Proteins in Tumor-Derived Plasma Extracellular Vesicles Indicate Tumor Origin.

Cancer-derived extracellular vesicles (EVs) promote tumorigenesis, premetastatic niche formation, and metastasis via their protein cargo. However, the proteins packaged by patient tumors into EVs cannot be determined in vivo because of the presence of EVs derived from other tissues. We therefore developed a cross-species proteomic method to quantify the human tumor-derived proteome of plasma EVs produced by patient-derived xenografts of four cancer types. Proteomic profiling revealed individualized packaging of novel protein cargo, and machine learning accurately classified the type of the underlying tumor.

Investigating predictive coding in younger and older children using MEG and a multi-feature auditory oddball paradigm.

There is mounting evidence for predictive coding theory from computational, neuroimaging, and psychological research. However, there remains a lack of research exploring how predictive brain function develops across childhood. To address this gap, we used pediatric magnetoencephalography to record the evoked magnetic fields of 18 younger children (M = 4.1 years) and 19 older children (M = 6.2 years) as they listened to a 12-min auditory oddball paradigm. For each child, we computed a mismatch field "MMF": an electrophysiological component that is widely interpreted as a neural signature of predictive coding. At the sensor level, the older children showed significantly larger MMF amplitudes relative to the younger children. At the source level, the older children showed a significantly larger MMF amplitude in the right inferior frontal gyrus relative to the younger children, P < 0.05. No differences were found in 2 other key regions (right primary auditory cortex and right superior temporal gyrus) thought to be involved in mismatch generation. These findings support the idea that predictive brain function develops during childhood, with increasing involvement of the frontal cortex in response to prediction errors. These findings contribute to a deeper understanding of the brain function underpinning child cognitive development.

Real-time, model-based magnetic field correction for moving, wearable MEG.

Most neuroimaging techniques require the participant to remain still for reliable recordings to be made. Optically pumped magnetometer (OPM) based magnetoencephalography (OP-MEG) however, is a neuroimaging technique which can be used to measure neural signals during large participant movement (approximately 1 m) within a magnetically shielded room (MSR) (Boto et al., 2018; Seymour et al., 2021). Nevertheless, environmental magnetic fields vary both spatially and temporally and OPMs can only operate within a limited magnetic field range, which constrains participant movement. Here we implement real-time updates to electromagnetic coils mounted on-board of the OPMs, to cancel out the changing background magnetic fields. The coil currents were chosen based on a continually updating harmonic model of the background magnetic field, effectively implementing homogeneous field correction (HFC) in real-time (Tierney et al., 2021). During a stationary, empty room recording, we show an improvement in very low frequency noise of 24 dB. In an auditory paradigm, during participant movement of up to 2 m within a magnetically shielded room, introduction of the real-time correction more than doubled the proportion of trials in which no sensor saturated recorded outside of a 50 cm radius from the optimally-shielded centre of the room. The main advantage of such model-based (rather than direct) feedback is that it could allow one to correct field components along unmeasured OPM axes, potentially mitigating sensor gain and calibration issues (Borna et al., 2022).

Interference suppression techniques for OPM-based MEG: Opportunities and challenges.

One of the primary technical challenges facing magnetoencephalography (MEG) is that the magnitude of neuromagnetic fields is several orders of magnitude lower than interfering signals. Recently, a new type of sensor has been developed - the optically pumped magnetometer (OPM). These sensors can be placed directly on the scalp and move with the head during participant movement, making them wearable. This opens up a range of exciting experimental and clinical opportunities for OPM-based MEG experiments, including paediatric studies, and the incorporation of naturalistic movements into neuroimaging paradigms. However, OPMs face some unique challenges in terms of interference suppression, especially in situations involving mobile participants, and when OPMs are integrated with electrical equipment required for naturalistic paradigms, such as motion capture systems. Here we briefly review various hardware solutions for OPM interference suppression. We then outline several signal processing strategies aimed at increasing the signal from neuromagnetic sources. These include regression-based strategies, temporal filtering and spatial filtering approaches. The focus is on the practical application of these signal processing algorithms to OPM data. In a similar vein, we include two worked-through experiments using OPM data collected from a whole-head sensor array. These tutorial-style examples illustrate how the steps for suppressing external interference can be implemented, including the associated data and code so that researchers can try the pipelines for themselves. With the popularity of OPM-based MEG rising, there will be an increasing need to deal with interference suppression. We hope this practical paper provides a resource for OPM-based MEG researchers to build upon.

