Efficient depletion of host DNA contamination in malaria clinical sequencing.

The cost of whole-genome sequencing (WGS) is decreasing rapidly as next-generation sequencing technology continues to advance, and the prospect of making WGS available for public health applications is becoming a reality. So far, a number of studies have demonstrated the use of WGS as an epidemiological tool for typing and controlling outbreaks of microbial pathogens. Success of these applications is hugely dependent on efficient generation of clean genetic material that is free from host DNA contamination for rapid preparation of sequencing libraries. The presence of large amounts of host DNA severely affects the efficiency of characterizing pathogens using WGS and is therefore a serious impediment to clinical and epidemiological sequencing for health care and public health applications. We have developed a simple enzymatic treatment method that takes advantage of the methylation of human DNA to selectively deplete host contamination from clinical samples prior to sequencing. Using malaria clinical samples with over 80% human host DNA contamination, we show that the enzymatic treatment enriches Plasmodium falciparum DNA up to ∼9-fold and generates high-quality, nonbiased sequence reads covering >98% of 86,158 catalogued typeable single-nucleotide polymorphism loci

Can policy be risk-based? The cultural theory of risk and the case of livestock disease containment

This article explores the nature of calls for risk-based policy present in expert discourse from a cultural theory perspective. Semi-structured interviews with professionals engaged in the research and management of livestock disease control provide the data for a reading proposing that the real basis of policy relating to socio-technical hazards is deeply political and cannot be purified through ‘escape routes’ to objectivity. Scientists and risk managers are shown calling, on the one hand, for risk-based policy approaches while on the other acknowledging a range of policy drivers outside the scope of conventional quantitative risk analysis including group interests, eventualities such as outbreaks, historical antecedents, emergent scientific advances and other contingencies. Calls for risk-based policy are presented, following cultural theory, as ideals connected to a reductionist epistemology and serving particular professional interests over others rather than as realistic proposals for a paradigm shift

Paper Supercapacitor Developed Using a Manganese Dioxide/Carbon Black Composite and a Water Hyacinth Cellulose Nanofiber-Based Bilayer Separator

Flexible and green energy storage devices have a wide range of applications in prospective electronics and connected devices. In this study, a new eco-friendly bilayer separator and primary and secondary paper supercapacitors based on manganese dioxide (MnO2)/carbon black (CB) are developed. The bilayer separator is prepared via a two-step fabrication process involving freeze–thawing and nonsolvent-induced phase separation. The prepared bilayer separator exhibits superior porosity of 46%, wettability of 46.5°, and electrolyte uptake of 194% when compared with a Celgard 2320 trilayer separator (39%, 55.58°, and 110%). Moreover, lower bulk resistance yields a higher ionic conductivity of 0.52 mS cm–1 in comparison to 0.22 mS cm–1 for the Celgard separator. Furthermore, the bilayer separator exhibits improved mean efficiency of 0.44% and higher specific discharge capacitance of 13.53%. The anodic and cathodic electrodes are coated on a paper substrate using MnO2/CB and zinc metal-loaded CB composites. The paper supercapacitor demonstrates a high specific capacitance of 34.1 mF cm–2 and energy and power density of 1.70 μWh cm–2 and 204.8 μW cm–2 at 500 μA, respectively. In summary, the concept of an eco-friendly bilayer cellulose separator with paper-based supercapacitors offers an environmentally friendly alternative to traditional energy storage devices

Unraveling Twisty Linear Polarization Morphologies in Black Hole Images

We investigate general relativistic magnetohydrodynamic simulations (GRMHD) to determine the physical origin of the twisty patterns of linear polarization seen in spatially resolved black hole images and explain their morphological dependence on black hole spin. By characterising the observed emission with a simple analytic ring model, we find that the twisty morphology is determined by the magnetic field structure in the emitting region. Moreover, the dependence of this twisty pattern on spin can be attributed to changes in the magnetic field geometry that occur due to the frame dragging. By studying an analytic ring model, we find that the roles of Doppler boosting and lensing are subdominant. Faraday rotation may cause a systematic shift in the linear polarization pattern, but we find that its impact is subdominant for models with strong magnetic fields and modest ion-to-electron temperature ratios. Models with weaker magnetic fields are much more strongly affected by Faraday rotation and have more complicated emission geometries than can be captured by a ring model. However, these models are currently disfavoured by the recent EHT observations of M87*. Our results suggest that linear polarization maps can provide a probe of the underlying magnetic field structure around a black hole, which may then be usable to indirectly infer black hole spins. The generality of these results should be tested with alternative codes, initial conditions, and plasma physics prescriptions.Comment: 25 pages, 19 figure

