The selection of such parasites was first described by Jeffers (33) who showed that serial passage of oocysts through a chicken and collection at earlier and earlier time points post-challenge resulted in parasites

of attenuated virulence. Importantly, infection of chickens with these parasites induced a high level of immunity against a challenge with the parent line (34). Whilst initial attempts to derive further protective lines of precocious parasites failed (34,35), precocious lines were click here eventually described for all seven species of Eimeria (11). Characteristically, precocious parasites of Eimeria have a marked reduction in oocyst reproduction and pathogenicity, and yet are still highly immunogenic. Studies also demonstrated the genetic stability of precocious lines, where precociousness was retained through serial passage without selection for early maturation of oocysts (36); buy Osimertinib thus, lines do not revert back to virulence. With this inherent improvement in safety, and parasites being more predictable and reliable than embryo-adapted lines, precocious

lines of Eimeria became the basis of the development of the first attenuated anticoccidial vaccine, Paracox® (Intervet/Schering Plough Animal Health, Milton Keynes, UK). Paracox® was launched in 1989, to protect laying and breeding hens and it contained precocious lines of

all seven species of Eimeria, including two lines of E. maxima due to antigenic variation seen in this species (6,37,38). As its introduction, several other formulations and attenuated vaccines have become commercially available for use in different poultry flocks. Generally, E. maxima, E. tenella and E. acervulina are the only species included in vaccines for broiler birds as younger flocks rarely encounter the pathogenic species E. brunetti Methocarbamol or E. necatrix (5,39). In 2003, EIMERIAVAX 4m, was the first live coccidiosis vaccine registered for use in Australian poultry. It is comprised of drug-sensitive, precocious lines of E. acervulina, E. maxima, E. tenella and E. necatrix, each isolated from backyard flocks of Australian chickens (40,41). Field trials showed that the vaccine could protect broiler breeders, broilers, free range and barn flocks of egg laying hens by eye-drop or coarse aerosol application (42). Efforts continue to be directed towards the derivation of further vaccines based on precociousness and it is probably fair to say that reliance on these type of vaccines will, if anything, increase in years to come (36). An anticoccidial vaccine composed of protective antigens, either native or recombinant, has been pursued as an alternative to live vaccines and the problems and costs associated with them.

32 In a recent study, urinary N-acetyl-beta-D-glucosaminidase (NAG) levels at 24 h post kidney transplantation predicted 12 month glomerular filtration rate (GFR) of less than 45 mL/h with a receiver operating characteristic (ROC) value of 0.73.33 C-X-C motif chemokine 10 (CXCL-10/IP-10) is a chemoattractant that promotes adhesion of macrophages, T cells, natural killer cells and dendritic cells to endothelial cells.34,35 CXCL-10 is secreted by TEC, monocytes, endothelial cells and fibroblasts upon IFN-γ induction.16 Elevated CXCL-10 levels have been reported in the urine of kidney transplant patients with impending AR episodes within the first 4 weeks post transplantation

and are predictive of restricted graft function at 6 months.36 Interestingly, administration of antibodies against the CXCR3 chemokine receptor Selleckchem NVP-LDE225 prolonged cardiac graft survival in a murine model37 with donor-derived CXCL-10 playing the major role.38 Expression of other chemokines such as RANTES, Mig and MCP-1 were reported during cell-mediated kidney transplant rejection, and may be useful in diagnosing AR.39–41 However, CXCL-10

has also been studied in baboon models, providing promising results as an indicator for AR.37,42 Kidney injury molecule-1 is a type I cell membrane glycoprotein upregulated in kidney TEC and shed into the urine following kidney injury in both human and rodent experimental models.43 More recently, KIM-1 was shown to be expressed selectively by injured proximal tubules44 and its urinary level strongly correlated with tubular expression.29 Kidney MLN0128 solubility dmso transplant patients with AR showed higher expression of KIM-1 molecules on their biopsy specimens.45 Urinary excretion of KIM-1 has been proposed as an independent predictor of long-term graft loss.29 A recent study demonstrated that urinary KIM-1 and NAG levels showed a significant

