A Real-Time Quantitative PCR Protocol for the Quantification of Plasmid Copy Number in Lactococcus lactis.

The determination of the number of plasmid copies in each cell of Lactococcus lactis is critical for the control and regulation of the production of recombinant proteins and plasmids. This protocol describes a method for the determination of the plasmid copy number per genome of L. lactis, which is based on the detection by real-time quantitative PCR of the number of plasmid molecules and the number of chromosomes and subsequently their ratio after calculating the amplification efficiency.

Real-Time PCR Method for Assessment of ParA-Mediated Recombination Efficiency in Minicircle Production.

The in vivo intramolecular recombination of a parental plasmid allows excising prokaryotic backbone from the eukaryotic cassette of interest, leading to the formation of, respectively, a miniplasmid and a minicircle. Here we describe a real-time PCR protocol suitable for the determination of recombination efficiency of parental plasmids with multimer resolution sites (MRS). The protocol was successfully applied to purified DNA samples obtained from E.

Bioengineered Mesenchymal-Stromal-Cell-Derived Extracellular Vesicles as an Improved Drug Delivery System: Methods and Applications.

Extracellular vesicles (EVs) are cell-derived nano-sized lipid membranous structures that modulate cell-cell communication by transporting a variety of biologically active cellular components. The potential of EVs in delivering functional cargos to targeted cells, their capacity to cross biological barriers, as well as their high modification flexibility, make them promising drug delivery vehicles for cell-free therapies. Mesenchymal stromal cells (MSCs) are known for their great paracrine trophic activity, which is largely sustained by the secretion of EVs.

Hydrodynamic Effects on Biofilm Development and Recombinant Protein Expression.

Hydrodynamics play an important role in the rate of cell attachment and nutrient and oxygen transfer, which can affect biofilm development and the level of recombinant protein production. In the present study, the effects of different flow conditions on the development of biofilms and the expression of a model recombinant protein (enhanced green fluorescent protein, eGFP) were examined. Planktonic and biofilm cells were grown at two different flow rates in a recirculating flow cell system for 7 days: 255 and 128 L h (corresponding to a Reynolds number of 4600 and 2300, respectively).

The Effect of Recombinant Protein Production in Transcriptome and Proteome.

is a food-grade, and generally recognized as safe, bacterium, which making it ideal for producing plasmid DNA (pDNA) or recombinant proteins for industrial or pharmaceutical applications. The present paper reviews the major findings from transcriptome and proteome studies, with an overexpression of native or recombinant proteins. These studies should provide important insights on how to engineer the plasmid vectors and/or the strains in order to achieve high pDNA or recombinant proteins yields, with high quality standards.

Mesenchymal Stromal Cells (MSCs): A Promising Tool for Cell-Based Angiogenic Therapy.

The Mesenchymal stromal cells (MSCs) are a diverse subset of adult multipotent precursors, known for their potential therapeutic properties in regenerative medicine mainly sustained by paracrine effects through secretion of a variety of biologically active molecules. MSC secretome includes a wide range of soluble protein factors, composed of growth factors and cytokines, and vesicular components, which transfer proteins and genetic material modulating the host microenvironment. In particular, MSC-derived secretome mediates the different steps of the angiogenic process, inducing endothelial cell functions in vitro and promoting angiogenesis in vivo.

Recombination efficiency measurement by real-time PCR: A strategy to evaluate ParA-mediated minicircle production.

Minicircles (MCs) are DNA molecules that are produced in Escherichia coli by replicating a parental plasmid (PP) and inducing its site-specific intramolecular recombination into miniplasmid (MP; containing the prokaryotic backbone) and MC molecules (comprised by the eukaryotic cassette). The determination of the recombination efficiency and the monitoring of PP, MC and MP species during processing and in the final product are critical aspects of MC manufacturing. This work describes a real-time PCR method for the specific identification of PP, MP or MC that uses sets of primers specific for each species.

Minicircle-based expression of vascular endothelial growth factor in mesenchymal stromal cells from diverse human tissues.

Background: Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. Despite bone marrow (BM) being the most studied tissue source, cells with similar intrinsic properties can be isolated from adipose tissue (AT) and umbilical cord matrix (UCM). The present study aims to compare the angiogenic potential of MSC obtained from BM, AT and UCM that were genetically modified with vascular endothelial growth factor (VEGF)-encoding minicircle (MC) vectors.

Plasmid Replicons for the Production of Pharmaceutical-Grade pDNA, Proteins and Antigens by Cell Factories.

The bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination.

Effects of Equine Chorionic Gonadotropin on Ovulatory and Luteal Characteristics of Mares Submitted to an P4-Based Protocol of Ovulation Induction With hCG.

The aim of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) at the end of progesterone (P4) treatment on follicular and luteal characteristics during transition period (TP) and reproductive breeding season (RP). A total of 13 crossbred mares were distributed in two experimental groups in the spring and summer (n = 26). The animals received intravaginal P4 (1.

Conditioned Medium From Azurin-Expressing Human Mesenchymal Stromal Cells Demonstrates Antitumor Activity Against Breast and Lung Cancer Cell Lines.

Recently, cell-based therapies have been explored as a strategy to enhance the specificity of anticancer therapeutic agents. In this perspective, human mesenchymal stromal cells (MSC) hold a promising future as cell delivery systems for anticancer proteins due to their unique biological features. In this study, we engineered human MSC to secrete a human codon-optimized version of azurin (hazu), a bacterial protein that has demonstrated anticancer activity toward different cancer models both and .

