Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.

Ribonucleic acid sequencing can allow us to monitor the RNAs present in a sample. This enables us to detect the presence and nucleotide sequence of viruses, or to build a picture of how active transcriptional processes are changing – information that is useful for understanding the status and function of a sample. Oxford Nanopore Technologies’ sequencing technology is capable of electronically analysing a sample’s DNA directly, and in real-time. In this manuscript we demonstrate the ability of an array of nanopores to sequence RNA directly, and we apply it to a range of biological situations. Nanopore technology is the only available sequencing technology that can sequence RNA directly, rather than depending on reverse transcription and PCR. There are several potential advantages of this approach over other RNA-seq strategies, including the absence of amplification and reverse transcription biases, the ability to detect nucleotide analogues and the ability to generate full-length, strand-specific RNA sequences. Direct RNA sequencing is a completely new way of analysing the sequence of RNA samples and it will improve the ease and speed of RNA analysis, while yielding richer biological information.

There is profound interest in knowing the degree to which China's institutions are capable of protecting its natural forests and biodiversity in the face of economic and political change. China's 2 most important forest-protection policies are its National Forest Protection Program (NFPP) and its nationallevel nature reserves (NNRs

Development times of eggs, larvae and pupae of vectors of onchocerciasis (Simulium spp.) and of Onchocerca volvulus larvae within the adult females of the vectors decrease with increasing temperature. At and above 25°C, the parasite could reach its infective stage in less than 7 days when vectors could transmit after only two gonotrophic cycles. After incorporating exponential functions for vector development into a novel blackfly population model, it was predicted that fly numbers in Liberia and Ghana would peak at air temperatures of 29°C and 34°C, about 3°C and 7°C above current monthly averages, respectively; parous rates of forest flies (Liberia) would peak at 29°C and of savannah flies (Ghana) at 30°C. Small temperature increases (less than 2°C) might lead to changes in geographical distributions of different vector taxa. When the new model was linked to an existing framework for the population dynamics of onchocerciasis in humans and vectors, transmission rates and worm loads were projected to increase with temperature to at least 33°C. By contrast, analyses of field data on forest flies in Liberia and savannah flies in Ghana, in relation to regional climate change predictions, suggested, on the basis of simple regressions, that 13–41% decreases in fly numbers would be expected between the present and before 2040. Further research is needed to reconcile these conflicting conclusions.

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BackgroundThe number of Anopheles arabiensis (Diptera: Culicidae) and Anopheles pharoensis caught by human and cattle baits was investigated experimentally in the Arba Minch district of southern Ethiopia to determine if attraction to humans, indoors or outdoors, was affected by the presence or absence of cattle.MethodsField studies were made of the effect of a surrounding ring (10 m radius) of 20 cattle on the numbers of mosquitoes collected by human-baited sampling methods (i) inside or (ii) outside a hut.ResultsThe numbers of An. arabiensis caught outdoors by a human landing catch (HLC) with or without a ring of cattle were not significantly different (2 × 2 Latin square comparisons: means = 24.8 and 37.2 mosquitoes/night, respectively; n = 12, P > 0.22, Tukey HSD), whereas, the numbers of An. pharoensis caught were significantly reduced (44%) by a ring of cattle (4.9 vs. 8.7; n = 12, P < 0.05). The catch of An. arabiensis in human-baited traps (HBT) was 25 times greater than in cattle-baited traps (CBT) (34.0 vs. 1.3, n = 24; P < 0.001) whereas, for An. pharoensis there was no significant difference. Furthermore, HBT and CBT catches were unaffected by a ring of cattle (4 × 4 Latin square comparison) for either An. arabiensis (n = 48; P > 0.999) or An. pharoensis (n = 48, P > 0.870). The HLC catches indoors vs. outdoors were not significantly different for either An. arabiensis or An. pharoensis (n = 12, P > 0.969), but for An. arabiensis only, the indoor catch was reduced significantly by 49% when the hut was surrounded by cattle (Tukey HSD, n = 12, P > 0.01).ConclusionsOutdoors, a preponderance of cattle (20:1, cattle:humans) does not provide any material zooprophylactic effect against biting by An. arabiensis. For a human indoors, the presence of cattle outdoors nearly halved the catch. Unfortunately, this level of reduction would not have an appreciable impact on malaria incidence in an area with typically > 1 infective bite/person/night. For An. pharoensis, cattle significantly reduced the human catch indoors and outdoors, but still only by about half. These results suggest that even for traditional pastoralist communities of East Africa, the presence of large numbers of cattle does not confer effective zooprophylaxis against malaria transmitted by An. arabiensis or An. pharoensis.

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We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into ruffles at the periphery, but not in the center of a mature cytolytic NK cell IS. Time-lapse imaging showed that the membrane ruffles formed at the initial point of contact between NK cells and target cells and then spread radialy across the intercellular contact as the size of the IS increased, becoming absent from the center of the mature synapse. Understanding the role of such extensive membrane ruffling in the assembly of cytolytic synapses is an intriguing new goal.