HIV-1 proviral DNA excision using an evolved recombinase

HIV-1 integrates into the host chromosome and persists as a provirus flanked by long terminal repeats (LTRs). To date, treatment regimens primarily target the virus enzymes or virus-cell fusion, but not the integrated provirus. We report here the substrate-linked protein evolution of a tailored recombinase that recognizes an asymmetric sequence within an HIV-1 LTR. This evolved recombinase efficiently excised integrated HIV proviral DNA from the genome of infected cells. Although a long way from use in the clinic, we speculate that this type of technology might be adapted in future antiretroviral therapies, among other possible uses.     

Prioritizing marine invasive alien species in the European Union through horizon scanning

1. The disproportionately low presence of marine species in the list of invasive alien species (IAS) of Union concern of the European Union (EU) Regulation 1143/2014 does not fully acknowledge the threat they pose to the EU marine environment.
2. In this study, the first EU‐scale Horizon Scanning (HS) focusing on marine alien species was performed, aiming to deliver a ranked list of species that should be of high priority for risk assessment (Article 5 of the EU IAS Regulation).
3. Species absent from or with a limited distribution in EU marine waters were targeted. In total, 363 alien species were initially screened for HS by a panel of experts, including a broad range of taxonomic groups. Species were scored for their likelihood of arrival, establishment, spread, and impact in EU waters.
4. A consensus workshop ranked 267 species, including a subset of 26 prioritized species. These species are considered to be mainly introduced by shipping (fouling and ballast water), via the Suez Canal, and aquaculture activities. The 26 priority species were also scrutinized in terms of feasibility of their management; 18 of them were suggested for performing risk assessments on the basis of the EU IAS Regulation.
5. Since biological invasions are dynamic and connected with accelerated globalization and diversified human activities, we recommend HS to be repeated periodically to review the species already listed and assess new ones.

     

Seroprevalence of Toxoplasma gondii and Neospora caninum infection of cats in Hungary

Blood samples were collected from 330 cats in Hungary in order to evaluate their seroconversion to Toxoplasma gondii and Neospora caninum using the indirect fluorescent antibody test (IFAT). The overall prevalence of toxoplasmosis was 47.6%, the prevalence being 22.4% among urban, 50% among suburban and 61.3% among rural animals. Significantly more cats had high IFAT titres (1:640 to 1:5120) in the countryside. Female cats were more frequently infected with T. gondii than males (53.3% vs. 39.3%), and seropositivity increased with the age of animals. The prevalence (0.6%) and titre (1:40) of antibodies to N. caninum was low. Sixty-two cats were also screened for seroconversion to feline infectious peritonitis (FIP) virus. Higher titres to T. gondii were more frequently detected among FIP-positive cats, but this difference was non-significant due to the small number of cats with concurrent infection.     

Verbleib des 15N nach einmaliger Verabreichung von markiertem Düngerstickstoff im Dauerversuch Ewiger Roggenbau Halle

Fate of ¹⁵N after one single application of labelled N fertilizer in the long-term experiment Eternal Rye Cropping Halle To elucidate the fate of N in the long-term field experiment with continuous rye cropping in Halle, Germany (Haplic Phaeozem derived from sandy loess; mean annual precipitation: 466 mm) micro plots (9 m²) were established within the NPK main plot in 1993 and 1994 and the routine N dose (60 kg N ha^—1 a^—1) was once applied as ¹⁵N labelled fertilizer. In the subsequent four years each micro plot was analyzed for the ¹⁵N withdrawn with grain and straw and that remaining in the soil after harvest. Parallel to this, micro plots on the unfertilized main plot received a single dose of 5 kg ha^—1 of high-label ¹⁵N in order to achieve labelling of the annual N input from the air (˜40 kg ha^—1 a^—1) and to follow its fate. — In the NPK treatment about one third of the labelled fertilizer N applied (= *N) was taken up by the rye in the first year, while nearly half of it remained in the soil (0—100 cm), and 10—15% was not recovered (most likely lost via leaching). During the following three years the amount of *N remaining in the soil decreased from 32.7 kg ha^—1 to 21.5 kg ha^—1. However, less than half of the remobilized *N had been taken up by the crop. On the unfertilized plot the uptake of *N was lower at the end of the experiment compared to the NPK plot, while the loss exceeded the comparative figure correspondingly.     

Comparative cytotoxicity evaluation of lanthanide nanomaterials on mouse and human cell lines with metabolic and DNA-quantification assays

Lanthanide nanomaterials are considered a less toxic alternative to quantum dots for bioimaging applications. This study evaluated the cytotoxicity of terbium (Tb)-doped gadolinium oxide (Gd(2)O(3)) and dysprosium oxide (Dy(2)O(3)) nanoparticles exposed to human (BEAS-2B) and mouse (L929) cell lines at a concentration range of 200-2000?μg/ml for 48 h. Two assay methods were utilized-WST-8 assay (colorimetric) based on mitochondrial metabolic activity and Pico-Green assay (fluorescence), which measures total DNA content. The authors' data showed that Tb-doped Gd(2)O(3) nanoparticles were consistently more toxic than Tb-doped Dy(2)O(3) nanoparticles. However, exposure to these nanomaterials caused a decrease in proliferation rate for both cell lines rather than a net loss of viable cells after 48 h of exposure. Additionally, there was some degree of discrepancy observed with the two assay methods. For the mouse L929 cell line, the WST-8 assay yielded consistently lower proliferation rates compared to the Pico-Green assay, whereas the opposite trend was observed for the human BEAS-2B cell line. This could arise because of the differential effects of these nanoparticles on the metabolism of L929 and BEAS-2B cells, which in turn may translate to differences in their postexposure proliferation rates. Hence, the Pico-Green assay could have an advantage over the WST-8 assay because it is not skewed by the differential effects of nanomaterials on cellular metabolism.     

