Biology subjects

Levy, R.

Publications and source records attributed to Levy, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Comparison between optical tissue clearing methods for detecting administered mesenchymal stromal cells in mouse lungs

Optical tissue clearing of lung tissue enables the intact lung to be imaged using fluorescence microscopy. Several clearing protocols have been developed in recent years, including the Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC), stabilised 3D imaging of solvent-cleared organs (s-DISCO) and Ethyl cinnamate (ECi) methods. Here we compared these protocols with the aim of determining the biodistribution of mesenchymal stroma cells (MSCs) and understanding how they interact with host cells in the mouse lung. First, we evaluated how each method affected the size, morphology, and transparency of the lungs. Then, we compared the preservation of the fluorescence of the protein tdTomato expressed by the MSCs, and of the organic dye Evans Blue which labels the vasculature. In addition, we tested the compatibility of the methods with immunofluorescence staining. We found that CUBIC clearing is the only method that enables direct imaging of fluorescently labelled MSCs in the lungs thereby allowing the study of the MSC interaction with endothelial and immune cells when combined with immunofluorescence staining. Overall, 3D imaging of CUBIC cleared lungs confirmed that injected MSCs are initially retained in the pulmonary microvasculature, and that most cells are eliminated from the lungs within the first 24 h. Summary statementWe present a tissue clearing approach to visualize exogenous MSCs in the mouse lung and study their effects in the host.

physiology↗

bioRxiv2022

Computed tomography lacks sensitivity to image gold labelled mesenchymal stromal cells in vivo as evidenced by multispectral optoacoustic tomography.

Elucidating the mechanisms of action and long-term safety of cell therapies is necessary for their clinical translation. Non-invasive imaging technologies such as bioluminescence imaging (BLI), computed tomography (CT) and multispectral optoacoustic tomography (MSOT) have been proposed as tools for longitudinal cell monitoring but their performances have not been compared. Here, we evaluate combinations of these modalities to track the in vivo distribution of gold-labelled mesenchymal stromal cells (MSCs). We found that injected MSCs labelled with gold nanoparticles and expressing the reporter gene firefly luciferase could be detected with BLI and MSOT but not CT. We conclude that the MSCs did not carry enough contrast agent to be tracked by CT, demonstrating that CT tracking of gold-labelled cells is not a practical approach as high amounts of gold, which might impair cell viability, are necessary.

cell biology↗

bioRxiv2022

Inhibition of advanced glycation end product formation and serum protein infiltration in bioprosthetic heart valve leaflets: Investigations of anti-glycation agents.

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated heterograft tissue, such as bovine pericardium (BP) are the most commonly used heart valve replacements. However, BHV durability is limited by structural valve degeneration (SVD) resulting from both calcification and advanced glycation end product (AGE) deposition together with serum protein infiltration. In the present study we investigated the hypothesis that anti-AGE agents, Aminoguanidine (AG), Pyridoxamine (PYR), and N-Acetylcysteine (NAC) could mitigate AGE-serum protein mechanisms in model studies, both in vitro and in vivo, using rat subdermal implants of BP. In vitro studies demonstrated that each of these agents could significantly inhibit AGE formation in BP. However, in rat 28 days BP subdermal implants, only PYR demonstrated both significant inhibition of AGE and serum albumin accumulation per immunostaining. BHV calcification was not mitigated by PYR. It is concluded that AGE-serum protein pathophysiology contributing to SVD can be ameliorated by PYR.

biochemistry↗

bioRxiv2021

Time course of EEG power during creative problem-solving with insight or remote thinking

Problem-solving often requires creativity and is critical in everyday life. However, the neurocognitive mechanisms underlying creative problem-solving remain poorly understood. Two mechanisms have been highlighted: forming new connections from and between the problem elements and insight solving (with a sudden realization of a solution). We examined EEG activity during an adapted version of a classical insight problem task, the Remote Associates Test, that requires finding a word connecting three words. It allowed us to explore remoteness in semantic connections (by varying the remoteness of the solution word across trials) and insight solving (identified as a "Eureka" moment reported by the participants). Semantic remoteness was associated with a power increase in alpha band (8-12Hz) in a left parieto-temporal cluster, beta band (13-30Hz) in a right fronto-temporal cluster in the early phase of the task, and theta band (3-7Hz) in frontal cluster before the participants responded. Insight solving was associated with power increase preceding the response in alpha and gamma band (31-60Hz) in left temporal clusters and theta band in a frontal cluster. Source reconstructions show the brain regions associated with these clusters. Overall, our findings shed new light on the dynamic of some of the mechanisms involved in creative problem-solving.

