Biology subjects

Agarwal, R.

Publications and source records attributed to Agarwal, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

GPRC6A is a Potential Therapeutic Target for Metformin Regulation of Glucose Homeostasis in Mice

Understanding the mechanism of metformin actions in treating type 2 diabetes is limited by an incomplete knowledge of the specific protein targets mediating its metabolic effects. Metformin has structural similarities to L-Arginine (2-amino-5-guanidinopentanoic acid), which is a ligand for GPRC6A, a Family C G-protein coupled receptor that regulates energy metabolism. Ligand activation of GPRC6A results in lowering of blood glucose and other metabolic changes resembling the therapeutic effect of metformin. In the current study, we tested if metformin activates GPRC6A. We used Alphafold2 to develop a structural model for L-Arginine (L-Arg) binding to the extracellu-lar bilobed venus flytrap domain (VFT) of GPRC6A. We found that metformin docked to the site in the VFT that overlaps the binding site for L-Arg. Metformin resulted in a dose-dependent stimulation of GPRC6A activity in HEK-293 cells transfected with full-length wild-type GPRC6A but not in untransfected control cells. In addition, metformin failed to activate an alternatively spliced GPRC6A isoform lacking the putative binding site in the VFT. More specifically, mutation of the predicted metformin key binding residues Glu170 and Asp303 in the GPRC6A VFT resulted in loss of metformin receptor activation in vitro. The in vivo role of GPRC6A in mediating the effects of metformin was tested in Gprc6a-/- mice. Administration of therapeutic doses of metformin lowered blood glucose levels following a glucose tolerance test in wild-type but not Gprc6a-/- mice. Finally, we EN300, created by adding a carboxymethyl group from L-Arg to the biguanide backbone of metformin. EN300 showed dose-dependent stimulation of GPRC6A activity in vitro with greater potency than L-Arginine, but less than met-formin. Thus, we suggest that GPRC6A is a potential molecular target for metformin which may be used to understand the therapeutic actions of metformin and develop novel small molecules to treat T2D.

physiology↗

bioRxiv2023

Chikungunya-virus-specific CD4+ T cells are associated with chronic chikungunya viral arthritic disease in humans

Chikungunya virus (CHIKV) is a mosquito-borne re-emerging viral infection that can cause chronic chikungunya viral arthritic disease (CHIKVD), which is characterized by incapacitating arthralgia and inflammation that can last for months to years following infection. Despite CHIKV outbreaks occurring worldwide and several vaccines currently in development, immune responses to CHIKV in humans remains largely understudied, and targets of T cell response are currently unknown. In this study, we tested peripheral blood mononuclear cells (PBMCs) collected from patients diagnosed with CHIKV infection during the 2014-2015 outbreak in Colombia against pools of overlapping peptides sequentially spanning each CHIKV protein. Using high-resolution flow cytometry, we detected robust CHIKV-specific CD4+, but not CD8+ T cell responses, in these patients. Patients still experiencing disease symptoms six years after infection displayed significantly stronger CHIKV- specific CD4+ T cell responses against nsP1, nsP2 and E2 proteins, compared to patients that resolved the infection. CHIKV-specific CD4+ T cells in symptomatic patients displayed a significantly lower Th1 CD4+ helper T cell responses and were enriched within the Th17 CD4+ helper subset, identifying tumor necrosis factor-alpha (TNF) as the predominantly produced cytokine. In conclusion, this study comprehensively characterizes the T cell response against CHIKV in humans during the chronic phase and provides insights into the role of T cells and possible treatment of CHIKVD.

immunology↗

bioRxiv2023

Behavioral adaptation to changing energy constraints via altered frequency of movement selection

Animal behavior is strongly constrained by energy consumption. A natural manipulation which provides insight into this constraint is development, where an animal must adapt its movement to a changing energy landscape as its body grows. Unlike many other animals, for fish it is relatively easy to estimate the energy consumed by their movements via fluid mechanics. Here we simulated the fluid mechanics of >100,000 experimentally-recorded movement bouts from larval zebrafish across different ages and fluid conditions as they hunted Paramecia. We find that these fish adapt to their changing relationship with the fluid environment as they grow by adjusting the frequency with which they select different types of movements, so that more expensive movements are chosen less often. This strategy was preserved when fish were raised in an unnaturally viscous environment. This work suggests a general principle by which animals could minimize energy consumption in the face of changing energy costs over development.