Robust estimation of 1/f activity improves oscillatory burst detection.

Neural oscillations often occur as transient bursts with variable amplitude and frequency dynamics. Quantifying these effects is important for understanding brain-behaviour relationships, especially in continuous datasets. To robustly measure bursts, rhythmical periods of oscillatory activity must be separated from arrhythmical background 1/f activity, which is ubiquitous in electrophysiological recordings. The Better OSCillation (BOSC) framework achieves this by defining a power threshold above the estimated background 1/f activity, combined with a duration threshold. Here we introduce a modification to this approach called fBOSC, which uses a spectral parametrisation tool to accurately model background 1/f activity in neural data. fBOSC (which is openly available as a MATLAB toolbox) is robust to power spectra with oscillatory peaks and can also model non-linear spectra. Through a series of simulations, we show that fBOSC more accurately models the 1/f power spectrum compared with existing methods. fBOSC was especially beneficial where power spectra contained a 'knee' below ~.5-10 Hz, which is typical in neural data. We also found that, unlike other methods, fBOSC was unaffected by oscillatory peaks in the neural power spectrum. Moreover, by robustly modelling background 1/f activity, the sensitivity for detecting oscillatory bursts was standardised across frequencies (e.g., theta- and alpha-bands). Finally, using openly available resting state magnetoencephalography and intracranial electrophysiology datasets, we demonstrate the application of fBOSC for oscillatory burst detection in the theta-band. These simulations and empirical analyses highlight the value of fBOSC in detecting oscillatory bursts, including in datasets that are long and continuous with no distinct experimental trials.

"Submergence" of Western equine encephalitis virus: Evidence of positive selection argues against genetic drift and fitness reductions.

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.

ProteoSushi: A Software Tool to Biologically Annotate and Quantify Modification-Specific, Peptide-Centric Proteomics Data Sets.

Large-scale proteomic profiling of protein post-translational modifications has provided important insights into the regulation of cell signaling and disease. These modification-specific proteomics workflows nearly universally enrich modified peptides prior to mass spectrometry analysis, but protein-centric proteomic software tools have many limitations evaluating and interpreting these peptide-centric data sets. We, therefore, developed ProteoSushi, a software tool tailored to analysis of each modified site in peptide-centric proteomic data sets that is compatible with any post-translational modification or chemical label. ProteoSushi uses a unique approach to assign identified peptides to shared proteins and genes, minimizing redundancy by prioritizing shared assignments based on UniProt annotation score and optional user-supplied protein/gene lists. ProteoSushi simplifies quantitation by summing or averaging intensities for each modified site, merging overlapping peptide charge states, missed cleavages, spectral matches, and variable modifications into a single value. ProteoSushi also annotates each PTM site with the most up-to-date biological information available from UniProt, such as functional roles or known modifications, the protein domain in which the site resides, the protein's subcellular location and function, and more. ProteoSushi has a graphical user interface for ease of use. ProteoSushi's flexibility and combination of analysis features streamlines peptide-centric data processing and knowledge mining of large modification-specific proteomics data sets.

Using OPMs to measure neural activity in standing, mobile participants.

