Systems genetic analysis of lignin biosynthesis in Populus tremula.

The genetic control underlying natural variation in lignin content and composition in trees is not fully understood. We performed a systems genetic analysis to uncover the genetic regulation of lignin biosynthesis in a natural 'SwAsp' population of aspen (Populus tremula) trees. We analyzed gene expression by RNA sequencing (RNA-seq) in differentiating xylem tissues, and lignin content and composition using Pyrolysis-GC-MS in mature wood of 268 trees from 99 genotypes. Abundant variation was observed for lignin content and composition, and genome-wide association study identified proteins in the pentose phosphate pathway and arabinogalactan protein glycosylation among the top-ranked genes that are associated with these traits. Variation in gene expression and the associated genetic polymorphism was revealed through the identification of 312 705 local and 292 003 distant expression quantitative trait loci (eQTL). A co-expression network analysis suggested modularization of lignin biosynthesis and novel functions for the lignin-biosynthetic CINNAMYL ALCOHOL DEHYDROGENASE 2 and CAFFEOYL-CoA O-METHYLTRANSFERASE 3. PHENYLALANINE AMMONIA LYASE 3 was co-expressed with HOMEOBOX PROTEIN 5 (HB5), and the role of HB5 in stimulating lignification was demonstrated in transgenic trees. The systems genetic approach allowed linking natural variation in lignin biosynthesis to trees´ responses to external cues such as mechanical stimulus and nutrient availability.

NMR structures of small molecules bound to a model of an RNA CUG repeat expansion.

Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntington's disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to the structured RNAs. Both Huntington's disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1×1 nucleotide UU loops (5'CUG/3'GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBP) and hence the source of its gain-of-function. Here, we report NMR-refined structures of single 5'CUG/3'GUC motifs in complex with three different small molecules, a di-guandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1, 2, and 3 formed stacking and hydrogen bonding interactions with the 5'CUG/3'GUC motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of RNA r(CUG) repeats bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.

NMR structures of small molecules bound to a model of a CUG RNA repeat expansion.

Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntington's disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to these structured RNAs. Both Huntington's disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1 × 1 nucleotide UU loops (5'CUG/3'GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBPs) and hence the source of its gain-of-function. Here, we report nuclear magnetic resonance (NMR)-refined structures of single 5'CUG/3'GUC motifs in complex with three different small molecules, a di-guandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using NMR spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1, 2, and 3 formed stacking and hydrogen bonding interactions with the 5'CUG/3'GUC motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of a r(CUG) repeat bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.

The effect of nitrogen source and levels on hybrid aspen tree physiology and wood formation.

Nitrogen can be taken up by trees in the form of nitrate, ammonium and amino acids, but the influence of the different forms on tree growth and development is poorly understood in angiosperm species like Populus. We studied the effects of both organic and inorganic forms of nitrogen on growth and wood formation of hybrid aspen trees in experimental conditions that allowed growth under four distinct steady-state nitrogen levels. Increased nitrogen availability had a positive influence on biomass accumulation and the radial dimensions of both xylem vessels and fibers, and a negative influence on wood density. An optimal level of nitrogen availability was identified where increases in biomass accumulation outweighed decreases in wood density. None of these responses depended on the source of nitrogen except for shoot biomass accumulation, which was stimulated more by treatments complemented with nitrate than by ammonium alone or the organic source arginine. The most striking difference between the nitrogen sources was the effect on lignin composition, whereby the abundance of H-type lignin increased only in the presence of nitrate. The differential effect of nitrate is possibly related to the well-known role of nitrate as a signaling compound. RNA-sequencing revealed that while the lignin-biosynthetic genes did not significantly (FDR <0.01) respond to added NO3 - , the expression of several laccases, catalysing lignin polymerization, was dependent on N-availability. These results reveal a unique role of nitrate in wood formation and contribute to the knowledge basis for decision-making in utilizing hybrid aspen as a bioresource.

Discovery of Tumor-Targeted 6-Methyl Substituted Pemetrexed and Related Antifolates with Selective Loss of RFC Transport.