animal behavior and cognition↗

bioRxiv2023

Pose prediction accuracy in ligand docking to RNA

Structure-based virtual high-throughput screening is used in early-stage drug discovery. Over the years, docking protocols and scoring functions for protein-ligand complexes have evolved to improve accuracy in the computation of binding strengths and poses. In the last decade, RNA has also emerged as a target class for new small molecule drugs. However, most ligand docking programs have been validated and tested for proteins and not RNA. Here, we test the docking power (pose prediction accuracy) of three state-of-the-art docking protocols on [~]173 RNA-small molecule crystal structures. The programs are AutoDock4 (AD4) and AutoDock Vina (Vina), which were designed for protein targets, and rDock, which was designed for both protein and nucleic acid targets. AD4 performed relatively poorly. For RNA targets for which a crystal structure of a bound ligand is available, and the goal is to identify new molecules for the same pocket, rDock performs slightly better than Vina. However, in the more common type of early-stage drug discovery setting, in which no structure of a ligand:target complex is known, rDock performed similar to Vina, with a low success rate of [~]27 %. Vina was found to bias for ligands with certain physicochemical properties whereas rDock performs similarly for all ligand properties. Thus, for projects where no ligand:protein structure already exists, Vina and rDock are both applicable. However, the relatively poor performance of all methods relative to protein target docking illustrates a need for further methods refinement.

biophysics↗

bioRxiv2023

Genomic perspectives of SARS CoV-2 in liver disease patients with its clinical correlation: A single centre retrospective study

BackgroundSevere Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2), is a causative agent of current global pandemic of Coronavirus disease-19 (COVID-19). Due to propagated outbreak and global vaccination drive an immense immunological selection pressure has been exerted on SARS CoV-2 leading to evolution of new variants. This study was performed to compare the mutational and clinical profile of liver disease patients infected with different variants of SARS CoV-2. MethodologyThis was a single-centre, retrospective, cohort study in which clinicogenomic analysis of liver disease (LD) patients infected with SARS CoV-2 was performed. Complete demographic and clinical details were retrieved from Hospital Information System (HIS). QC-threshold passed FASTA files containing sequences from COVID-19 patients (n=174) were compared with a reference genome of SARS-CoV-2 isolate named Wuhan-Hu-1 (NCBI Reference Sequence: NC_045512.2) for mutational analysis. ResultsOut of 232 finally analysed patients 137 (59.1%) were LD-CoV (+) and 95 (40.9%) were LD-CoV(-). LD patients with comorbidities were affected more with COVID-19 (p=0.002). On comparing the outcome in the terms of mortality, LD-CoV (+) had 2.29 times (OR 2.29, CI 95%, 1.25-4.29) higher of odds of succumbing to COVID-19 (p=0.006). Multivariate regression analysis revealed, abdominal distention (p=0.05), severe COVID-19 pneumonia (p=0.046) and the change in serum bilirubin levels (p=0.005) as well as Alkaline phosphatase (ALP) levels (p=0.003) to have an association with adverse outcome in LD patients with COVID-19. In Delta (22%) and Omicron (48%) groups, Spike gene harboured maximum mutations. On comparing the mutations between LD-CoV(+/D) and LD-CoV(+/O) a total of nine genes had more mutations in LD-CoV(+/O) whereas three genes had more mutations in LD-CoV(+/D). ConclusionWe concluded that LD patients are more susceptible to COVID-19 as compared to a healthy adult with associated adverse clinical outcomes in terms of mortality and morbidity. Therefore this special group should be given priority while devising and introducing new vaccination and vaccination policies. The infection with different variants did not result in different outcome in our group of patients.