Optically pumped magnetometer-based magnetoencephalography (OP-MEG) can be used to measure neuromagnetic fields while participants move in a magnetically shielded room. Head movements in previous OP-MEG studies have been up to 20 cm translation and ∼30° rotation in a sitting position. While this represents a step-change over stationary MEG systems, naturalistic head movement is likely to exceed these limits, particularly when participants are standing up. In this proof-of-concept study, we sought to push the movement limits of OP-MEG even further. Using a 90 channel (45-sensor) whole-head OP-MEG system and concurrent motion capture, we recorded auditory evoked fields while participants were: (i) sitting still, (ii) standing up and still, and (iii) standing up and making large natural head movements continuously throughout the recording - maximum translation 120 cm, maximum rotation 198°. Following pre-processing, movement artefacts were substantially reduced but not eliminated. However, upon utilisation of a beamformer, the M100 event-related field localised to primary auditory regions. Furthermore, the event-related fields from auditory cortex were remarkably consistent across the three conditions. These results suggest that a wide range of movement is possible with current OP-MEG systems. This in turn underscores the exciting potential of OP-MEG for recording neural activity during naturalistic paradigms that involve movement (e.g. navigation), and for scanning populations who are difficult to study with stationary MEG (e.g. young children).

Modelling optically pumped magnetometer interference in MEG as a spatially homogeneous magnetic field.

Here we propose that much of the magnetic interference observed when using optically pumped magnetometers for MEG experiments can be modeled as a spatially homogeneous magnetic field. We show that this approximation reduces sensor level variance and substantially improves statistical power. This model does not require knowledge of the underlying neuroanatomy nor the sensor positions. It only needs information about the sensor orientation. Due to the model's low rank there is little risk of removing substantial neural signal. However, we provide a framework to assess this risk for any sensor number, design or subject neuroanatomy. We find that the risk of unintentionally removing neural signal is reduced when multi-axis recordings are performed. We validated the method using a binaural auditory evoked response paradigm and demonstrated that removing the homogeneous magnetic field increases sensor level SNR by a factor of 3. Considering the model's simplicity and efficacy, we suggest that this homogeneous field correction can be a powerful preprocessing step for arrays of optically pumped magnetometers.

Oral administration of dermatan sulphate reduces venous thrombus formation in vivo: potential use as a formulation for venous thromboembolism.

Dermatan sulphate (DS) is a sulphated polysaccharide that displays complexity in constituent sulphated disaccharides and interacts with proteins and signalling molecules to modulate numerous biological processes, including inhibition of the coagulation cascade and regulation of blood clotting and fibrinolysis. This study shows the antithrombotic and anticoagulant effects of DS prepared from bovine collagen waste liquor following oral and intravenous administrations in a deep vein thrombosis (DVT) rabbit model. In vitro, the prothrombin time, activated partial thromboplastin time, and thrombin citrated plasma clotting assays revealed that bovine DS had strong antithrombotic and anticoagulant effects comparable to low-molecular-weight heparin [Clexane® (enoxaparin sodium)]. In a DVT rabbit model, animals received intravenous and oral administrations of bovine DS and Clexane® providing further evidence that both agents had strong antithrombotic and anticoagulant effects by significantly reducing or preventing clot formation. Thromboelastography (TEG) assays revealed further that both bovine DS and Clexane® substantially prolonged the clotting time of recalcified citrated whole blood, but only bovine DS could retain clot strength suggesting that bovine DS had less effect on platelet-fibrin interactions. In conclusion, this is the first report that oral administration of DS from bovine collagen waste liquor reduces experimental venous thrombus formation warranting further research into bovine DS as an oral antithrombotic therapeutic.

Neuromagnetic activation and oscillatory dynamics of stimulus-locked processing during naturalistic viewing.

