ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases

<p>Abstract</p> <p>Background</p> <p>During the last 15 years the internal transcribed spacer (ITS) of nuclear DNA has been used as a target for analyzing fungal diversity in environmental samples, and has recently been selected as the standard marker for fungal DNA barcoding. In this study we explored the potential amplification biases that various commonly utilized ITS primers might introduce during amplification of different parts of the ITS region in samples containing mixed templates ('environmental barcoding'). We performed <it>in silico </it>PCR analyses with commonly used primer combinations using various ITS datasets obtained from public databases as templates.</p> <p>Results</p> <p>Some of the ITS primers, such as ITS1-F, were hampered with a high proportion of mismatches relative to the target sequences, and most of them appeared to introduce taxonomic biases during PCR. Some primers, e.g. ITS1-F, ITS1 and ITS5, were biased towards amplification of basidiomycetes, whereas others, e.g. ITS2, ITS3 and ITS4, were biased towards ascomycetes. The assumed basidiomycete-specific primer ITS4-B only amplified a minor proportion of basidiomycete ITS sequences, even under relaxed PCR conditions. Due to systematic length differences in the ITS2 region as well as the entire ITS, we found that ascomycetes will more easily amplify than basidiomycetes using these regions as targets. This bias can be avoided by using primers amplifying ITS1 only, but this would imply preferential amplification of 'non-dikarya' fungi.</p> <p>Conclusions</p> <p>We conclude that ITS primers have to be selected carefully, especially when used for high-throughput sequencing of environmental samples. We suggest that different primer combinations or different parts of the ITS region should be analyzed in parallel, or that alternative ITS primers should be searched for.</p

Damaged Spent Nuclear Fuel at U.S. DOE Facilities Experience and Lessons Learned

From a handling perspective, any spent nuclear fuel (SNF) that has lost its original technical and functional design capabilities with regard to handling and confinement can be considered as damaged. Some SNF was damaged as a result of experimental activities and destructive examinations; incidents during packaging, handling, and transportation; or degradation that has occurred during storage. Some SNF was mechanically destroyed to protect proprietary SNF designs. Examples of damage to the SNF include failed cladding, failed fuel meat, sectioned test specimens, partially reprocessed SNFs, over-heated elements, dismantled assemblies, and assemblies with lifting fixtures removed. In spite of the challenges involved with handling and storage of damaged SNF, the SNF has been safely handled and stored for many years at DOE storage facilities. This report summarizes a variety of challenges encountered at DOE facilities during interim storage and handling operations along with strategies and solutions that are planned or were implemented to ameliorate those challenges. A discussion of proposed paths forward for moving damaged and nondamaged SNF from interim storage to final disposition in the geologic repository is also presented

Differential microbicidal effects of human histone proteins H2A and H2B on Leishmania promastigotes and amastigotes

Recent studies have shown that histone proteins can act as antimicrobial peptides in host defense against extracellular bacteria, fungi, and Leishmania promastigotes. In this study, we used human recombinant histone proteins to further study their leishmaniacidal effects and the underlying mechanisms. We found that the histones H2A and H2B (but not H1(0)) could directly and efficiently kill promastigotes of Leishmania amazonensis, L. major, L. braziliensis, and L. mexicana in a treatment dose-dependent manner. Scanning electron microscopy revealed surface disruption of histone-treated promastigotes. More importantly, the preexposure of promastigotes to histone proteins markedly decreased the infectivity of promastigotes to murine macrophages (Mφs) in vitro. However, axenic and lesion-derived amastigotes of L. amazonensis and L. mexicana were relatively resistant to histone treatment, which correlated with the low levels of intracellular H2A in treated amastigotes. To understand the mechanisms underlying these differential responses, we investigated the role of promastigote surface molecules in histone-mediated killing. Compared with the corresponding controls, transgenic L. amazonensis promastigotes expressing lower levels of surface gp63 proteins were more susceptible to histone H2A, while L. major and L. mexicana promastigotes with targeted deletion of the lipophosphoglycan 2 (lpg2) gene (but not the lpg1 gene) were more resistant to histone H2A. We discuss the influence of promastigote major surface molecules in the leishmaniacidal effect of histone proteins. This study provides new information on host innate immunity to different developmental stages of Leishmania parasites

Cross-Sectional Comparison of Small Animal [F-18]-Florbetaben Amyloid-PET between Transgenic AD Mouse Models

