Translate text to:
In JoVE (1)
- Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy
Other Publications (26)
- Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics
- Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics
- Journal of Immunology (Baltimore, Md. : 1950)
- The American Journal of Pathology
- Physical Review Letters
- Nature Neuroscience
- Chaos (Woodbury, N.Y.)
- Physical Review Letters
- Ecological Applications : a Publication of the Ecological Society of America
- Biophysical Journal
- Journal of Biomedical Optics
- Journal of Environmental Management
- American Journal of Public Health
- Journal of Neuroscience Methods
- Methods in Molecular Biology (Clifton, N.J.)
- American Journal of Preventive Medicine
- PLoS Genetics
- Conservation Biology : the Journal of the Society for Conservation Biology
- PloS One
- PloS One
- Journal of Virology
- Regional Environmental Change
- Frontiers in Cellular Neuroscience
Articles by Ryan McAllister in JoVE
Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy
Parnika Kadam1,2, Ryan McAllister3, Jeffrey S. Urbach3, Kathryn Sandberg1,2, Susette C. Mueller4
1Department of Biochemistry, Georgetown University Medical Center, 2Department of Medicine, Georgetown University Medical Center, 3Department of Physics, Georgetown University Medical Center, 4Department of Oncology, Georgetown University Medical Center
Other articles by Ryan McAllister on PubMed
Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Aug, 2002 | Pubmed ID: 12241276
The complex dynamics resulting from electronic feedback of a laser's intensity are explored and characterized. Distinct stable and chaotic regimes can be elicited from the laser by tuning the bias of the feedback loop. An additional branch of the feedback loop, containing a derivative filter, provides access to new kinds of dynamics, including a more gradual transition to chaos. The whole feedback network together allows the laser dynamics to be selected from among a wide range of chaotic wave forms distinguished by statistical or spectral information. In other words, this laser system can be used as a tunable generator of chaotic functions.
Nature. Jan, 2003 | Pubmed ID: 12520296
Turbulent flows are highly intermittent--for example, they exhibit intense bursts of vorticity and strain. Kolmogorov theory describes such behaviour in the form of energy cascades from large to small spatial and temporal scales, where energy is dissipated as heat. But the causes of high intermittency in turbulence, which show non-gaussian statistics, are not well understood. Such intermittency can be important, for example, for enhancing the mixing of chemicals, by producing sharp drops in local pressure that can induce cavitation (damaging mechanical components and biological organisms), and by causing intense vortices in atmospheric flows. Here we present observations of the three components of velocity and all nine velocity gradients within a small volume, which allow us to determine simultaneously the dissipation (a measure of strain) and enstrophy (a measure of rotational energy) of a turbulent flow. Combining the statistics of all measurements and the evolution of individual bursts, we find that a typical sequence for intense events begins with rapid strain growth, followed by rising vorticity and a final sudden decline in stretching. We suggest two mechanisms which can produce these characteristics, depending whether they are due to the advection of coherent structures through our observed volume or caused locally.
Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Jan, 2003 | Pubmed ID: 12636548
Competition between two distinct driving frequencies to phase synchronize the intensity dynamics of a chaotic laser has been observed. The phase of the chaotic intensity signal is constructed using the complex analytic signal. Competing frequencies alternately show phase locking and phase slipping. Competition has been quantified by calculating the portion of time the laser phase locks to each of the driving frequencies and their average.
Aod1 Controlling Day 3 Thymectomy-induced Autoimmune Ovarian Dysgenesis in Mice Encompasses Two Linked Quantitative Trait Loci with Opposing Allelic Effects on Disease Susceptibility
Journal of Immunology (Baltimore, Md. : 1950). Jun, 2003 | Pubmed ID: 12794114
Day 3 thymectomy (D3Tx) leads to a paucity of CD4(+)CD25(+) suppressor T cells, a loss of peripheral tolerance, and the development of organ-specific autoimmune disease in adult mice. Importantly, D3Tx does not lead to autoimmune disease in all mouse strains, indicating that this process is genetically controlled. Previously, we reported linkage of D3Tx-induced autoimmune ovarian dysgenesis (AOD) and its intermediate phenotypes, antiovarian autoantibody responsiveness, oophoritis, and atrophy, to five quantitative trait loci (QTL), designated Aod1 through Aod5. We also showed interaction between these QTL and H2 as well as Gasa2, a QTL controlling susceptibility to D3Tx-induced autoimmune gastritis. To physically map Aod1, interval-specific bidirectional recombinant congenic strains of mice were generated and studied for susceptibility to D3Tx-induced AOD. Congenic mapping studies revealed that Aod1 controls susceptibility to oophoritis and comprises two linked QTL with opposing allelic effects. Aod1a resides between D16Mit211 (23.3 cM) and D16Mit51 (66.75 cM) on chromosome 16. Aod1b maps proximal of Aod1a between D16Mit89 (20.9 cM) and D16Mit211 (23.3 cM) and includes the candidate genes stefin A1, A2, and A3 (Stfa1-Stfa3), inhibitors of cathepsin S, a cysteine protease required for autoantigen presentation, and the development of autoimmune disease of the salivary and lacrimal glands following D3Tx. cDNA sequencing revealed the existence of structural polymorphisms for both Stfa1 and Stfa2. Given the roles of cathepsins in Ag processing and presentation, Stfa1 and Stfa2 alleles have the potential to control susceptibility to autoimmune disease at the level of both CD4(+)CD25(+) suppressor and CD4(+)CD25(-) effector T cells.
The American Journal of Pathology. Nov, 2003 | Pubmed ID: 14578173
Anthrax lethal toxin (LT) is the principal virulence factor associated with lethal pathologies following infection with Bacillus anthracis. Macrophages are the primary effector cells mediating lethality since macrophage-depleted mice are resistant to LT challenge. Recently, Ltxs1, the gene controlling differential susceptibility of murine macrophages to cytolysis following in vitro exposure to LT, was identified as Kif1c. To directly assess the in vivo role of Kif1c alleles in mortality, we studied a panel of interval-specific recombinant congenic lines carrying various segments of central chromosome 11 derived from LT-resistant DBA/2 mice on the LT-susceptible BALB/c background. The results of this study reveal that mortality is controlled by three linked quantitative trait loci (QTL): Ltxs1/Kif1c (42-43 cM), Ltxs2 (35-37 cM), and Ltxs3 (45-47 cM). The Ltxs3 interval encompasses Nos2, which is an attractive candidate gene for Ltxs3. In this regard, we demonstrate that selective, pharmacologically based inhibition of Nos2 activity in vivo partially overrides genetic resistance to LT and that Nos2 expression as determined by reverse transcription-polymerase chain reaction differs significantly between DBA/2 and BALB/c macrophages. Additionally, to recapitulate dominant resistance to mortality as seen in (BALB/c x DBA/2) F(1) hybrids, DBA/2 alleles are required at all three QTL.
Physical Review Letters. Oct, 2003 | Pubmed ID: 14611348
We demonstrate generalized synchronization of chaos in a two-mode laser system. The total intensity of the laser output (the sum of the individual mode intensities) is used as the drive signal. This lumped variable transmitted to the identical response system does not generate identical synchronization. Generalized synchronization is observed instead of identical synchronization because of the hidden internal degrees of freedom.
Nature Neuroscience. Jun, 2004 | Pubmed ID: 15162167
Axonal chemotaxis is believed to be important in wiring up the developing and regenerating nervous system, but little is known about how axons actually respond to molecular gradients. We report a new quantitative assay that allows the long-term response of axons to gradients of known and controllable shape to be examined in a three-dimensional gel. Using this assay, we show that axons may be nature's most-sensitive gradient detectors, but this sensitivity exists only within a narrow range of ligand concentrations. This assay should also be applicable to other biological processes that are controlled by molecular gradients, such as cell migration and morphogenesis.
Chaos (Woodbury, N.Y.). Dec, 2004 | Pubmed ID: 15568905
Physical Review Letters. Dec, 2004 | Pubmed ID: 15697817
The consistency of a nonlinear system's response to a repeated complex waveform drive signal is an important consideration in classical and quantum systems as diverse as lasers, neuronal networks, and manufacturing plants. We show from a consideration of different characteristic waveforms that there is typically an optimal drive amplitude for the most consistent response; internal noise sources dominate for small amplitude driving while deterministic system nonlinearity reduces consistency for large amplitudes. We test this general concept and its measurement experimentally and numerically on the specific example of a laser system.
Ecological Applications : a Publication of the Ecological Society of America. Apr, 2006 | Pubmed ID: 16711045
We explore the response of pastoralists to rangeland resource variation in time and space, focusing on regions where high variation makes it unlikely that an economically viable herd can be maintained on a single management unit. In such regions, the need to move stock to find forage in at least some years has led to the evolution of nomadism and transhumance, and reciprocal grazing agreements among the holders of common-property rangeland. The role of such informal institutions in buffering resource variation is well documented in some Asian and African rangelands, but in societies with formally established private-property regimes, where we focus, such institutions have received little attention. We examine agistment networks, which play an important role in buffering resource variation in modern-day Australia. Agistment is a commercial arrangement between pastoralists who have less forage than they believe they require and pastoralists who believe they have more. Agistment facilitates the movement of livestock via a network based largely on trust. We are concerned exclusively with the link between the characteristics of biophysical variation and human aspects of agistment networks, and we developed a model to test the hypothesis that such a link could exist. Our model builds on game theory literature, which explains cooperation between strangers based on the ability of players to learn whom they can trust. Our game is played on a highly stylized landscape that allows us to control and isolate the degree of spatial variation and spatial covariation. We found that agistment networks are more effective where spatial variation in resource availability is high, and generally more effective when spatial covariation is low. Policy design that seeks to work with existing social networks in rangelands has potential, but this potential varies depending on localized characteristics of the biophysical variability.
Biophysical Journal. Dec, 2006 | Pubmed ID: 16950838
We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.
Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 1. Quantification of Hemozoin Development for Drug Sensitive Versus Resistant Malaria
Biochemistry. Oct, 2006 | Pubmed ID: 17029396
We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites.
Spinning Disk Confocal Microscopy of Live, Intraerythrocytic Malarial Parasites. 2. Altered Vacuolar Volume Regulation in Drug Resistant Malaria
Biochemistry. Oct, 2006 | Pubmed ID: 17029397
In the previous paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12400-12410], we reported on a customized Nipkow spinning disk confocal microscopy (SDCM) system and its initial application to DIC imaging of hemozoin within live, synchronized, intraerythrocytic Plasmodium falciparum malarial parasites. In this paper, we probe the biogenesis as well as the volume and pH regulation of the parasite digestive vacuole (DV), using the fluorescence imaging capabilities of the system. Several previous reports have suggested that mutant PfCRT protein, which causes chloroquine resistance (CQR) in P. falciparum, also causes increased acidification of the DV. Since pH and volume regulation are often linked, we wondered whether DV volume differences might be associated with CQR. Using fast acquisition of SDCM z stacks for synchronized parasites with OGd internalized into the DV, followed by iterative deconvolution using experimental point spread functions, we quantify the volume of the DV for live, intraerythrocytic HB3 (CQS), Dd2 (CQR via drug selection), GCO3 (CQS), and GCO3/C3(Dd2) (CQR via transfection with mutant pfcrt) malarial parasites as they develop within the human red blood cell. We find that relative to both CQS strains, both CQR strains show significantly increased DV volume as the organelle forms upon entry into the trophozoite stage of development and that this persists until the trophozoite-schizont boundary. A more acidic DV pH is found for CQR parasites as soon as the organelle forms and persists throughout the trophozoite stage. We probe DV volume and pH changes upon ATP depletion, hypo- and hypertonic shock, and rapid withdrawal of perfusate chloride. Taken together, these data suggest that the PfCRT mutations that cause CQR also lead to altered DV volume regulation.
Journal of Biomedical Optics. Sep-Oct, 2008 | Pubmed ID: 19021437
We describe the principles, design, and systems integration of a flexible, high-speed, high-sensitivity, high-resolution confocal spinning disk microscopy (SDCM) system. We present several artifacts unique to high-speed SDCM along with techniques to minimize them. We show example experimental results from a specific implementation capable of generating 3-D image stacks containing 30 2-D slices at 30 stacks per second. This implementation also includes optics for differential interference contrast (DIC), phase, and bright-field imaging, as well as an optical trap with sensitive force and position measurement.
Legalizing Markets and the Consequences for Poaching of Wildlife Species: the Vicuña As a Case Study
Journal of Environmental Management. Jan, 2009 | Pubmed ID: 18082928
Vicuña provide an excellent case study for examining the sustainable use of wildlife outside protected areas: the community-based conservation approach. Vicuña populations in the high Andes of Argentina, Bolivia, Chile, Ecuador and Perú fell to a critically low level, but a Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) ban on trade in their fiber has seen numbers recover dramatically, and now live shearing of vicuña for a high-value international market is being promoted as a mechanism to secure both sustainable vicuña populations and local livelihoods. We used a dynamic optimization model to explore the consequences of legalizing markets, including the consequences for poaching which is critical in vicuña dynamics. Using parameters obtained from the literature and expert knowledge, we explored different scenarios for the Argentine region of Cieneguillas. Our results showed that the role of the international market is ambiguous; live shearing for an international market can provide the very best of outcomes for both vicuña and local people, with large herds generating high revenues. But an international market also creates a market for poached vicuña fiber; as a result, vicuña numbers risk once again falling to critically low levels, resulting also in minimal revenues from sale of fiber. The message for the international community is that if community-based conservation is not implemented carefully then its impact can easily be perverse.
American Journal of Public Health. Apr, 2009 | Pubmed ID: 19150892
Journal of Neuroscience Methods. May, 2009 | Pubmed ID: 19428538
We demonstrate effective guidance of neurites extending from PC12 cells in a three-dimensional collagen matrix using a focused infrared laser. Processes can be redirected in an arbitrarily chosen direction in the imaging plane in approximately 30 min with an 80% success rate. In addition, the application of the laser beam significantly increases the rate of neurite outgrowth. These results extend previous observations on 2D coated glass coverslips. We find that the morphology of growth cones is very different in 3D than in 2D, and that this difference suggests that the filopodia play a key role in optical guidance. This powerful, flexible, non-contact guidance technique has potentially broad applications in tissues and engineered environments.
Methods in Molecular Biology (Clifton, N.J.). 2009 | Pubmed ID: 19763971
During development a variety of cell types are guided by molecular concentration gradients to form tissues and organ systems. In the nervous system, the migration and neuronal pathfinding that occurs during development is organized and driven by "guidance cues." Some of these cues are substrate bound or nondiffusible, while many are diffusible and form gradients within the developing embryo to guide neurons and neurites to their appropriate destination. There have been many approaches used to discover and characterize the multitude of guidance cues, their cognate receptors, and how these cues and receptors are regulated to achieve the highly detailed connections found in the nervous system. Here we present a method for creating precisely controlled gradients of molecular factors within a three-dimensional culture environment. The method is based on a non contact mediated delivery of biomolecules to the surface of a collagen gel. The factors are printed in a pattern on the top of a gel containing the tissue or cell type of interest embedded in the gel. The formation of the gradient is dependent upon the diffusion of the printed molecule in the gel. The concentration of the factor within the gel becomes independent of depth rapidly, and the gradient becomes smooth on a similar time scale. The gradients formed can remain relatively stable for a day or more. Moreover, the steepness and molar concentration of tropic or trophic factors within the gradient can be controlled.
