Biology subjects

Soni, R.

Publications and source records attributed to Soni, R..

11 recordsLinked to original sources

bioRxiv2026

Protein Surface Site Determines the Evolutionary Accessibility of Allosteric Regulation

Domain recombination is a major source of new allosteric regulation in both evolved and engineered proteins. However, the sequence and structural features that govern where new allostery may emerge remain poorly understood. Here, we test the hypothesis that the evolutionary accessibility of allosteric regulation following domain insertion is constrained by local surface context, specifically association with pre-existing cooperative networks known as protein sectors. We began with two synthetic domain fusions wherein the Avena sativa light-oxygen-voltage (LOV2) domain was inserted into Escherichia coli dihydrofolate reductase (DHFR) at either a sector connected or non-sector connected surface. The insertion sites are only separated by five residues and both DHFR enzymes retain similar catalytic activity, yet the sector connected version exhibits a light-dependent allosteric phenotype, while the non-sector connected version does not. Using deep mutational scanning, we measured the effect of nearly all single point mutations on allostery in each chimera. The sector-connected DL121 was significantly more evolvable, possessing numerous allostery-tuning single mutants. In contrast, DL116 lacked statistically significant mutants that introduce allosteric regulation, suggesting the protein surface used by DL116 may be an evolutionary "dead end" for a regulatory phenotype. Surprisingly, DL116 did not show cooperative unfolding at temperatures up to 80 {degrees}C, suggesting that enhanced protein stability does not promote the evolvability of allosteric regulation as it does with other phenotypes. Together, our findings show that protein surface context influences the mutational pathways available for allosteric regulation, consistent with the view that sector-connected surface sites harbor a latent capacity for allostery while other locations are more evolutionarily inert.

biophysics↗

bioRxiv2026

Evaluating codon optimization strategies for mammalian glycoprotein production with an open-source expression vector

Efficient production of human proteins for the development of tool compounds and biologics depends on a detailed understanding of the protein expression machinery in mammalian cells. Codon optimization is widely believed to enhance protein yield, yet its impact in homologous mammalian systems remains poorly defined. Here, we systematically compare five codon usage strategies reflecting common assumptions about rare codons, RNA stability, and synthesis efficiency. We developed pTipi, an efficient open-source mammalian expression vector, and evaluated its performance in antibody production. We generated plasmids for common epitope tag antibodies such as V5, anti-biotin and anti-His for distribution by Addgene. To compare codon usage schemes, we performed a bake-off of 18 human and murine Wnt pathway glycoproteins in mammalian cells. Small-scale expression screens revealed that codon optimization did not provide a general advantage over native coding sequences, while strategies prioritizing RNA stability consistently reduced expression. Interestingly, a skewed codon scheme using the most abundant codons produced yields comparable to native sequences and occasionally enhanced protein output. To enable flexible evaluation of codon strategies, we implemented a Golden Gate-compatible pTipi platform for efficient synthetic gene incorporation. We conclude that native codons are sufficient for robust homologous mammalian expression of glycoproteins, while selective codon skewing can be beneficial for some targets.

molecular biology↗

bioRxiv2025

A workflow using diaPASEF global quantitative proteomic analysis reveals extracellular vesicle biomarker candidates for non-invasive diagnostics in non-small cell lung cancer

Lung cancer is the second most diagnosed cancer in the world. Non-small cell lung cancer is the most common type of lung cancer in the United States. Tissue biopsy is the gold standard for detecting lung cancer but is highly invasive as it necessitates the extraction of a sample of tissue for histologic analysis. It also carries risks of bleeding and/or infection and is inconvenient from a patient perspective. The development of a minimally invasive test, such as one utilizing a blood or urine sample, and capable of providing accurate results for lung cancer detection and/or subtyping, would significantly enhance the clinical landscape and streamline patient care. In this study we utilize A549 and H1299 human lung cancer cell lines, differing in cell type, location within the lung, and genetic composition (Kras & p53 status), and employ diaPASEF for global quantitative proteomic analysis. We demonstrate that extracellular vesicle protein content provides enhanced resolution to differentiate between these two cell lines relative to protein lysate content and reveals alterations in signaling. Protein clusters are identified showing enrichment for distinct biological processes representing multiple gene ontology categories unique to each lung cancer subtype-oxidative phosphorylation, apical junction, and epithelial-mesenchymal transition. We subsequently delineate a short list of urine-detectable protein candidates that is prognostic in a second cohort of lung cancer patients. This list of protein candidates may be useful for the development of a non-invasive test to distinguish between these two subtypes of human lung cancer.

pathology↗

bioRxiv2024

Macrophage WDFY3, a protector against autoimmunity

Efficient efferocytosis is essential for maintaining homeostasis. Excessive apoptotic cell (AC) death and impaired macrophage efferocytosis lead to autoantigen release and autoantibody production, immune activation, and organ damage. It remains unclear whether these immunogenic autoantigens are the sole cause of increased autoimmunity or if efferocytosis of ACs directly influences macrophage function, impacting their ability to activate T cells and potentially amplifying autoimmune responses. Additionally, it has not been established if enhancing macrophage efferocytosis or modulating macrophage responses to AC engulfment can be protective in autoimmune-like disorders. Our previous work showed WDFY3 is crucial for efficient macrophage efferocytosis. This study reveals that myeloid knockout of Wdfy3 exacerbates autoimmunity in young mice with increased AC burden by systemic injections of ACs and in middle-aged mice developing spontaneous autoimmunity, whereas ectopic overexpression of WDFY3 suppresses autoimmunity in these models. Macrophages, as efferocytes, can activate T cells and the inflammasome upon engulfing ACs, which are suppressed by overexpressing WDFY3. This work uncovered the role of WDFY3 as a protector against autoimmunity by promoting macrophage efferocytosis thus limiting autoantigen production, as well as mitigating T cell activation and inflammasome activation.

immunology↗

bioRxiv2023

Chemogenetic activation of corticotropin-releasing factor-expressing neurons in the anterior bed nucleus of the stria terminalis reduces effortful motivation behaviors

Corticotropin-releasing factor (CRF) in the anterior bed nucleus of the stria terminalis (aBNST) is associated with chronic stress and avoidance behavior. However, CRF+ BNST neurons project to reward- and motivation-related brain regions, suggesting a potential role in motivated behavior. We used chemogenetics to selectively activate CRF+ aBNST neurons in male and female CRF-ires-Cre mice during an effort-related choice task and a concurrent choice task. In both tasks, mice were given the option either to exert effort for high value rewards or to choose freely available low value rewards. Acute chemogenetic activation of CRF+ aBNST neurons reduced barrier climbing for a high value reward in the effort-related choice task in both males and females. Furthermore, acute activation of CRF+ aBNST neurons also reduced effortful lever pressing in high-performing males in the concurrent choice task. These data suggest a novel role for CRF+ aBNST neurons in effort-based decision and motivated behavior.

neuroscience↗

bioRxiv2023

Automatic Abdominal Multi Organ Segmentationusing Residual UNet

Automated segmentation of abdominal organs plays an important role in supporting computer-assisted diagnosis, radiotherapy, biomarker extraction, surgery navigation, and treatment planning. Segmenting multiple abdominal organs using a single algorithm would improve model development efficiency and accelerate model deployment into clinical workflows. To achieve broadly generalized performance, we trained a residual UNet using 500 CT/MRI scans collected from multi-center, multi-vendor, multi-phase, multi-disease patients, each with voxel-level annotation of 15 abdominal organs. Using the model trained on multimodality (CT/MRI), we achieved an average dice of 0.8990 in the held-out test dataset with only CT scans (N=100). An average dice of 0.8948 was achieved in the held-out test dataset with both CT and MRI scans (N=120. Our results demonstrate broad generalization of the model.

bioengineering↗

bioRxiv2023

Automatic Whole Body FDG PET/CT Lesion Segmentation using Residual UNet and Adaptive Ensemble

