ABSTRACT
Gene behavior is governed by activity of other genes in an ecosystem as well as context-specific cues including cell type, microenvironment, and prior exposure to therapy. Here, we developed the Algorithm for Linking Activity Networks (ALAN) to compare gene behavior purely based on patient -omic data. The types of gene behaviors identifiable by ALAN include co-regulators of a signaling pathway, protein-protein interactions, or any set of genes that function similarly. ALAN identified direct protein-protein interactions in prostate cancer (AR, HOXB13, and FOXA1). We found differential and complex ALAN networks associated with the proto-oncogene MYC as prostate tumors develop and become metastatic, between different cancer types, and within cancer subtypes. We discovered that resistant genes in prostate cancer shared an ALAN ecosystem and activated similar oncogenic signaling pathways. Altogether, ALAN represents an informatics approach for developing gene signatures, identifying gene targets, and interpreting mechanisms of progression or therapy resistance.
Subject(s)
Ecosystem , Prostatic Neoplasms , Male , Humans , Prostatic Neoplasms/pathology , Genes, myc , Genomics , Signal Transduction/genetics , Tumor Microenvironment/geneticsABSTRACT
Alkane monooxygenase (AlkB) is a widely occurring integral membrane metalloenzyme that catalyzes the initial step in the functionalization of recalcitrant alkanes with high terminal selectivity. AlkB enables diverse microorganisms to use alkanes as their sole carbon and energy source. Here we present the 48.6-kDa cryo-electron microscopy structure of a natural fusion from Fontimonas thermophila between AlkB and its electron donor AlkG at 2.76 Å resolution. The AlkB portion contains six transmembrane helices with an alkane entry tunnel within its transmembrane domain. A dodecane substrate is oriented by hydrophobic tunnel-lining residues to present a terminal C-H bond toward a diiron active site. AlkG, an [Fe-4S] rubredoxin, docks via electrostatic interactions and sequentially transfers electrons to the diiron center. The archetypal structural complex presented reveals the basis for terminal C-H selectivity and functionalization within this broadly distributed evolutionary class of enzymes.
Subject(s)
Alkanes , Cryoelectron Microscopy , Cytochrome P-450 CYP4A/chemistry , Alkanes/chemistryABSTRACT
BACKGROUND: A prognostic risk score (Halabi score) in metastatic castration-resistant prostate cancer (mCRPC) accurately predicts overall survival, but its association with quality of life (QOL) has not been defined. We hypothesize that a higher pretreatment Halabi score is associated with worse QOL outcomes over time in mCRPC patients. METHODS: Patient-level data from the docetaxel plus prednisone control arm of Mainsail, a Phase 3 clinical trial in mCRPC were accessed via ProjectDataSphere. Pretreatment Halabi score included disease-related factors: metastatic site, opioid use, Eastern Cooperative Oncology Group performance status (ECOG-PS), alkaline phosphatase, albumin, hemoglobin, lactic acid dehydrogenase, and PSA, with higher score indicating worse survival. Three QOL scales were created: Functional Assessment of Cancer Therapy-Prostate (FACT-P, higher score = better QOL), Brief Pain Inventory-Short Form Severity score (BPI-SFSS, higher score = higher pain severity), and BPI-SF Interference score (BPI-SFIS, higher score = greater pain interference). Mixed linear model was used to estimate the associations between Halabi score and QOL scores assessed at different time points (baseline, 2 months, and 6 months). RESULTS: This analysis included 412 mCRPC patients (median age = 68 years, 82% white, 5% Black, median log PSA = 4.4 ng/mL). After multivariable adjustment, Halabi score was significantly associated with QOL scores at all time points. At 6 months, multivariable adjusted FACT-P decreased by 10.0 points (worsening), BPI-SFSS increased by 0.8 points (worsening), and BPI-SFIS increased by 0.9 points (worsening) for each unit increase in Halabi risk score. In multivariable analysis of individual components, ECOG-PS, site of metastasis, and opioid use were significantly associated with worse QOL scores at baseline. CONCLUSIONS: Chemotherapy-naïve mCRPC patients with poorer Halabi prognostic risk scores have poorer QOL and greater pain intensity and interference at baseline and during follow-up.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Quality of Life , Male , Humans , Aged , Prostatic Neoplasms, Castration-Resistant/pathology , Prognosis , Prostate-Specific Antigen/therapeutic use , Analgesics, Opioid/therapeutic use , Prednisone/therapeutic use , Docetaxel/therapeutic use , Pain/drug therapy , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Treatment OutcomeABSTRACT
The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 1012â photonsâ s-1), micro-focus (7â µm × 5â µm), low-divergence (1â mrad × 0.35â mrad) energy-tunable (5-18â keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented.