Pemetrexed and related 5-substituted pyrrolo[2,3-d]pyrimidine antifolates are substrates for the ubiquitously expressed reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT) and folate receptors (FRs) which are more tumor-selective. A long-standing goal has been to discover tumor-targeted therapeutics that draw from one-carbon metabolic vulnerabilities of cancer cells and are selective for transport by FRs and PCFT over RFC. We discovered that a methyl group at the 6-position of the pyrrole ring in the bicyclic scaffold of 5-substituted 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidine antifolates 1-4 (including pemetrexed) abolished transport by RFC with modest impacts on FRs or PCFT. From molecular modeling, loss of RFC transport involves steric repulsion in the scaffold binding site due to the 6-methyl moiety. 6-Methyl substitution preserved antiproliferative activities toward human tumor cells (KB, IGROV3) with selectivity over IOSE 7576 normal ovary cells and inhibition of de novo purine biosynthesis. Thus, adding a 6-methyl moiety to 5-substituted pyrrolo[2,3-d]pyrimidine antifolates affords tumor transport selectivity while preserving antitumor efficacy.

Development of LB244, an Irreversible STING Antagonist.

The cGMP-AMP Synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway plays a critical role in sensing dsDNA localized to the cytosol, resulting in the activation of a robust inflammatory response. While cGAS-STING signaling is essential for antiviral immunity, aberrant STING activation is observed in amyotrophic lateral sclerosis (ALS), lupus, and autoinflammatory diseases such as Aicardi-Goutières syndrome (AGS) and STING associated vasculopathy with onset in infancy (SAVI). Significant efforts have therefore focused on the development of STING inhibitors. In a concurrent submission, we reported that BB-Cl-amidine inhibits STING-dependent signaling in the nanomolar range, both in vitro and in vivo. Considering this discovery, we sought to generate analogs with higher potency and proteome-wide selectivity. Herein, we report the development of LB244, which displays nanomolar potency and inhibits STING signaling with markedly enhanced proteome-wide selectivity. Moreover, LB244 mirrored the efficacy of BB-Cl-amidine in vivo. In summary, our data identify novel chemical entities that inhibit STING signaling and provide a scaffold for the development of therapeutics for treating STING-dependent inflammatory diseases.

The Influence of a Wearable-Based Reward Program on Health Care Costs: Retrospective, Propensity Score-Matched Cohort Study.

BACKGROUND: Mobile health (mHealth) technology holds great promise as an easily accessible and effective solution to improve population health at scale. Despite the abundance of mHealth offerings, only a minority are grounded in evidence-based practice, whereas even fewer have line of sight into population-level health care spending, limiting the clinical utility of such tools. OBJECTIVE: This study aimed to explore the influence of a health plan-sponsored, wearable-based, and reward-driven digital health intervention (DHI) on health care spending over 1 year. The DHI was delivered through a smartphone-based mHealth app available only to members of a large commercial health plan and leveraged a combination of behavioral economics, user-generated sensor data from the connected wearable device, and claims history to create personalized, evidence-based recommendations for each user. METHODS: This study deployed a propensity score-matched, 2-group, and pre-post observational design. Adults (≥18 years of age) enrolled in a large, national commercial health plan and self-enlisted in the DHI for ≥7 months were allocated to the intervention group (n=56,816). Members who were eligible for the DHI but did not enlist were propensity score-matched to the comparison group (n=56,816). Average (and relative change from baseline) medical and pharmacy spending per user per month was computed for each member of the intervention and comparison groups during the pre- (ie, 12 months) and postenlistment (ie, 7-12 months) periods using claims data. RESULTS: Baseline characteristics and medical spending were similar between groups (P=.89). On average, the total included sample population (N=113,632) consisted of young to middle-age (mean age 38.81 years), mostly White (n=55,562, 48.90%), male (n=46,731, 41.12%) and female (n=66,482, 58.51%) participants. Compared to a propensity score-matched cohort, DHI users demonstrated approximately US $10 per user per month lower average medical spending (P=.02) with a concomitant increase in preventive care activities and decrease in nonemergent emergency department admissions. These savings translated to approximately US $6.8 million in avoidable health care costs over the course of 1 year. CONCLUSIONS: This employer-sponsored, digital health engagement program has a high likelihood for return on investment within 1 year owing to clinically meaningful changes in health-seeking behaviors and downstream medical cost savings. Future research should aim to elucidate health behavior-related mechanisms in support of these findings and continue to explore novel strategies to ensure equitable access of DHIs to underserved populations that stand to benefit the most.

Simple monocyclic pyrimidine analogs as microtubule targeting agents binding to the colchicine site.