neuroscience↗

bioRxiv2021

Determinants of functional synaptic connectivity among amygdala-projecting prefrontal cortical neurons

The medial prefrontal cortex (mPFC) mediates a variety of complex cognitive functions via its vast and diverse connections with cortical and subcortical structures. Understanding the patterns of synaptic connectivity that comprise the mPFC local network is crucial for deciphering how this circuit processes information and relays it to downstream structures. To elucidate the synaptic organization of the mPFC, we developed a high-throughput optogenetic method for mapping large-scale functional synaptic connectivity. We show that mPFC neurons that project to the basolateral amygdala display unique spatial patterns of local-circuit synaptic connectivity within the mPFC, which distinguish them from the general mPFC cell population. Moreover, the intrinsic properties of the postsynaptic mPFC cell and anatomical position of both cells jointly account for ~7.5% of the variation in probability of connection between mPFC neurons, with anatomical distance and laminar position explaining most of this fraction in variation. Our findings demonstrate a functional segregation of mPFC excitatory neuron subnetworks, and reveal the factors determining connectivity in the mPFC.

neuroscience↗

bioRxiv2021

Development of amyloid beta gold nanorod aggregates as optoacoustic probes

Propagation of small amyloid beta (A{beta}) aggregates (or seeds) has been suggested as a potential mechanism of Alzheimers disease progression. Monitoring the propagation of A{beta} seeds in an organism would enable testing of this hypothesis and, if confirmed, provide mechanistic insights. This requires a contrast agent for long-term tracking of the seeds. Gold nanorods combine several attractive features for this challenging task, in particular, their strong absorbance in the infrared (enabling optoacoustic imaging) and the availability of several established protocols for surface functionalization. In this work, polymer-coated gold nanorods were conjugated with anti-A{beta} antibodies and specifically attached to pre-formed A{beta} seeds. The resulting complexes were characterized for their optical properties by UV/Vis spectroscopy and multispectral optoacoustic tomography. The complexes retained their biophysical properties, i.e. their ability to seed A{beta} fibril formation. They remained stable in biological media for at least 2 days and showed no toxicity to SH-SY5Y neuroblastoma cells up to 1.5 nM and 6 M of gold nanorods and A{beta} seeds, respectively. Taken together, this study describes the first steps in the development of probes for monitoring the spread of A{beta} seeds in animal models.

biochemistry↗

bioRxiv2021

The role of epistasis in determining the fitness landscape of HIV proteins

The rapid evolution of HIV is constrained by interactions between mutations which affect viral fitness. In this work, we explore the role of epistasis in determining the fitness landscape of HIV for multiple drug target proteins, including Protease, Reverse Transcriptase, and Integrase. Epistatic interactions between residues modulate the mutation patterns involved in drug resistance with unambiguous signatures of epistasis best seen in the comparison of a maximum entropy sequence co-variation (Potts) model predicted and experimental HIV sequence "prevalences" when expressed as higher-order marginals (beyond triplets) of the sequence probability distribution. In contrast, the evidence for epistasis based on experimental measures of fitness such as replicative capacity is weak; the correspondence with Potts model "prevalence"-based predictions is obscured by site conservation and limited precision. Double mutant cycles provide in principle one of the best ways to probe epistatic interactions experimentally without reference to a particular background, and we find they reveal that the most strongly interacting mutations in HIV involve correlated sets of drug-resistance-associated residues, however the analysis is complicated by the small dynamic range of measurements. The use of correlated models for the design of experiments to probe viral fitness can help identify the epistatic interactions involved in mutational escape, and lead to better inhibitor therapies. Author summaryProtein covariation models provide an alternative to experimental measures for estimating the fitness of mutations in proteins from across a variety of organisms. Yet, for viral proteins, it has been shown that models including epistatic couplings between residues, or other machine learning models perform no better or even worse than a simpler independent model devoid of such epistatic couplings in estimating viral fitness measurements such as replicative capacities, providing weak or ambiguous evidence for epistasis. We show that the evidence for long-range epistasis is strong by the analysis of the high-order marginals of the MSA distribution (up to subsequences of length 14), which are accurately captured by a correlated Potts sequence-covariation model but not by an independent model. While double mutant cycles in principle provide well-established biophysical probes for epistatic interactions, we demonstrate that the analysis and comparison between model and experiment is difficult due to the much smaller dynamic range of the measurements, making them more susceptible to noise.

