Generation and characterization of single-chain antibody fragments specific against transmembrane envelope glycoprotein gp46 of maedi-visna virus

A single-chain antibody fragments (scFv) was developed directed against transmembrane envelope glycoprotein gp46 of the virus maedi-visna, by the application of the antibody phage display library. To get specific scFv binders, the library was panned against the biotinylated peptide of 20 amino acids corresponding to the principal immunodominant domain of gp46 protein. The number of positively binding scFvs was evaluated by scFv-phage ELISA, BstN1 fingerprinting and DNA sequencing. The scFvs were expressed in soluble form and purified by immobilized metal affinity chromatography (IMAC) with a yield of 2-2.5 mg/l. Two scFvs have shown to recognize gp46 and gp150 proteins in Western blot analysis. The scFvs also recognized the virus in infected cells as shown by immunofluorescence assay. The affinity of the obtained antibody fragments to gp46 peptide was measured by surface plasmon resonance, and the resulting K(A) was in the 10(6)-10(7)lmol(-1) range. The application of characterized scFvs for expression as intrabodies in intracellular immunization against virus maedi-visna infection and for the diagnosis of this virus is discussed.     

An overview of the <Emphasis Type="Italic">Gyrodactylus</Emphasis> (Monogenea: Gyrodactylidae) species parasitizing African catfishes,and their morphological and molecular diversity

An overview of Gyrodactylus infecting catfishes from the African continent is provided, including new data from Sudan, Senegal, Kenya and Mozambique. Haptoral sclerite morphometry and nuclear ribosomal DNA sequences revealed the presence of eight Gyrodactylus species. On Senegalese Synodontis nigrita, Gyrodactylus synodonti n. sp. and Gyrodactylus nigritae n. sp. are described. These are the first reports of gyrodactylid parasites from mochokid hosts. From the fins of North African catfish Clarias gariepinus collected in Mozambique, Gyrodactylus alekosi n. sp. and Gyrodactylus rysavyi were identified. G. rysavyi was also reported from Kenyan C. gariepinus and Senegalese Clarias anguillaris. From the fins of C. anguillaris studied in Senegal, two more species, Gyrodactylus transvaalensis and Gyrodactylus gelnari n. sp. were recognised. In addition, Gyrodactylus turkanaensis n. sp. from the gills of Kenyan C. gariepinus was described and an undescribed Gyrodactylus sp. was recorded from Sudanese representatives of the same host. Detailed morphometrical and molecular comparisons of the species are presented and discussed. The study highlights the hitherto understudied diversity of viviparous monogenean parasites throughout Africa.     

High Archaea diversity in Varvara hot spring, Bulgaria

The phylogeny of the latest recognized domain, Archaea, is still complicated and it is largely based on environmental sequences. A culture independent molecular phylogenetic analysis revealed high Archaea diversity in a terrestrial hot spring, village Varvara, Bulgaria. A total of 35 archaeal operational taxonomic units (OTUs) belonging to three of the classified five Archaea phyla were identified. Most of the sequences were affiliated with the phylum Crenarchaeota (23), grouped in four branches. The rest of the sequences showed highest similarity to the unidentified archaeal clones (9), Euryarchaeota (2), and "Korarchaeota " (1). Eight (23%) of the sequenced 16S rDNAs didn't have known close relatives and represented new and diverse OTUs, four of them forming a new archaeal subgroup without close described sequences or culturable relatives. A sequence affiliated with "Korarchaeota " showed low similarity (90%) to the closest neighbor and both sequences formed unique branch in this phylum. Consequently, the constructed archaeal libraries are characterized by (1) high proportion of OTUs representing uncultivated archaeal phylogroups, (2) the abundance of novel phylotype sequences, (3) the presence of high proportions of Crenarchaeota phylotypes unrelated to cultivated organisms and (4) the presence of a sequence only distantly related to "Korarchaeota " phylum.     