Subject(s)
Synchrotrons , Crystallography, X-Ray , Macromolecular Substances/chemistryABSTRACT
De novo structure determination from single-wavelength anomalous diffraction using native sulfur or phospho-rus in biomolecules (native-SAD) is an appealing method to mitigate the labor-intensive production of heavy-atom derivatives and seleno-methio-nyl substitutions. The native-SAD method is particularly attractive for membrane proteins, which are difficult to produce and often recalcitrant to grow into decent-sized crystals. Native-SAD uses lower-energy X-rays to enhance anomalous signals from sulfur or phospho-rus. However, at lower energies, the scattering and absorption of air contribute to the background noise, reduce the signals and are thus adverse to native-SAD phasing. We have previously demonstrated native-SAD phasing at an energy of 5â keV in air at the NSLS-II FMX beamline. Here, the use of a helium path developed to reduce both the noise from background scattering and the air absorption of the diffracted X-ray beam are described. The helium path was used for collection of anomalous diffraction data at 5â keV for two proteins: thaumatin and the membrane protein TehA. Although anomalous signals from each individual crystal are very weak, robust anomalous signals are obtained from data assembled from micrometre-sized crystals. The thaumatin structure was determined from 15 microcrystals and the TehA structure from 18 microcrystals. These results demonstrate the usefulness of a helium environment in support of native-SAD phasing at 5â keV.
ABSTRACT
The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for the development of novel antiviral therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic tem-per-ature or room tem-per-ature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple tem-per-atures from cryogenic to physiological, and another at high humidity. We inter-rogate these data sets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a perturbation-dependent conformational landscape for Mpro, including a mobile zinc ion inter-leaved between the catalytic dyad, mercurial conformational heterogeneity at various sites including a key substrate-binding loop, and a far-reaching intra-molecular network bridging the active site and dimer inter-face. Our results may inspire new strategies for antiviral drug development to aid preparation for future coronavirus pandemics.
ABSTRACT
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.
Subject(s)
COVID-19 Drug Treatment , Antiviral Agents/therapeutic use , Coronavirus 3C Proteases , Cysteine Endopeptidases/metabolism , Hepacivirus/metabolism , Humans , Molecular Docking Simulation , Protease Inhibitors/chemistry , SARS-CoV-2 , Viral Nonstructural Proteins/geneticsABSTRACT
KAMO and BLEND provide particularly effective tools to automatically manage the merging of large numbers of data sets from serial crystallography. The requirement for manual intervention in the process can be reduced by extending BLEND to support additional clustering options such as the use of more accurate cell distance metrics and the use of reflection-intensity correlation coefficients to infer `distances' among sets of reflections. This increases the sensitivity to differences in unit-cell parameters and allows clustering to assemble nearly complete data sets on the basis of intensity or amplitude differences. If the data sets are already sufficiently complete to permit it, one applies KAMO once and clusters the data using intensities only. When starting from incomplete data sets, one applies KAMO twice, first using unit-cell parameters. In this step, either the simple cell vector distance of the original BLEND or the more sensitive NCDist is used. This step tends to find clusters of sufficient size such that, when merged, each cluster is sufficiently complete to allow reflection intensities or amplitudes to be compared. One then uses KAMO again using the correlation between reflections with a common hkl to merge clusters in a way that is sensitive to structural differences that may not have perturbed the unit-cell parameters sufficiently to make meaningful clusters. Many groups have developed effective clustering algorithms that use a measurable physical parameter from each diffraction still or wedge to cluster the data into categories which then can be merged, one hopes, to yield the electron density from a single protein form. Since these physical parameters are often largely independent of one another, it should be possible to greatly improve the efficacy of data-clustering software by using a multi-stage partitioning strategy. Here, one possible approach to multi-stage data clustering is demonstrated. The strategy is to use unit-cell clustering until the merged data are sufficiently complete and then to use intensity-based clustering. Using this strategy, it is demonstrated that it is possible to accurately cluster data sets from crystals that have subtle differences.
