Visualizing the Beta Interferon Response in Mice during Infection with Influenza A Viruses Expressing or Lacking Nonstructural Protein 1

The innate host defense against influenza virus is largely dependent on the type I interferon (IFN) system. However, surprisingly little is known about the cellular source of IFN in the infected lung. To clarify this question, we employed a reporter mouse that contains the firefly luciferase gene in place of the IFN-β-coding region. IFN-β-producing cells were identified either by simultaneous immunostaining of lungs for luciferase and cellular markers or by generating conditional reporter mice that express luciferase exclusively in defined cell types. Two different strains of influenza A virus were employed that either do or do not code for nonstructural protein 1 (NS1), which strongly suppresses innate immune responses of infected cells. We found that epithelial cells and lung macrophages, which represent the prime host cells for influenza viruses, showed vigorous IFN-β responses which, however, were severely reduced and delayed if the infecting virus was able to produce NS1. Interestingly, CD11c⁺ cell populations that were either expressing or lacking macrophage markers produced the bulk of IFN-β at 48 h after infection with wild-type influenza A virus. Our results demonstrate that the virus-encoded IFN-antagonistic factor NS1 disarms specifically epithelial cells and lung macrophages, which otherwise would serve as main mediators of the early response against infection by influenza virus.     

Comparative binding of Cry1Ab and Cry1F Bacillus thuringiensis toxins to brush border membrane proteins from Ostrinia nubilalis, Ostrinia furnacalis and Diatraea saccharalis (Lepidoptera: Crambidae) midgut tissue

The European (Ostrinia nubilalis Hübner) and Asian corn borers (Ostrinia furnacalis Guenée) are closely related and display similar sensitivity to Cry1 toxins. In this study, we compared the binding patterns of Cry1Ab and Cry1F toxins between both Ostrinia spp., as well as the expression of putative cadherin- and aminopeptidase-N (APN)-like protein receptors. Additionally, cDNA sequences of these putative toxin receptors from both Ostrinia species were compared. Ligand blots for both species indicated a similar binding pattern for Cry1Ab with the strongest immunoreactive band at 260 kDa in both species. In addition, similar expression of the putative cadherin- and APN-like protein receptors were observed at 260 and 135 kDa, respectively. A high degree of similarity (98% amino acid sequence identity) of cDNA sequences for both putative receptor sequences was observed. The Cry1F ligand blot revealed that O. furnacalis and O. nubilalis BBMV exhibited slightly different binding patterns, with strong binding to putative proteins at 150 and 140 kDa, respectively. Both proteins appeared to also bind Cry1Ab, although the signal intensity was much reduced with Cry1Ab. O. furnacalis showed an additional but weaker band at 210 kDa relative to the 150 kDa band. Diatraea saccharalis (Fabricius), which was used as an outgroup species, exhibited different binding patterns than either Ostrinia species, with both Cry1Ab and Cry1F toxins binding to a 210 kDa protein. These results support the previous experiments indicating that O. nubilalis and O. furnacalis share similar patterns of susceptibility to Cry toxins.     

Signatures of diversifying selection at EST‐SSR loci and association with climate in natural Eucalyptus populations

Understanding the environmental parameters that drive adaptation among populations is important in predicting how species may respond to global climatic changes and how gene pools might be managed to conserve adaptive genetic diversity. Here, we used Bayesian F_ST outlier tests and allele–climate association analyses to reveal two Eucalyptus EST‐SSR loci as strong candidates for diversifying selection in natural populations of a southwestern Australian forest tree, Eucalyptus gomphocephala (Myrtaceae). The Eucalyptus homolog of a CONSTANS‐like gene was an F_ST outlier, and allelic variation showed significant latitudinal clinal associations with annual and winter solar radiation, potential evaporation, summer precipitation and aridity. A second F_ST outlier locus, homologous to quinone oxidoreductase, was significantly associated with measures of temperature range, high summer temperature and summer solar radiation, with important implications for predicting the effect of temperature on natural populations in the context of climate change. We complemented these data with investigations into neutral population genetic structure and diversity throughout the species range. This study provides an investigation into selection signatures at gene‐homologous EST‐SSRs in natural Eucalyptus populations, and contributes to our understanding of the relationship between climate and adaptive genetic variation, informing the conservation of both putatively neutral and adaptive components of genetic diversity.     

Performance of Lead-Free versus Lead-Based Hunting Ammunition in Ballistic Soap

Background

Lead-free hunting bullets are an alternative to lead-containing bullets which cause health risks for humans and endangered scavenging raptors through lead ingestion. However, doubts concerning the effectiveness of lead-free hunting bullets hinder the wide-spread acceptance in the hunting and wildlife management community.

Methods

We performed terminal ballistic experiments under standardized conditions with ballistic soap as surrogate for game animal tissue to characterize dimensionally stable, partially fragmenting, and deforming lead-free bullets and one commonly used lead-containing bullet. The permanent cavities created in soap blocks are used as a measure for the potential wound damage. The soap blocks were imaged using computed tomography to assess the volume and shape of the cavity and the number of fragments. Shots were performed at different impact speeds, covering a realistic shooting range. Using 3D image segmentation, cavity volume, metal fragment count, deflection angle, and depth of maximum damage were determined. Shots were repeated to investigate the reproducibility of ballistic soap experiments.

