Biology subjects

Rubin, J.

Publications and source records attributed to Rubin, J..

29 records · Page 2Linked to original sources

bioRxiv2021

Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26)

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against SARS-CoV-2 and HIV-1. Yet, its primary receptor portfolio remains controversial, potentially including sialic acid, CAR, integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned by the inability to co-crystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domains CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (IM) injection, but not after intranasal (IN) administration. Ad26 transduction was 10-fold lower than Ad5 after intratumoral (IT) injection of CD46-expressing tumors. Ad26 transduction of liver was 1000-fold lower than Ad5 after intravenous (IV) injection. These data demonstrate the use of CD46 by Ad26 under certain situations, but also show that the receptor has little consequence by other routes of administration. Finally, IV injection of high doses of Ad26 into CD46 mice induced release of liver enzymes in the bloodstream and reduced white blood cell counts, but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during COVID-19 vaccination with this serotype of adenovirus. IMPORTANCEHuman species D Ad26 is being pursued as a low seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection, and its role in virus biology, vaccine efficacy, and importantly, in vaccine safety.

pharmacology and toxicology↗

bioRxiv2021

Comparison of Mucosal and Intramuscular Immunization against SARS-CoV-2 with Replication-Defective and Replicating Single-cycle Adenovirus Vaccines

SARS-CoV-2 enters the body at mucosal surfaces, such as the nose and lungs. These events involve a small number of virions at these mucosal barriers and are therefore a strategic point to stop a COVID-19 infection before it starts. Despite this, most vaccines against COVID-19 are being injected into the muscle where they will not generate the highest levels of mucosal protection. The vaccines that are approved for use in humans are all replication-defective (RD) mRNA, DNA, or adenovirus (Ad) vaccines that do not amplify antigen transgenes. We developed single cycle adenovirus (SC-Ad) vectors that replicate antigen genes up to 10,000-fold in human cells, but that are disabled from producing infectious Ad particles. We show here that SC-Ad expressing the full-length SARS-CoV-2 spike protein produces 100-fold more spike protein than a matched RD-Ad-Spike vector. When Ad-permissive hamsters were immunized with these vaccines by intranasal (IN) or intramuscular (IM) routes, SC-Ad produced significantly stronger antibody responses as compared to RD-Ad against the spike protein that rose over 14 weeks after one immunization. Single IN or IM immunizations generated significant antibody responses in serum and in bronchoalveolar lavages (BALs). IN priming, but not IM priming, generated HLA-restricted CD8 T cell responses in BALs. SC-Ad-Spike generated antibodies that retain binding to spike receptor binding domains (RBDs) with mutations from new viral variants. These data suggest empowering the genomes of gene-based vaccines with the ability to amplify antigen genes can increase potency. This may be particularly advantageous when applying mucosal vaccines to combat mucosal pathogens like SARS-CoV-2. One Sentence SummaryArming adenovirus vaccines with the ability to replicate vaccine antigen genes may increase potency for systemic, or more importantly, mucosal immunization against mucosal pathogens.

immunology↗

bioRxiv2020

Dynamic decision policy reconfiguration under outcome uncertainty

In uncertain or unstable environments, sometimes the best decision is to change your mind. To shed light on this flexibility, we evaluated how the underlying decision policy adapts when the most rewarding action changes. Human participants performed a dynamic two-armed bandit task that manipulated the certainty in relative reward (conflict) and the reliability of action-outcomes (volatility). Continuous estimates of conflict and volatility contributed to shifts in exploratory states by changing both the rate of evidence accumulation (drift rate) and the amount of evidence needed to make a decision (boundary height), respectively. At the trialwise level, following a switch in the optimal choice, the drift rate plummets and the boundary height weakly spikes, leading to a slow exploratory state. We find that the drift rate drives most of this response, with an unreliable contribution of boundary height across experiments. Surprisingly, we find no evidence that pupillary responses associated with decision policy changes. We conclude that humans show a stereotypical shift in their decision policies in response to environmental changes.