An Effective Method to Purify Plasmodium falciparum DNA Directly from Clinical Blood Samples for Whole Genome High-Throughput Sequencing

Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of “natural” parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ∼40-fold coverage of the genome was observed per lane for samples with ≤30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ∼40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum

Characterization of Within-Host Plasmodium falciparum Diversity Using Next-Generation Sequence Data

Our understanding of the composition of multi-clonal malarial infections and the epidemiological factors which shape their diversity remain poorly understood. Traditionally within-host diversity has been defined in terms of the multiplicity of infection (MOI) derived by PCR-based genotyping. Massively parallel, single molecule sequencing technologies now enable individual read counts to be derived on genome-wide datasets facilitating the development of new statistical approaches to describe within-host diversity. In this class of measures the FWS metric characterizes within-host diversity and its relationship to population level diversity. Utilizing P. falciparum field isolates from patients in West Africa we here explore the relationship between the traditional MOI and FWS approaches. FWS statistics were derived from read count data at 86,158 SNPs in 64 samples sequenced on the Illumina GA platform. MOI estimates were derived by PCR at the msp-1 and -2 loci. Significant correlations were observed between the two measures, particularly with the msp-1 locus (P = 5.92×10−5). The FWS metric should be more robust than the PCR-based approach owing to reduced sensitivity to potential locus-specific artifacts. Furthermore the FWS metric captures information on a range of parameters which influence out-crossing risk including the number of clones (MOI), their relative proportions and genetic divergence. This approach should provide novel insights into the factors which correlate with, and shape within-host diversity

Bat pluripotent stem cells reveal unique entanglement between host and viruses

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat (Rhinolophus ferrumequinum) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats’ unique traits.N

Whole-genome scans provide evidence of adaptive evolution in Malawian Plasmodium falciparum isolates

BACKGROUND  Selection by host immunity and antimalarial drugs has driven extensive adaptive evolution in Plasmodium falciparum, and continues to produce ever-changing landscapes of genetic variation. METHODS  We carried out whole-genome sequencing of 69 P. falciparum isolates from Malawi and used population genetics approaches to investigate genetic diversity and population structure, and identify loci under selection. RESULTS  High genetic diversity (π=2.4 x 10(-4)), moderately high multiplicity of infection (2.7), and low linkage disequilibrium (500-bp) were observed in Chikhwawa District, Malawi, an area of high malaria transmission. Allele frequency-based tests provided evidence of recent population growth in Malawi and detected potential targets of host immunity and candidate vaccine antigens. Comparing the sequence variation between isolates from Malawi and those from 5 geographically dispersed countries (Kenya, Burkina Faso, Mali, Cambodia, and Thailand) detected population genetic differences between Africa and Asia, within Southeast Asia, and within Africa. Applying haplotype-based tests of selection to sequence data from all 6 populations identified signals of directional selection at known drug-resistance loci, including pfcrt, pfdhps, pfmdr1, and pfgch1. CONCLUSIONS  The sequence variations observed at drug-resistance loci reflect differences in each country's historical use of antimalarial drugs, and may be useful in formulating local malaria treatment guidelines

Imputation-based meta-analysis of severe malaria in three African populations.

Combining data from genome-wide association studies (GWAS) conducted at different locations, using genotype imputation and fixed-effects meta-analysis, has been a powerful approach for dissecting complex disease genetics in populations of European ancestry. Here we investigate the feasibility of applying the same approach in Africa, where genetic diversity, both within and between populations, is far more extensive. We analyse genome-wide data from approximately 5,000 individuals with severe malaria and 7,000 population controls from three different locations in Africa. Our results show that the standard approach is well powered to detect known malaria susceptibility loci when sample sizes are large, and that modern methods for association analysis can control the potential confounding effects of population structure. We show that pattern of association around the haemoglobin S allele differs substantially across populations due to differences in haplotype structure. Motivated by these observations we consider new approaches to association analysis that might prove valuable for multicentre GWAS in Africa: we relax the assumptions of SNP-based fixed effect analysis; we apply Bayesian approaches to allow for heterogeneity in the effect of an allele on risk across studies; and we introduce a region-based test to allow for heterogeneity in the location of causal alleles

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome