Characterizing Mosquito Biting Behavior Using the BiteOscope.

The biteOscope enables the high-resolution monitoring and video recording of blood-feeding mosquitoes. Mosquito biting is induced by combining host cues, an artificial bloodmeal, a membrane, and a transparent heater in a transparent behavioral arena. Machine vision techniques enable the tracking and pose estimation of individual mosquitoes to discern behavior and resolve individual feeding events. The workflow allows multiple replicates and large amounts of imaging data to be generated rapidly. These data are suitable for downstream analysis using machine learning tools for behavioral analysis, allowing subtle behavioral effects to be characterized.

Characterizing Mosquito Biting Behavior at High Resolution.

Blood feeding is a critical event in the life cycle of female mosquitoes. In addition to providing nutrients to the mosquito, blood feeding facilitates the transmission of parasites and viruses to hosts, potentially having devastating health consequences. Our understanding of these short, yet important, bouts of behavior is incomplete. How and where a mosquito decides to bite and the success of feeding can influence the transmission of pathogens. A more thorough understanding of these processes may allow the development of interventions that reduce or prevent infections. Here, we present an overview of strategies for studying mosquito biting behavior and introduce the biteOscope, which provides an opportunity to observe and understand this behavior at unprecedented spatial and temporal resolution under tightly controlled conditions. The biteOscope combines recent advances in computer vision and automated tracking with designs for behavioral arenas and controllable artificial host cues that use low-cost and readily available materials.

A minimal 3D model of mosquito flight behaviour around the human baited bed net.

BACKGROUND: Advances in digitized video-tracking and behavioural analysis have enabled accurate recording and quantification of mosquito flight and host-seeking behaviours, facilitating development of individual (agent) based models at much finer spatial scales than previously possible. METHODS: Quantified behavioural parameters were used to create a novel virtual testing model, capable of accurately simulating indoor flight behaviour by a virtual population of host-seeking mosquitoes as they interact with and respond to simulated stimuli from a human-occupied bed net. The model is described, including base mosquito behaviour, state transitions, environmental representation and host stimulus representation. RESULTS: In the absence of a bed net and human host bait, flight distribution of the model population was relatively uniform throughout the arena. Introducing an unbaited untreated bed net induced a change in distribution with an increase in landing events on the net surface, predominantly on the sides of the net. Adding the presence of a simulated human bait dramatically impacted flight distribution patterns, exploratory foraging and, the number and distribution of landing positions on the net, which were determined largely by the orientation of the human within. The model replicates experimental results with free-flying living mosquitoes at human-occupied bed nets, where contact occurs predominantly on the top surface of the net. This accuracy is important as it quantifies exposure to the lethal insecticide residues that may be unique to the net roof (or theoretically any other surface). Number of net contacts and height of contacts decreased with increasing attractant dispersal noise. CONCLUSIONS: Results generated by the model are an accurate representation of actual mosquito behaviour recorded at and around a human-occupied bed net in untreated and insecticide-treated nets. This fine-grained model is highly flexible and has significant potential for in silico screening of novel bed net designs, potentially reducing time and cost and accelerating the deployment of new and more effective tools for protecting against malaria in sub-Saharan Africa.

Diffuse retro-reflective imaging for improved video tracking of mosquitoes at human baited bednets.

Robust imaging techniques for tracking insects have been essential tools in numerous laboratory and field studies on pests, beneficial insects and model systems. Recent innovations in optical imaging systems and associated signal processing have enabled detailed characterization of nocturnal mosquito behaviour around bednets and improvements in bednet design, a global essential for protecting populations against malaria. Nonetheless, there remain challenges around ease of use for large-scale in situ recordings and extracting data reliably in the critical areas of the bednet where the optical signal is attenuated. Here, we introduce a retro-reflective screen at the back of the measurement volume, which can simultaneously provide diffuse illumination, and remove optical alignment issues while requiring only one-sided access to the measurement space. The illumination becomes significantly more uniform, although noise removal algorithms are needed to reduce the effects of shot noise, particularly across low-intensity bednet regions. By systematically introducing mosquitoes in front of and behind the bednet in laboratory experiments, we are able to demonstrate robust tracking in these challenging areas. Overall, the retro-reflective imaging set-up delivers mosquito segmentation rates in excess of 90% compared to less than 70% with backlit systems.

Barrier bednets target malaria vectors and expand the range of usable insecticides.