neuroscience↗

bioRxiv2020

PyInteraph2 and PyInKnife2 to analyze networks in protein structural ensembles

MotivationProtein dynamic is essential for cellular functions. Due to the complex nature of non-covalent interactions and their long-range effects, the analysis of protein conformations using network theory can be enlightening. Protein Structure Networks (PSNs) rely on different philosophies, and the currently available tools suffer from limitations in terms of input formats, supported network models, and version control. Another issue is the precise definition of cutoffs for the network calculations and the assessment of the stability of the parameters, which ultimately affect the outcome of the analyses. ResultsWe provide two open-source software packages, i.e., PyInteraph2 and PyInKnife2, to implement and analyze PSNs in a harmonized, reproducible, and documented manner. PyInteraph2 interfaces with multiple formats for protein ensembles and calculates a diverse range of network models with the possibility to integrate them into a macro-network and perform further downstream graph analyses. PyInKnife2 is a standalone package that supports the network models implemented in PyInteraph2. It employs a jackknife resampling approach to estimate the convergence of network properties and streamline the selection of distance cutoffs. Several functionalities are based on MDAnalysis and NetworkX, including parallelization, and are available for Python 3.7. PyInteraph2 underwent a massive restructuring in terms of setup, installation, and test support compared to the original PyInteraph software. ConclusionsWe foresee that the modular structure of the code and the version control system of GitHub will promote the transition to a community-driven effort, boost reproducibility, and establish harmonized protocols in the PSN field. As developers, we will guarantee the introduction of new functionalities, assistance, training of new contributors, and maintenance of the package. AvailabilityThe packages are available at https://github.com/ELELAB/pyinteraph2 and https://github.com/ELELAB/PyInKnife2 with guides provided within the packages.

bioinformatics↗

bioRxiv2020

Lamin A/C functions independently from mechanical signaling during adipogenesis

Mesenchymal stem cells (MSC) maintain the musculoskeletal system by differentiating into multiple cell types including osteocytes and adipocytes. Mechanical signals, including strain and low intensity vibration (LIV), are important regulators of MSC differentiation. Lamin A/C is a vital protein for nuclear architecture that supports chromatin organization, as well as mechanical integrity and mechano-sensitivity of the nucleus in MSCs. Here, we investigated whether Lamin A/C and mechano-responsiveness are functionally coupled during adipogenesis. Lamin depletion in MSCs using siRNA increased nuclear area, height and volume and decreased circularity and stiffness, while phosphorylation of focal adhesions and dynamic substrate strain in response to LIV remained intact. Lamin A/C depletion decelerates adipogenesis as reflected by delayed appearance of key biomarkers (e.g., adiponectin/ADIPOQ). Based on RNA-seq data, reduced Lamin A/C levels decrease the activation of the adipocyte transcriptome that is normally observed in response to adipogenic cues mediating differentiation of MSCs. Mechanical stimulation via daily LIV application reduced the expression levels of ADIPOQ in both control and Lamin A/C depleted cells. Yet, treatment with LIV did not induce major transcriptome changes in either control or Lamin A/C depleted MSCs, suggesting that the biological effects of LIV on adipogenesis may not occur at the transcriptional level. We conclude that while Lamin A/C activation is essential for normal adipogenesis, it is dispensible for activation of focal adhesions by dynamic vibration induced mechanical signals.

bioengineering↗

bioRxiv2019

A Homogeneous SIRPα-CD47 Cell-Based, Ligand-Binding Assay: Utility for Small Molecule Drug Development in Immuno-oncology.

CD47 is an immune checkpoint protein that downregulates both the innate and adaptive anti-tumor immune response via its counter receptor SIRP. Biologics, including humanized CD47 monoclonal antibodies and decoy SIRP receptors, that block the SIRP-CD47 interaction, are currently being developed as cancer immunotherapy agents. However, adverse side effects and limited penetration of tumor tissue associated with their structure and large size may impede their clinical application. We recently developed a quantitative high throughput screening assay platform to identify small molecules that disrupt the binding of SIRP and CD47 as an alternative approach to these protein-based therapeutics. Here, we report on the development and optimization of a cell-based binding assay to validate active small molecules from our biochemical screening effort. This assay has a low volume, high capacity homogenous format that relies on laser scanning cytometry (LSC) and associated techniques to enhance signal to noise measurement of cell surface binding. The LSC assay is specific, concentration dependent, and validated for the two major human SIRP variants (V1 and V2), with results that parallel those of our biochemical data as well as published studies. The LSC assay also confirmed published studies, such as the inhibition of amino-terminal pyroglutamate formation in CD47 disrupts SIRP binding. SIRP-CD47 interaction could be quantitatively measured in live and fixed tumor cells. Use of fixed cells reduces the burden of cell maintenance and provides stable cell standards to control for inter- and intra-assay variations. We also demonstrate the utility of the assay to characterize the activity of the first reported small molecule antagonists of the SIRP-CD47 interaction. This assay will support the screening of thousands of compounds to identify or validate active small molecules as hits, develop structure activity relationships and assist in the optimization of hits to leads by a typical iterative medicinal chemistry campaign.

