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Phan, C.

Publications and source records attributed to Phan, C..

12 recordsLinked to original sources

Priors for perceived speed combine supramodally for visual-tactile motion but not for audio-visual motion.

Perception combines incoming sensory evidence with priors learned from preceding input. One example is the central tendency effect in which stimulus estimates regress towards a prior based on the mean of the stimulus distribution. Whether such priors are specific to each modality or shared across modalities remains unresolved. Here, we examined this question using speed perception in the visual, auditory and tactile modalities and used central tendency as a behavioral probe. By giving each modality its own speed range, clear predictions emerge when modalities are interleaved: if priors are modality specific, each modality should maintain its own central tendency bias whereas a supramodal prior would exhibit a different mean based on the pooled set of speeds. Moreover, a supra-modal prior should exhibit precision weighting, with the more reliable cue receiving more weight. For vision and touch, speed estimates converged when interleaved: both modalities shifted but the less reliable one shifted more, consistent with a precision-weighted supramodal prior. For vision and audition, the priors remained largely distinct: audition (the less reliable modality) showed a partial shift but did not converge on the precision weighted, supramodal mean. This was not explained by the large audiovisual reliability difference: it persisted when visual precision was reduced. These results show that in the motion domain, visual and tactile signals can combine crossmodally, but auditory and visual signals do not. Supramodal processing of motion therefore depends on which senses are involved and is likely explained by the lack of an early spatial mapping in audition.

neuroscience↗

Heterotypic interactions and sequence features modulate cellular reflectin condensate dynamics

Reflectin proteins drive dynamic structural coloration in cephalopods by organizing into dense intracellular lamellar structures that dictate local refractive index. While reconstituted reflectins readily undergo liquid-liquid phase separation in vitro, these assemblies frequently undergo dynamic arrest, vitrifying into non-dynamic condensates. Here, we investigate how primary sequence features, post-translational modifications, and heterotypic interactions regulate the material properties of reflectin condensates within the environment of mammalian HeLa cells. Using confocal microscopy and fluorescence recovery after photobleaching (FRAP), we demonstrate that canonical block copolymeric A-type reflectins readily form dynamically arrested condensates, with the linker blocks primarily responsible for the observed arrest. In contrast, non-canonical B/C reflectin variants exhibit significantly greater fluidity and rapid recovery kinetics. We show that phosphomimetic substitutions progressively fluidize some reflectin condensates. Lastly, we find that heterotypic condensates composed of canonical and non-canonical reflectins in combinations associated with reversible iridescence in squid substantially enhance canonical mobility. Our findings establish a biophysical framework in which phosphorylation and heterotypic mixing cooperatively suppress dynamic arrest, enabling the reversible material transitions required for active cephalopod camouflage and communication.

biophysics↗

P-Rex2 exhibits unique structural features and regulatory mechanisms distinct from the closely related RhoGEF P-Rex1

Rho guanine-nucleotide exchange factors (RhoGEFs) activate small GTPases to drive cytoskeletal rearrangement, cell motility, and proliferation. The phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent Rac exchanger (P-Rex) subfamily of RhoGEFs includes P-Rex1 and P-Rex2 which, when misregulated, contribute to cancer progression and metastasis. P-Rex activity is controlled by accessory domains that maintain the protein in a cytosolic, autoinhibited state until activated by the lipid PIP3 and G protein {beta}{gamma} subunits. While P-Rex1 autoinhibition has been structurally and biochemically characterized, P-Rex2 has remained largely unexplored. Furthermore, despite high sequence similarity and domain conservation, P-Rex homologs differ in substrate specificity and regulatory interactions, and the molecular basis for these divergences is unknown. Here, we have taken an integrative structural biology approach to investigate these gaps. Using cryo-EM, we determined the first structure of full-length P-Rex2 to moderate resolution, revealing that, while the overall structure closely resembles that of P-Rex1, there is a substantial repositioning of the N-terminal module relative to the C-terminal core. This may play a key role in precluding the intramolecular interactions between the N- and C-terminal domains that are observed in autoinhibited P-Rex1. Hydrogen-deuterium exchange mass spectrometry revealed that, unlike P-Rex1, P-Rex2 dynamics are unaffected by IP4, the headgroup of PIP3. SEC-SAXS data support that the N-terminal module itself is less dynamic, and biochemical assays show that P-Rex2 may be more tightly regulated by autoinhibition, likely through a mechanism different from P-Rex1. These findings uncover unique features in the molecular mechanisms of P-Rex2 regulation.

biochemistry↗

Actin and myosin dynamics during epithelial remodeling in avian gastrulation

Epithelial remodeling is powered by contractile forces exerted by the actomyosin cytoskeleton. In invertebrates, pulsatile contractile flows of the medio-apical actomyosin cortex have been shown to be critical in promoting junction contraction that ultimately drive cell rearrangements, apical constriction, and cell extrusion. However, how actomyosin dynamics drives epithelial remodeling in amniotes remains poorly understood. In this study, we generated transgenic quail lines reporting actin and myosin and investigate their dynamics in gastrulating embryos. We show that during this process, epithelial remodeling events are closely associated with the contraction of junctional myosin. Although we observe medio-apical contractile flows, those appear to contribute to junction contraction by promoting junctional myosin recruitment. Notably, by characterizing live and apoptotic cell extrusions and their associated actomyosin dynamics, we provide new insights into the cellular processes underlying primitive streak formation and the emergence of germ layers.

developmental biology↗

Live imaging and functional characterization of the avian hypoblast redefine the mechanisms of primitive streak induction

In birds and mammals, the formation of the primitive streak, the hallmark of the primary axis and site of gastrulation, is thought to occur when an anterior displacement of the hypoblast (visceral endoderm in mice) lifts its inhibition on the posterior epiblast, enabling the activation of NODAL signaling. Although the anterior movement of the murine visceral endoderm is well documented, the dynamics of the avian hypoblast remain poorly understood. Here, using live imaging and quantitative image analysis, we find that the hypoblast is mechanically coupled to the epiblast and does not migrate away from its posterior end. Instead, the hypoblast moves and deforms passively, in response to the forces transmitted from the epiblast that shape the primitive streak, after its induction. Furthermore, we show that the posterior hypoblast does not exert an inhibitory effect on the epiblast but instead expresses NODAL, which activates primitive streak formation. NODAL concomitantly regulates gene expression in the hypoblast, patterning it along the anteroposterior axis. Our results thus redefine how the primary axis is established in avians, demonstrating that the displacement of the hypoblast and its concomitant anteroposterior patterning are consequences -- rather than drivers -- of primitive streak induction, downstream of NODAL signaling. Essential summaryNODAL asymmetric expression in the hypoblast activates primitive streak formation in the avian embryo.

developmental biology↗

Direct measurements of active forces and material properties unveil the active mechanics of early embryogenesis

Despite progress in probing tissue mechanics, direct long-term measurements in live embryonic epithelia are lacking. This limits our understanding of amniote embryonic morphogenesis, which takes place over hours or days. Here, we introduce a combined technological and analytical framework to directly measure active forces and material properties in developing quail embryos, in a minimally invasive way. We find that the embryonic epithelium behaves elastically on short timescales with a modulus [~] 2 nN {middle dot} m-1 but flows over longer timescales with a viscosity [~] 4 N {middle dot} s {middle dot} m-1, and that both properties are under active regulation. Notably, we demonstrate that cell division is an active epithelial fluidizer, with division rates correlating with tissue fluidity. This fluidization is crucial for the emergence of the primary embryonic axis, which we show is shaped by a force [~] 2 N. Altogether, our approach delivers a complete and quantitative view of early embryonic mechanics, and opens new avenues for biomechanical studies in live embryos and tissues.

developmental biology↗

Assessing the impact of autologous neutralizing antibodies on rebound dynamics in postnatally SHIV-infected ART-treated infant Rhesus macaques