negative correlation with subsequent 6 and 12 month allograft function after kidney transplant as early as 24 h post transplant.33,46 Neutrophil gelatinase lipocalin is a 25 kDa protein involved in iron shuttling from the extracellular environment to the intracellular compartment. It is upregulated and released by kidney TEC during inflammation and ischaemic injury.47,48 NGAL is one of the earliest click here induced proteins in the kidney undergoing nephrotoxic or ischaemic damage,49 being detectable within 3 h in a rodent model of ischaemic renal injury.50 Urinary NGAL and IL-18 predict delayed graft function and poor graft survival post transplant with high sensitivity (90%).51 A high level of NGAL could be detected from day 0 post operation in the urine of patient with less favourable graft function.51 With a baseline estimated GFR of 60 mL/min and above, urinary NGAL may be used to predict acute kidney injury within 6 h after a patient is admitted to the intensive care unit with a ROC value of 0.68.

IRF-8 was originally identified as a repressor of IFN-stimulated response elements and through its ability to inhibit the transcriptional activation R788 of other IRFs [50, 51]. Yet, studies of human monocytes and murine cDCs found that IRF-8 promoted type I IFN production [35, 52]. Current findings show that IRF-8 is a strong negative regulator of CpG-driven IFN-β and IL-6 production by human pDCs (Fig. 4B). This is an important observation, as pDCs constitutively express high levels of IRF-8 [13] and IRF-8 KO mice

fail to generate pDCs [36]. Taken together, current findings demonstrate that IRF-8 expression plays a role in negatively regulating pro-inflammatory and IFN responses following TLR9 stimulation of pDCs. We are in the process of examining whether the elevated levels of IRF-8 in the nucleus of unstimulated pDCs (Fig. 2) reflect a constitutive role for IRF-8 in the regulation of gene activation and whether IRF-8 interacts with IRF-5. Several findings support the technical reliability of results from the knockdown experiments upon which these conclusions are largely based. First, no off-target (i.e. nonspecific) Atezolizumab manufacturer activity was detected

with any of the siRNAs tested (Fig. 3A and C and 4A, and Supporting Information Fig. 2A–C). Second, cells transfected with siRNA were not stimulated unless CpG ODN was added (in contrast to the report by Hornung et al. [34]) (Supporting Information Fig. 2D and E). Third, siRNA administration significantly reduced the level of expression of both mRNA and protein of the targeted gene (Fig. 3A and C and 4A, Supporting Information Fig. 2A–C). Finally, siRNA knockdown of MyD88 and TRAF6 blocked the induction of IFN-β and IL-6 mRNA by CpG-stimulated

pDCs, consistent with earlier reports (Fig. 3B; [15, 31, 32]). K” ODN triggered the rapid translocation of NF-κB p50 and p65 (RelA) from the cytoplasm to the nucleus in CAL-1 cells and human pDCs (Fig. 2D, 6, and 7). Interestingly, the knockdown of p105/p50 but not p65 significantly reduced IFN-β production (Fig. 3D), whereas both p105/p50 and p65 contributed to the induction of IL-6. Accumulating evidence indicates that IκBξ (also known as MAIL, a nuclear ankyrin repeat protein) is required for TLR-dependent upregulation of IL-6 [53, 54]. As IκBξ associates with both p50 and Adenylyl cyclase p65 [55], current findings suggest that eliminating either impairs IκBξ-dependent induction of IL-6. K” ODN induced the rapid nuclear translocation of both IRF-5 and NF-κB p50 (Fig. 2, 6, and 7). PLA, a technique used to identify protein–protein interactions under physiologic conditions, was employed to examine whether these transcriptional factors associated upon stimulation [40]. Only proteins in close proximity (<40 nM) are visualized by PLA, yielding results comparable to resonance energy transfer techniques (such as fluorescence resonance energy transfer analysis).

We observed a significant increase in the production of anti-MSP-119 IgG antibody

in normal and heterozygous children during the 12 months of follow-up, but LEE011 in vitro not in homozygous mutants. Normal children had a significantly lower malaria incidence rate compared to other genotypes (χ2 = 115.59; P < 0.01). We conclude that the presence of the c.1264 T>G mutation that leads to CD36 deficiency is closely associated with reduced IgG production and higher malaria incidence. It is most likely that deficiency of CD36 which is known to modulate dendritic cell function suppresses the production of protective IgG antibodies directed to Plasmodium falciparum MSP-119 antigen, which predisposes to the acquisition of clinical malaria in children. Adhesion molecules are proteins located on the cell surface, involved in binding