immunology↗

bioRxiv2019

Prevalence of beta-lactam drug-resistance genes in commensal Escherichia coli contaminating ready-to-eat lettuce

The objective of this study was to evaluate the prevalence of antibiotic resistance and beta-lactam drug resistance genes in Escherichia coli isolated from ready-to-eat lettuce, obtained from local supermarkets in Northern California. Bags of lettuce were purchased from 4 chain supermarkets during three different periods--Oct 2018-Jan 2019, Feb 2019-Apr 2019 and May 2019-July 2019. From 91 packages of lettuce, we recovered 34 E. coli isolates from 22 (24%) lettuce samples. All E. coli isolates were genotyped by multilocus sequence typing (MLST), and we found 15 distinct sequence types (STs). Five of these genotypes (ST2819, ST4600, ST2432, ST1198 and ST5143) have been reported to cause infection in humans. Twenty (59%) E. coli isolates were found resistant to at least one of the antibacterial drugs. They included resistance to ampicillin (AMP, 85%) and ampicillin/sulbactam (SAM, 50%), cefoxitin (FOX, 40%) and cefuroxime (CXM, 35%). We found 8 (40%) of 20 beta-lactam resistant E. coli isolates to carry blaCTX-M; 5 (25%) tested positive for blaSHV, while only 4 (20%) tested positive for blaTEM. Additionally, we identified a class A broad-spectrum beta-lactamase SED-1 gene, blaSED, reported by others in Citrobacter sedlakii isolated from bile of a patient. This study found that a large proportion of fresh lettuce carry beta-lactam drug-resistant E. coli, which could serve as a reservoir for drug resistance genes that could potentially enter pathogens to cause human infections.

microbiology↗

bioRxiv2019

Prevalence of antimicrobial resistance genes and integrons in commensal Gram-negative bacteria in a college community

Although the human intestinal microbiome has been shown to harbor antimicrobial drug-resistance genes (ARG), the prevalence of such genes in a healthy population and their impact on extraintestinal infections that occur in that community are not well established. This study sought to identify ARG prevalence and their mobile elements in the intestines of a healthy community population at a California university, and compared these genes to those found in uropathogenic Escherichia coli isolated from patients with community-acquired urinary tract infection (CA-UTI). We isolated Gram-negative bacteria (GNB) from fecal samples of healthy volunteers and screened them by polymerase chain reaction (PCR) for ARG encoding resistance against ampicillin (AMP), trimethoprim-sulfamethoxazole (TMP-SMX), gentamicin (GENT), and colistin (COL). We found antimicrobial resistant GNB from 85 (83%) of 102 non-redundant rectal swab samples. Sixty-seven (66%) of these samples contained {beta}-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA, blaOXY), dihydrofolate reductase (DHFR) genes (dhfr-A17, dhfr-A7, dhfr-A5, dhfr-A21, dhfr-A1, dhfr-A15, and dhfr-B3), and aminoglycoside resistance genes (aadA5, aadA1, and aadB). Integron sequences were found in 37 fecal samples. These genes were found in 11 different GNB species isolated from the fecal samples. The same ARG were found in E. coli strains isolated from patients with CA-UTI examined at the college outpatient health clinic. The high prevalence of clinically-common ARG and integrons harbored by GNB in the intestine of a healthy population suggest that human intestines may serve as a major reservoir of these mobile ARG that appear in E. coli strains causing extraintestinal infections in the same community.\n\nImportanceIncreasing frequency of antimicrobial resistance (AMR) in human pathogenic bacteria has compromised our ability to treat infections. Since mobile antibiotic resistance genes (ARG) are readily exchanged between different species of bacteria through horizontal gene transfer, there is interest in investigating sources of these genes. The normal intestinal flora has been shown to contain a wide variety of ARG, which may have been introduced via food-containing AMR bacteria. We sought to assess the prevalence of ARG carriage in the intestines of a healthy population and determine if these ARG are found in E. coli strains that cause community-acquired urinary tract infection (CA-UTI) in the same community. Our findings indicate that the human intestine may serve as an important reservoir as well as a site in which ARG are transferred into E. coli that cause UTI. Further research is needed to reduce ARG carriage and devise new strategies to prevent AMR infections.

microbiology↗

bioRxiv2019

Corticostriatal synaptic weight evolution in a two-alternative forced choice task

