Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations

Wound infections are considered a major cause for wound-associated morbidity. There is a high demand for alternative, robust, and affordable methods that can provide relatable and repro-ducible results when testing topical treatments, both in research and in the pharmaceutical industry. Here we present an ex vivo wound infection model using porcine skin and a burn wounding method, allowing for the efficacy evaluation of topical antimicrobial formulations. Utilizing this model, we demonstrate the potential of topical treatments after infecting the wounds with clinically significant bacteria, P. aeruginosa and S. aureus. We show that the method is compatible with several analytical tools used to analyze infection and antimicrobial effects. Both bacterial strains success-fully infected the wound surface, as well as deeper regions of the tissue. Quantification of viable bacteria on the wound surface and in the tissue, longitudinal measurements of bioluminescence, fluorescence microscopy, and scanning electron microscopy were used to confirm the effects of an-tibacterial treatments. Furthermore, we show that biofilms are formed on the wound surface, indi-cating that the demonstrated method mirrors typical in vivo infections

Porcine synapsin 1: SYN1 gene analysis and functional characterization of the promoter

AbstractSynapsin 1 (SYN1) is a phosphoprotein involved in nerve signal transmission. The porcine SYN1 promoter orthologue was cloned and characterized to provide a means of expressing a transgene specifically in neurons. The nucleotide sequence of the promoter displayed a high degree of conservation of elements responsible for neuron-specific expression. Expression analysis of SYN1 demonstrated presence of transcript during embryonic development. Analysis of GFP expression in transgenic zebrafish embryos suggests that the pig SYN1 promoter directs expression in neuronal cells. Thus, the SYN1 promoter is a good candidate for use in the generation of pig models of human neurodegenerative disorders

Comparison of efficacy between subcutaneous and intravenous application of moss‐aGal in the mouse model of Fabry disease

Abstract Fabry disease (FD, OMIM 301500) is a rare X‐linked inherited lysosomal storage disorder associated with reduced activities of α‐galactosidase A (aGal, EC 3.2.1.22). The current standard of care for FD is based on enzyme replacement therapy (ERT), in which a recombinantly produced version of αGal is intravenously (iv) applied to Fabry patients in biweekly intervals. Though the iv application is clinically efficacious, periodical infusions are inconvenient, time‐ and resource‐consuming and they negatively impact the patients’ quality of life. Subcutaneous (sc) injection, in contrast, is an established route of administration for treatment of chronic conditions. It opens the beneficial option of self‐administration, thereby improving patients’ quality of life and at the same time reducing treatment costs. We have previously shown that Moss‐α‐Galactosidase (moss‐aGal), recombinantly produced in the moss Physcomitrium patens, is efficient in degrading accumulated Gb3 in target organs of murine model of FD and in the phase I clinical study, we obtained first efficacy evidence in human patients following single iv infusion. Here, we tested the efficacy of subcutaneous administration of moss‐aGal and compared it with the results observed following iv infusion in Fabry mice. The obtained findings demonstrate that subcutaneously applied moss‐aGal is correctly transported to target organs and efficacious in degrading Gb3 deposits there and thus suggest the possibility of using this route of administration for therapy of Fabry disease

Probing Skin Barrier Recovery on Molecular Level Following Acute Wounds : An In Vivo/Ex Vivo Study on Pigs.

Proper skin barrier function is paramount for our survival, and, suffering injury, there is an acute need to restore the lost barrier and prevent development of a chronic wound. We hypothesize that rapid wound closure is more important than immediate perfection of the barrier, whereas specific treatment may facilitate perfection. The aim of the current project was therefore to evaluate the quality of restored tissue down to the molecular level. We used Göttingen minipigs with a multi-technique approach correlating wound healing progression in vivo over three weeks, monitored by classical methods (e.g., histology, trans-epidermal water loss (TEWL), pH) and subsequent physicochemical characterization of barrier recovery (i.e., small and wide-angle X-ray diffraction (SWAXD), polarization transfer solid-state NMR (PTssNMR), dynamic vapor sorption (DVS), Fourier transform infrared (FTIR)), providing a unique insight into molecular aspects of healing. We conclude that although acute wounds sealed within two weeks as expected, molecular investigation of stratum corneum (SC) revealed a poorly developed keratin organization and deviations in lipid lamellae formation. A higher lipid fluidity was also observed in regenerated tissue. This may have been due to incomplete lipid conversion during barrier recovery as glycosphingolipids, normally not present in SC, were indicated by infrared FTIR spectroscopy. Evidently, a molecular approach to skin barrier recovery could be a valuable tool in future development of products targeting wound healing

Molecular Cloning and Characterization of Porcine Na⁺/K⁺-ATPase Isoforms α1, α2, α3 and the ATP1A3 Promoter

<div><p>Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1–α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1–α3 genes, <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i>, their chromosomal localization, and expression patterns. Our <i>ATP1A1</i> sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine <i>ATP1A1</i> sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i> mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i> transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine <i>ATP1A3</i> promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine <i>ATP1A3</i> promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the <i>ATP1A3</i> promoter in attempts to generate transgenic porcine models of neurological diseases caused by <i>ATP1A3</i> mutations.</p></div

Functional importance of Arg841.