biophysics↗

bioRxiv2021

Unique Features of Different Classes of G-Protein-Coupled Receptors Revealed from Sequence Coevolutionary and Structural Analysis

G-protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and represent the primary targets of about one third of currently marketed drugs. Despite the critical importance, experimental structures have been determined for only a limited portion of GPCRs and functional mechanisms of GPCRs remain poorly understood. Here, we have constructed novel sequence coevolutionary models of the A and B classes of GPCRs and compared them with residue contact frequency maps generated with available experimental structures. Significant portions of structural residue contacts were successfully detected in the sequence-based covariational models. "Exception" residue contacts predicted from sequence coevolutionary models but not available structures added missing links that were important for GPCR activation and allosteric modulation. Moreover, we identified distinct residue contacts involving different sets of functional motifs for GPCR activation, such as the Na+ pocket, CWxP, DRY, PIF and NPxxY motifs in the class A and the HETx and PxxG motifs in the class B. Finally, we systematically uncovered critical residue contacts tuned by allosteric modulation in the two classes of GPCRs, including those from the activation motifs and particularly the extracellular and intracellular loops in class A GPCRs. These findings provide a promising framework for rational design of ligands to regulate GPCR activation and allosteric modulation. SignificanceG-protein-coupled receptors (GPCRs) play key roles in cellular signaling and serve as the primary targets of [~]1/3 of currently marketed drugs. In this work, we have presented the first analysis of both residue sequence coevolution and structural contact maps in different classes of GPCRs. We have inferred pathways for GPCR signal transduction that could not be determined from structural analysis alone. Distinct residue contacts have been identified in the signaling pathways of class A and B GPCRs. Our combined sequence coevolutionary and structural contact analysis has thus revealed important insights into the mechanism of GPCR signal transduction, which is expected to facilitate rational drug design of the GPCRs.

bioinformatics↗

bioRxiv2021

Recurrent connectivity underlies lateralized temporal processing differences in Auditory Cortex

Brain asymmetry in the sensitivity to spectrotemporal modulation is an established functional feature that underlies the perception of speech and music. Recent studies in humans suggest separable neural mechanisms must underlie these cognitively complex functions. The left Auditory Cortex (ACx) is believed to specialize in processing fast temporal components of speech sounds, and the right ACx slower components. However, the circuit features and neural computations behind these lateralized spectrotemporal processes are poorly understood. To answer these mechanistic questions we use mice, an animal model that captures some relevant features of human communication systems. In this study, we screened for circuit features that could subserve temporal integration differences between the left and right ACx. We mapped excitatory input to principal neurons in all cortical layers and found significantly stronger recurrent connections in the superficial layers of the right ACx compared to the left. We hypothesized that the underlying recurrent neural dynamics would exhibit differential characteristic timescales corresponding to their hemispheric specialization. To investigate, we recorded spike trains from awake mice and estimated the network time constants using a quasi-Bayesian method to combine evidence from multiple weak signal-to-noise ratio neurons. We found longer temporal integration windows in the right ACx compared to the left as predicted by stronger recurrent excitation. Our study shows direct evidence that stronger recurrent synaptic connections lead to longer network time scales. These findings support speech processing theories that purport asymmetric integration time constants is a crucial feature of lateralization in auditory processing.

neuroscience↗

bioRxiv2021

An mRNA SARS-CoV-2 vaccine employing a novel delivery vehicle with a TLR-9 agonist induces neutralizing antibodies and T cell memory

The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with Lipid Nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general. Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called Charge-Altering Releasable Transporters (CARTs). Using these inherently nonimmunogenic vehicles we can tailor the vaccine immunogenicity by inclusion of co-formulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of RBD-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long lasting RBD-specific TH1 T cell responses including CD4+ and CD8+ T cell memory.

immunology↗

bioRxiv2021

Methylation-mediated retuning on the enhancer-to-silencer activity scale of networked regulatory elements guides driver-gene misregulation