Subject(s)
Algorithms , Software , Cluster Analysis , Crystallography, X-Ray , Proteins/chemistryABSTRACT
Metastatic castration-resistant prostate cancers (mCRPCs) are treated with therapies that antagonize the androgen receptor (AR). Nearly all patients develop resistance to AR-targeted therapies (ARTs). Our previous work identified CREB5 as an upregulated target gene in human mCRPC that promoted resistance to all clinically approved ART. The mechanisms by which CREB5 promotes progression of mCRPC or other cancers remains elusive. Integrating ChIP-seq and rapid immunoprecipitation and mass spectroscopy of endogenous proteins, we report that cells overexpressing CREB5 demonstrate extensive reprogramming of nuclear protein-protein interactions in response to the ART agent enzalutamide. Specifically, CREB5 physically interacts with AR, the pioneering actor FOXA1, and other known co-factors of AR and FOXA1 at transcription regulatory elements recently found to be active in mCRPC patients. We identified a subset of CREB5/FOXA1 co-interacting nuclear factors that have critical functions for AR transcription (GRHL2, HOXB13) while others (TBX3, NFIC) regulated cell viability and ART resistance and were amplified or overexpressed in mCRPC. Upon examining the nuclear protein interactions and the impact of CREB5 expression on the mCRPC patient transcriptome, we found that CREB5 was associated with Wnt signaling and epithelial to mesenchymal transitions, implicating these pathways in CREB5/FOXA1-mediated ART resistance. Overall, these observations define the molecular interactions among CREB5, FOXA1, and pathways that promote ART resistance.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Receptors, Androgen , Cell Line, Tumor , Cell Nucleus/metabolism , Cyclic AMP Response Element-Binding Protein A , Hepatocyte Nuclear Factor 3-alpha/genetics , Humans , Male , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolismABSTRACT
Purpose: The Retzius-sparing (RS) approach represents an important surgical innovation in how robot-assisted laparoscopic prostatectomy (RALP) is performed. The aim of this study was to examine to what extent its development has followed the idea, development, evolution, assessment, and long-term study (IDEAL) recommendations. Materials and Methods: We conducted a comprehensive literature search for studies up to the 18th of March 2021. Abstracted data points included authorship, year of publication, study design, reported endpoints, and length of follow-up. We mapped each study to the five IDEAL stages of surgical innovation using published criteria. Results: Of 415 references, 118 were included in our analysis. Five academic centers authored >50% of all study reports, with the groups from Seoul (24; 20.3%), Milan (15; 12.7%), and Ninjang (10; 8.5%) being the main contributors. Approximately 40% of studies (50/118) were reported as full-text publications. Most of the reports mapped to retrospective studies (97/118; 82.2%) with approximately one-third (31/97; 32.0%) reporting the use of prospectively collected data. Cumulatively, 17,974 were reported on RS-RALP. Of those, 13,929 were unique cases. Approximately 23% of cases were reported in multiple publications (4045/17,974). We mapped 2, 12, and 3 studies to the idea, assessment, and long-term study stages, respectively, and no study to the development and evaluation stages. Conclusions: Few reported studies followed the IDEAL stages for surgical innovation; none addressed the stages of development and evaluation. Future systematic prospectively planned assessments would be helpful to refine the approach and address issues related to the surgical learning curve.