<p>The rat α1 Na⁺/K⁺-ATPase Arg843 homologous to pig Arg841 was replaced by alanine (“mutant”) and the functional consequences analyzed (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079127#s3" target="_blank">Methods</a>). Wild type, <i>closed circles</i>; mutant, <i>open circles</i>. The standard errors are indicated as error bars (seen only when larger than the size of the symbols). <b>A.</b> Na⁺ dependence of phosphorylation. Phosphorylation was carried out for 10 s at 0°C in the presence of 2 µM [γ-³²P]ATP in P-medium with oligomycin and the indicated concentrations of Na⁺. Each <i>line</i> shows the best fit of the Hill equation, giving <i>K</i>_0.5(Na⁺) values of 0.50±0.01 mM for wild type and 1.07±0.04 mM for the mutant. <b>B.</b> K⁺ dependence of Na⁺/K⁺-ATPase activity. The ATPase activity was measured at 37°C in A-medium with 40 mM Na⁺, 3 mM ATP, and the indicated concentrations of K⁺. Each <i>line</i> shows the best fit of the Hill equation, giving <i>K</i>_0.5(K⁺) values of 0.67±0.01 mM for wild type and 0.50±0.02 mM for the mutant. <b>C.</b> ATP dependence of Na⁺/K⁺-ATPase activity. The ATPase activity was measured at 37°C in A-medium with 130 mM Na⁺, 20 mM K⁺, and the indicated concentrations of ATP. Each <i>line</i> shows the best fit of the Hill equation, giving <i>K</i>_0.5(ATP) values of 0.50±0.03 mM for wild type and 0.43±0.04 mM for the mutant. <b>D.</b> Vanadate dependence of Na⁺/K⁺-ATPase activity. The ATPase activity was measured at 37°C in A-medium with 130 mM Na⁺, 20 mM K⁺, 3 mM ATP, and the indicated concentrations of vanadate. Each <i>line</i> shows the best fit of the Hill equation for inhibition, giving <i>K</i>_0.5(vanadate) values of 2.2±0.1 µM for wild type and 2.4±0.1 µM for the mutant. <b>E.</b> Distribution of phosphoenzyme intermediates between E1P and E2P. Phosphorylation was carried out for 10 s at 0°C in the presence of 2 µM [γ-³²P]ATP in P-medium with 20 mM Na⁺. Dephosphorylation was initiated by addition of 1 mM non-radioactive ATP and 2.5 mM ADP and terminated by acid quenching at the indicated times. Each <i>line</i> shows the best fit of a bi-exponential decay function giving amplitudes (corresponding to E2P) for the slow phase of 63±4% for wild type and 84±8% for the mutant. <b>F.</b> Rate of E1P→E2P interconversion. Phosphorylation was carried out for 15 s at 0°C in the presence of 2 µM [γ-³²P]ATP in P-medium with 600 mM Na⁺. Dephosphorylation was initiated by addition of a chase solution producing final concentrations of 600 mM Na⁺, 20 mM K⁺, and 1 mM non-radioactive ATP in addition to the components in the P-medium, and terminated by acid quenching at the indicated times. Each <i>line</i> shows the best fit by a bi-exponential decay function giving rate constants for the slow phase (corresponding to the E1P→E2P interconversion) of 0.14±0.05 s⁻¹ for wild type and 0.43±0.18 s⁻¹ for the mutant.</p

Comparative expression levels of porcine <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i> mRNA in different organs and tissues from adult pigs.

<p><i>β-actin</i> (b_ACT) is used as endogenous reference. Each column represents the mean expression of a triplicate from three different pigs. The considerable biological variation between the animals represented in each column is indicated by error bars showing the standard deviation. FCO: frontal cortex, CBE: cerebellum, HIP: hippocampus, BST: brain stem, HEA: heart, LDO: longissimus dorsi, BFE: biceps femoris, KID: kidney.</p

Relative expression pattern of porcine <i>ATP1A1</i>, <i>ATP1A2</i>, and <i>ATP1A3</i> mRNA in different organs and tissues from adult pigs and from brain tissues at different stages of embryonic development.

<p><i>GAPDH</i> is used as endogenous reference. Each column represents the mean expression of a triplicate from three different pigs. The considerable biological variation between the animals represented in each column is indicated by error bars showing the standard deviation. KID: kidney, LUN: lung, LIV: liver, HEA: heart, THG: thyroid gland, LDO: longissimus dorsi, PGL: pituitary gland, SPC: spinal cord, FCO: frontal cortex, CBE: cerebellum; BST: brain stem, HIP: hippocampus, BSG: basal ganglia, D60: embryo of day 60, D80: embryo of day 80, D100: embryo of day 100, D115: embryo of day 115.</p