Naturalistic stimuli such as watching a movie while in the scanner provide an ecologically valid paradigm that has the potential of extracting valuable information on how the brain processes complex stimuli in realistic visual and auditory contexts. Naturalistic viewing is also easier to conduct with challenging participant groups including patients and children. Given the high temporal resolution of MEG, in the present study, we demonstrate how a short movie clip can be used to map distinguishable activation and connectivity dynamics underlying the processing of specific classes of visual stimuli such as face and hand manipulations, as well as contrasting activation dynamics for auditory words and non-words. MEG data were collected from 22 healthy volunteers (6 females, 3 left handed, mean age - 27.7 â€‹± â€‹5.28 years) during the presentation of naturalistic audiovisual stimuli. The MEG data were split into trials with the onset of the stimuli belonging to classes of interest (words, non-words, faces, hand manipulations). Based on the components of the averaged sensor ERFs time-locked to the visual and auditory stimulus onset, four and three time-windows, respectively, were defined to explore brain activation dynamics. Pseudo-Z, defined as the ratio of the source-projected time-locked power to the projected noise power for each vertex, was computed and used as a proxy of time-locked brain activation. Statistical testing using the mean-centered Partial Least Squares analysis indicated periods where a given visual or auditory stimuli had higher activation. Based on peak pseudo-Z differences between the visual conditions, time-frequency resolved analyses were performed to assess beta band desynchronization in motor-related areas, and inter-trial phase synchronization between face processing areas. Our results provide the first evidence that activation and connectivity dynamics in canonical brain regions associated with the processing of particular classes of visual and auditory stimuli can be reliably mapped using MEG during presentation of naturalistic stimuli. Given the strength of MEG for brain mapping in temporal and frequency domains, the use of naturalistic stimuli may open new techniques in analyzing brain dynamics during ecologically valid sensation and perception.

Dysregulated oscillatory connectivity in the visual system in autism spectrum disorder.

Autism spectrum disorder is increasingly associated with atypical perceptual and sensory symptoms. Here we explore the hypothesis that aberrant sensory processing in autism spectrum disorder could be linked to atypical intra- (local) and interregional (global) brain connectivity. To elucidate oscillatory dynamics and connectivity in the visual domain we used magnetoencephalography and a simple visual grating paradigm with a group of 18 adolescent autistic participants and 18 typically developing control subjects. Both groups showed similar increases in gamma (40-80 Hz) and decreases in alpha (8-13 Hz) frequency power in occipital cortex. However, systematic group differences emerged when analysing intra- and interregional connectivity in detail. First, directed connectivity was estimated using non-parametric Granger causality between visual areas V1 and V4. Feedforward V1-to-V4 connectivity, mediated by gamma oscillations, was equivalent between autism spectrum disorder and control groups, but importantly, feedback V4-to-V1 connectivity, mediated by alpha (8-13 Hz) oscillations, was significantly reduced in the autism spectrum disorder group. This reduction was positively correlated with autistic quotient scores, consistent with an atypical visual hierarchy in autism, characterized by reduced top-down modulation of visual input via alpha-band oscillations. Second, at the local level in V1, coupling of alpha-phase to gamma amplitude (alpha-gamma phase amplitude coupling) was reduced in the autism spectrum disorder group. This implies dysregulated local visual processing, with gamma oscillations decoupled from patterns of wider alpha-band phase synchrony (i.e. reduced phase amplitude coupling), possibly due to an excitation-inhibition imbalance. More generally, these results are in agreement with predictive coding accounts of neurotypical perception and indicate that visual processes in autism are less modulated by contextual feedback information.

Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge.