AO_SCPLOWBSTRACTC_SCPLOWThe presence of antibodies against HIV in infected children is associated with a greater capacity to control viremia in the absence of therapy. While the benefits of early antiretroviral treatment (ART) in infants are well documented, early ART may interfere with the development of antibody responses. In contrast to adults, early treated children lack detectable HIV-specific antibodies, suggesting a fundamental difference in HIV pathogenesis. Despite this potential adverse effect, early ART may decrease the size of the latent reservoir established early in infection in infants, which can be beneficial in viral control. Understanding the virologic and immunologic aspects of pediatric HIV is crucial to inform innovative targeted strategies for treating children living with HIV. In this study, we investigate how ART initiation time sets the stage for trade-offs in the latent reservoir establishment and the development of humoral immunity and how these, in turn, affect post-treatment dynamics. We also elucidate the biological function of antibodies in pediatric HIV. We employ mathematical modeling coupled with experimental data from an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model. Infant Rhesus macaques (RMs) were orally challenged with SHIV.C.CH505 375H dCT four weeks after birth and started treatment at different times after infection. In addition to viral load measurements, antibody responses and latent reservoir sizes were measured. We estimate model parameters by fitting viral load measurements to the standard HIV viral dynamics model within a nonlinear fixed effects framework. This approach allows us to capture differences between rhesus macaques (RMs) that develop antibody responses or exhibit high latent reservoir sizes compared to those that do not. We find that neutralizing antibody responses are associated with increased viral clearance and decreased viral infectivity but decreased death rate of infected cells. In addition, the presence of detectable latent reservoir is associated with less robust immune responses. These results demonstrate that both immune response and latent reservoir dynamics are needed to understand post-rebound dynamics and point to the necessity of a comprehensive approach in tailoring personalized medical interventions.

immunology↗

Comparative Analysis of Within-Host Dynamics of Acute Infection and Viral Rebound Dynamics in Postnatally SHIV-Infected ART-Treated Infant Rhesus Macaques

Viral dynamics of acute HIV infection and HIV rebound following suspension of antiretroviral therapy may be qualitatively similar but must differ given, for one, development of adaptive immune responses. Understanding the differences of acute HIV infection and viral rebound dynamics in pediatric populations may provide insights into the mechanisms of viral control with potential implications for vaccine design and the development of effective targeted therapeutics for infants and children. Mathematical models have been a crucial tool to elucidate the complex processes driving viral infections within the host. Traditionally, acute HIV infection has been modeled with a standard model of viral dynamics initially developed to explore viral decay during treatment, while viral rebound has necessitated extensions of that standard model to incorporate explicit immune responses. Previous efforts to fit these models to viral load data have underscored differences between the two infection stages, such as increased viral clearance rate and increased death rate of infected cells during rebound. However, these findings have been predicated on viral load measurements from disparate adult individuals. In this study, we aim to bridge this gap, in infants, by comparing the dynamics of acute infection and viral rebound within the same individuals by leveraging an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model. Ten infant Rhesus macaques (RMs) orally challenged with SHIV.C.CH505 375H dCT and given ART at 8 weeks post-infection. These infants were then monitored for up to 60 months post-infection with serial viral load and immune measurements. We use the HIV standard viral dynamics model fitted to viral load measurements in a nonlinear mixed effects framework. We find that the primary difference between acute infection and rebound is the increased death rate of infected cells during rebound. We use these findings to generate hypotheses on the effects of adaptive immune responses. We leverage these findings to formulate hypotheses to elucidate the observed results and provide arguments to support the notion that delayed viral rebound is characterized by a stronger CD8+ T cell response.

immunology↗

Exploring serial crystallography for drug discovery

Structure-based drug design is highly dependent on the availability of structures of the protein of interest in complex with lead compounds. Ideally, this information can be used to guide the chemical optimization of a compound into a pharmaceutical drug candidate. A limitation of the main structural method used today, rotational cryo-crystallography, is that it only provides structural information of the protein-complex in its frozen state. Serial crystallography is a relatively new approach that offers the possibility to study protein structures at room-temperature. Here, we explore the use of serial crystallography to determine structures of the pharmaceutical target soluble epoxide hydrolase. We introduce a new method to screen for optimal microcrystallization conditions suitable for use in serial crystallography and present a number of room-temperature ligand-bound structures of our target protein. From a comparison between the room-temperature structural data and previously published cryo-temperature structures, we describe an example of a temperature-dependent difference in ligand-binding mode and observe that flexible loops are better resolved at ambient temperature. Finally, we discuss current limitations and potential future advances of serial crystallography for use within pharmaceutical drug discovery.