with other cells or with the extracellular matrix in a process called cytoadherence, and which occurs as a result of the interaction between parasite ligands and host receptors. Selleckchem MK 2206 Cytoadherence of infected erythrocytes containing late developmental stages of the malaria parasites (trophozoites and schizonts) to the endothelium of capillaries and venules is characteristic of Plasmodium falciparum infections [1]. Cytoadherence is an important mechanism in the pathogenesis of malaria. Polymorphisms in more than 30 human genes determining the immune response have been associated with susceptibility to malaria [2], particularly in genes

involved in cell adhesion and immunity. Cell adhesion molecules (CAM) have been identified as receptors for infected red blood cell and are associated with susceptibility to malaria. The adhesion molecules include CD36, intercellular adhesion molecule 1 (ICAM-1, CD54), platelet/endothelial cell adhesion molecule1 (PECAM-1, CD31), vascular cell adhesion molecule1 (VCAM-1), thrombospondin, E-selectin, P-selectin and chondrotin sulphate A [3]. Most P. falciparum antigens bind to CD36 molecules, which Oxymatrine are thus considered to be the major endothelial receptors for sequestration [4]. Mutations in the CD36 gene may, therefore, influence the outcome of malaria. CD36 is an 88-kDa member of the class B scavenger receptor family of cell surface glycoproteins. CD36 is broadly expressed on monocytes, macrophages, dendritic cells, fat cells, muscle cells, capillary endothelial cells and platelets [5]. CD36 acts as a facilitator of fatty acid uptake and is a receptor for a wide range of ligands including collagen, thrombospondin, anionic phospholipids, oxidized low density lipoprotein and erythrocytes parasitized with P. falciparum [6–8], and participate in macrophage fusion induced by IL-4 cytokines [9]. In vitro studies carried out by Urban and colleagues revealed that P.

Perren, I.

Bravi, L. Jennen, A. Feuchtinger, J. Drouin, F. Roncaroli and N. S. Pellegata (2013) Neuropathology and Applied Neurobiology39, 256–269 Characterization of MENX-associated pituitary tumours Aims: The aim of this study is to evaluate the pathological features, serum hormone levels and ex vivo cultures of pituitary adenomas that occur in rats affected by MENX syndrome. MENX is multiple endocrine neoplasia syndrome caused by a germline Erlotinib in vivo mutation in the cell cycle inhibitor p27. Characterization of MENX adenomas is a prerequisite to exploit this animal model for molecular and translational studies of pituitary adenomas. Methods: We investigated MENX pituitary adenomas with immunohistochemistry, double immunofluorescence, electron microscopy, reverse transcription

polymerase chain reaction (RT-PCR), measurement of serum hormone levels and ex vivo cultures. Results: Adenomas BAY 57-1293 concentration in MENX rats belong to the gonadotroph lineage. They start from 4 months of age as multiple neoplastic nodules and progress to become large lesions that efface the gland. Adenomas are composed of chromophobic cells predominantly expressing the glycoprotein alpha-subunit (αGSU). They show mitotic activity and high Ki67 labelling. A few neoplastic cells co-express gonadotropins and the transcription factor steroidogenic factor 1, together with growth hormone or prolactin and Pit-1, suggesting that they are not fully committed to one Ribonucleotide reductase cell lineage. Ex vivo cultures show features similar to the primary tumour. Conclusions: Our results suggest that p27 function is critical to regulate gonadotroph cells growth. The MENX syndrome represents a unique model to elucidate the physiological and molecular mechanisms mediating the pathogenesis of gonadotroph adenomas. “
“Intracranial malignant solitary fibrous tumor (SFT) is very rare. It was identified in a 39-year-old female patient who underwent malignant transformation over 6 months. MRI revealed an 8 × 5 × 6 cm mass with heterogenous strong enhancement in the left occipital lobe. Histologic findings and immunophenotype (positive for CD34, bcl-2 and vimentin, and negative for epithelial membrane

antigen or S100 protein) of the primary tumor were typical of SFT. However, there was a focal area (<10% of tumor volume) showing hypercellularity, nuclear pleomorphism and increased Ki-67 labeling index (LI) of 10% (average, 1%). At the second operation, the recurrent tumor revealed full-blown histologic features of malignant SFT, such as infiltrative brain invasion, marked nuclear pleomorphism, frequent mitotic figures (15/10 high power fields), and high Ki-67 LI (25%). The presence of atypical histologic finding or increased Ki-67 LI in the typical SFT, although it is focal, needs to be mentioned in the diagnosis and also may require more aggressive surgical management. "
“Circumventricular organs (CVOs) are specialized ventricular structures around the third and fourth ventricles of the brain.