Most alphaviruses are mosquito borne and exhibit a broad host range, infecting many different vertebrates, including birds, rodents, equids, humans, and nonhuman primates. Recently, a host-restricted, mosquito-borne alphavirus, Eilat virus (EILV), was described with an inability to infect vertebrate cells based on defective attachment and/or entry, as well as a lack of genomic RNA replication. We investigated the utilization of EILV recombinant technology as a vaccine platform against eastern (EEEV) and Venezuelan equine encephalitis viruses (VEEV), two important pathogens of humans and domesticated animals. EILV chimeras containing structural proteins of EEEV or VEEV were engineered and successfully rescued in Aedes albopictus cells. Cryo-electron microscopy reconstructions at 8 and 11 Å of EILV/VEEV and EILV/EEEV, respectively, showed virion and glycoprotein spike structures similar to those of VEEV-TC83 and other alphaviruses. The chimeras were unable to replicate in vertebrate cell lines or in brains of newborn mice when injected intracranially. Histopathologic examinations of the brain tissues showed no evidence of pathological lesions and were indistinguishable from those of mock-infected animals. A single-dose immunization of either monovalent or multivalent EILV chimera(s) generated neutralizing antibody responses and protected animals against lethal challenge 70 days later. Lastly, a single dose of monovalent EILV chimeras generated protective responses as early as day 1 postvaccination and partial or complete protection by day 6. These data demonstrate the safety, immunogenicity, and efficacy of novel insect-specific EILV-based chimeras as potential EEEV and VEEV vaccines.IMPORTANCE Mostly in the last decade, insect-specific viruses have been discovered in several arbovirus families. However, most of these viruses are not well studied and largely have been ignored. We explored the use of the mosquito-specific alphavirus EILV as an alphavirus vaccine platform in well-established disease models for eastern (EEE) and Venezuelan equine encephalitis (VEE). EILV-based chimeras replicated to high titers in a mosquito cell line yet retained their host range restriction in vertebrates both in vitro and in vivo In addition, the chimeras generated immune responses that were higher than those of other human and/or equine vaccines. These findings indicate the feasibility of producing a safe, efficacious, mono- or multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV. Lastly, these data demonstrate how host-restricted, insect-specific viruses can be engineered to develop vaccines against related pathogenic arboviruses that cause severe disease in humans and domesticated animals.

Recombinant Isfahan Virus and Vesicular Stomatitis Virus Vaccine Vectors Provide Durable, Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge.

The demonstrated clinical efficacy of a recombinant vesicular stomatitis virus (rVSV) vaccine vector has stimulated the investigation of additional serologically distinct Vesiculovirus vectors as therapeutic and/or prophylactic vaccine vectors to combat emerging viral diseases. Among these viral threats are the encephalitic alphaviruses Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV), which have demonstrated potential for natural disease outbreaks, yet no licensed vaccines are available in the event of an epidemic. Here we report the rescue of recombinant Isfahan virus (rISFV) from genomic cDNA as a potential new vaccine vector platform. The rISFV genome was modified to attenuate virulence and express the VEEV and EEEV E2/E1 surface glycoproteins as vaccine antigens. A single dose of the rISFV vaccine vectors elicited neutralizing antibody responses and protected mice from lethal VEEV and EEEV challenges at 1 month postvaccination as well as lethal VEEV challenge at 8 months postvaccination. A mixture of rISFV vectors expressing the VEEV and EEEV E2/E1 glycoproteins also provided durable, single-dose protection from lethal VEEV and EEEV challenges, demonstrating the potential for a multivalent vaccine formulation. These findings were paralleled in studies with an attenuated form of rVSV expressing the VEEV E2/E1 glycoproteins. Both the rVSV and rISFV vectors were attenuated by using an approach that has demonstrated safety in human trials of an rVSV/HIV-1 vaccine. Vaccines based on either of these vaccine vector platforms may present a safe and effective approach to prevent alphavirus-induced disease in humans.IMPORTANCE This work introduces rISFV as a novel vaccine vector platform that is serologically distinct and phylogenetically distant from VSV. The rISFV vector has been attenuated by an approach used for an rVSV vector that has demonstrated safety in clinical studies. The vaccine potential of the rISFV vector was investigated in a well-established alphavirus disease model. The findings indicate the feasibility of producing a safe, efficacious, multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV, both of which can cause fatal disease. This work also demonstrates the efficacy of an attenuated rVSV vector that has already demonstrated safety and immunogenicity in multiple HIV-1 phase I clinical studies. The absence of serological cross-reactivity between rVSV and rISFV and their phylogenetic divergence within the Vesiculovirus genus indicate potential for two stand-alone vaccine vector platforms that could be used to target multiple bacterial and/or viral agents in successive immunization campaigns or as heterologous prime-boost agents.