Comprehensive seroprofiling of sixteen B. burgdorferi OspC: Implications for Lyme disease diagnostics design

Early diagnosis of Lyme disease (LD) is critical to successful treatment. However, current serodiagnostic tests do not reliably detect antibodies during early infection. OspC induces a potent early immune response and is also one of the most diverse proteins in the Borrelia proteome. Yet, at least 70% of the amino acid sequence is conserved among all 21 known OspC types. We performed a series of comprehensive seroprofiling studies to select the OspC types that have the most cross-reactive immunodominant epitopes. We found that proteins belonging to seven OspC types detect antibodies from all three infected host species regardless of the OspC genotype of the infecting strain. Although no one OspC type identifies all seropositive human samples, combinations of as few as two OspC proteins identified all patients that had anti-OspC antibodies.     

Typical and atypical neurovascular relations of the trigeminal nerve in the cerebellopontine angle: an anatomical study

The aim of the present study was to anatomically evaluate in adults the neurovascular trigeminal relations in the cerebellopontine angle (CPA), from a morphological and topographical perspective and thus to improve, detail and debate the pre-existing information, with educational and surgical implications. For the present anatomical study we performed bilateral dissections on 20 human adult skull bases, in formalin-fixed cadavers, at the level of the cerebellopontine angle, using the anatomical superior approach; we also studied 20 additional drawn specimens—cerebellum and brainstems, from autopsied cadavers, in order to better document the vasculature at the trigeminal root entry zone (REZ). The most constant but not exclusive neurovascular relations of the trigeminal nerves were those with the superior cerebellar artery (SCA) and the superior petrosal vein (the petrosal vein of Dandy). The regular possibility for the SCA to appear divided into a medial and a lateral branch and these to represent individual trigeminal relations at the level of the pontine cistern or REZ must not be neglected. The petrosal vein tributaries can also represent superior, inferior, or interradicular trigeminal relations. Arterioles emerging from the SCA or the anterior inferior cerebellar artery (AICA) represented trigeminal relations either at the REZ or were coursing between the trigeminal roots. A dissected specimen presented a radicular trigeminal artery emerging from the basilar artery and entering the trigeminal cavum inferior to the nerve. Another specimen presented two bony lamellae superior to the trigeminal nerve at the entrance in the trigeminal cavum—these lamellae were embedded within the lateral border of tentorium cerebelli and the posterior petroclinoid ligament. So we bring here an evidence-based support extremely useful not only for specialists dealing with this area but also for educational purposes. It appears important not only to consider the typical anatomy at this level but also to take into account the atypical and hardly predictable morphologies that may alter the diagnoses and the specific surgical procedures.     

Increased proportion of CD56bright natural killer cells in active and inactive systemic lupus erythematosus

Natural killer (NK) cells belong to the innate immune system but can also affect adaptive immune reactions. This immune regulatory function is often ascribed to the CD56(bright) subpopulation of NK cells that is prevalent in secondary lymphoid tissues and has potent cytokine-producing ability. The NK cells have been described as affecting autoimmune disease and stimulating B-cell production of antibodies, but their role in systemic lupus erythematosus (SLE) pathology has not been extensively studied. We have studied NK cells in SLE, a B-cell-driven systemic autoimmune disease, and phenotypically characterized peripheral blood NK cells in comparison to NK cells from patients with immunoglobulin A nephritis, rheumatoid arthritis and healthy individuals. We have found an increased proportion of CD56(bright) NK cells in SLE, regardless of disease activity. We detected a somewhat increased expression of the activating receptor NKp46/CD335 on NK cells from SLE patients, although neither the percentage of NK cells of all lymphocytes nor the expression of other NK receptors analysed (LIR-1/CD85j, CD94, NKG2C/CD159c, NKG2D/CD314, NKp30/CD337, NKp44/CD336, CD69) differed between patient groups. We show that type I interferon, a proinflammatory cytokine known to be abundant in SLE, can cause increases of CD56(bright) NK cells in vitro. We confirmed that serum levels of interferon-alpha were increased in active, but not in inactive, disease in the SLE patient group. In conclusion, we found an increased proportion of CD56(bright) NK cells in the blood of SLE patients, although it remains to be examined whether and how this relates to the disease process.     