Subject(s)
Laparoscopy , Robotic Surgical Procedures , Robotics , Humans , Male , Prostatectomy , Retrospective Studies , Treatment OutcomeABSTRACT
AAA+ proteases regulate numerous physiological and cellular processes through tightly regulated proteolytic cleavage of protein substrates driven by ATP hydrolysis. FtsH is the only known family of membrane-anchored AAA+ proteases essential for membrane protein quality control. Although a spiral staircase rotation mechanism for substrate translocation across the FtsH pore has been proposed, the detailed conformational changes among various states have not been clear due to absence of FtsH structures in these states. We report here the cryo-EM structure for Thermotoga maritima FtsH (TmFtsH) in a fully ADP-bound symmetric state. Comparisons of the ADP-state structure with its apo-state and a substrate-engaged yeast YME1 structure show conformational changes in the ATPase domains, rather than the protease domains. A reconstruction of the full-length TmFtsH provides structural insights for the dynamic transmembrane and the periplasmic domains. Our structural analyses expand the understanding of conformational switches between different nucleotide states in ATP hydrolysis by FtsH.
Subject(s)
ATP-Dependent Proteases , Thermotoga maritima , ATP-Dependent Proteases/metabolism , ATPases Associated with Diverse Cellular Activities/chemistry , ATPases Associated with Diverse Cellular Activities/metabolism , Adenosine Diphosphate , Adenosine Triphosphate/metabolism , Cryoelectron Microscopy , Models, Molecular , Protein ConformationABSTRACT
A correction in the paper by Lazo et al. [(2021). J. Synchrotron Rad. 28, 1649-1661] is made.
ABSTRACT
Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and apply it to screen 61 eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functionally competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.
Subject(s)
High-Throughput Screening Assays , Membrane Proteins , Cell-Free System/metabolism , Detergents/chemistry , Eukaryota , Lipids , Membrane Proteins/chemistry , Membranes/metabolismABSTRACT
The androgen receptor (AR) signaling pathway regulates the progression of prostate cancer (PC). Metastatic castration-resistant prostate cancer (mCRPC) patients generally receive AR-targeted therapies (ART) or androgen-deprivation therapies (ADT) with the initial response; however, resistance is inevitably observed. Prior studies have shown activity and upregulation of a family of androgen production, uptake, and conversion - APUC genes - based on genomic analyses of patient germlines. Genetic variants of some APUC genes, such as the conversion gene, HSD3B1, predict response to second-generation androgen-targeted therapies. Studies have begun to elucidate the overall role of APUC genes, each with unique actionable enzymatic activity, in mCRPC patient outcomes. The current role and knowledge of the genetic and genomic features of APUC genes in advanced prostate cancer and beyond are discussed in this review. These studies inform of how interpreting behavior of APUC genes through genomic tools will impact the treatment of advanced prostate cancer.
ABSTRACT
Here we present two robotic sample changers integrated into the experimental stations for the macromolecular crystallography (MX) beamlines AMX and FMX, and the biological small-angle scattering (bioSAXS) beamline LiX. They enable fully automated unattended data collection and remote access to the beamlines. The system designs incorporate high-throughput, versatility, high-capacity, resource sharing and robustness. All systems are centered around a six-axis industrial robotic arm coupled with a force torque sensor and in-house end effectors (grippers). They have the same software architecture and the facility standard EPICS-based BEAST alarm system. The MX system is compatible with SPINE bases and Unipucks. It comprises a liquid nitrogen dewar holding 384 samples (24 Unipucks) and a stay-cold gripper, and utilizes machine vision software to track the sample during operations and to calculate the final mount position on the goniometer. The bioSAXS system has an in-house engineered sample storage unit that can hold up to 360 samples (20 sample holders) which keeps samples at a user-set temperature (277â K to 300â K). The MX systems were deployed in early 2017 and the bioSAXS system in early 2019.
Subject(s)
Crystallography, X-Ray/methods , Macromolecular Substances/chemistry , Robotics/methods , Equipment Design , Scattering, Small Angle , Software , Synchrotrons , X-RaysABSTRACT
The trade in falsified medicine has increased significantly and it is estimated that global falsified sales have reached $100 billion in 2020. The EU Falsified Medicines Directive states that falsified medicines do not only reach patients through illegal routes but also via the legal supply chain. Falsified medicines can contain harmful ingredients. They can also contain too little or too much active ingredient or no active ingredient at all. BARDS (Broadband Acoustic Resonance Dissolution Spectroscopy) harnesses an acoustic phenomenon associated with the dissolution of a sample (tablet or powder). The resulting acoustic spectrum is unique and intrinsic to the sample and can be used as an identifier or signature profile. BARDS was evaluated in this study to determine whether a product is falsified or genuine in a rapid manner and at lower cost than many existing technologies. A range of genuine and falsified medicines, including falsified antimalarial tablets from south-east Asia, were tested, and compared to their counterpart genuine products. Significant differences between genuine and falsified doses were found in their acoustic signatures as they disintegrate and dissolve. Principal component analysis was employed to differentiate between the genuine and falsified medicines. This demonstrates that the tablets and capsules included here have intrinsic acoustic signatures which could be used to screen the quality of medicines.