Down but not out in posterior cingulate cortex: Deactivation yet functional coupling with prefrontal cortex during demanding semantic cognition.

The posterior cingulate cortex (pCC) often deactivates during complex tasks, and at rest is often only weakly correlated with regions that play a general role in the control of cognition. These observations led to the hypothesis that pCC contributes to automatic aspects of memory retrieval and cognition. Recent work, however, has suggested that the pCC may support both automatic and controlled forms of memory processing and may do so by changing its communication with regions that are important in the control of cognition across multiple domains. The current study examined these alternative views by characterising the functional coupling of the pCC in easy semantic decisions (based on strong global associations) and in harder semantic tasks (matching words on the basis of specific non-dominant features). Increasingly difficult semantic decisions led to the expected pattern of deactivation in the pCC; however, psychophysiological interaction analysis revealed that, under these conditions, the pCC exhibited greater connectivity with dorsolateral prefrontal cortex (PFC), relative to both easier semantic decisions and to a period of rest. In a second experiment using different participants, we found that functional coupling at rest between the pCC and the same region of dorsolateral PFC was stronger for participants who were more efficient at semantic tasks when assessed in a subsequent laboratory session. Thus, although overall levels of activity in the pCC are reduced during external tasks, this region may show greater coupling with executive control regions when information is retrieved from memory in a goal-directed manner.

Chikungunya vaccine candidate is highly attenuated and protects nonhuman primates against telemetrically monitored disease following a single dose.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes major epidemics of rash, fever, and debilitating arthritis. Currently, there are no vaccines or antivirals available for prevention or treatment. We therefore generated 2 live-attenuated vaccine candidates based on the insertion of a picornavirus internal ribosome entry site (IRES) sequence into the genome of CHIKV. Vaccination of cynomolgus macaques with a single dose of either vaccine produced no signs of disease but was highly immunogenic. After challenge with a subcutaneous inoculation of wild-type CHIKV, both vaccine candidates prevented the development of detectable viremia. Protected animals also exhibited no significant changes in core body temperature or cardiovascular rhythm, whereas sham-vaccinated animals showed hyperthermia, followed by sustained hypothermia, as well as significant changes in heart rate. These CHIKV/IRES vaccine candidates appear to be safe and efficacious, supporting their strong potential as human vaccines to protect against CHIKV infection and reduce transmission and further spread.

A chimeric vesiculo/alphavirus is an effective alphavirus vaccine.

While a large number of mosquito-transmitted alphaviruses are known to cause serious human diseases, there are no licensed vaccines that protect against alphavirus infections. The alphavirus chikungunya virus (CHIKV) has caused multiple recent outbreaks of chikungunya fever. This virus has the potential to cause a worldwide epidemic and has generated strong interest in development of a prophylactic CHIKV vaccine. We report here on the development of a potent experimental vaccine for CHIKV based on a chimeric vesicular stomatitis virus (VSV) expressing the entire CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G). These VSVΔG-CHIKV chimeras incorporated functional CHIKV glycoproteins into the viral envelope in place of VSV G. The chimeric viruses were attenuated for growth in tissue culture but could be propagated to high titers without VSV G complementation. They also generated robust neutralizing antibody and cellular immune responses to CHIKV in mice after a single dose and protected mice against CHIKV infection. VSVΔG-alphavirus chimeras could have general applicability as alphavirus vaccines.

Vector-borne transmission imposes a severe bottleneck on an RNA virus population.

