Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis

Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic characterization using 100 fastidious Gram negative bacteria. Seventy-five strains represented 21 of the 26 taxa included in the Vitek 2 NH database and 25 strains represented related species not included in the database. Of the 100 strains, 31 were the type strains of the species. Vitek 2 NH identification results: 48 of 75 database strains were correctly identified, 11 strains gave `low discrimination´, seven strains were unidentified, and nine strains were misidentified. Identification of 25 non-database strains resulted in 14 strains incorrectly identified as belonging to species in the database. Partial 16S rRNA gene sequence analysis results: For 76 strains phenotypic and sequencing identifications were identical, for 23 strains the sequencing identifications were either probable or possible, and for one strain only the genus was confirmed. Thus, the Vitek 2 NH system identifies most of the commonly occurring species included in the database. Some strains of rarely occurring species and strains of non-database species closely related to database species cause problems. Partial 16S rRNA gene sequence analysis performs well, but does not always suffice, additional phenotypical characterization being useful for final identification

Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology

DNA Sequencing Historical Lichen Specimens

Biological specimens in natural history collections worldwide are increasingly being used in biogeographical, environmental, and taxonomic studies. For their meaningful use, correct species identification is crucial. For example, clarifying if a species is new to science requires an overview of what has already been described. This includes comparisons with existing authoritative specimens (types). Most type specimens are rather old and their DNA expected to be degraded to various extents. Comparative DNA sequence analysis is in regular use in taxonomic research of today and is essential for identifying and delimiting species. In this study, we focus on lichenized fungi (lichens), in which many species groups are highly inconspicuous and impossible to identify to species based on morphology alone. Our aim was to test the non-mutually exclusive hypotheses that DNA quality of lichens depends on (1) time since collection, (2) taxonomic affinity, and/or (3) habitat/ecology. We included two species from each of four different lichen genera (i.e., Cladonia, Nephroma, Peltigera, and Ramalina), each species pair with a different autecology. For each species, we included samples from approximately every 25 years from present to about 150 years back in time. We used a two-step PCR-based approach followed by sequencing on an Ion Torrent PGM to produce target sequences (mtSSU) of degraded DNA. We received satisfactory DNA sequence information for 54 of 56 specimens. We recovered full-length sequences for several more than 100-years-old specimens, including a 127-years-old specimen, and retrieved enough sequence information for species identification of a 150-years-old specimen. As expected, sequencing success was negatively correlated with age of the specimens. It also varied with taxonomic affinity. We found no significant correlation between sequencing success and habitat ecology of the investigated specimens. The herein tested Ion Torrent sequencing approach outperformed Sanger sequencing with regard to sequencing success and efficiency. We find the protocol used herein highly suitable for obtaining sequences from both young and old lichen specimens and discuss potential improvements to it

Absence of an Intron Splicing Silencer in Porcine Smn1 Intron 7 Confers Immunity to the Exon Skipping Mutation in Human SMN2

Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system

DNA Metabarcoding for Quality Control of Basil, Oregano, and Paprika

Herbs and spices are some of the most vulnerable products in terms of fraud and adulteration in the food sector. Although standard analytical methods are accurate for quality control of specific lead or marker compounds, they cannot accurately assess the entire species composition of many marketed products. Complementary analytical approaches are thus often used for comprehensive screening of herbs and spices. In this study we evaluate DNA metabarcoding for the identification and authentication of 62 products, containing basil, oregano, and paprika collected from different retailers and importers in Norway. Our results show varying degrees of discrepancy between the constituent species and those listed on the product labels, despite high product authenticity. We suggest the false positives result from the sensitivity of DNA metabarcoding and filtering thresholds should be integrated into protocols to reduce false positives. Our results highlight how integrating DNA metabarcoding into the toolbox of analytical methods for quality control of fresh and/or processed plant-based food can improve product quality

The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells

<div><p>DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The <em>Sleeping Beauty</em> (SB), <em>piggyBac</em> (PB), and <em>Tol2</em> transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and <em>Tol2</em> transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.</p> </div

Insulation of SB, PB, and Tol2 transposon vectors in ARPE-19 cells.

<p>(<b>A</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones carrying insulated transposon vectors. Measurements were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048421#pone-0048421-g002" target="_blank">Figure 2a</a>. (<b>B</b>) Comparison of mean MFI levels for insulated and uninsulated clones. Stably transfected ARPE-19 cell clones were grown for 8 weeks in the absence of selection, and eGFP expression levels were measured by flow cytometry at day 0 and day 56 of passage. (<b>C</b>) Comparison of mean MFI levels for insulated and uninsulated clones carrying 1-3 transposon insertions. (<b>D</b>) Comparison of mean MFI levels for insulated and uninsulated clones carrying 9 or more transposon insertions.</p