Similarly, bleomycin-induced fibrosis of the skin was enhanced in Lsp−/− mice [26]. Fibrocytes from patients with thermal burns and those from normal donors have substantially less capacity for collagen production than do dermal fibroblasts [27]. When conditioned medium from fibrocytes derived from burned individuals was incubated

with dermal fibroblasts, they exhibited accelerated proliferation when compared to those incubated in medium from control fibrocytes. These effects could be blocked with TGF-β neutralizing antibodies [27]. These same investigators have shown that IFN-α2b can reduce scar formation following selleck kinase inhibitor thermal injury by attenuating fibrocyte activity and reducing their numbers [28]. With regard to the kidney, the participation of bone marrow-derived stem cells remains controversial Selleckchem Belinostat [29]. Results generated in a number of models of renal injury suggest that these stem cells can localize to specific areas of the kidney and might facilitate tissue regeneration. Thus, their therapeutic potential in several forms of human kidney dysfunction is under evaluation. The outcome of such studies will probably influence the research being conducted in allied

disease processes involving other organs and tissues. Graves’ disease represents an autoimmune process where the thyroid becomes enlarged and overactive [30]. The basis for the over-production of thyroid hormones and gland enlargement in this disease involves the production and activity of autoantibodies targeting the thyrotrophin (aka thyroid-stimulating

hormone) receptor (TSHR). In addition, the IGF-1 receptor (IGF-1R) is over-expressed by orbital fibroblasts Morin Hydrate [31], B [32] and T cells [33,34] in patients with the disease. IGF-1R represents a second potentially pathogenic autoantigen that may account for abnormal thyroid enlargement and underlie the trafficking of lymphocytes to affected tissues, including the pretibial skin and orbit. Pritchard et al. [31] have suggested that T cell trafficking to the orbit in Graves’ disease might be mediated through fibroblast responses to IGF-1 and Graves’ disease-immunoglobulin G (GD-IgG). When exposed to either agent, these fibroblasts express high levels of the T cell chemoattractants, IL-16 and regulated on activation normal T cell expressed and secreted (RANTES). The fibroblast response is mediated through IGF-1R activation and post-receptor signalling through the FRAP/mTor/Akt/p70s6k pathway. It is absent in fibroblasts derived from healthy donors [35]. In addition to the generation of chemoattractants, thyroid-associated ophthalmopathy (TAO) orbital fibroblasts synthesize high levels of hyaluronan, in response to either GD-IgGs or IGF-1. Hyaluronan is a non-sulphated glycosaminoglycan, the accumulation of which is thought to result in tissue oedema [36]. Orbital connective tissue derives in large part from neural ectoderm [37]. This tissue has a special propensity for inflammation.

In this report, we applied NNAlign to peptide–MHC class II binding data for five HLA-DP and six HLA-DQ molecules to characterize their specificities and binding motifs. The binding data were obtained from the publication by Wang et al.7 They comprise a total of 17 092 measured peptide–MHC affinities, with an average of over 1500 measurements per allelic variant. Each data set was split in five random subsets and, each time excluding MG-132 datasheet one subset, a network was trained on the remaining four subsets. We set the motif length to nine amino acids, and for all the remaining parameters we used the default values of the NNAlign web server: sequences were presented to the networks using Blosum encoding,13

hidden layers were composed of three neurons, training lasted 500 iterations per training example, starting from five different initial configurations for each cross-validation

find protocol fold, subsets for cross-validation were created using a homology clustering at 80% to reduce similarity between subsets, using the best four networks for each cross-validation step. The resulting 20 networks in each ensemble, trained on different subsets of the data and from alternative initial conditions, capture motifs that can be different from each other to some extent. They often place the alignment core in a different register, and might disagree on the exact boundaries of the motif. The offset correction algorithm described by Andreatta et al.12 proved extremely efficient in correcting for this disagreement, allowing re-alignment of different networks to a common core. This alignment procedure creates a position-specific scoring matrix (PSSM) representation of the motif of