In natural environments, mammals can efficiently select actions based on noisy sensory signals and quickly adapt to unexpected outcomes to better exploit opportunities that arise in the future. Such feedback-based changes in behavior rely on long term plasticity within cortico-basal-ganglia-thalamic networks, driven by dopaminergic modulation of cortical inputs to the direct and indirect pathway neurons of the striatum. While the firing rates of corticostriatal neurons have been shown to adapt across a range of feedback conditions, it remains difficult to directly assess the corticostriatal synaptic weight changes that contribute to these adaptive firing rates. In this work, we simulate a computational model for the evolution of corticostriatal synaptic weights based on a spike timing-dependent plasticity rule driven by dopamine signaling that is induced by outcomes of actions in the context of a two-alternative forced choice task. Results show that plasticity predominantly impacts direct pathway weights, which evolve to drive action selection toward a more-rewarded action in settings with deterministic reward outcomes. After the model is tuned based on such fixed reward scenarios, its performance agrees with the results of behavioral experiments carried out with probabilistic reward paradigms.

animal behavior and cognition↗

bioRxiv2019

Robustness of respiratory rhythm generation across dynamic regimes

A central issue in the study of the neural generation of respiratory rhythms is the role of the intrinsic pacemaking capabilities that some respiratory neurons exhibit. The debate on this issue has occurred in parallel to investigations of interactions among respiratory network neurons and how these contribute to respiratory behavior. In this computational study, we demonstrate how these two issues are inextricably linked. We use simulations and dynamical systems analysis to show that once a conditional respiratory pacemaker, which can be tuned across oscillatory and non-oscillatory dynamic regimes in isolation, is embedded into a respiratory network, its dynamics become masked: the network exhibits similar dynamic properties regardless of the conditional pacemaker nodes tuning, and that nodes outputs are dominated by network influences. Furthermore, the outputs of the respiratory central pattern generator as a whole are invariant to these changes of dynamical properties, which ensures flexible and robust performance over a wide dynamic range. Author summaryBreathing movements in mammals are generated by brainstem respiratory central pattern generator (CPG) networks, which incorporate an excitatory oscillator located in the pre-Botzinger Complex (preBotC) that can exhibit autorhythmic behavior. To understand how these autorhythmic properties impact CPG network dynamical performance, we performed computational studies with an established modeling framework to systematically analyze network behavior when the preBotC excitatory neurons intrinsic dynamics are tuned to operate in autorhythmic versus non-autorhythmic regimes. Both of these regimes enable rhythmic activity of the CPG network, and we show that the rhythm and its responses to various manipulations are preserved across the tunings of intrinsic properties of the preBotC component. Correspondingly, the emergence of behaviorally appropriate rhythmic patterns of network activity is maintained across preBotC regimes, accompanied by an expansion of the ranges of network output frequencies and amplitudes beyond those attainable with either preBotC regime alone. These results lead to the novel conclusion and concept that the dynamical operation of the CPG is functionally highly robust with respect to the rhythmogenic state of the preBotC excitatory circuits, which could represent a key property for preserved respiratory function across varying conditions and demands on network performance.

neuroscience↗

bioRxiv2018

Reward-driven changes in striatal pathway competition shape evidence evaluation in decision-making

Cortico-basal-ganglia-thalamic (CBGT) networks are critical for adaptive decision-making, yet how changes to circuit-level properties impact cognitive algorithms remains unclear. Here we explore how dopaminergic plasticity at corticostriatal synapses alters competition between striatal pathways, impacting the evidence accumulation process during decision-making. Spike-timing dependent plasticity simulations showed that dopaminergic feedback based on rewards modified the ratio of direct and indirect corticostriatal weights within opposing action channels. Using the learned weight ratios in a full spiking CBGT network model, we simulated neural dynamics and decision outcomes in a reward-driven decision task and fit them with a drift diffusion model. Fits revealed that the rate of evidence accumulation varied with inter-channel differences in direct pathway activity while boundary height varied with overall indirect pathway activity. This multi-level modeling approach demonstrates how complementary learning and decision computations can emerge from corticostriatal plasticity. Author summaryCognitive process models such as reinforcement learning (RL) and the drift diffusion model (DDM) have helped to elucidate the basic algorithms underlying error-corrective learning and the evaluation of accumulating decision evidence leading up to a choice. While these relatively abstract models help to guide experimental and theoretical probes into associated phenomena, they remain uninformative about the actual physical mechanics by which learning and decision algorithms are carried out in a neurobiological substrate during adaptive choice behavior. Here we present an "upwards mapping" approach to bridging neural and cognitive models of value-based decision-making, showing how dopaminergic feedback alters the network-level dynamics of cortico-basal-ganglia-thalamic (CBGT) pathways during learning to bias behavioral choice towards more rewarding actions. By mapping "up" the levels of analysis, this approach yields specific predictions about aspects of neuronal activity that map to the quantities appearing in the cognitive decision-making framework.

neuroscience↗