RNA viruses typically occur in genetically diverse populations due to their error-prone genome replication. Genetic diversity is thought to be important in allowing RNA viruses to explore sequence space, facilitating adaptation to changing environments and hosts. Some arboviruses that infect both a mosquito vector and a mammalian host are known to experience population bottlenecks in their vectors, which may constrain their genetic diversity and could potentially lead to extinction events via Muller's ratchet. To examine this potential challenge of bottlenecks for arbovirus perpetuation, we studied Venezuelan equine encephalitis virus (VEEV) enzootic subtype IE and its natural vector Culex (Melanoconion) taeniopus, as an example of a virus-vector interaction with a long evolutionary history. Using a mixture of marked VEEV clones to infect C. taeniopus and real-time RT-PCR to track these clones during mosquito infection and dissemination, we observed severe bottleneck events that resulted in a significant drop in the number of clones present. At higher initial doses, the midgut was readily infected and there was a severe bottleneck at the midgut escape. Following a lower initial dose, the major bottleneck occurred at initial midgut infection. A second, less severe bottleneck was identified at the salivary gland infection stage following intrathoracic inoculation. Our results suggest that VEEV consistently encounters bottlenecks during infection, dissemination and transmission by its natural enzootic vector. The potential impacts of these bottlenecks on viral fitness and transmission, and the viral mechanisms that prevent genetic drift leading to extinction, deserve further study.

Under-dominance constrains the evolution of negative autoregulation in diploids.

Regulatory networks have evolved to allow gene expression to rapidly track changes in the environment as well as to buffer perturbations and maintain cellular homeostasis in the absence of change. Theoretical work and empirical investigation in Escherichia coli have shown that negative autoregulation confers both rapid response times and reduced intrinsic noise, which is reflected in the fact that almost half of Escherichia coli transcription factors are negatively autoregulated. However, negative autoregulation is rare amongst the transcription factors of Saccharomyces cerevisiae. This difference is surprising because E. coli and S. cerevisiae otherwise have similar profiles of network motifs. In this study we investigate regulatory interactions amongst the transcription factors of Drosophila melanogaster and humans, and show that they have a similar dearth of negative autoregulation to that seen in S. cerevisiae. We then present a model demonstrating that this striking difference in the noise reduction strategies used amongst species can be explained by constraints on the evolution of negative autoregulation in diploids. We show that regulatory interactions between pairs of homologous genes within the same cell can lead to under-dominance--mutations which result in stronger autoregulation, and decrease noise in homozygotes, paradoxically can cause increased noise in heterozygotes. This severely limits a diploid's ability to evolve negative autoregulation as a noise reduction mechanism. Our work offers a simple and general explanation for a previously unexplained difference between the regulatory architectures of E. coli and yeast, Drosophila and humans. It also demonstrates that the effects of diploidy in gene networks can have counter-intuitive consequences that may profoundly influence the course of evolution.

Warning signals of regime shifts as intrinsic properties of endogenous dynamics.

Ecosystem dynamics can exhibit large, nonlinear changes after small changes in an environmental parameter that passes a critical threshold. These regime shifts are often associated with loss of biodiversity and ecosystem services. Because critical thresholds for regime shifts are hard to determine with precision, some recent studies have focused on deriving signals from dynamics leading up to the thresholds. Models in these studies depend on using noise terms independent of system parameters and variables to add stochasticity. However, demographic stochasticity, an important source of random variability, arises directly from system dynamics. In this study, a framework is developed for modeling demographic stochasticity in a mechanistic way, incorporating system variables and parameters. This framework is applied to a deterministic, dynamic model of a coral reef benthos. The resulting stochastic model indicates that increasing variance-but not skewness-is consistently found in system dynamics approaching a critical threshold of grazing pressure. Even if the threshold is breached, attraction of transient dynamics by a saddle point provides an opportunity for regime shift reversal by management intervention. These results suggest that early warning signals of regime shifts can arise intrinsically in endogenous dynamics and can be detected without reliance on random environmental forcings.