each network, and then aligns the matrices to maximize the information content of the combined core. We used a slightly modified version of the algorithm described in detail in a previous publication,12 where PSSMs are extended at both ends with background frequencies before alignment, so allowing the PSSMs to be aligned on a window Dichloromethane dehalogenase of the same length as the matrices. This process assigns to each PSSM, and its relative network, an offset value that quantifies the shift distance from other networks. Note that the alignment procedure does not guarantee that the final combined register corresponds to the biologically correct register (in the case of peptide–MHC binding, the nine-amino-acid stretch bound in the MHC binding cleft), but rather to the window with the maximum information content. In most of the cases informative positions are also biologically important positions, so the core register would be in the correct place. However, if either terminal of the core has very weak information content (i.e. no particular amino acid preference at terminal positions), the sequences might possibly, although aligned correctly, all be shifted by one or more positions with respect to the biologically correct core register.

22, paired two-tailed Student’s t-test). This suggests selleck chemical that stability in general is a better indicator of immunogenicity than affinity is. The above comparison of immunogenic peptides and peptides of unknown immunogenicity is potentially flawed. First, these peptides have been selected for purposes other than the present study and

do not necessarily represent a random, representative and unbiased sample of the peptide space. Second, the data on these peptides are not particularly homogenous, since the database entries on immunogenicity are the result of the work over several decades by many different scientists using many different techniques. Third, the data might have be skewed due to the frequent use of predictions based on more or less complicated MHC-I-binding motifs, which may have led to an oversampling of peptides carrying perfect motif matches resulting in a likely overrepresentation of high-affinity and -stability binders. Fourth, the data are not error free. The immunogenic peptide sequences identified by synthesis and functional analysis do not necessarily represent the final stimulatory moieties (as first noted by

Ploegh and colleagues [[21]]). Also, in most cases it has not been examined whether the peptide sequences used here as control peptides are truly nonimmunogenic (albeit the frequency of random peptides Mitomycin C research buy being immunogenic a priori is low [[22]]). Thus, one should be cautious when interpreting the data obtained with this panel of peptides. To circumvent the above problem and reliably evaluate how affinity and/or stability Clostridium perfringens alpha toxin correlate with immunogenicity, one should ideally perform a systematic and unbiased

analysis of all possible overlapping peptides from a model antigen or organism; however, the resources required would be prohibitive. As a work-round, we analyzed the stability of peptide-HLA-A*02:01 complexes reported in a recent study by Sette and colleagues on the T-cell specificities recognized after infecting HLA-A*02:01 transgenic mice with vaccinia virus [[6]]. This is one of the most comprehensive and careful studies of its kind: it used a very broad HLA-A*02:01 motif definition to capture an estimated 99.8% of all possible 9- and 10-mer binders from a large collection of proteins known to be targeted by CTLs; and it examined the immunogenicity of a representative sample of high-affinity binding peptides both following vaccinia infection as well as after peptide immunization.

Human diagnostic muscle biopsies that failed to show histological alterations (n = 3) and from patients with a molecular diagnosis of DM1 (n = 3) and DM2

(n = 3) were used, with approval by the Ethical Committee of Tor Vergata University Hospital. Molecular diagnosis selleck chemicals llc of DM2 was performed as previously described [34]. Animal work conformed to the guidelines of the Institutional Board for the care and utilization of laboratory animals. Adult male Sprague-Dawley rats (Harlan, Milano, Italy) were maintained under routine conditions on a standard commercial diet. For immunofluorescence (IF) and Western blot (WB) studies, rats (n = 3) were sacrificed by an i.p. overdose of sodium thiopental, and organs and tissues were dissected and immediately frozen in liquid nitrogen-cooled isopentane. In order to examine ZNF9 distribution in the brain, two additional rats were transcardially perfused with 60 ml of saline solution containing 0.05 ml heparin, followed by 200 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were removed and postfixed overnight at +4°C, cryoprotected in 20% sucrose/10% glycerol solution with 0.02% sodium azide for 48 h at 4°C [35]. Polyclonal anti-ZNF9 antibodies (Abs) were obtained by immunization of rabbit with a 20 amino acid peptide (CYRCGESGHLARECTIEATA) from the C-terminus of human find protocol ZNF9, which includes