A classification scheme for earthworm populations (Lumbricidae) in cultivated agricultural soils in Brandenburg,Germany

Earthworm activity is observed at long‐term monitoring sites as an indicator of soil function to assess changes resulting from environmental and management conditions. In order to assess changes, characteristic values of earthworm populations under different site conditions have to be known. Therefore, a classification scheme for site‐specific earthworm populations was developed for soil in agricultural use, taking interactions between earthworm populations and soil properties into account. Characteristics of sites grouped by means of a cluster analysis after principal‐component analysis served as a basis for the derivation of the classification scheme. Soil variables found to characterize site differences with respect to earthworm populations were the texture of the topsoil, the texture of the subsoil, and the soil organic‐matter (SOM) content. The textural classes of the topsoil were divided into five groups comprising sandy soils (Ss), silty sand soils (Su), slightly loamy sand soils (Sl2), medium to strongly loamy sand soils (Sl3/Sl4), and loam and clay soils. Soil organic matter was divided into grades of equal size in a range from <1%, 1%–2% up to >6%. The variables “earthworm abundance” and “earthworm species” were selected to represent earthworm populations and were divided into six groups ranging from very low to extremely high. Defined groups of earthworm populations showed a clear structure in relation to soil textural groups and the content of SOM. From this distribution, a classification scheme was derived as basis for prognostic values of site‐specific earthworm populations, thus enabling the interpretation of changes over time. For some soil textural groups, selected variables appeared to enable the derivations of expected earthworm densities and species composition outside the range of the given database, but for some soil textural groups, broader databases will be needed to specify these derivations.     

Tracking Collembola feeding strategies by the natural C signal of fatty acids in an arable soil with different fertilizer regimes

Compound specific stable isotope analysis (¹³C/¹²C ratio of fatty acids) was used to assess the allocation of plant carbon in soil microbiota, and to identify the trophic links to microbial grazers in an arable field with long-term mineral and organic fertilizer amendments. The feeding strategy of two dominant Collembola species, epedaphic Isotoma viridis and euedaphic Willemia anophthalma was determined. The investigation was conducted following a shift to amaranth, a C₄ plant, after 27 years of continuous C₃ crop rotation. The influence of new C₄ plant carbon was observed in microbial phospholipids (PLFAs) with higher δ¹³C recorded in C₄ amaranth than in C₃ clover soils. The strongest enrichment occurred in the fungal PLFA 18:2ω6,9c and bacterial PLFA 18:1ω9t with 11.2‰ and 6.6‰, respectively. However, other bacterial PLFAs showed no isotopic change, suggesting that the microbial community simultaneously utilized “new” and “old” plant carbon. The δ¹³C of Collembola fatty acids displayed species specific lipid pattern, which was affected by crop type, but not fertilizer amendments. Isotopic separation of Collembola lipids from amaranth and clover plots was more distinct in I. viridis than W. anophthalma. With up to 18‰, the enrichment in Collembola lipids was stronger than in microbial PLFAs, pointing to a distinct incorporation of carbon resources originating from the actual plant residues. The δ¹³C pattern in I. viridis indicated trophic links with bacteria, saprotrophic fungi and plant tissues, while saprotrophic fungi and plant tissues were accountable for the patterns observed in W. anophthalma.     

Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: Characterization of lung pathology,persistence of variable surface protein antigens and local immune response

Background

Mycoplasma bovis is associated with pneumonia in calves characterized by the development of chronic caseonecrotic lesions with the agent persisting within the lesion. The purposes of this study were to characterize the morphology of lung lesions, examine the presence of M. bovis variable surface protein (Vsp) antigens and study the local immune responses in calves after infection with M. bovis strain 1067.

Methods

Lung tissue samples from eight calves euthanased three weeks after experimental infection with M. bovis were examined by bacteriology and pathology. Lung lesions were evaluated by immunohistochemical (IHC) staining for wide spectrum cytokeratin and for M. bovis Vsp antigens and pMB67 antigen. IHC identification and quantitative evaluation of CD4⁺ and CD8⁺ T lymphocytes and immunoglobulin (IgG1, IgG2, IgM, IgA)-containing plasma cells was performed. Additionally, expression of major histocompatibility complex class II (MHC class II) was studied by IHC.

Results

Suppurative pneumonic lesions were found in all calves. In two calves with caseonecrotic pneumonia, necrotic foci were surrounded by epithelial cells resembling bronchial or bronchiolar epithelium. In all calves, M. bovis Vsp antigens were constantly present in the cytoplasm of macrophages and were also present extracellularly at the periphery of necrotic foci. There was a considerable increase in numbers of IgG1- and IgG2-positive plasma cells among which IgG1-containing plasma cells clearly predominated. Statistical evaluation of the numbers of CD4⁺ and CD8⁺ T cells, however, did not reveal statistically significant differences between inoculated and control calves. In M. bovis infected calves, hyperplasia of bronchus-associated lymphoid tissue (BALT) was characterized by strong MHC class II expression of lymphoid cells, but only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II.

Conclusions

The results from this study show that infection of calves with M. bovis results in various lung lesions including caseonecrotic pneumonia originating from bronchioli and bronchi. There is long-term persistence of M. bovis as demonstrated by bacteriology and immunohistochemistry for M. bovis antigens, i.e. Vsp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from local downregulation of antigen presenting mechanisms and an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins.