Vivo Dynamics Research Articles

In Vivo Dynamics of HIV-1 Infection With Impaired Antibody Immunity and Three General Infection Mechanisms

In this paper we investigate two generalized human immunodeficiency virus type-1 (HIV-1) dynamics models with impaired antibody immunity. The models include both latently and actively infected cells. Three infection mechanisms are incorporated into the models, viral infection mechanism (VIM), latent cellular infection mechanism (CIM) and active CIM. The three infection rates are provided by generic nonlinear functions. The second model includes three types of distributed time delays. We find that our models are biologically feasible. The global stability analysis of equilibria are performed and found the basic reproduction ratio (R0) as a threshold parameter. Using Lyapunov method we show that, the virus-free equilibrium is globally asymptotically stable when R0≤1 and the virus-persistence equilibrium is globally asymptotically stable when R0>1. Sensitivity analysis on R0 is studied. To support our theoretical results we provide some numerical simulations. We have demonstrated that R0 is influenced by all three of the infection types, and that if one of them were ignored, R0 would be underestimated. This might lead to inadequate medication effectiveness that aims to remove HIV-1 from the body. The effects of time delay and impaired antibody immunity on HIV-1 progression are examined. According to our research, lowered immunity is a significant factor in the infection's growth. Furthermore, time delays might drastically reduce R0, which would prevent HIV-1 from replicating. The information provided by our research in this work can improve our comprehension of HIV-1 dynamics within-host and provide guidance for the creation of novel pharmacological treatments.

Use of Fluorescence Recovery After Photobleaching (FRAP) to Measure In Vivo Dynamics of Cell Junction-Associated Polarity Proteins.

Here, we present a detailed protocol for fluorescence recovery after photobleaching (FRAP) to measure the dynamics of junctional populations of proteins in living tissue. Specifically, we describe how to perform FRAP in Drosophila pupal wings on fluorescently tagged core planar polarity proteins, which exhibit relatively slow junctional turnover. We provide a step-by-step practical guide to performing FRAP, and list a series of controls and optimizations to do before conducting a FRAP experiment. Finally, we describe how to present the FRAP data for publication.

In Vivo Dynamics of the Latent Reservoir for HIV-1: New Insights and Implications for Cure.

Although antiretroviral therapy (ART) can reduce viremia to below the limit of detection and allow persons living with HIV-1 (PLWH) to lead relatively normal lives, viremia rebounds when treatment is interrupted. Rebound reflects viral persistence in a stable latent reservoir in resting CD4+ T cells. This reservoir is now recognized as the major barrier to cure and is the focus of intense international research efforts. Strategies to cure HIV-1 infection include interventions to eliminate this reservoir, to prevent viral rebound from the reservoir, or to enhance immune responses such that viral replication is effectively controlled. Here we consider recent developments in understanding the composition of the reservoir and how it can be measured in clinical studies. We also discuss exciting new insights into the in vivo dynamics of the reservoir and the reasons for its remarkable stability. Finally we discuss recent discoveries on the complex processes that govern viral rebound.

On the Nature of Murine Radiation-Induced Subcapsular Cataracts: Optical Coherence Tomography-Based Fine Classification, In Vivo Dynamics and Impact on Visual Acuity.

Ionizing radiation is widely known to induce various kinds of lens cataracts, of which posterior subcapsular cataracts (PSCs) have the highest prevalence. Despite some studies regarding the epidemiology and biology of radiation-induced PSCs, the mechanism underscoring the formation of this type of lesions and their dose dependency remain uncertain. Within the current study, our team investigated the in vivo characteristics of PSCs in B6C3F1 mice (F1-hybrids of BL6 × C3H) that received 0.5-2 Gy γ-ray irradiation after postnatal day 70. For purposes of assessing lenticular damages, spectral domain optical coherence tomography was utilized, and the visual acuity of the mice was measured to analyze their levels of visual impairment, and histological sections were then prepared in to characterize in vivo phenotypes. Three varying in vivo phenotype anterior and posterior lesions were thus revealed and correlated with the applied doses to understand their marginal influence on the visual acuity of the studied mice. Histological data indicated no significantly increased odds ratios for PSCs below a dose of 1 Gy at the end of the observation time. Furthermore, our team demonstrated that when the frequencies of the posterior and anterior lesions were calculated at early time points, their responses were in accordance with a deterministic model, whereas at later time points, their responses were better described via a stochastic model. The current study will aid in honing the current understanding of radiation-induced cataract formation and contributes greatly to addressing the fundamental questions of lens dose response within the field of radiation biology.

Pulsed SILAM Reveals In Vivo Dynamics of Murine Brain Protein Translation.

Identification of proteins that are synthesized de novo in response to specific microenvironmental cues is critical for understanding molecular mechanisms that underpin vital physiological processes and pathologies. Here, we report that a brief period of SILAM (Stable Isotope Labeling of Mammals) diet enables the determination of biological functions corresponding to actively translating proteins in the mouse brain. Our results demonstrate that the synapse, dendrite, and myelin sheath are highly active neuronal structures that display rapid protein synthesis, producing key mediators of chemical signaling as well as nutrient sensing, lipid metabolism, and amyloid precursor protein processing/stability. Together, these findings confirm that protein metabolic activity varies significantly between brain functional units in vivo. Our data indicate that pulsed SILAM approaches can unravel complex protein expression dynamics in the murine brain and identify active synthetic pathways and associated functions that are likely impaired in neurodegenerative diseases.

Exploring In Vivo Dynamics of Bovine Milk Derived Gangliosides

Gangliosides are glycosphingolipids present in mammalian cell membranes, playing important structural and functional roles. Human studies on the health benefits of gangliosides are increasing, but knowledge gaps regarding ganglioside analysis exist. The study aimed to investigate blood sample type (serum/plasma), storage conditions, diurnal, day-to-day variation and acute effects of consuming bovine-derived gangliosides on circulating monosialylated gangliosides. Seventy-one women (18–40 yrs, 20–≤30.0 kg/m2) were enrolled and 61 completed the intervention. They visited the clinic three times following overnight fasting. Serum/plasma gangliosides were analyzed over 2 h (visit-1), 8 h (visit-2) and 8 h following either zero or high ganglioside meals (visit-3). Samples stored at −20 °C and −70 °C were analyzed at 3-, 6-, 12- and 18-months. Plasma and serum GM3-gangliosides did not differ, plasma GM3 did not change diurnally, from day-to-day, in response to a high vs. low ganglioside meal or after 7-days low ganglioside vs. habitual diet (P > 0.05). GM3 concentrations were lower in samples stored at −70 °C vs. −20 °C from 6-months onwards and decreased over time with lowest levels at 12- and 18-months stored at −70 °C. In conclusion, either serum/plasma stored at −20- or −70 °C for up to 6 months, are acceptable for GM3-ganglioside analysis. Blood samples can be collected at any time of the day and participants do not have to be in the fasted state.

Visualizing in Vivo Dynamics of Designer Nanoscaffolds.

Enzymes of natural biochemical pathways are routinely subcellularly organized in space and time in order to improve pathway efficacy and control. Designer scaffolding platforms are under development to confer similar benefits upon engineered pathways. Herein, we evaluate bacterial microcompartment shell (pfam0936-domain) proteins as modules for constructing well-defined nanometer scale scaffolds in vivo. We use a suite of visualization techniques to evaluate scaffold assembly and dynamics. We demonstrate recruitment of target cargo molecules onto assembled scaffolds by appending reciprocally interacting adaptor domains. These interactions can be refined by fine-tuning the scaffold expression level. Real-time observation of this system reveals a nucleation-limited step where multiple scaffolds initially form within a cell. Over time, nucleated scaffolds reorganize into a single intracellular assembly, likely due to interscaffold competition for protein subunits. Our results suggest design considerations for using self-assembling proteins as building blocks to construct nanoscaffolds, while also providing a platform to visualize scaffold-cargo dynamics in vivo.

Biomechanics of the Tricuspid Annulus: A Review of the Annulus' In Vivo Dynamics With Emphasis on Ovine Data.

The tricuspid annulus forms the boundary between the tricuspid valve leaflets and their surrounding perivalvular tissue of the right atrioventricular junction. Its shape changes throughout the cardiac cycle in response to the forces from the contracting right heart myocardium and the blood-valve interaction. Alterations to annular shape and dynamics in disease lead to valvular dysfunctions such as tricuspid regurgitation from which millions of patients suffer. Successful treatment of such dysfunction requires an in-depth understanding of the normal shape and dynamics of the tricuspid annulus and of the changes following disease and subsequent repair. In this manuscript we review what we know about the shape and dynamics of the normal tricuspid annulus and about the effects of both disease and repair based on non-invasive imaging studies and invasive fiduciary marker-based studies. We further show, by means of ovine data, that detailed engineering analyses of the tricuspid annulus provide regionally-resolved insight into the kinematics of the annulus which would remain hidden if limiting analyses to simple geometric metrics.