We aimed to compare [F-18]-florbetaben PET imaging in four transgenic mouse strains modelling Alzheimer's disease (AD), with the main focus on APPswe/PS2 mice and C57Bl/6 mice serving as controls (WT). A consistent PET protocol (N = 82 PET scans) was used, with cortical standardized uptake value ratio (SUVR) relative to cerebellum as the endpoint. We correlated methoxy-X04 staining of beta-amyloid with PET results, and undertook ex vivo autoradiography for further validation of a partial volume effect correction (PVEC) of PET data. The SUVR in APPswe/PS2 increased from 0.95 +/- 0.04 at five months (N = 5) and 1.04 +/- 0.03 (p < 0.05) at eight months (N = 7) to 1.07 +/- 0.04 (p < 0.005) at ten months (N = 6), 1.28 +/- 0.06 (p < 0.001) at 16 months (N = 6) and 1.39 +/- 0.09 (p < 0.001) at 19 months (N = 6). SUVR was 0.95 +/- 0.03 in WT mice of all ages (N = 22). In APPswe/PS1G384A mice, the SUVR was 0.93/0.98 at five months (N = 2) and 1.11 at 16 months (N = 1). In APPswe/PS1dE9 mice, the SUVR declined from 0.96/0.96 at 12 months (N = 2) to 0.91/0.92 at 24 months (N = 2), due to beta-amyloid plaques in cerebellum. PVEC reduced the discrepancy between SUVR-PET and autoradiography from -22% to +2% and increased the differences between young and aged transgenic animals. SUVR and plaque load correlated highly between strains for uncorrected (R = 0.94, p < 0.001) and PVE-corrected (R = 0.95, p < 0.001) data. We find that APPswe/PS2 mice may be optimal for longitudinal amyloid-PET monitoring in planned interventions studies

CD4+ T Cell-Dependent Macrophage Activation Modulates Sustained PS Exposure on Intracellular Amastigotes of Leishmania amazonensis

Leishmania amazonensis amastigotes can make use of surface-exposed phosphatidylserine (PS) molecules to promote infection and non-classical activation of macrophages (MΦ), leading to uncontrolled intracellular proliferation of the parasites. This mechanism was quoted as apoptotic mimicry. Moreover, the amount of PS molecules exposed on the surface of amastigotes correlates with the susceptibility of the host. In this study, we tested whether host cellular responses influence PS expression on intracellular amastigotes. We found that the level of PS exposure on intracellular amastigotes was modulated by CD4+ T cell and MΦ activation status in vitro and in vivo. L. amazonensis infection generated a Th1/Th2-mixed cytokine profile, providing the optimal MΦ stimulation that favored PS exposure on intracellular amastigotes. Maintenance of PS exposed on the parasite was dependent on low, but sustained, levels of nitric oxide and polyamine production. Amastigotes obtained from lymphopenic nude mice did not expose PS on their surface, and adoptive transfer of CD4+ T cells reversed this phenotype. In addition, histopathological analysis of mice treated with anti-PS antibodies showed increased inflammation and similarities to nude mouse lesions. Collectively, our data confirm the role of pathogenic CD4+ T cells for disease progression and point to PS as a critical parasite strategy to subvert host immune responses

NiN-basiskartlegging av utvalgte verneområder i Nordland, 2022

NIBIO har i samarbeid med Sállir Natur AS og Natur og Samfunn AS kartlagt naturtyper og arter i fem verneområder i Nordland i 2022 etter kartleggingsmetodikken Natur i Norge (NiN). Rapporten oppsummerer forhold som kommer dårlig frem i kartobjekter og egenskapsdata som har blitt registrert og rapportert via NiNapp. Rapporten inneholder generelle faglige vurderinger, eventuelle observerte forvaltningsrelevante problemstillinger, praktiske utfordringer i felt, eventuell usikkerhet knyttet til kartleggingsenheter og viser noen utvalgte bilder for verneområdene.NiN-basiskartlegging av utvalgte verneområder i Nordland, 2022publishedVersio

A population based time series analysis of asthma hospitalisations in Ontario, Canada: 1988 to 2000

BACKGROUND: Asthma is a common yet incompletely understood health problem associated with a high morbidity burden. A wide variety of seasonally variable environmental stimuli such as viruses and air pollution are believed to influence asthma morbidity. This study set out to examine the seasonal patterns of asthma hospitalisations in relation to age and gender for the province of Ontario over a period of 12 years. METHODS: A retrospective, population-based study design was used to assess temporal patterns in hospitalisations for asthma from April 1, 1988 to March 31, 2000. Approximately 14 million residents of Ontario eligible for universal healthcare coverage during this time were included for analysis. Time series analyses were conducted on monthly aggregations of hospitalisations. RESULTS: There is strong evidence of an autumn peak and summer trough seasonal pattern occurring every year over the 12-year period (Fisher-Kappa (FK) = 23.93, p > 0.01; Bartlett Kolmogorov Smirnov (BKS) = 0.459, p < 0.01). This pattern was observed in both sexes. However, young males (0–4 years) were hospitalised at two to three times the rate of females of the same age. Rates were much lower in the older age groups. A downward trend in asthma hospitalisations was observed in the total population over the twelve-year period (beta = -0.980, p < 0.01). CONCLUSIONS: A clear and consistent seasonal pattern was observed in this study for asthma hospitalisations. These findings have important implications for the development of effective management and prevention strategies