Topographic prominence as a method for cluster identification in single‐molecule localisation data

Single‐molecule localisation based super‐resolution fluorescence imaging produces maps of the coordinates of fluorescent molecules in a region of interest. Cluster analysis algorithms provide information concerning the clustering characteristics of these molecules, often through the generation of cluster heat maps based on local molecular density. The goal of this study was to generate a new cluster analysis method based on a topographic approach. In particular, a topographic map of the level of clustering across a region is generated based on Getis' variant of Ripley's K‐function. By using the relative heights (topographic prominence, TP) of the peaks in the map, cluster characteristics can be identified more accurately than by using previously demonstrated height thresholds. Analogous to geological TP, the concepts of wet and dry TP and topographic isolation are introduced to generate binary maps. The algorithm is validated using simulated and experimental data and found to significantly outperform previous cluster identification methods.

Illustration of the topographic prominence based cluster analysis algorithm.     

Bed Net Durability Assessments: Exploring a Composite Measure of Net Damage

Background

The durability of Long Lasting Insecticidal Nets (LLINs) in field conditions is of great importance for malaria prevention and control efforts; however, the physical integrity of the net fabric is not well understood making it challenging to determine overall effectiveness of nets as they age. The 2011 World Health Organization Pesticide Evaluation Scheme (WHOPES) guidelines provide a simple, standardized method using a proportional hole index (PHI) for assessing net damage with the intent to provide national malaria control programs with guidelines to assess the useful life of LLINS and estimate the rate of replacement.

Methods

We evaluated the utility of the PHI measure using 409 LLINs collected over three years in Nampula Province, Mozambique following a mass distribution campaign in 2008. For each LLIN the diameter and distance from the bottom of the net were recorded for every hole. Holes were classified into four size categories and a PHI was calculated following WHOPES guidelines. We investigate how the size, shape, and location of holes influence the PHI. The areas of the WHOPES defined categories were compared to circular and elliptical areas based on approximate shape and actual measured axes of each hole and the PHI was compared to cumulative damaged surface area of the LLIN.

Results

The damaged area of small, medium, large, and extra-large holes was overestimated using the WHOPES categories compared to elliptical areas using the actual measured axes. Similar results were found when comparing to circular areas except for extra-large holes which were underestimated. (Wilcoxon signed rank test of differences p< 0.0001 for all sizes). Approximating holes as circular overestimated hole surface area by 1.5 to 2 times or more. There was a significant difference in the mean number of holes < 0.5 cm by brand and there were more holes of all sizes on the bottom of nets than the top. For a range of hypothetical PHI thresholds used to designate a “failed LLIN”, roughly 75 to 80% of failed LLINs were detected by considering large and extra-large holes alone, but sensitivity varied by brand.

Conclusions

Future studies may refine the PHI to better approximate overall damaged surface area. Furthermore, research is needed to identify whether or not appropriate PHI thresholds can be used to deem a net no longer protective. Once a cutoff is selected, simpler methods of determining the effective lifespan of LLINs can help guide replacement strategies for malaria control programs.     

Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis

The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.     

Studying sources of incongruence in arthropod molecular phylogenies: Sea spiders (Pycnogonida) as a case study

In this report, we analyze the phylogeny of Pycnogonida using the three nuclear and three mitochondrial markers currently sequenced for studying inter- and intrafamilial relationships within Arthropoda: 18S and 28S rRNA genes, Histone H3, cytochrome c oxidase subunit 1 (CO1), 12S and 16S rRNA genes. We identify several problems in previous studies, due to the use of inappropriate sequences (taxonomic misidentification, DNA contamination, sequencing errors, missing data) or taxa (outgroup choice). Our analyses show that most markers are not powerful to study the phylogeny of sea spiders. The results suggest however a recent diversification of the group (Mesozoic rather than Paleozoic) and the early divergence of Austrodecidae, followed by Colossendeidae, Pycnogonidae and Rhynchothoracidae. Except Ammotheidae and Callipallenidae, all other families were recovered as monophyletic. Analyses of synonymous sites in CO1 sequences reveal an extreme heterogeneity of nucleotide composition within sea spiders, as six unrelated species show a reverse strand-specific bias. We therefore suggest that several independent reversals of asymmetric mutational constraints occurred during the evolution of Pycnogonida, as a consequence of genomic inversions involving either the control region or a fragment containing the CO1 gene. These hypotheses are supported by the comparison of two complete mitochondrial genomes of sea spiders (Achelia bituberculata and Nymphon gracile) with that of Limulus.     