Transmission of Plasmodium falciparum malaria parasites occurs when nocturnal Anopheles mosquito vectors feed on human blood. In Africa, where malaria burden is highest, bednets treated with pyrethroid insecticide were highly effective in preventing mosquito bites and reducing transmission, and essential to achieving unprecedented reductions in malaria until 2015 (ref. 1). Since then, progress has stalled2, and with insecticidal bednets losing efficacy against pyrethroid-resistant Anopheles vectors3,4, methods that restore performance are urgently needed to eliminate any risk of malaria returning to the levels seen before their widespread use throughout sub-Saharan Africa5. Here, we show that the primary malaria vector Anopheles gambiae is targeted and killed by small insecticidal net barriers positioned above a standard bednet in a spatial region of high mosquito activity but zero contact with sleepers, opening the way for deploying many more insecticides on bednets than is currently possible. Tested against wild pyrethroid-resistant A. gambiae in Burkina Faso, pyrethroid bednets with organophosphate barriers achieved significantly higher killing rates than bednets alone. Treated barriers on untreated bednets were equally effective, without significant loss of personal protection. Mathematical modelling of transmission dynamics predicted reductions in clinical malaria incidence with barrier bednets that matched those of 'next-generation' nets recommended by the World Health Organization against resistant vectors. Mathematical models of mosquito-barrier interactions identified alternative barrier designs to increase performance. Barrier bednets that overcome insecticide resistance are feasible using existing insecticides and production technology, and early implementation of affordable vector control tools is a realistic prospect.

Molecular Evidence of Genome Editing in a Mouse Model of Immunodeficiency.

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors.

Small-molecule-regulated gene expression offers the promise of titrating the dose and duration of action of DNA-based therapies. To this end, we show that engineered zinc-finger protein transcription factors (ZFP TFs) can be coupled with a drug-inducible regulatory domain to permit small-molecule control of endogenous gene transcription. We constructed a drug-responsive ZFP TF via the fusion of a ZFP DNA-binding domain (DBD) targeting the human VEGF-A gene and an effector domain containing a truncated progesterone receptor ligand-binding domain linked to the NFkappaB p65 activation domain. Introduction of this engineered ZFP TF into human or murine cells allowed expression of the chromosomal VEGF-A gene to be induced upon addition of mifepristone, a synthetic steroid analog. Mifepristone-dependent VEGF-A induction was rapid, dose-dependent and reversible. Moreover, stable lines expressing the drug-responsive ZFP TF could be maintained in a state of continuous induction for at least 30 days without loss of viability. Potent VEGF-A induction was demonstrated using different engineered ZFP DBDs, thus this approach may represent a general solution to small-molecule regulation of targeted endogenous genes.

Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.

Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.

Targeted regulation of imprinted genes by synthetic zinc-finger transcription factors.

Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2-H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for 'transcription therapy' of human disease.

A transient histone hyperacetylation signal marks nucleosomes for remodeling at the PHO8 promoter in vivo.

Chromatin remodeling of the yeast PHO8 promoter requires the SAGA histone acetyltransferase complex. We report here that SAGA is necessary and sufficient to establish an activator-dependent hyperacetylation peak over the PHO8 promoter that is restricted to those nucleosomes that are remodeled upon activation. This local hyperacetylated state is observed upon activation in the absence of the SWI/SNF complex when the remodeling process is frozen subsequent to activator binding. Hyperacetylation is lost, however, if remodeling is permitted to go to completion. Thus, a transient histone hyperacetylation signal is shown to be a prerequisite for, and determinant of, the domain of nucleosome remodeling in vivo.

Histone acetylation and chromatin remodeling.

Chromatin represents a repressive barrier to the process of transcription. This molecular obstacle is a highly dynamic structure, able to compact the DNA of the entire genome into the confines of a nucleus, and yet it allows access to the genetic information held within. The acetylation of histones has emerged as a regulatory mechanism capable of modulating the properties of chromatin and thus the competence of the DNA template for transcriptional activation. The role of acetylation in chromatin remodeling is therefore of paramount importance to our understanding of gene regulation in vivo.

Transcription and chromatin converge: lessons from yeast genetics.

The control of transcription through the modification of chromatin has been a subject of intense study over the past year. The increasing use of genome-wide approaches to examine the role of chromatin and the complexes able to modify it is providing a global perspective that is profoundly altering our view of the transcription process.

Chromatin remodelling at the PHO8 promoter requires SWI-SNF and SAGA at a step subsequent to activator binding.