Multimodal Positron Emission Tomography/Computed Tomography (PET/CT) plays a key role in the diagnosis, staging, restaging, treatment response assessment, and radiotherapy planning of malignant tumors. The complementary nature of high-resolution anatomic CT and high sensitivity/specificity molecular PET imaging provides accurate assessment of disease status [14] In oncology, 18-fluorodeoxyglucose (FDG) PET/CT is the most widely used method to identify and analyze metabolically active tumors. In particular, FDG uptake allows for more accurate detection of both nodal and distant forms of metastatic disease. Accurate quantification and staging of tumors is the most important prognostic factor for predicting the survival of patients and for designing personalized patient management plans. [8,3] Analyzing PET/CT quantitatively by experienced medical imaging experts/radiologists is timeconsuming and error-prone. Automated quantitative analysis by deep learning algorithms to segment tumor lesions will enable accurate feature extraction, tumor staging, radiotherapy planning, and treatment response assessment. The AutoPET Challenge 2022 provided an opensource platform to develop and benchmark deep learning models for automated PET lesion segmentation by providing large open-source wholebody FDG-PET/CT data. Using the multimodal PET/CT data from 900 subjects with 1014 studies provided by the AutoPET MICCAI 2022 Challenge, we applied fivefold cross-validation on residual UNETs to automatically segment lesions. We then utilized the output from adaptive ensemble highly contributive models as the final segmentation. Our method achieved a 10th ranking with a dice score of 0.5541 in the heldout test dataset (N=150 studies).

bioengineering↗

bioRxiv2022

SUMO2 Protects Against Tau-induced Synaptic and Cognitive Dysfunction

Abnormal intracellular accumulation of Tau aggregates is a hallmark of Alzheimers disease (AD) and other Tauopathies, such as Frontotemporal dementia (FTD), which can be caused by mutations of Tau. Mutated and pathological Tau can undergo a range of post-translational modifications (PTMs) that might trigger or modulate disease pathology. Recent studies indicate that modification of wild type Tau by Small ubiquitin-like modifier SUMO isoform 1 (SUMO1) controls Tau hyperphosphorylation and aggregation, suggesting that SUMOylation acts as a central regulator of Taus biochemical properties. Besides SUMO1, Tau is modified by SUMO2/3, however the consequences of this modification have not been investigated. Here, using viral approaches on primary hippocampal neurons, transgenic mice expressing mutant Tau and SUMO2, and iPSC-derived neurons from FTD patients, we evaluated whether SUMO2/3 conjugation modifies the neurodegenerative disease pathology associated with the aggregation-prone mutant Tau P301L, P301S, and R406W variants. We found that mutant forms of Tau are targets of SUMO2/3, and SUMO2/3 conjugation is neuroprotective. Importantly, expression of mutant Tau is accompanied by a significant reduction of SUMO2/3 conjugation levels, and restoring levels of SUMO2 reduces mutant Tau aggregation and phosphorylation in all model systems Furthermore, overexpression of SUMO2 restores levels of pre- and post-synaptic markers, associated with a complete rescue of the LTP and memory deficits in transgenic mice expressing mutant Tau. These findings bring to light the potential therapeutic implication of manipulating SUMO conjugation to detoxify Tau through PTM-based approaches.

neuroscience↗

bioRxiv2022

The CD58:CD2 axis is co-regulated with PD-L1 via CMTM6 and governs anti-tumor immunity

The cell autonomous balance of immune-inhibitory and -stimulatory signals is a critical yet poorly understood process in cancer immune evasion. Using patient-derived co-culture models and humanized mouse models, we show that an intact CD58:CD2 interaction is necessary for anti-tumor immunity. Defects in this axis lead to multi-faceted immune evasion through impaired CD2-dependent T cell polyfunctionality, T cell exclusion, impaired intra-tumoral proliferation, and concurrent protein stabilization of PD-L1. We performed genome-scale CRISPR-Cas9 and CD58 coimmunoprecipitation mass spectrometry screens identifying CMTM6 as a key stabilizer of CD58, and show that CMTM6 is required for concurrent upregulation of PD-L1 in CD58 loss. Single-cell RNA-seq analysis of patient melanoma samples demonstrates that most TILs lack expression of primary costimulatory signals required for response to PD-1 blockade (e.g. CD28), but maintain strong CD2 expression, thus providing an opportunity to mobilize a so far therapeutically untapped pool of TILs for anti-tumor immunity. We identify two potential therapeutic avenues, including rescued activation of human CD2-expressing TILs using recombinant CD58 protein, and targeted disruption of PD-L1/CMTM6 interactions. Our work identifies an underappreciated yet critical axis at the nexus of cancer immunity and evasion, uncovers a fundamental mechanism of co-inhibitory and -stimulatory signal balancing, and provides new approaches to improving cancer immunotherapies.