Links between archaeal community structure, vegetation type and methanogenic pathway in Alaskan peatlands

Although northern peatlands contribute significantly to natural methane emissions, recent studies of the importance and type of methanogenesis in these systems have provided conflicting results. Mechanisms controlling methanogenesis in northern peatlands remain poorly understood, despite the importance of methane as a greenhouse gas. We used 16S rRNA gene retrieval and denaturing gradient gel electrophoresis (DGGE) to analyse archaeal communities in 15 high-latitude peatland sites in Alaska and three mid-latitude peatland sites in Massachusetts. Archaeal community composition was analysed in the context of environmental, vegetation and biogeochemical factors characterized in a parallel study. Phylogenetic analysis revealed that Alaskan sites were dominated by a cluster of uncultivated crenarchaeotes and members of the families Methanomicrobiaceae and Methanobacteriaceae, which are not acetoclastic. Members of the acetoclastic family Methanosarcinaceae were not detected, whereas those of the family Methanosaetaceae were either not detected or were minor. These results are consistent with biogeochemical evidence that acetoclastic methanogenesis is not a predominant terminal decomposition pathway in most of the sites analysed. Ordination analyses indicated a link between vegetation type and archaeal community composition, suggesting that plants (and/or the environmental conditions that control their distribution) influence both archaeal community activity and dynamics.     

The blood contains multiple distinct progenitor populations with clonogenic B and T lineage potential

The thymus is seeded by bone marrow-derived progenitors that circulate in the blood. Multiple cell types can be found in the thymus early after i.v. administration or in steady state, but most fail to satisfy the known characteristics of true T progenitors. Cells that do conform to classical definitions retain multilineage potential, but surprisingly, cannot make B cells. Because acquisition of the T lineage fate among noncommitted progenitors is a lengthy process, the absence of B cell potential in early thymocytes suggests that B and T lineages diverge prethymically. To test this suggestion, we screened numerous presumptive progenitor populations for T cell growth and differentiation potential, as well as for clonogenic T or B cell development. We find that blood and marrow each contain multiple distinct subsets that display growth and differentiation potential consistent with being canonical T progenitors. Assessment of clonogenic potential further shows that although all blood and marrow populations have high T cell cloning potential, no T/non-B cells are apparent. These data suggest that either true thymic reconstitution potential derives from a small T/non-B cell subset of one of these populations, or that most of the cells defined as canonical progenitors within the thymus do not, in fact, reside in the mainstream of T progenitor differentiation.     

Crystal structure of bacteriophage T4 5' nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.     

DCIS and aromatase inhibitors

In patients with hormone receptor positive DCIS tamoxifen reduces recurrence rates by almost 50%. Few data are available with aromatase inhibitors from randomised studies. In the ATAC study there were three DCIS lesions in the anastrozole arm and four in the tamoxifen arm in the women with ER positive invasive cancer. In the MA17 study which randomised patients to up to 5 years of letrozole or placebo there was only one DCIS event in the contralateral breast in patients taking letrozole and five on placebo. There were also four patients in this study who had DCIS in the conserved breast on placebo and none in the letrozole treated group. The few clinical data that are available therefore suggest the aromatase inhibitors are likely to be effective in DCIS. A histological review of a study of 206 postmenopausal women with invasive oestrogen receptor positive breast cancer who were randomised as part of a 14 day preoperative study to receive 2.5 mg of letrozole or 1 mg of anastrozole identified 27 patients with 28 pairs of tumours in whom there was sufficient ER positive DCIS in invasive cancer in the initial core biopsy and in the subsequent surgery specimen, to evaluate for PgR activity and proliferation. Within the DCIS both aromatase inhibitors significantly reduced PgR expression and both drugs also produced a significant fall in proliferation. There was a moderate degree of agreement between the fall in PgR in both the invasive cancer and DCIS (Kappa = 0.5; p = 0.0013) and between the fall in proliferation and between the invasive and in situ components (correlation coefficient = 0.68; p < 0.001). This study has shown significant effects of aromatase inhibitors on DCIS indicating that these agents are therapeutically active in this condition.     

Microbial community diversity in the phycosphere of natural populations of the toxic alga, Alexandrium fundyense

The dinoflagellate Alexandrium fundyense is the major causative organism of paralytic shellfish poisoning in the Gulf of Maine. While laboratory studies have shown that A. fundyense population dynamics can be affected dramatically by co-occurring bacteria, little is known about these interactions in nature. Because A. fundyense is typically a minor Gulf of Maine phytoplankton community member, analyses of the bulk community cannot be used to address bacterium-A. fundyense associations. Therefore, an immunomagnetic bead method was used to selectively capture A. fundyense cells, and the bacteria attached to them, from complex natural samples. Bulk particle-associated and free-living bacterial communities were collected simultaneously. DNA was extracted from all sample types and subjected to 16S rRNA gene fragment amplification, denaturing gradient gel electrophoresis (DGGE) and sequence analysis. Ordination analysis of DGGE profiles confirmed that A. fundyense-associated bacteria community profiles were distinct from bulk bacterial community profiles, indicating selection of specific phylotypes in the A. fundyense phycosphere. Phylogenetic analyses confirmed that Alexandrium-associates were distinct from bulk particle-associated bacteria and that they included a greater prevalence and broader diversity of Gammaproteobacteria than previously thought to be associated with toxic algae. Phylogenetic groups known to be associated with dinoflagellates were also found, including members of the families Alteromonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae and Flavobacteraceae.