The SWI-SNF and SAGA complexes possess ATP-dependent nucleosome remodelling activity and histone acetyltransferase (HAT) activity, respectively. Mutations that eliminate the ATPase activity of the SWI-SNF complex, or the HAT activity of SAGA, abolish proper chromatin remodelling at the PHO8 promoter in vivo. These effects are mechanistically distinct, since the absence of SWI-SNF freezes chromatin in the repressed state, while the absence of Gcn5 permits a localized perturbation of chromatin structure immediately adjacent to the upstream transactivator binding site. However, this remodelling is not propagated to the proximal promoter, and no activation is observed under all conditions. Furthermore, Pho4 is bound to the PHO8 promoter in the absence of Snf2 or Gcn5, confirming a role for SWI-SNF and SAGA in chromatin remodelling independent of activator binding. These data provide new insights into the roles of the SWI-SNF and SAGA complexes in chromatin remodelling in vivo.

Analyzing chromatin structure and transcription factor binding in yeast.

The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatin in vivo. The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cells in vivo to determine the binding of factors to cis elements in the locus of interest. Specific examples illustrating the techniques described are given from our work on the regulation of the yeast PHO8 gene, but have also been successfully and reliably applied to the study of many other yeast loci. These procedures make it possible to correlate the binding of a transactivator with an altered or perturbed chromatin organization at a specific locus.

Absence of Gcn5 HAT activity defines a novel state in the opening of chromatin at the PHO5 promoter in yeast.

Histone acetyltransferase (HAT) activity has been demonstrated for several transcriptional activators, formally connecting chromatin modification with gene regulation. However, no effect on chromatin has been demonstrated. We have investigated the role of the HAT Gcn5 at the nucleosomally regulated PHO5 promoter. Under conditions of constitutive submaximal activation (i.e., in the absence of the negative regulator Pho80), deletion of Gcn5 determines a novel randomized nucleosomal organization across the promoter and leads to a dramatic reduction in activity. Furthermore, mutation of amino acids critical for Gcn5 HAT activity is sufficient to generate this structure. This intermediate state in chromatin opening gives way to the fully open structure upon maximal induction (phosphate starvation), even in the absence of Gcn5. Thus, Gcn5 is shown to affect directly the remodeling of chromatin in vivo.

Life with nucleosomes: chromatin remodelling in gene regulation.

In the past year, the role of chromatin has emerged at the forefront of transcription research. Discovery and characterisation of the chromatin modifying machinery have significantly advanced our understanding of the molecular activities that establish a transcriptionally competent substrate in vivo, and have underscored the importance of the part played by chromatin in the regulation of transcription.

Chromatin and transcription--how transcription factors battle with a repressive chromatin environment.

The last year has seen much progress in our understanding of chromatin and transcription. Transcriptionally active chromatin has long been correlated with a higher level of histone acetlyation. The discovery of a nuclear histone acetyltransferase activity encoded by factors with a role in transcription raises the possibility that the cell is able to dynamically modulate the (local) level of histone acteylation, switching chromatin templates from inactive to transcriptionally active states. Furthermore, histone acetylation states have shown to play a role in determining the efficacy of transcriptionally silenced chromatin in both yeast and Drosophila. The advances in our knowledge regarding the role of the origin-recognition complex in the establishment of silencing, and the requirement for a locally concentrated zone of the silence information regulator proteins in the nucleus has provided insights into the complex architecture of silenced chromatin. The goal of understanding the mechanisms by which the cell is able to 'open' repressive chromatin structures has prompted the discovery of multiple chromatin remodelling activities. These large protein complexes identified from organisms as diverse as yeast, mouse, fly and man demonstrate the ubiquity and fundamental importance of the ability to perturb the structure of chromatin allowing transcription of the desired genes. These data provide the latest and potentially most significant demonstration of the importance of the nucleosome in the regulation of transcription.

Studies of the repressor (BlaI) of beta-lactamase synthesis in Staphylococcus aureus.

Purified BlaI, the putative repressor of the beta-lactamase operon in Staphylococcus aureus, binds specifically to two regions of dyad symmetry (operators) located in the blaZ-blaR1 intergenic region. BlaI binds with similar affinity to the two regions and to the related sequence upstream of the mec gene found in methicillin-resistant strains of S. aureus, providing physical evidence for the cross-talk previously observed between these systems. A change from a lysine in the N-terminus of BlaI to an alanine or deletion of the C-terminal 23 amino acids severely reduces its DNA-binding ability, demonstrating the functional importance of both the N- and C-termini. An operator DNA-protein complex observed with crude cell lysates from repressed cells, indistinguishable from that observed with purified BlaI, was eliminated by induction of the beta-lactamase operon. Furthermore, BlaI is proteolytically cleaved in response to the addition of inducer in a blaR1-dependent manner, providing primary evidence for the molecular basis of induction. Thus, BlaI is shown to be the repressor of the beta-lactamase system.