microbiology↗

bioRxiv2023

The lateral septum plays a transforming role in acute stress-induced analgesia

AbstractStress is a potent modulator of pain. Specifically, acute stress due to physical restraint induces stress-induced analgesia (SIA). However, where and how acute stress and pain pathways interface in the brain are poorly understood. Here, we describe how the dorsal lateral septum (dLS), a forebrain limbic nucleus, facilitates SIA through its downstream targets in the lateral hypothalamic area (LHA) of mice. Taking advantage of transsynaptic viral-genetic, optogenetic, and chemogenetic techniques, we show that the dLS[->]LHA circuitry is sufficient to drive analgesia and is required for SIA. Further, our results reveal that the dLS[->]LHA pathway is opioid-dependent and modulates pain through the pro-nociceptive neurons in the rostral ventromedial medulla (RVM). Remarkably, we found that the inhibitory dLS neurons are recruited specifically when the mice struggle to escape under restraint and, in turn, inhibit excitatory LHA neurons. As a result, the RVM neurons downstream of LHA are disengaged, thus suppressing nociception. Together, we delineate a poly-synaptic pathway that can transform escape behavior in mice under restraint to acute stress into analgesia.

neuroscience↗

bioRxiv2023

Lung-mimicking hydrogel culture system reproduces several tuberculosis phenotypes and demonstrates pyrazinamide efficacy

Faithful mimics of tuberculosis (TB) infection are needed to provide mechanistic insights into the complex host-pathogen interactions and accelerate drug discovery. Current in vitro models only allow short investigation durations, present divergent transcriptional signatures to human infections, and are unreliable drug discovery platforms. We developed a 3D collagen culture system mimicking the lung microenvironment (collagen fibres, pore size and stiffness), where we incorporated Mycobacterium tuberculosis (Mtb) infected human THP-1 or primary monocytes. Dual RNA-sequencing revealed high mammalian gene expression similarity with patient samples compared to 2D macrophage infections. Similarly, gene expression of bacteria was much more representative to in vivo gene expression compared to bacteria in 2D cultures (114 genes in 3D vs 21 genes in 2D). Key phenotypes observed in humans, such as foamy macrophages and mycobacterial cords (never seen in any other in vitro culture system), were reproduced in our model. Our system overcomes many challenges associated with the traditional platforms, including showing remarkable efficacy with clinically relevant concentrations of first-line anti-TB drug pyrazinamide, not seen in any other in vitro model, making it reliable, readily adoptable for tuberculosis studies and drug screening. Significance statementMtb is a slow-growing pathogen which modulates host response over time. The current in vitro platforms offer a very short study duration to study, are unreliable as drug discovery platforms, and the phenotypic and genotypic traits of the host and pathogen differ. The collagen-I hydrogel culture system developed in this study addresses these challenges by successfully recapitulating several key phenotypes observed in human infections. Dual RNA sequence also showed excellent gene expression similarities for both the host and the bacteria. Furthermore, remarkable efficacy with the antibiotic Pyrazinamide was demonstrated, a first for in vitro cultures despite over 50 years of clinical use of the drug. We expect our platform to be exploited widely for drug discovery and understanding host-pathogen interactions.

bioengineering↗

bioRxiv2023

Sustained release Resolvin D1 liposomes are effective in the treatment of osteoarthritis in obese mice

Osteoarthritis (OA) is the most common joint disorder and currently affects > 500 million patients worldwide, with ~60% of them also suffering from obesity. There is no drug approved for human use that changes the course of OA progression. OA is one of the most common comorbidities of obesity, and obesity-related OA (ObOA) is a serious health concern because it shows heightened severity of tissue damage and also predominantly affects the working population. Unresolved inflammation is a major driver of ObOA, thus, resolving disease-associated inflammation is a viable strategy to treat ObOA. Resolvins are highly potent molecules that play a role in the resolution of inflammation and promote tissue healing. However, small molecules (like Resolvin D1; RvD1) have to be administered frequently or prior to injury because they lose their in vivo activity rapidly either by lymphatic clearance, or oxidation-mediated deactivation. In this study, we have encapsulated RvD1 in liposomes and established its efficacy in the mouse model of ObOA at much lower dosages than freely administered RvD1. Liposomal RvD1 (lipo-RvD1) acted as a source of the RvD1 molecules for ~11 days in vitro in synovial fluid derived from patients. When administered prophylactically or therapeutically, lipo-RvD1 suppressed cartilage damage in male C57BL/6 mice compared to untreated and free RvD1 treatments. This efficacy was achieved by increasing the proportion of the proresolution M2 macrophages over proinflammatory M1 macrophages in the synovial membrane. These results show the potential of lipo-RvD1 as an anti-OA agent. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/525015v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@141d889org.highwire.dtl.DTLVardef@1ef1753org.highwire.dtl.DTLVardef@1e463c1org.highwire.dtl.DTLVardef@15138c6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Mechanism of working of lipo-RvD1 in ObOA joint. C_FIG