American Journal of Preventive Medicine. Nov, 2010 | Pubmed ID: 20965388
Identification of Orch3, a Locus Controlling Dominant Resistance to Autoimmune Orchitis, As Kinesin Family Member 1C
PLoS Genetics. Dec, 2012 | Pubmed ID: 23300462
Experimental autoimmune orchitis (EAO), the principal model of non-infectious testicular inflammatory disease, can be induced in susceptible mouse strains by immunization with autologous testicular homogenate and appropriate adjuvants. As previously established, the genome of DBA/2J mice encodes genes that are capable of conferring dominant resistance to EAO, while the genome of BALB/cByJ mice does not and they are therefore susceptible to EAO. In a genome scan, we previously identified Orch3 as the major quantitative trait locus controlling dominant resistance to EAO and mapped it to chromosome 11. Here, by utilizing a forward genetic approach, we identified kinesin family member 1C (Kif1c) as a positional candidate for Orch3 and, using a transgenic approach, demonstrated that Kif1c is Orch3. Mechanistically, we showed that the resistant Kif1c(D2) allele leads to a reduced antigen-specific T cell proliferative response as a consequence of decreased MHC class II expression by antigen presenting cells, and that the L(578) â†’ P(578) and S(1027) â†’ P(1027) polymorphisms distinguishing the BALB/cByJ and DBA/2J alleles, respectively, can play a role in transcriptional regulation. These findings may provide mechanistic insight into how polymorphism in other kinesins such as KIF21B and KIF5A influence susceptibility and resistance to human autoimmune diseases.
Conservation Biology : the Journal of the Society for Conservation Biology. Feb, 2013 | Pubmed ID: 23305381
Many of the challenges conservation professionals face can be framed as scale mismatches. The problem of scale mismatch occurs when the planning for and implementation of conservation actions is at a scale that does not reflect the scale of the conservation problem. The challenges in conservation planning related to scale mismatch include ecosystem or ecological process transcendence of governance boundaries; limited availability of fine-resolution data; lack of operational capacity for implementation; lack of understanding of social-ecological system components; threats to ecological diversity that operate at diverse spatial and temporal scales; mismatch between funding and the long-term nature of ecological processes; rate of action implementation that does not reflect the rate of change of the ecological system; lack of appropriate indicators for monitoring activities; and occurrence of ecological change at scales smaller or larger than the scale of implementation or monitoring. Not recognizing and accounting for these challenges when planning for conservation can result in actions that do not address the multiscale nature of conservation problems and that do not achieve conservation objectives. Social networks link organizations and individuals across space and time and determine the scale of conservation actions; thus, an understanding of the social networks associated with conservation planning will help determine the potential for implementing conservation actions at the required scales. Social-network analyses can be used to explore whether these networks constrain or enable key social processes and how multiple scales of action are linked. Results of network analyses can be used to mitigate scale mismatches in assessing, planning, implementing, and monitoring conservation projects.
Phenotypic Differences in Virulence and Immune Response in Closely Related Clinical Isolates of Influenza A 2009 H1N1 Pandemic Viruses in Mice
PloS One. 2013 | Pubmed ID: 23441208
To capture the possible genotypic and phenotypic differences of the 2009 influenza A virus H1N1 pandemic (H1N1pdm) strains circulating in adult hospitalized patients, we isolated and sequenced nine H1N1pdm viruses from patients hospitalized during 2009-2010 with severe influenza pneumonia in Kentucky. Each viral isolate was characterized in mice along with two additional H1N1 pandemic strains and one seasonal strain to assess replication and virulence. All isolates showed similar levels of replication in nasal turbinates and lung, but varied in their ability to cause morbidity. Further differences were identified in cytokine and chemokine responses. IL-6 and KC were expressed early in mice infected with strains associated with higher virulence. Strains that showed lower pathogenicity in mice had greater IFNγ, MIG, and IL-10 responses. A principal component analysis (PCA) of the cytokine and chemokine profiles revealed 4 immune response phenotypes that correlated with the severity of disease. A/KY/180/10, which showed the greatest virulence with a rapid onset of disease progression, was compared in additional studies with A/KY/136/09, which showed low virulence in mice. Analyses comparing a low (KY/136) versus a high (KY/180) virulent isolate showed a significant difference in the kinetics of infection within the lower respiratory tract and immune responses. Notably by 4 DPI, virus titers within the lung, bronchoalveolar lavage fluid (BALf), and cells within the BAL (BALc) revealed that the KY/136 replicated in BALc, while KY/180 replication persisted in lungs and BALc. In summary, our studies suggest four phenotypic groups based on immune responses that result in different virulence outcomes in H1N1pdm isolates with a high degree of genetic similarity. In vitro studies with two of these isolates suggested that the more virulent isolate, KY/180, replicates productively in macrophages and this may be a key determinant in tipping the response toward a more severe disease progression.