Results

All bullets showed an increasing cavity volume with increasing deposited energy. The dimensionally stable and fragmenting lead-free bullets achieved a constant conversion ratio while the deforming copper and lead-containing bullets showed a ratio, which increases linearly with the total deposited energy. The lead-containing bullet created hundreds of fragments and significantly more fragments than the lead-free bullets. The deflection angle was significantly higher for the dimensionally stable bullet due to its tumbling behavior and was similarly low for the other bullets. The deforming bullets achieved higher reproducibility than the fragmenting and dimensionally stable bullets.

Conclusion

The deforming lead-free bullet closely resembled the deforming lead-containing bullet in terms of energy conversion, deflection angle, cavity shape, and reproducibility, showing that similar terminal ballistic behavior can be achieved. Furthermore, the volumetric image processing allowed superior analysis compared to methods that involve cutting of the soap blocks.     

Host tree utilization by epiphytic orchids in different land-use intensities in Kathmandu Valley, Nepal

We studied the influence of site conditions on epiphytic orchids under a subtropical climate in the Kathmandu Valley, Nepal. We analysed 96 systematically distributed grid points situated in Kathmandu Valley across a land-use intensity gradient (national park to urbanised city area). Geographical Information System (GIS) and remote sensing were used for classification of land-use types. We identified 23 species of epiphytic orchids, within 13 genera, from 42 different host tree species. Host preference is obvious for some orchid species (e.g., Dendrobium nobile), with certain tree species (e.g., Schima wallichii, Ficus religiosa) hosting more orchid species than others. The orchid Rhynchostylis retusa was the most common species found on many different host tree species across the land-use intensity gradient. Host species and host bark characteristics (e.g., rugosity, pH and exposure to wind) played a vital role for orchid distribution, with lower abundance in areas of higher impact. Under strong human impact (urban city area), F. religiosa was the dominant host tree, with large individual trees (mean diameter in breast height, dbh?=?1.3?m) providing the habitat for considerable populations of R. retusa individuals. In general, epiphytic orchids were found on larger host trees in urban areas than in areas of lower human impact. We found that some hosts are more likely to harbour orchid species, especially native host species. Older larger trees with rougher bark, low pH, exposed to wind and reduced human impact provided better habitats for orchids. We suggest these characteristics should be considered in urban planning to reduce human impact on the associated orchid epiphytic community.     

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-)γ(c) (-/-) mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+), MHCII(+), CD11a(+) and CD79αcy(+). PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.     

Field of Dreams: Restitution of Pollinator Services in Restored Bird‐Pollinated Plant Populations

Ecosystem functionality is an increasingly important objective of ecological restoration. Despite this, a few studies have rigorously assessed reproductive functionality within restored plant populations, and it is largely assumed that pollinators follow restoration of plant communities—“build it and they will come.” Here, we applied an ecological genetic approach to determine the impact of spatial separation on mating in Banksia menziesii (Proteaceae), a dominant bird‐pollinated species of Banksia woodlands of Western Australia. All plants at three post‐mining restored sites (n = 72 [13 years old], n = 21 [8 years old], and n = 20 [9 years old]), as well as a sample from an adjacent natural reference site (n = 42), were genotyped at nine microsatellite loci. Seed set, mating system parameters, realized pollen dispersal through the assignment of paternity to seed, and avian pollinator species composition, abundance and behavior, were assessed. All patches displayed equivalent heterozygosity (H_e = 0.53–0.59) and very weak genetic divergence (F_ST ≤ 0.01). Seed of plants within restored sites showed complete outcrossing and relatively high seed set, 26% of which were sired by pollen donors located beyond the local patch. Similar abundance and movement of nectar‐feeding birds was observed in restored and natural sites, despite lower bird species diversity in the restored site, where a smaller, less aggressive species was dominant. Our results demonstrate the restitution of wide outcrossing in these restored Banksia patches within an active mine‐site, and suggest that restored bird‐pollinated Banksia populations are resilient to human impacts, due largely to their generalist pollinator requirements and highly‐mobile avian pollinators.     

Fluorescence techniques in biotechnology

The high specificity and sensitivity of fluorescence techniques have made them important analytical tools in medicine and biotechnology. Besides monitoring and quantitative detection of biomolecules these methods can be used for controlling bacterial activities or for measuring physiological states of cells or tissues. Three topics of importance in biotechnology — immunoassays, photosynthesis and fermentation — are treated in detail.     

Effect of Circulating Forms of Soy Isoflavones on the Oxidation of Low Density Lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.     

Distribution and antifungal susceptibility of yeasts isolates from intensive care unit patients

Yeasts frequently colonize non-sterile sites in the body. The aim of the study was to determine distribution in clinical samples and antifungal susceptibility to five antifungals. From January 2013 through June 2015, 800 isolates were obtained from intensive care unit patients. Candida albicans (58.9%), Candida glabrata (20.4%), Candida krusei (8.6%), and Candida parapsilosis (3.6%) were the leading species. Majority of the C. albicans isolates were susceptible to the fluconazole. Elevated voriconazole minimal inhibitory concentrations (MICs) were observed in isolates exhibiting high fluconazole MICs, most frequently in C. glabrata. Isolates with echinocandins MICs suggesting reduced susceptibility were only sporadic cases with the exception of Trichosporon spp. The amphotericin B MICs were slightly higher for some C. krusei.