Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

Mortality,Morbidity and Health-Seeking Behaviour during the Ebola Epidemic 2014–2015 in Monrovia Results from a Mobile Phone Survey

Between March 2014 and July 2015 at least 10,500 Ebola cases including more than 4,800 deaths occurred in Liberia, the majority in Monrovia. However, official numbers may have underestimated the size of the outbreak. Closure of health facilities and mistrust in existing structures may have additionally impacted on all-cause morbidity and mortality. To quantify mortality and morbidity and describe health-seeking behaviour in Monrovia, Médecins sans Frontières (MSF) conducted a mobile phone survey from December 2014 to March 2015. We drew a random sample of households in Monrovia and conducted structured mobile phone interviews, covering morbidity, mortality and health-seeking behaviour from 14 May 2014 until the day of the survey. We defined an Ebola-related death as any death meeting the Liberian Ebola case definition. We calculated all-cause and Ebola-specific mortality rates. The sample consisted of 6,813 household members in 905 households. We estimated a crude mortality rate (CMR) of 0.33/10,000 persons/day (95%CI:0.25–0.43) and an Ebola-specific mortality rate of 0.06/10,000 persons/day (95%-CI:0.03–0.11). During the recall period, 17 Ebola cases were reported including those who died. In the 30 days prior to the survey 277 household members were reported sick; malaria accounted for 54% (150/277). Of the sick household members, 43% (122/276) did not visit any health care facility. The mobile phone-based survey was found to be a feasible and acceptable alternative method when data collection in the community is impossible. CMR was estimated well below the emergency threshold of 1/10,000 persons/day. Non-Ebola-related mortality in Monrovia was not higher than previous national estimates of mortality for Liberia. However, excess mortality directly resulting from Ebola did occur in the population. Importantly, the small proportion of sick household members presenting to official health facilities when sick might pose a challenge for future outbreak detection and mitigation. Substantial reported health-seeking behaviour outside of health facilities may also suggest the need for adapted health messaging and improved access to health care.     

Ecosystem Services of Woody Crop Production Systems

Short-rotation woody crops are an integral component of regional and national energy portfolios, as well as providing essential ecosystem services such as biomass supplies, carbon sinks, clean water, and healthy soils. We review recent USDA Forest Service Research and Development efforts from the USDA Biomass Research Centers on the provisioning of these ecosystem services from woody crop production systems. For biomass, we highlight productivity and yield potential, pest susceptibility, and bioenergy siting applications. We describe carbon storage in aboveground woody biomass and studies assessing the provision of clean and plentiful water. Soil protection and wildlife habitat are also mentioned, in the context of converting lands from traditional row-crop agriculture to woody production systems.     

Cadmium reduces the efficiency of Sindbis virus replication in human cells and promotes their survival by inhibiting apoptosis

Arthritogenic alphaviruses are emerging arthropod-borne viruses that occasionally cause sporadic to global outbreaks all over the world. Many environmental factors including xenobiotics have been identified as capable of influencing the spread, the susceptibility and the outcome of viral infection. Among them cadmium is a toxic non-essential heavy metal and a prevalent environmental contaminant. In the present study we evaluated the effect of cadmium exposure on alphavirus infection in vitro. We infected Human Embryonic Kidney (HEK) 293 cells in the presence of cadmium chloride (CdCl₂) with Sindbis virus. Cell viability, apoptosis and viral growth were then examined. Our data show that effective doses of cadmium decreased the virus mediated-cell death by inhibition of apoptosis. Moreover, virus growth in HEK 293 cells was also reduced by CdCl₂ treatment. Altogether our results demonstrate that cadmium triggers a protective response which renders HEK 293 cells resistant against Sindbis virus infection.     

Tumor-Stroma Mechanics Coordinate Amino Acid Availability to Sustain Tumor Growth and Malignancy

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Unlocking biodiversity and conservation studies in high‐diversity environments using environmental DNA (eDNA): A test with Guianese freshwater fishes

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and nondestructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large‐scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity.     

Contribution of Flooded Soils to Sediment and Nutrient Fluxes in a Hydropower Reservoir (Sarrans,Central France)

Ecosystems - When a water reservoir is created, the pre-existing soils and vegetation are flooded. Here, we took advantage of the complete emptying of the Sarrans Reservoir, which was flooded...     

Simultaneous Regulation of Cytokinetic Furrow and Nucleus Positions by Cortical Tension Contributes to Proper DNA Segregation during Late Mitosis

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Nutrient depletion may trigger the Yersinia pestis OmpR‐EnvZ regulatory system to promote flea‐borne plague transmission

The flea’s lumen gut is a poorly documented environment where the agent of flea‐borne plague, Yersinia pestis, must replicate to produce a transmissible infection. Here, we report that both the acidic pH and osmolarity of the lumen’s contents display simple harmonic oscillations with different periods. Since an acidic pH and osmolarity are two of three known stimuli of the OmpR‐EnvZ two‐component system in bacteria, we investigated the role and function of this Y. pestis system in fleas. By monitoring the in vivo expression pattern of three OmpR‐EnvZ‐regulated genes, we concluded that the flea gut environment triggers OmpR‐EnvZ. This activation was not, however, correlated with changes in pH and osmolarity but matched the pattern of nutrient depletion (the third known stimulus for OmpR‐EnvZ). Lastly, we found that the OmpR‐EnvZ and the OmpF porin are needed to produce the biofilm that ultimately obstructs the flea’s gut and thus hastens the flea‐borne transmission of plague. Taken as a whole, our data suggest that the flea gut is a complex, fluctuating environment in which Y. pestis senses nutrient depletion via OmpR‐EnvZ. Once activated, the latter triggers a molecular program (including at least OmpF) that produces the biofilm required for efficient plague transmission.