PloS One. 2013 | Pubmed ID: 23536784
New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM) environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA) surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1) increased the strength of cell-induced forces on the ECM, 2) did not significantly change migration speed, and 3) increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity.
The Murine Model for Hantaan Virus-induced Lethal Disease Shows Two Distinct Paths in Viral Evolutionary Trajectory with and Without Ribavirin Treatment
Journal of Virology. Oct, 2013 | Pubmed ID: 23903835
In vitro, ribavirin acts as a lethal mutagen in Hantaan virus (HTNV)-infected Vero E6 cells, resulting in an increased mutation load and viral population extinction. In this study, we asked whether ribavirin treatment in the lethal, suckling mouse model of HTNV infection would act similarly. The HTNV genomic RNA (vRNA) copy number and infectious virus were measured in lungs of untreated and ribavirin-treated mice. In untreated, HTNV-infected mice, the vRNA copy number increased for 10 days postinfection (dpi) and thereafter remained constant through 26 dpi. Surprisingly, in ribavirin-treated, HTNV-infected mice, vRNA levels were similar to those in untreated mice between 10 and 26 dpi. Infectious virus levels, however, were different: in ribavirin-treated mice, the amount of infectious HTNV was significantly decreased relative to that in untreated mice, suggesting that ribavirin reduced the specific infectivity of the virus (amount of infectious virus produced per vRNA copy). Mutational analysis revealed a ribavirin-associated elevation in mutation frequency in HTNV vRNA similar to that previously reported in vitro. Codon-based analyses of rates of nonsynonymous (dN) and synonymous (dS) substitutions in the S segment revealed a positive selection for codons within the HTNV N protein gene in the ribavirin-treated vRNA population. In contrast, the vRNA population in untreated, HTNV-infected mice showed a lower level of diversity, reflecting purifying selection for the wild-type genome. In summary, these experiments show two different evolutionary paths that Hantavirus may take during infection in a lethal murine model of disease, as well as the importance of the in vivo host environment in the evolution of the virus, which was not apparent in our prior in vitro model system.
Regional Environmental Change. 2014 | Pubmed ID: 25821400
Frontiers in Cellular Neuroscience. 2015 | Pubmed ID: 26578882
Actively generated mechanical forces play a central role in axon growth and guidance, but the mechanisms that underly force generation and regulation in growing axons remain poorly understood. We report measurements of the dynamics of traction stresses from growth cones of actively advancing axons from postnatal rat DRG neurons. By tracking the movement of the growth cone and analyzing the traction stress field from a reference frame that moves with it, we are able to show that there is a clear and consistent average stress field that underlies the complex spatial stresses present at any one time. The average stress field has strong maxima on the sides of the growth cone, directed inward toward the growth cone neck. This pattern represents a contractile stress contained within the growth cone, and a net force that is balanced by the axon tension. Using high time-resolution measurements of the growth cone traction stresses, we show that the stress field is composed of fluctuating local stress peaks, with a large number peaks that live for a short time, a population of peaks whose lifetime distribution follows an exponential decay, and a small number of very long-lived peaks. We show that the high time-resolution data also reveal that the tension appears to vary randomly over short time scales, roughly consistent with the lifetime of the stress peaks, suggesting that the tension fluctuations originate from stochastic adhesion dynamics.