High-Dimensional Single-Cell Mapping of Central Nervous System Immune Cells Reveals Distinct Myeloid Subsets in Health,Aging, and Disease

1. Download high-res image (303KB)
2. Download full-size image

     

Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

Eukaryotes can have thousands of 45S ribosomal RNA (rRNA) genes, many of which are silenced during development. Using fluorescence-activated sorting techniques, we show that active rRNA genes in Arabidopsis thaliana are present within sorted nucleoli, whereas silenced rRNA genes are excluded. DNA methyltransferase (met1), histone deacetylase (hda6), or chromatin assembly (caf1) mutants that disrupt silencing abrogate this nucleoplasmic–nucleolar partitioning. Bisulfite sequencing data indicate that active nucleolar rRNA genes are nearly completely demethylated at promoter CGs, whereas silenced genes are nearly fully methylated. Collectively, the data reveal that rRNA genes occupy distinct but changeable nuclear territories according to their epigenetic state.     

Probing the biofunctionality of biotinylated hyaluronan and chondroitin sulfate by hyaluronidase degradation and aggrecan interaction

Molecular interactions involving glycosaminoglycans (GAGs) are important for biological processes in the extracellular matrix (ECM) and at cell surfaces, and also in biotechnological applications. Enzymes in the ECM constantly modulate the molecular structure and the amount of GAGs in our tissues. Specifically, the changeable sulfation patterns of many GAGs are expected to be important in interactions with proteins. Biotinylation is a convenient method for immobilizing molecules to surfaces. When studying interactions at the molecular, cell and tissue level, the native properties of the immobilized molecule, i.e. its biofunctionality, need to be retained upon immobilization. Here, the GAGs hyaluronan (HA) and chondroitin sulfate (CS), and synthetically sulfated derivatives of the two, were immobilized using biotin-streptavidin binding. The degree of biotinylation and the placement of biotin groups (end-on/side-on) were varied. The introduction of biotin groups could have unwanted effects on the studied molecule, but this aspect that is not always straightforward to evaluate. Hyaluronidase, an enzyme that degrades HA and CS in the ECM, was investigated as a probe to evaluate the biofunctionality of the immobilized GAGs, using both quartz crystal microbalance and high-performance liquid chromatography. Our results showed that end-on biotinylated HA was efficiently degraded by hyaluronidase, whereas already a low degree of side-on biotinylation destroyed the degrading ability of the enzyme. Synthetically introduced sulfate groups also had this effect. Hence hyaluronidase degradation is a cheap and easy way to investigate how molecular function is influenced by the introduced functional groups. Binding experiments with the proteoglycan aggrecan emphasized the influence of protein size and surface orientation of the GAGs for in-depth studies of GAG behavior.     

Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.Invasive infections caused by mucormycetes started to occur more frequently in the last decade and are connected with rapid progression and high mortality rates. Early diagnostics and targeted treatment are crucial. Most mucormycosis cases (over 90%) are caused by Rhizopus spp., followed by Mucor spp., Lichtheimia spp., Rhizomucor pusillus, and, rarely, some other species (2, 9, 11, 16).Definitive diagnosis of mucormycosis is usually made after histopathological proof of mucormycete-like hyphae in involved tissue; the causative agent can be determined only by culture (13). So far, no serological test is available and radiological methods are nonspecific.Molecular detection of mucormycetes is complicated by several factors, and we still do not have any standard protocol. Few methods for the detection of mucormycetes have been published, and only some have been evaluated using clinical samples (1, 5, 10, 14, 15, 17) or samples from animal models (6, 7).The aim of this study was to develop a rapid and sensitive technique for the detection and identification of clinically important mucormycetes. We adopted primers from a qualitative method previously published by Bialek et al. (1) that is specific for members of the order Mucorales targeting 18S ribosomal DNA (rDNA). We modified it to seminested real-time PCR with EvaGreen dye, followed by species distinction by high-resolution melt (HRM) analysis. HRM analysis uses amplification of DNA in the presence of intercalation dye. Fluorescence is measured during a controlled melting of PCR product that results in a melt curve that depends mainly on GC content, length, and sequence of the PCR product. This simple method can be used for genotyping or mutation scanning without the need for time-consuming sequencing (4, 12).DNA was isolated from 50 μl of fungal culture (inoculum was prepared by covering sporulating colonies with approximately 2 ml of sterile 0.85% saline) or a piece of fresh tissue (2 by 1 mm) using the ZR fungal/bacterial DNA kit (Zymo Research). Tissue samples were incubated in lysis buffer overnight, and cultures were immediately processed according to the manufacturer''s protocol. Disruption was extended to 15 min (Disruptor Genie; Scientific Industries). DNA from formalin-fixed, paraffin wax-embedded (FFPE) tissue samples was isolated from 2 or 3 scrolls (5 to 10 μm each) of paraffin block using a DNeasy blood and tissue kit (Qiagen). Paraffin was dissolved in 1 ml of xylene, and then the tissue was washed two times using 1 ml of 96% ethanol and incubated in 180 μl of ATL buffer (Qiagen) and 20 μl of proteinase K (600 mAU/ml solution, where one mAU represents the activity of proteinase K that releases folin-positive amino acids and peptides corresponding to 1 μmol of tyrosine per min) at 55°C overnight and then at 90°C for 1 h. The next steps were done in accordance with the manufacturer''s protocol. DNA isolation from clinical samples was done in a biological safety cabinet. An aliquot of sterile water was processed with each set of samples as a control of potential contamination during the isolation process.Five microliters of DNA was amplified in 25 μl of amplification mixture that contained a 0.2 μM concentration each of primers ZM1 and ZM2 (1), 120 μM deoxynucleoside triphosphates (dNTPs; Roche, Germany), 2.5 mM MgCl₂, 1× GeneAmp PCR Gold buffer, and 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The cycling conditions were 10 min at 95°C, 16 cycles of 30 s at 94°C, 30 s at 50°C, and 60 s at 72°C, and 7 min at 72°C. One microliter of PCR product from the external round was then amplified in duplicate using Rotorgene 6000 (Corbett Research, Australia). Twenty-five microliters of the amplification mixture contained a 0.4 μM concentration each of primers ZM1 and ZM3 (1), 12.5 μl of SensiMix HRM, and 1 μl of EvaGreen (both from a SensiMix HRM kit; Quantace, United Kingdom). The cycling conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 20 s at 60°C, and 30 s at 72°C (acquired on the green channel), followed by HRM analysis (ramp from 74°C to 79.5°C, rising by 0.1°C each cycle, acquired on the HRM channel). Rotorgene 6000 series software (version 1.7) was used for analysis of the results. All positive results were confirmed by sequencing of the PCR product. DNA was purified using a QIAquick PCR purification kit (Qiagen, Germany) and sequenced using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) on an ABI Prism 310 genetic analyzer (Applied Biosystems). Sequences were analyzed using the BLAST alignment program of the GenBank database.We used DNA extracted from five mucormycete cultures diluted in Tris-EDTA (TE) buffer as positive controls in every run. A DNA isolation control (sterile water processed with clinical samples) and a negative control of PCR (sterile water) were added to each run as well.In this study, we tested 31 fungal isolates, comprising 10 mucormycete isolates and 21 isolates from other filamentous fungal groups (Department of Clinical Microbiology, University Hospital Brno and Czech Collection of Microorganisms, Czech Republic). All mucormycete isolates were correctly identified. The melting temperatures (T_m) for each species were as follows: for Rhizopus microsporus, 76.46°C; for Rhizopus oryzae, 76.59°C; for Mucor racemosus, 76.78°C; for Mucor circinelloides, 76.98°C; for Rhizomucor pusillus, 77.87°C; and for Lichtheimia corymbifera, 78.56°C. Representative HRM curves for six different mucormycetes are shown in Fig. ​Fig.1.1. All HRM analysis results were confirmed by sequencing. None of the nonmucormycete fungi were positively tested. The results are summarized in Table ​Table11.Open in a separate windowFIG. 1.Representative result of high-resolution melt (HRM) analysis. Shown are HRM curves for six mucormycete isolates (black curves) and one negative and one positive tissue sample (gray curves).

TABLE 1.

List of fungal isolates used in this study and results of HRM analysis^a