In Vivo Dynamics and Phase State of Natural Lipid Droplets

All organisms store lipids as energy resource for metabolism. Such lipids are accumulated in the form of intracellular droplets. The structure of the lipid droplet is complex. The hydrophobic core represents a mix of un- and saturated triglycerides, enclosed by an amphiphilic shell of phospholipids with embedded proteins. Uncovering intrinsic biophysical properties of lipid droplets in living cells of various organisms is crucial for metabolism manipulation or reducing lipid storage. The type and organization of lipids affect their phase state and microscopic dynamics in the droplet. We characterized lipid droplets in the cells of fresh human and porcine subcutaneous fat tissues, as well as yeast cells using quasi-elastic neutron scattering, probing the molecular motions in a time scale of 6-400 picoseconds and a length scale of 3-20 Angstroms. The detected two-component dynamics in the droplet is associated with lipid unrestricted diffusion (D ∼ 0.006 A2/ps) and motions of its hydrocarbon chains (D ∼ 0.2 A2/ps) in a restricted volume (5-12 A). The dynamics of lipids is significantly reduced below 305 K and 266 K in porcine and human tissues, respectively, due to a first-order phase transition of lipids from fluid to gel (similar to synthetic lipid bilayer). The lipid phase change in human tissue is confirmed and enhanced at applied pressure of 90 bars. However, lipid droplets in the yeast cells remain in a fluid-like state within range of 280-310 K. We believe that phase behavior of lipid droplets is different in the cells of tissues and microorganism due to lipid composition, which was investigated by mass spectrometry. Lipid packing in the droplet is tight in the tissues, having mostly saturated lipids, and loose in the yeast droplet, having equal proportions of un- and saturated lipids.

Modelling the Effects of Immune Response and Time Delay on HIV-1 in Vivo Dynamics in the Presence of Chemotherapy

Numerous models of mathematics have existed to pronounce the immunological response to contagion by human immunodeficiency virus (HIV-1). The models have been used to envisage the regression of HIV-1 in vitro and in vivo dynamics. Ordinarily the studies have been on the interface of HIV virions, CD4+T-cells and Antiretroviral (ARV). In this study, time delay, chemotherapy and role of CD8+T-cells is considered in the HIV-1 in-vivo dynamics. The delay is used to account for the latent time that elapses between exposure of a host cell to HIV-1 and the production of contagious virus from the host cell. This is the period needed to cause HIV-1 to replicate within the host cell in adequate number to become transmittable. Chemotherapy is by use of combination of Reverse transcriptase inhibitor and Protease inhibitor. CD8+T-cells is innate immune response. The model has six variables: Healthy CD4+T-cells, Sick CD4+T-cells, Infectious virus, Non-infectious virus, used CD8+T-cells and unused CD8+T-cells. Positivity and boundedness of the solutions to the model equations is proved. In addition, Reproduction number (R0) is derived from Next Generation Matrix approach. The stability of disease free equilibrium is checked by use of linearization of the model equation. We show that the Disease Free Equilibrium is locally stable if and only if R0 0, Disease Free Equilibrium is stable when τ =0.67. This stability is only achieved if drug efficacy is administered. The results show that when drug efficacy of α1=0.723 and α2=0.723 the DFE is achieved.

Visualizing and Quantifying In Vivo Cortical Cytoskeleton Structure and Dynamics.

The cortical microtubule and actin meshworks play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM), spinning disk confocal microscopy (SDCM), and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of free computing tools based on the publicly available ImageJ software package, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.

A Photoactivatable Far-Red/Near-Infrared BODIPY To Monitor Cellular Dynamics in Vivo.

A mechanism to photoactivate far-red/near-infrared fluorescence with infinite contrast and under mild visible illumination was designed around the photophysical properties of borondipyrromethene (BODIPY) dyes and the photochemical behavior of oxazine heterocycles. Specifically, the photoinduced and irreversible cleavage of an oxazine ring with a laser line at 405 nm extends the electronic conjugation of a BODIPY chromophore over a 3 H-indole auxochrome with a 2-(4-methoxyphenyl)ethenyl substituent in position 5. This structural transformation shifts bathochromically the main absorption band of the BODIPY component to allow the selective excitation of the photochemical product with a laser line of 633 nm and produce fluorescence between 600 and 850 nm. This combination of activation, excitation, and emission wavelengths permits the visualization of the cellular blastoderm of developing Drosophila melanogaster embryos with optimal contrast and essentially no autofluorescence from the biological specimen. Furthermore, the sequential acquisition of images, after the photoactivation event, enables the tracking of individual cells within the embryos in real time. Thus, our structural design and operating principles for the photoactivation of far-red/near-infrared fluorescence can evolve into invaluable probes to monitor cellular dynamics in vivo.