the seventh zinc finger. The raw antiserum was purified to obtain either an high

pressure liquid of chromatography-purified or an affinity-purified polyclonal Ab (Syntem, Nimes, France). Given that in preliminary experiments both Abs had shown a complete antigen specificity both in Xenopus laevis and in a Balb/3T3 murine cell line, the high pressure liquid chromatography-purified Ab (K20) was used in the following experiments as it showed a greater sensitivity. Rat tissues were homogenized using a Dounce homogenizer in cold lysis buffer (10 mM NaCl, 2 mM EGTA, 10 mM MgCl2, 10 mM Tris–HCl pH 7.5) containing protease inhibitors (1 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin, pepstatin A 1 µg/ml) and 1% NP40. After 5 min of centrifugation at 16000 g, nuclei were discarded and protein concentration was determined using the Bio-Rad Protein assay. For SDS-PAGE, extracts were adjusted to 20% glycerol, 3% 2-mercaptoethanol, 4% SDS, 25 mM Tris-Cl, pH 6.8 and boiled for 5 min. Human muscle samples from control, DM1 and DM2 patients were homogenized using a Dounce homogenizer in cold lysis buffer also containing protease inhibitors without detergent. The cytoplasmic fraction was further purified by centrifugation for 15 min at 20 000 g to pellet-insoluble cell membranes. Homogenates (50 µg/lane) were separated on a 15% polyacrylamide gel and transferred to nitrocellulose Immobilon membrane (Millipore, Milano, Italy). Membranes were incubated with K20 (diluted 1:1000) or anti-eIF2α Ab (Santa Cruz, Biotechnology, Inc.

To ensure an effective DNA isolation from nail clippings, the

lysis buffer of QIAamp® DNA Mini Kit was exchanged by the buffers L, N and proteinase K which were part of the multiplex PCR kit and applied as described by the manufacturer (Biotype Diagnostic GmbH, Dresden, Germany). Purified DNA from fungal cultures was quantified via UV–VIS spectrometry using a NanoDrop® ND-1000 (PEQLAB Biotechnologie GmbH, Erlangen, Germany). PCR was performed with Mentype® MycodermQS PCR Amplification Kit (Biotype Diagnostic) according to the instructions of the manufacturer. Briefly, Selleck Ensartinib the kit consists of all reagents to perform two separate multiplex PCRs (Table 2 and Fig. 1). Primer mix 1 contains specific PCR primer pairs for E. floccosum, M. canis,

Microsporum gypseum, Trichosporon cutaneum, S. brevicaulis, Aspergillus spp., Candida spp. and an unrelated internal amplification control (QS, quality sensor). Primer mix 2 supplies specific PCR primer pairs for the amplification of T. rubrum, T. interdigitale, Trichophyton spp. and QS. The calculated amplicon size of QS is 1231 bp. Aliquots of 7 or 4 μl purified DNA solution from clinical samples were applied to PCR 1 or PCR 2, respectively, in a final volume of 25 μl. The thermocyclers GeneAmp 9700 (Applied Biosystems Deutschland GmbH, Darmstadt, Germany), Eppendorf Mastercycler ep-S (Eppendorf PXD101 supplier AG, Hamburg,

Germany) and Biometra T1 (Biometra GmbH, Göttingen, Germany) were used for analytical validation. The enzyme reaction consisted of 4 min at 96 °C followed by five cycles of 30 s at 94 °C, 60 s at 62 °C and 90 s at 72 °C, and 35 cycles of 30 s at 94 °C, 60 s at 60 °C for 60 s and 90 s at 72 °C. Dermatophyte-specific PCR results were partially confirmed by PCR second with alternative primer pairs as described.[1, 20-22] After PCR, 10 μl was mixed with 2 μl sixfold gel-loading buffer (Applichem GmbH, Darmstadt, Germany) and subjected to 2% agarose gel electrophoresis in onefold TBE-buffer using the iMupid Mini Agarose Gel Electrophoresis System (Helixx Technologies Inc., Toronto, ON, Canada) at 100 V until the bromphenol marker reached the end of the 7 cm isolating distance. The gel was stained for 20 min with GelRed™ (Biotium Inc., Hayward, CA, USA) and analysed with the gel documentation system BioVision 3000 (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany) equipped with a 312-nm UV light source, a 590-nm emission filter and the software Bio1D. The PCR kit is provided with reference ladders for PCR 1 and 2, respectively, which were applied in separate wells in electrophoresis and used as size standards for gel analysis (Fig. 1 and Fig. 2).