Reproductive characteristics of bulls from two breed compositions and their correlations with infrared thermography.

The objective was to evaluate reproductive characteristics of crossbred Girolando (Gyr x Holstein) bulls from two breed compositions and correlate these results with infrared thermography data. Evaluations were performed considering sperm motility, vigor and morphology; scrotal circumference; body morphology and temperament. Infrared thermography was performed to determine surface temperatures of ocular and scrotal areas.

Plasmid Copy Number of pTRKH3 in Lactococcus lactis is Increased by Modification of the repDE Ribosome-Binding Site.

Plasmids for DNA vaccination are exclusively produced in the Gram-negative Escherichia coli. One important drawback of this system is the presence of lipopolysaccharides. The generally recognized as safe Lactococcus lactis (L.

Effect of Using Two Cryopreservation Methods on Viability and Fertility of Frozen Stallion Sperm.

Studies involving different methods and techniques of cryopreservation and its interactions with the conception rates in artificial insemination (AI) programs are reported in the literature. This study evaluated the sperm kinetics, plasma membrane integrity, and fertility rates of mares inseminated with cryopreserved stallion semen subjected to different freezing methods. For this, four ejaculates from five stallions were collected and frozen in conventional (Styrofoam box) or automated system in Mini-Digitcool ZH 400.

Engineering of Human Mesenchymal Stem/Stromal Cells with Vascular Endothelial Growth Factor-Encoding Minicircles for Angiogenic Ex Vivo Gene Therapy.

Peripheral artery disease (PAD) is a debilitating and prevalent condition characterized by blockage of the arteries, leading to limb amputation in more severe cases. Mesenchymal stem/stromal cells (MSC) are known to have intrinsic regenerative properties that can be potentiated by the introduction of pro-angiogenic genes such as the vascular endothelial growth factor (VEGF). Herein, the use of human bone marrow MSC transiently transfected with minicircles encoding for VEGF is proposed as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients.

Production and Purification of Supercoiled Minicircles by a Combination of In Vitro Endonuclease Nicking and Hydrophobic Interaction Chromatography.

A wider application of minicircle (MC) vectors in gene therapy research depends critically on the ability to purify supercoiled (sc) MC from related miniplasmid (MP) and parental plasmid (PP) impurities. This protocol describes a purification strategy that combines the in vitro enzymatic relaxation of sc MP and PP impurities by a nicking endonuclease, and topoisomer separation and RNA clearance by hydrophobic interaction chromatography. The time required to follow the full protocol, from production to isolation of sc MC, is approximately 50 h.

Characterizing Slow Photochemical Reaction Kinetics by Enhanced Sampling of Rare Events with Capillary Optical Fibers and Kramers' Theory.

Characterization of slow chemical reactions is essential for assessing catalytic efficiency in chemistry and biology. Traditionally, chemical reaction rates are obtained from population relaxation kinetics measurements and the Arrhenius equation. Unfortunately, it is difficult to use this approach to characterize reactions wherein concentrations change slowly.

Draft Genome Sequence of the Plasmid-Free subsp. Strain LMG 19460.

We report here the draft genome sequence of the plasmid-free subsp. strain LMG 19460. This strain has potential application for a cost-effective production of food-grade plasmid DNA to use in DNA vaccines, produce recombinant proteins, and be used as a mucosal delivery vehicle of therapeutic molecules.

Design and characterization of plasmids encoding antigenic peptides of Aha1 from Aeromonas hydrophila as prospective fish vaccines.

The current investigation aimed at designing DNA vaccines against Aeromonas hydrophila infections. The DNA vaccine candidates were designed to express two antigenic outer membrane protein (Aha1) peptides and to be delivered by a nanoparticle-based delivery system. Gene sequences of conserved regions of antigenic Aha1 [aha1(211-381), aha1(211-381)opt, aha1(703-999) and aha1(703-999)opt] were cloned into pVAX-GFP expression vector.

Towards effective non-viral gene delivery vector.

Despite very good safety records, clinical trials using plasmid DNA failed due to low transfection efficiency and brief transgene expression. Although this failure is both due to poor plasmid design and to inefficient delivery methods, here we will focus on the former. The DNA elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats showed detrimental effects on plasmids' performance as biopharmaceuticals.

Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase.

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used.

DNA Vaccines Against Maedi-Visna Virus.

Maedi-visna virus (MVV) is an ovine retrovirus of the Lentivirus genus, responsible for a chronic and progressive disease of sheep with a high prevalence all over the world. Therefore, measures aiming at the control of MVV infection are necessary, and the development of DNA vaccines may be the ideal approach. A DNA vaccine is an antigen-encoding bacterial plasmid designed to mimic infections safely, with ability to generate both humoral and cellular long-lasting immune responses once it is delivered to the host.

Development of a nicking endonuclease-assisted method for the purification of minicircles.

Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which is superior to the one afforded by conventional plasmids. MC vectors are produced by replicating a parental plasmid (PP) and promoting its recombination in Escherichia coli. This generates a MC with the expression cassette, and a miniplasmid (MP) with the replication segment.

Strategies to improve the fertility of fresh and frozen donkey semen.

Fertility rates of donkey semen in jennies are lower compared to mares. The aims of this study were to evaluate different sperm cryopreservation methods and insemination strategies to improve the fertility of donkey semen in jennies. Three experiments were performed: (1) the comparison of two freezing methods of donkey semen (conventional method and automated method); (2) the determination of a suitable insemination dose of fresh donkey semen for jennies and mares; and (3) the influence of the semen deposition site on fertility of jennies inseminated with frozen donkey semen.