Subject(s)
Counterfeit Drugs/analysis , Spectrum Analysis/methods , Powders/analysis , Tablets/analysisABSTRACT
The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence.
Subject(s)
Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/metabolism , Intercellular Junctions/metabolism , Membrane Proteins/metabolism , Nucleoside-Phosphate Kinase/metabolism , Amino Acid Sequence , Coronavirus Envelope Proteins/ultrastructure , Cryoelectron Microscopy , Humans , Protein Domains , SARS-CoV-2/physiology , Structural Homology, Protein , Structure-Activity RelationshipABSTRACT
The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these datasets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a temperature-dependent conformational landscape for Mpro, including mobile solvent interleaved between the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to counter-punch COVID-19 and combat future coronavirus pandemics.
ABSTRACT
Genetic variations in androgen metabolism may influence prostate cancer (PC) prognosis. Clinical studies consistently linked PC prognosis with four single nucleotide polymorphisms (SNPs) in the critical androgen-regulating genes: 3-beta-hydroxysteroid dehydrogenase (HSD3B1) rs1047303, 5-alpha-reductase 2 (SRD5A2) rs523349, and solute carrier organic ion (SLCO2B1) rs1789693 and rs12422149. We tested the association of four androgen-regulating SNPs, individually and combined, with PC-specific mortality in the ARIC population-based prospective cohort. Men diagnosed with PC (N = 622; 79% White, 21% Black) were followed for death (N = 350) including PC death (N = 74). Cox proportional hazards regression was used to estimate hazard ratios (HR) and 95%CI adjusting for center, age, stage, and grade at diagnosis using separate hazards for races. A priori genetic risk score (GRS) was created as the unweighted sum of risk alleles in the four pre-selected SNPs. The gain-of-function rs1047303C allele was associated PC-specific mortality among men with metastatic PC at diagnosis (HR = 4.89 per risk allele, p = 0.01). Higher GRS was associated with PC-specific mortality (per risk allele: HR = 1.26, p = 0.03). We confirmed that the gain-of-function allele in HSD3B1 rs1047303 is associated with greater PC mortality in men with metastatic disease. Additionally, our findings suggest a cumulative effect of androgen-regulating genes on PC-specific mortality; however, further validation is required.
ABSTRACT
Stability testing is essential in the pharmaceutical industry to determine product shelf- life and the conditions under which drug products should be stored. Stability testing involves a complex set of procedures, considerable cost, time, and scientific expertise to build quality, efficacy and safety in a drug formulation. This paper highlights a new complementary approach to stability testing called Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS). BARDS measurements are based on reproducible changes in the compressibility of a solvent during dissolution. It is monitored acoustically via associated changes in the frequency of induced acoustic resonances. This study presents a novel approach to track the change of various drug formulations to determine the formulation's stability. Pellets, tablet and multiple-unit pellet system (MUPS) formulations were investigated to examine the effect of polymer coating and formulation core degradation over time. In combination with minimal usage of Ultra Violet - Visible Spectroscopy, BARDS can effectively track these changes. The technique offers a rapid approach to characterizing pharmaceutical formulations. BARDS can enable rapid development of solid drug formulation dissolution and disintegration testing as an In-Process Control test and drug stability analysis. The data show that a solid oral dose formulation has an intrinsic acoustic signature specific to the method of manufacture, excipient composition and elapsed time since the production of a product. BARDS data are also indicative of which aspect of a formulation may be unstable, whether a coating, sub-coating or core. It is potentially a time-efficient, cost-effective and greener approach to testing coating stability, disintegration and overall formulation stability.