Cancers arise when particular disease-driving genes adopt abnormal functions, but analyses of coding and regulatory sequences leave many of these abnormalities unexplained. We developed a strategy to explore alternations in the regulatory effects of silencers and enhancers in cancer tumors. Applying the method to 177 gene regulatory domains in human glioblastomas, we produced a driver-gene wide dataset of gene-associated, functional regulatory elements. Many genes were controlled by cis-regulatory networks composed of multiple regulatory elements, each of them providing a defined positive or negative input to the overall regulatory output of the network. Surprisingly, DNA methylation induces enhancers and silencers to acquire new activity setpoints within wide ranges of potential regulatory effects, varying between strong transcriptional enhancing to strong silencing. Extensive analysis of methylation-expression associations reveals the organization of domain-wide cis-regulatory networks, and highlighted key regulatory sites which provide pivotal contributions to the network outputs. Consideration of these effects through mathematical models of gene expression variations signified prime molecular events underlying cancer-genes misregulation in hitherto unexplained tumors. Of the observed gene-malfunctioning events, gene misregulation due to epigenetic retuning of networked enhancers and silencers dominated driver-genes mutagenesis, compared with other types of mutation including coding or regulatory sequence alterations. Elucidation of this gene-transformation mechanism may open the way for methodological disclosing of the driving forces behind cancers and other diseases.

genomics↗

bioRxiv2021

In vitro evaluation of the activity of terpenes and cannabidiol against Human Coronavirus E229

The activity of a new, terpene-based formulation, code-named NT-VRL-1, against Human Coronavirus (HCoV) strain 229E was evaluated in human lung fibroblasts (MRC-5 cells), with and without the addition of cannabidiol (CBD). The tested formulation exhibited an antiviral effect when it was pre-incubated with the host cells prior to virus infection. The combination of NT-VRL-1 with CBD potentiated the antiviral effect better than the positive controls pyrazofurin and glycyrrhizin. There was a strong correlation between the quantitative results from a cell-viability assay and the cytopathic effect seen under the microscope after 72 h. To the best of our knowledge, this is the first report of activity of a combination of terpenes and CBD against a coronavirus.

microbiology↗

bioRxiv2021

Optogenetic silencing of neurotransmitter release with a naturally occurring invertebrate rhodopsin

Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways in cognitive and behavioral processes requires temporally precise manipulation of their activity in vivo. However, existing optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homologue of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro as well as in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents leads to an ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at synaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

neuroscience↗

bioRxiv2020

Exploring the Free Energy Landscape and Thermodynamics of Protein-Protein AssociationAssociation: HIV-1 Integrase Multimerization Induced by an Allosteric Inhibitor

We report the free energy landscape and thermodynamics of the protein-protein association responsible for the drug-induced multimerization of HIV-1 integrase (IN). Allosteric HIV-1 integrase inhibitors (ALLINIs) promote aberrant IN multimerization by bridging IN-IN intermolecular interactions. However, the thermodynamic driving forces and kinetics of the multimerization remain largely unknown. Here we explore the early steps in the IN multimerization by using umbrella sampling and unbiased molecular dynamics simulations in explicit solvent. In direct simulations, the two initially separated dimers spontaneously associate to form near-native complexes that resemble the crystal structure of the aberrant tetramer. Most strikingly, the effective interaction of the protein-protein association is very short-ranged: the two dimers associate rapidly within tens of nanoseconds when their binding surfaces are separated by d ≤ 4.3 Å (less than two water diameters). Beyond this distance, the oligomerization kinetics appears to be diffusion controlled with a much longer association time. The free energy profile also captured the crucial role of ALLINI in promoting multimerization, and explained why several CTD mutations are remarkably resistant to the drug-induced multimerization. The results also show that at small separation the protein-protein binding process contains two consecutive phases with distinct thermodynamic signatures. First, inter-protein water molecules are expelled to the bulk resulting in a small increase in entropy, as the solvent entropy gain from the water release is nearly cancelled by the loss of side chain entropies as the two proteins approach each other. At shorter distances, the two dry binding surfaces adapt to each other to optimize their interaction energy at the expense of further protein configurational entropy loss. While the binding interfaces feature clusters of hydrophobic residues, overall, the protein-protein association in this system is driven by enthalpy and opposed by entropy.Statement of Significance Elucidating the energetics and thermodynamic aspects of protein-protein association is important for understanding this fundamental biophysical process. This study provided a more complete physical picture of the protein-protein association responsible for the drug-induced HIV-1 integrase multimerization. The results captured the critical role of the inhibitor, and accounted for the effects of mutations on the protein association. Remarkably, the effective range of the protein-protein attractive funnel is found to be very short, at less than two layers of water, despite the fact that the two binding partners carry opposite net charges. Lastly, entropy/enthalpy decomposition shows that the solvent release from the inter-protein region into the bulk is more than offset by the loss of the solute configurational entropy due to complexation.View Full Text

biophysics↗

bioRxiv2020

Incremental language comprehension difficulty predicts activity in the language network but not the multiple demand network