biochemistry↗

Ultra High-Plex Spatial Proteogenomic Investigation of Giant Cell Glioblastoma Multiforme Immune Infiltrates Reveals Distinct Protein and RNA Expression Profiles

A deeper understanding of complex biological processes, including tumor development and immune response, requires ultra high-plex, spatial interrogation of multiple "omes". Here we present the development and implementation of a novel spatial proteogenomic (SPG) assay on the GeoMx(R) Digital Spatial Profiler platform with NGS readout that enables ultra high-plex digital quantitation of proteins (> 100-plex) and RNA (whole transcriptome, > 18,000-plex) from a single FFPE sample. This study highlighted the high concordance, R > 0.85, and <11% change in sensitivity between SPG assay and the single analyte -assays on various cell lines and tissues from human and mouse. Furthermore, we demonstrate that the SPG assay was reproducible across multiple users. When used in conjunction with advanced cellular neighborhood segmentation, distinct immune or tumor RNA and protein targets were spatially resolved within individual cell subpopulations in human colorectal cancer and non-small cell lung cancer. We used the SPG assay to interrogate 23 different glioblastoma multiforme samples across 4 pathologies. The study revealed distinct clustering of both RNA and protein based on pathology and anatomic location. The in-depth investigation of giant cell glioblastoma multiforme revealed distinct protein and RNA expression profiles compared to that of the more common glioblastoma multiforme. More importantly, the use of spatial proteogenomics allowed simultaneous interrogation of critical protein post-translational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods.

cancer biology↗

Distinct upper airway epithelium interferon-stimulated and profibrotic gene expression between adult and infant rhesus macaques infected with SARS-CoV-2

The global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease (COVID-19) has led to a pandemic of unprecedented scale. An intriguing feature of the infection is the minimal disease in most children, a demographic at higher risk for respiratory viral diseases. To elucidate age-dependent effects of SARS-CoV-2 pathogenesis, we inoculated two rhesus macaque monkey dam-infant pairs with SARS-CoV-2 and conducted virological and transcriptomic analysis of the respiratory tract and evaluated systemic cytokine and antibody responses. Viral RNA levels in all sampled mucosal secretions were comparable across dam-infant pairs in the respiratory tract. Despite comparable viral loads, adult macaques showed higher IL-6 in serum while CXCL10 was induced in all animals. Both groups mounted neutralizing antibody (nAb) responses, with infants showing a more rapid induction at day 7. Transcriptome analysis of tracheal tissue isolated at day 14 post-infection revealed significant upregulation of multiple interferon-stimulated genes in infants compared to adults. In contrast, a profibrotic transcriptomic signature with genes associated with cilia structure and function, extracellular matrix (ECM) composition and metabolism, coagulation, angiogenesis, and hypoxia was induced in adults compared to infants. Our observations suggest age-dependent differential airway responses to SARS-CoV-2 infection that could explain the distinction in pathogenesis between infants and adults.

immunology↗

A putative enoyl-CoA hydratase contributes to biofilm formation and the antibiotic tolerance of Achromobacter xylosoxidans

Achromobacter xylosoxidans has attracted increasing attention as an emerging pathogen in patients with cystic fibrosis. Intrinsic resistance to several classes of antimicrobials and the ability to form robust biofilms in vivo contribute to the clinical manifestations of persistent A. xylosoxidans infection. Still, much of A. xylosoxidans biofilm formation remains uncharacterized due to the scarcity of existing genetic tools. Here we demonstrate a promising genetic system for use in A. xylosoxidans; generating a transposon mutant library which was then used to identify genes involved in biofilm development in vitro. We further described the effects of one of the genes found in the mutagenesis screen, encoding a putative enoyl-CoA hydratase, on biofilm structure and tolerance to antimicrobials. Through additional analysis, we find that a cis-2 fatty acid signaling compound is essential to A. xylosoxidans biofilm ultrastructure and maintenance. This work describes methods for the genetic manipulation of A. xylosoxidans and demonstrated their use to improve our understanding of A. xylosoxidans pathophysiology.

microbiology↗