bioengineering↗

bioRxiv2022

Differential Boric Acid and Water Transport in Type I and Type II Pores of Arabidopsis Nodulin 26-Intrinsic Protein

Nodulin-26 intrinsic proteins (NIPs) are plant-specific multifunctional aquaporin-like channels that are phylogenetically and structurally segregated into three subfamilies: NIP I, II, and III. Each subfamily has a characteristic selectivity filter sequence (the "aromatic-arginine" region, or ar/R) that controls substrate transport specificity based on steric constraints, hydrophobicity, and the spatial orientation of hydrogen bonding moieties. All three NIP subfamilies transport metalloid hydroxides, both beneficial as well as toxic, but with different selectivities. Here we investigated the B, As, and water selectivity of representative Arabidopsis thaliana NIP I and II proteins as well as their ar/R mutants in transport assays as well as through B complementation analysis in the B sensitive nip5;1 mutant background. All NIP proteins, and their ar/R mutants, showed equal permeability to arsenite, but showed differences in boric acid and aquaporin activities that was linked to the amino acid at the helix 2 (H2) position of the ar/R filter (Ala for NIP II and Trp for NIP I). The presence of an alanine at this position in NIP II proteins enhances boric acid permeability and drastically reduces the aquaporin/water permeability of the channel. A NIP II structural model generated from the AlphaFold2 resource and evaluated by MD simulation shows that the alanine results in a wider ar/R pore that accommodates the trigonal boric acid molecule and may allow gating of the pore in a manner that affects water permeability. In contrast, NIP I proteins adopt a more classical aquaporin/glyceroporin arrangement in the ar/R that allows metalloid permeability, although with greater selectivity, as well as permeation by water.

biochemistry↗

bioRxiv2022

Host specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including SARS-CoV-2, cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8s ability to sense coronavirus 3CLpros, and instead enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intra-species variation in inflammasome-mediated viral sensing and immunopathology.

immunology↗

bioRxiv2022

Immunomodulatory effect of mycobacterial outer membrane vesicles coated nanoparticles

Tuberculosis (TB) is one of the most widely prevalent infectious diseases that cause significant mortality. Bacillus Calmette-Guerin (BCG), the current TB vaccine used in clinics, shows variable efficacy and has safety concerns for immunocompromised patients. There is a need to develop new and more effective TB vaccines. Outer membrane vesicles (OMVs) are vesicles released by Mycobacteria that contain several lipids and membrane proteins and act as a good source of antigens to prime immune response. However, the use of OMVs as vaccines has been hampered by their heterogeneous size and low stability. Here we report that mycobacterial OMVs can be stabilized by coating over uniform-sized 50 nm gold nanoparticles. The OMV-coated gold nanoparticles (OMV-AuNP) show enhanced uptake and activation of macrophages and dendritic cells. Proteinase K and TLR inhibitor studies demonstrated that the enhanced activation was attributed to proteins present on OMVs and was mediated primarily by TLR2 and TLR4. Mass spectrometry analysis revealed several potential membrane proteins that were common in both free OMVs and OMV-AuNP. Such strategies may open up new avenues and the utilization of novel antigens for developing TB vaccines.

bioengineering↗

bioRxiv2022

Structure-based discovery of selective histone deacetylase (HDAC) 3 and 4 inhibitors