What role do domain-general executive functions play in human language comprehension? To address this question, we examine the relationship between behavioral measures of comprehension and neural activity in the domain-general "multiple demand" (MD) network, which has been linked to constructs like attention, working memory, inhibitory control, and selection, and implicated in diverse goal-directed behaviors. Specifically, fMRI data collected during naturalistic story listening are compared to theory-neutral measures of online comprehension difficulty and incremental processing load (reading times and eye-fixation durations). Critically, to ensure that variance in these measures is driven by features of the linguistic stimulus rather than reflecting participant-or trial-level variability, the neuroimaging and behavioral datasets were collected in non-overlapping samples. We find no behavioral-neural link in functionally localized MD regions; instead, this link is found in the domain-specific, fronto-temporal "core language network", in both left hemispheric areas and their right hemispheric homologues. These results argue against strong involvement of domain-general executive circuits in language comprehension.

neuroscience↗

bioRxiv2020

Charge-Altering Releasable Transporters enable specific phenotypic manipulation of resting primary natural killer cells

Natural killer (NK) cells are capable of rapid and robust cytotoxicity, making them excellent tools for immunotherapy. However, their recalcitrance to standard transfection techniques has limited both mechanistic studies and clinical applications. Current approaches for NK cell manipulation rely on viral transduction or methods requiring NK cell activation, which can alter NK cell function. Here, we report that non-viral Charge-Altering Releasable Transporters (CARTs) efficiently transfect primary human NK cells with mRNA without relying on NK cell activation. Compared to electroporation, CARTs transfect NK cells two orders of magnitude more efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype and function. Finally, we use CARTs to generate highly cytotoxic primary human chimeric antigen receptor NK cells, indicating potential therapeutic utility of this technique. To our knowledge, CARTs represent the first efficacious transfection technique for resting primary NK cells that preserves NK cell phenotype, and can drive new biological discoveries and clinical applications of this understudied lymphocyte subset.

immunology↗

bioRxiv2019

Rest functional brain maturation during the first year of life

The first year of life is a key period of brain development, characterized by dramatic structural and functional modifications. Here, we measured rest cerebral blood flow (CBF) modifications throughout babies first year of life using arterial spin labeling magnetic resonance imaging sequence in 52 infants, from 3 to 12 months of age. Overall, global rest CBF significantly increased during this age span. In addition, we found marked regional differences in local functional brain maturation. While primary sensorimotor cortices and insula showed early maturation, temporal and prefrontal region presented great rest CBF increase across the first year of life. Moreover, we highlighted a late and remarkably synchronous maturation of the prefrontal and posterior superior temporal cortices. These different patterns of regional cortical rest CBF modifications reflect a timetable of local functional brain maturation and are consistent with babys cognitive development within the first year of life.

neuroscience↗

bioRxiv2019

Stem cell delivery to kidney via minimally invasive ultrasound-guided renal artery injection in mice

Cell-based therapies are promising treatments for various kidney diseases. However, the major hurdle in initiating therapeutic responses is the inefficiency of injection routes to deliver cells to the kidney parenchyma. Systemic injection, such as intravenous injection only delivers a small proportion of cells to the kidney. Whereas direct delivery, such as renal artery injection require surgical procedures. A minimally invasive renal artery injection was therefore developed to enhance cell delivery to kidney. In this study, luciferase expressing human adipocyte derived stem cells (ADSC) were labelled with gold nanorods (GNR) and injected into the renal artery using ultrasound guidance. The ADSCs were tracked using bioluminescence and photoacoustic imaging serially over 7 days. Imaging confirmed that the majority of signal was within the kidney, indicative of successful injection and that signal remained stable for 3 days. Histology showed co-localization of GNRs with ADSC staining throughout the kidney with no indication of injury caused by injection. These findings demonstrate that ultrasound-guided renal artery injection is feasible in mice and can successfully deliver a large proportion of cells which are retained within the kidney for 3 days. Therefore, the techniques developed here will be useful for optimising cell therapy in kidney diseases.

cell biology↗