191 - Mesenchymal Stem Cell Treatment and In Vivo Dynamics of Regulatory T-Cells in Steroid-Refractory Acute Graft-Versus-Host Disease

191 - Mesenchymal Stem Cell Treatment and In Vivo Dynamics of Regulatory T-Cells in Steroid-Refractory Acute Graft-Versus-Host Disease

Section III. Everything’s Moving: In Vivo Dynamics of Epigenetic Regulators: Kinetic Models Based on Ordinary Differential Equations

Section III. Everything’s Moving: In Vivo Dynamics of Epigenetic Regulators: Kinetic Models Based on Ordinary Differential Equations

Clonal Dynamics In Vivo of Virus Integration Sites of T Cells Expressing a Safety Switch

Safety switches are becoming relevant for the clinical translation of T-cell-based immunotherapies. In patients receiving an allogeneic hematopoietic stem cell transplant, the inducible caspase-9 gene (iC9) safety switch expressed by donor-derived T lymphocytes efficiently controls acute graft versus host disease (GvHD). However, in vivo elimination of iC9-T cells by the chemical inducer of dimerization (CID) that activates the iC9 protein is incomplete. To study this effect, we characterized the clonal diversity and dynamics of vector insertion sites (VIS) in iC9-T cells pre- and post-CID administration in four patients who developed GvHD. We identified 3,203 VIS among four patients and followed their in vivo clonal dynamics up to 161 days post-CID. VIS were categorized by their proximity to host genome elements, gene associations, and cis-modulatory relationship to mapped promoters. We found that VIS are preferentially located near open chromatin and promoter regions; furthermore, there was no evidence for selection bias among VIS surviving the CID treatment. The majority of iC9-T cells with high normalized VIS copy number at the time of GvHD onset were eliminated by CID, while iC9-T cells detectable post-CID generally have low normalized VIS copy number. We propose that suboptimal iC9 transgene expression is responsible for the incomplete elimination of iC9-T cells and illustrate here by simple model how cis-modulatory influences of local genome context and T-cell receptor activation status at time of CID treatment contribute to stochastic sparing of iC9-T cells.

Dynein Dynamics in Vivo: A Single-Molecule View on its Function in Intraflagellar Transport

IFT-dynein (cytoplasmic dynein 2) co-operates with kinesin-2 motors to assemble and maintain cilia in a process called intraflagellar transport (IFT). Relatively little is known about this dynein, while the related cytoplasmic dynein 1 has received a lot of attention of late. Here, we study the in vivo IFT-dynein dynamics at the ensemble and single-molecule level.To this end, we use fluorescence microscopy to visualize labeled IFT-dynein motors, expressed at endogenous levels, in the chemosensory cilia of living C. elegans. Fluorescence movies were processed to kymographs, from which location-dependent velocities and motor numbers were obtained using custom kymograph-analysis software. To obtain insight into the behavior of individual motors, we employed photoactivation of PA-GFP-labeled IFT-dynein, which allowed, for the first time, to track individual IFT-dynein motors in vivo.The data revealed that IFT-dynein moves in trains consisting tens of motor proteins. Retrograde trains are smaller but more frequent than anterograde (kinesin-driven) trains. Anterograde and retrograde IFT-dynein flux are equal along cilia, indicating that the cilium is a closed system for dynein. While the kinesin composition of a train varies along the track, the amount of dynein per train remains relatively constant. Remarkably, this does not result in directionality changes along the track, like in reported ‘tug-of-war’ motor systems, suggesting that retrograde and anterograde motor activities are carefully orchestrated in IFT.Single IFT-dynein measurements support the ensemble findings. Trajectories show distinct motility behavior: diffusion at the ciliary base, pauses, turns and directed motion. Pauses in retrograde or anterograde trajectories are never followed by a directional switch. Moreover, retrograde-to-anterograde turn events are rare.This combined ensemble and single-molecule approach has provided new insights into IFT-dynein transport dynamics in living organisms, shedding light on dynein function in general.