Histone deacetylases (HDACs) are important cancer drug targets. Existing FDA-approved drugs target the catalytic pocket of HDACs, which is conserved across subfamilies (classes) of HDAC. Here, we use molecular modeling approaches to identify and target potential novel pockets specific to Class IIA HDAC-HDAC4 at the interface between HDAC4 and the NCOR protein. These pockets were then targeted using an ensemble docking approach combined with consensus scoring to identify compounds with a different mechanism of binding than the currently known HDAC modulators. Using this approach, 18 compounds predicted in silico to bind to HDAC4s novel pockets were tested in vivo testing on two cancer cell lines. Of these, 5 compounds decreased cell viability to less than 60%. One inhibited the catalytic activity of HDAC4 but not HDAC3, which belongs to a different family of HDACs (Class I). The most potent compound has an IC50 comparable to the FDA-approved compound SAHA (Vorinostat). While there are currently no known inhibitors reported to bind highly selectively to HDAC4, the present result suggests potential mechanistic and chemical approaches for the development of selective HDAC4 modulators.

cancer biology↗

bioRxiv2022

Distribution and frequency of salivary gland tumours: an international multicenter study

BackgroundSalivary gland tumours (SGT) are a relatively rare group of neoplasms with a wide range of histopathological appearance and clinical features. To date, most of the epidemiological studies on salivary gland tumours are limited for a variety of reason including being out of date, extrapolated from either a single centre or country studies, or investigating either major or minor glands only. MethodsThis study aimed to mitigate these shortcomings by analysing epidemiological data including demographic, anatomical location and histological diagnoses of SGT from multiple centres across the world. The analysed data included age, gender, location and histological diagnosis from fifteen centres covering the majority of the world health organisation (WHO) geographical regions between 2006 and 2019. ResultsA total of 5798 cases were analysed including 65% benign and 35% malignant tumours. A slight female predilection (54%) and peak incidence between the fourth and seventh decade for both benign and malignant tumours was observed. The majority (69%) of the SGT presented in major and 31% in the minor glands. The parotid gland was the most common location (70%) for benign and minor glands (46%) for malignant tumours. Pleomorphic adenoma (70%), and Warthins tumour (17%), were the most common benign tumours whereas mucoepidermoid carcinoma (25%) and adenoid cystic carcinoma (16%) were the most frequent malignant tumours. ConclusionsThis multicentre investigation presents the largest cohort study to date analysing salivary gland tumour data from tertiary centres scattered across the globe. These findings should serve as a baseline for future studies evaluating the epidemiological landscape of these tumours.

pathology↗

bioRxiv2022

A natural broad-spectrum inhibitor of enveloped virus entry, effective against SARS-CoV-2 and Influenza A Virus in preclinical animal models.

The COVID-19 pandemic has highlighted the need for novel antivirals for pandemic management and preparedness. Targeting host processes that are co-opted by viruses is an attractive strategy for developing antivirals with a high resistance barrier. Picolinic acid (PA) is a byproduct of tryptophan metabolism, endogenously produced in humans and other mammals. Here we report broad-spectrum antiviral effects of PA against enveloped viruses, including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), Influenza A virus (IAV), Flaviviruses, Herpes Simplex Virus, and Human Parainfluenza Virus. We further demonstrate using animal models that PA is effective against SARS-CoV-2 and IAV, especially as an oral prophylactic. The mode of action studies revealed that PA inhibits viral entry of enveloped viruses, primarily by interfering with viral-cellular membrane fusion, inhibiting virus-mediated syncytia formation, and dysregulating cellular endocytosis. Overall, our data establish PA as a broad-spectrum antiviral agent, with promising preclinical efficacy against pandemic viruses SARS-CoV-2 and IAV.

microbiology↗

bioRxiv2022

Osteocalcin binds to the GPRC6A Venus fly trap allosteric site to positively modulate GPRC6A signaling

GPRC6A is a member of the Family C G-protein coupled receptors that is activated by cations, L-amino acids, the osteocalcin (Ocn) peptide, and testosterone. GPRC6A functions as a master regulator of energy metabolism and sex hormone production. Based on homology to the related receptors mGluR5 and CaSR, GPRC6As multiple ligand specificity is likely based on an orthosteric ligand binding site in the bilobed Venus fly trap (VFT) domain together with two positive allosteric modulator (PAM) sites, one in the VFT and the other in the 7TM domain. Here, we show that Ocn acts as a PAM for GPRC6A by binding to a site in the VFT that is distinct from the orthosteric site for calcium and L-amino acids. In agreement with this finding, alternatively spliced GPRC6A isoforms 2 and 3, which lack regions of the VFT, and mutations in the predicted Ocn binding site, K352E and H355P, prevent Ocn activation of GPRC6A. These observations provide a structural framework for understanding the ability of multiple distinct classes of compounds to activate GPRC6A and set the stage to develop novel small molecules to activate and inhibit this receptor.