cancer biology↗

bioRxiv2022

STK25 inhibits PKA signaling by phosphorylating PRKAR1A

In the heart, Protein Kinase A (PKA) is critical for activating calcium handling and sarcomeric proteins in response to beta adrenergic stimulation leading to increased myocardial contractility and performance. The catalytic activity of PKA is tightly regulated by regulatory subunits which inhibit the catalytic subunit until released by cAMP binding. Phosphorylation of Type II regulatory subunits promotes PKA activation, however the role of phosphorylation in Type I regulatory subunits remain uncertain. Here we utilized human induced pluripotent stem cell cardiomyocytes (iPSC-CM) to identify STK25 as a kinase of the Type Ia regulatory subunit PRKAR1A. Phosphorylation of PRKAR1A led to inhibition of PKA kinase activity and increased binding to the catalytic subunit in the presence of cAMP. Stk25 knockout in mice diminished Prkar1a phosphorylation, increased Pka activity and augmented contractile response to beta adrenergic stimulation. Together, these data support STK25 as a negative regulator of PKA signaling through phosphorylation of PRKAR1A.

physiology↗

bioRxiv2019

Vaping Perpetuates Cardiac Electrical Instability

BackgroundTobacco cigarette smoking is on the decline, but the usage of electronic nicotine delivery systems (ENDS) is gaining popularity, specifically in the teen and young adult age groups. While the cardiac toxicity of tobacco cigarette smoking has been widely studied and is well established, the possible cardiac toxicity of ENDS products and their design characteristics, such as added flavorings, are largely underexplored. Vaping, a form of electronic nicotine delivery, uses "e-liquid" to generate "e-vapor", a smoke-like aerosolized mixture of nicotine and flavors. Here, we tested the hypothesis that vaping results in cardiac electrophysiological instability and arrhythmogenesis. We thus investigated how e-liquids with different flavors affect cardiac in-vitro and in-vivo toxicity, in cell culture and in animals. MethodsThree e-liquids with vanilla, cinnamon or fruit flavors were studied. We quantified apoptosis and oxygen consumption rate in HL-1 cells cultured with e-vapors extracts. In human iPSC derived ventricular cardiomyocytes (hiPSC-CM) cultured with e-vapor extract, beating frequency and repolarization duration were measured using multiple electrode arrays (MEA). Mass spectrometry (GC-MS) was used to analyze the composition of the e-vapors. Telemetric ECGs were obtained in freely moving C57BL/6J mice exposed to vanilla flavored e-vapor for 10 weeks and heart rate variability was analyzed (HRV). In-vivo inducibility of ventricular tachycardia as well as optical mapping of voltage in isolated Langendorff-perfused hearts were also carried out. ResultsE-vapor caused a dose dependent increase in toxicity in Hl-1 myocytes and e-vapors containing vanilla and cinnamon flavorings, as indicated by GC-MS, were more toxic, and inhibited cellular respiration more than the fruit flavored e-vapor. In hiPSC-CM cultured with 25% cinnamon flavored e-vapor for 24 hours, beating frequency increased, and the field potential duration significantly increased compared to air control. Inhalation exposure to vanilla flavored e-vapor for 10 weeks caused significant effects on HRV. Additionally, inducible VT was significantly longer, and in optical mapping, the magnitude of ventricular action potential duration alternans was significantly larger in the exposed mice compared to control ConclusionThe widely popular flavored ENDS are not harm free, and they show potential toxicity to the heart, in-vitro, and in vivo. Further studies are needed to further assess their cardiac safety profile, and long-term health effects.

physiology↗