Noninvasive Tracking of Quiescent and Activated Muscle Stem Cell (MuSC) Engraftment Dynamics In Vivo.

Muscle stem cells play a central role in muscle regeneration. Most studies in the field of muscle regeneration focus on the unraveling of muscle stem cell biology to devise strategies for treating failing muscles as seen in aging and muscle-related diseases. However, the common method used in assessing stem cell function in vivo is laborious, as it involves time-consuming immunohistological analyses by microscopy on serial cryo-sections of the muscle post stem cell transplantation. Here we describe an alternative method, which adapts the bioluminescence imaging (BLI) technique to allow noninvasive tracking of engrafted stem-cell function in vivo in real-time. This assay system enables longitudinal studies in the same mice over time and reveals parameters, not feasible by traditional analysis, such as the magnitude and dynamics of engrafted muscle stem cell expansion in vivo in response to a particular drug treatment or muscle injury.

Modeling the Effects of Time Delay on HIV-1 in Vivo Dynamics in the Presence of ARVs

Mathematical models to describe in vivo and in vitro immunological response to infection in humans by HIV-1 have been of major concern due to the rich variety of parameters affecting its dynamics. In this paper, HIV-1 in vivo dynamics is studied to predict and describe its evolutions in presence of ARVs using delay differential equations. The delay is used to account for the latent period of time that elapsed between HIV – CD4⁺ T cell binding (infection) and production of infectious virus from this host cell. The model uses four variables: healthy CD4⁺T-cells (T), infected CD4⁺T-cells (T^*), infectious virus (V_I) and noninfectious virus (V_N). Of importance is effect of time delay and drug efficacy on stability of disease free and endemic equilibrium points. Analytical results showed that DFE is stable for all τ>0. On the other hand, there is a critical value of delay τ₁>0, such that for all τ>τ₁, the EEP is stable but unstable forτ<τ₁. The critical value of delayτ₁ is the bifurcation value where the HIV-1 in vivo dynamics undergoes a Hopf-bifurcation. This stability in both equilibria is achieved only if the drug efficacy 0≤ε≤1 is above a threshold value of ε_c. Numerical simulations show that this stability is achieved at the drug efficacy of ε_c=0.59 and time delay of τ₁=0.65 days. This ratifies the fact that if CD4⁺T cells remain inactive for long periods of time τ>τ₁ the HIV-1 viral materials will not be reproduced, and the immune system together with treatment will have enough time to clear the viral materials in the blood and thus the EEP is maintain.

Deciphering Transcriptional Dynamics in Vivo by Counting Nascent RNAs

Transcription of genes is the focus of most forms of regulation of gene expression. Even though careful biochemical experimentation has revealed the molecular mechanisms of transcription initiation for a number of different promoters in vitro, the dynamics of this process in cells is still poorly understood. One approach has been to measure the transcriptional output (fluorescently labeled messenger RNAs or proteins) from single cells in a genetically identical population, which could then be compared to predictions from models that incorporate different molecular mechanisms of transcription initiation. However, this approach suffers from the problem, that processes downstream from transcription can affect the measured output and therefore mask the signature of stochastic transcription initiation on the cell-to-cell variability of the transcriptional outputs. Here we show theoretically that measurements of the cell-to-cell variability in the number of nascent RNAs provide a more direct test of the mechanism of transcription initiation. We derive exact expressions for the first two moments of the distribution of nascent RNA molecules and apply our theory to published data for a collection of constitutively expressed yeast genes. We find that the measured nascent RNA distributions are inconsistent with transcription initiation proceeding via one rate-limiting step, which has been generally inferred from measurements of cytoplasmic messenger RNA. Instead, we propose a two-step mechanism of initiation, which is consistent with the available data. These findings for the yeast promoters highlight the utility of our theory for deciphering transcriptional dynamics in vivo from experiments that count nascent RNA molecules in single cells.

Stochastic Model for Langerhans Cells and HIV Dynamics in Vivo

Many aspects of the complex interaction between HIV and the human immune system remain elusive. Our objective is to study these interactions, focusing on the specific roles of langerhans cells (LCs) in HIV infection. In patients infected with HIV, a large amount of virus is associated with LCs in lymphoid tissue. To assess the influence of LCs on HIV viral dynamics during anti-retroviral therapy, we present and analyse a stochastic model describing the dynamics of HIV, CD4 T-cells, and LCs interactions under therapeutic intervention in vivo. We perform sensitivity analyses on the model to determine which parameters and/or which interaction mechanisms strongly affect infection dynamics.