molecular biology↗

bioRxiv2022

Genomic and phenotypic analyses suggest moderate fitness differences among Zika virus lineages

RNA viruses have short generation times and high mutation rates, allowing them to undergo rapid molecular evolution during epidemics. However, the extent of RNA virus phenotypic evolution within epidemics and the resulting effects on fitness and virulence remain mostly unknown. Here, we screened the 2015-2016 Zika epidemic in the Americas for lineage-specific fitness differences. We engineered a library of recombinant viruses representing twelve major Zika virus lineages and used them to measure replicative fitness within disease-relevant human primary cells and live mosquitoes. We found that two of these lineages conferred significant in vitro replicative fitness changes among human primary cells, but we did not find fitness changes in Aedes aegypti mosquitoes. Additionally, we found evidence for elevated levels of positive selection among five amino acid sites that define major Zika virus lineages. While our work suggests that Zika virus may have acquired several phenotypic changes during a short time scale, these changes were relatively moderate and do not appear to have enhanced virulence or transmission during the epidemic.

evolutionary biology↗

bioRxiv2021

Cationic inhalable particles for enhanced drug delivery to M. tuberculosis infected macrophages

Inhalable microparticle-based drug delivery platforms are being investigated extensively for Tuberculosis (TB) treatment as they offer efficient deposition in lungs and improved pharmacokinetics of the encapsulated cargo. However, the effect of physical parameters of microcarriers on interaction with Mycobacterium tuberculosis (Mtb) infected mammalian cells is underexplored. In this study, we report that Mtb-infected macrophages are highly phagocytic and microparticle surface charge plays a major role in particle internalization by infected cells. Microparticles of different sizes (0.5 - 2 m) were internalized in large numbers by Mtb-infected THP-1 macrophages and murine primary Bone Marrow Derived Macrophages in vitro. Drastic improvement in particle uptake was observed with cationic particles in vitro and in mice lungs. Rapid uptake of rifampicin-loaded cationic microparticles allowed high intracellular accumulation of the drug and lead to enhanced anti-bacterial function when compared to non-modified rifampicin-loaded microparticles. Cytocompatibility assay and histological analysis in vivo confirmed that the formulations were safe and did not elicit any adverse reaction. Additionally, pulmonary delivery of cationic particles in mice resulted in two-fold higher uptake in resident alveolar macrophages compared to non-modified particles. This study provides a framework for future design of drug carriers to improve delivery of anti-TB drugs inside Mtb-infected cells.

bioengineering↗

bioRxiv2021

Mars, a molecule archive suite for reproducible analysis and reporting of single molecule properties from bioimages

The rapid development of new imaging approaches is generating larger and more complex datasets revealing the time evolution of individual cells and biomolecules. Single-molecule techniques, in particular, provide access to rare intermediates in complex, multistage molecular pathways. However, few standards exist for processing these information-rich datasets, posing challenges for wider dissemination. Here, we present Mars, an open-source platform for storing and processing image-derived properties of biomolecules. Mars provides Fiji/ImageJ2 commands written in Java for common single-molecule analysis tasks using a Molecule Archive architecture that is easily adapted to complex, multistep analysis workflows. Three diverse workflows involving molecule tracking, multichannel fluorescence imaging, and force spectroscopy, demonstrate the range of analysis applications. A comprehensive graphical user interface written in JavaFX enhances biomolecule feature exploration by providing charting, tagging, region highlighting, scriptable dashboards, and interactive image views. The interoperability of ImageJ2 ensures Molecule Archives can easily be opened in multiple environments, including those written in Python using PyImageJ, for interactive scripting and visualization. Mars provides a flexible solution for reproducible analysis of image-derived properties, facilitating the discovery and quantitative classification of new biological phenomena with an open data format accessible to everyone.

biophysics↗