched for multiple genes associated with β-cell development/progenitor cells, proliferation, apoptosis, as well as genes coding for other islet cell hormones such as glucagon. We also demonstrated that the heterogeneity in β-cell maturation states previously observed in vitro, can also be found in vivo. Collectively, these experiments contribute to the understanding of maturation, dedifferentiation and plasticity of adult pancreatic β-cells. The results have significant implications for islet regeneration and for in vitro generation of functional β-cells to treat diabetes.
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http://dx.doi.org/10.4161/isl.3.4.15881DOI Listing
November 2011

A multi-parameter, high-content, high-throughput screening platform to identify natural compounds that modulate insulin and Pdx1 expression.

PLoS One 2010 Sep 23;5(9):e12958. Epub 2010 Sep 23.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012958PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944895PMC
September 2010

Maturation of adult beta-cells revealed using a Pdx1/insulin dual-reporter lentivirus.

Endocrinology 2009 Apr 18;150(4):1627-35. Epub 2008 Dec 18.

Michael Smith Laboratories, University of British Columbia, Vancouver, Canada.

The enigmatic process of beta-cell maturation has significant implications for diabetes pathogenesis, and potential diabetes therapies. This study examined the dynamics and heterogeneity of insulin and pancreatic duodenal homeobox (Pdx)-1 gene expression in adult beta-cells. Insulin and Pdx1 expression were monitored in human and mouse islet cells and MIN6 cells using a Pdx1-monomeric red fluorescent protein/insulin-enhanced green fluorescent protein dual-reporter lentivirus. The majority of fluorescent cells were highly positive for both Pdx1 and insulin. Cells expressing Pdx1 but little or no insulin (Pdx1(+)/Ins(low)) comprised 15-25% of the total population. Time-lapse imaging demonstrated that Pdx1(+)/Ins(low) primary beta-cells and MIN6 cells could convert to Pdx1(+)/Ins(+) cells without cell division. Genes involved in the mature beta-cell phenotype (Glut2, MafA) were expressed at higher levels in Pdx1(+)/Ins(+) cells relative to Pdx1(+)/Ins(low) cells. Conversely, genes implicated in early beta-cell development (MafB, Nkx2.2) were enriched in Pdx1(+)/Ins(low) cells. Sorted Pdx1(+)/Ins(low) MIN6 cells had a higher replication rate and secreted less insulin relative to double-positive cells. Long-term phenotype tracking of Pdx1(+)/Ins(low) cells showed two groups, one that matured into Pdx1(+)/Ins(+) cells and one that remained immature. These results demonstrate that adult beta-cells pass through distinct maturation states, which is consistent with previously observed heterogeneity in insulin and Pdx1 expression in adult beta-cells. At a given time, a proportion of adult beta-cells share similar characteristics to functionally immature embryonic beta-cell progenitors. The maturation of adult beta-cells recapitulates development in that Pdx1 expression precedes the robust expression of insulin and other mature beta-cell genes. These results have implications for harnessing the maturation process for therapeutic purposes.
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http://dx.doi.org/10.1210/en.2008-1224DOI Listing
April 2009

Current Status for High Titre Poxvirus Stock Preparation in CEF Under Serum-Free Medium Conditions: Implication for Vaccine Development.

Cytotechnology 2005 Jun;48(1-3):79-88

BioTherapeutics Research Group, Robarts Research Institute, 100 Perth Drive, P.O. Box 5015, N6A 5K8, London, ON, Canada.

In light of the recent detection of BSE in North America and its endemic nature in other regions of the world, there is a real need to employ cell culture conditions that do not require any animal-derived material. Here we report the use of an ultra-low protein serum-free medium (VP-SFM, Invitrogen) for the amplification of poxviruses in primary chicken embryo fibroblasts (CEF). We compared the amplification of four different poxviruses (canarypox, modified Ankara Virus (MVA), vaccinia virus strain Copenhagen and myxoma strain Lausanne) in three different media: DMEM 10%, DMEM 2% and serum-free medium VP-SFM. VP-SFM is a serum-free, ultra-low protein medium containing no proteins or peptides of human or animal origin designed to support the replication of viruses and the production of recombinant proteins and monoclonal antibodies. Our results show that high titre poxvirus stocks can be prepared in VP-SFM equivalent to that prepared in serum containing medium.
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http://dx.doi.org/10.1007/s10616-005-3795-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3449719PMC
June 2005