Complex natural products that bind to tubulin/microtubules come under the broad category of microtubule binding agents. The design of simplified analogs of previously reported bicyclic, microtubule depolymerizer, pyrrolo[2,3-d]pyrimidine, provided valuable structure-activity relationship data and led to the identification of novel monocyclic pyrimidine analogs of which 12 was 47-fold more potent (EC50 123 nM) for cellular microtubule depolymerization activity and 7.5-fold more potent (IC50 24.4 nM) at inhibiting the growth of MDA-MB-435 cancer cells, suggesting significantly better binding of the target within the colchicine site of tubulin compared to lead compound 1. This compound and others of this series of monocyclic pyrimidine analogs were able to overcome multidrug resistance due to the expression of the ßIII-isotype of tubulin and P-glycoprotein. In vivo evaluation of the most potent analog 12 in an MDA-MB-435 xenograft mouse model indicated, along with paclitaxel, that both compounds showed a trend towards lower tumor volume however neither compound showed significant antitumor activity in the trial. To our knowledge these are the first examples of simple substituted monocyclic pyrimidines as colchicine site binding antitubulin compounds with potent antitumor activity.

PM sensors as an indicator of overall air quality: Pre-COVID and COVID periods.

Nowadays, there has been a substantial proliferation in the use of low-cost particulate matter (PM) sensors and facilitating as an indicator of overall air quality. However, during COVID-19 epidemics, air pollution sources have been deteriorated significantly, and given offer to evaluate the impact of COVID-19 on air quality in the world's most polluted city: Delhi, India. To address low-cost PM sensors, this study aimed to a) conduct a long-term field inter-comparison of twenty-two (22) low-cost PM sensors with reference instruments over 10-month period (evaluation period) spanning months from May 2019 to February 2020; b) trend of PM mass and number count; and c) probable local and regional sources in Delhi during Pre-CVOID (P-COVID) periods. The comparison of low-cost PM sensors with reference instruments results found with R2 ranging between 0.74 and 0.95 for all sites and confirm that PM sensors can be a useful tool for PM monitoring network in Delhi. Relative reductions in PM2.5 and particle number count (PNC) due to COVID-outbreaks showed in the range between (2-5%) and (4-13%), respectively, as compared to the P-COVID periods. The cluster analysis reveals air masses originated ∼52% from local, while ∼48% from regional sources in P-COVID and PM levels are encountered 47% and 66-70% from local and regional sources, respectively. Overall results suggest that low-cost PM sensors can be used as an unprecedented aid in air quality applications, and improving non-attainment cities in India, and that policy makers can attempt to revise guidelines for clean air.

Assessment of Infectious Diseases Risks From Dental Aerosols in Real-World Settings.

Background: Infectious diseases physicians are leaders in assessing the health risks in a variety of community settings. An understudied area with substantial controversy is the safety of dental aerosols. Previous studies have used in vitro experimental designs and/or indirect measures to evaluate bacteria and viruses from dental surfaces. However, these findings may overestimate the occupational risks of dental aerosols. The purpose of this study was to directly measure dental aerosol composition to assess the health risks for dental healthcare personnel and patients. Methods: We used a variety of aerosol instruments to capture and measure the bacterial, viral, and inorganic composition of aerosols during a variety of common dental procedures and in a variety of dental office layouts. Equipment was placed in close proximity to dentists during each procedure to best approximate the health risk hazards from the perspective of dental healthcare personnel. Devices used to capture aerosols were set at physiologic respiration rates. Oral suction devices were per the discretion of the dentist. Results: We detected very few bacteria and no viruses in dental aerosols-regardless of office layout. The bacteria identified were most consistent with either environmental or oral microbiota, suggesting a low risk of transmission of viable pathogens from patients to dental healthcare personnel. When analyzing restorative procedures involving amalgam removal, we detected inorganic elements consistent with amalgam fillings. Conclusions: Aerosols generating from dental procedures pose a low health risk for bacterial and likely viral pathogens when common aerosol mitigation interventions, such as suction devices, are employed.

Transcriptome-Wide Mapping of Small-Molecule RNA-Binding Sites in Cells Informs an Isoform-Specific Degrader of QSOX1 mRNA.

The interactions between cellular RNAs in MDA-MB-231 triple negative breast cancer cells and a panel of small molecules appended with a diazirine cross-linking moiety and an alkyne tag were probed transcriptome-wide in live cells. The alkyne tag allows for facile pull-down of cellular RNAs bound by each small molecule, and the enrichment of each RNA target defines the compound's molecular footprint. Among the 34 chemically diverse small molecules studied, six bound and enriched cellular RNAs. The most highly enriched interaction occurs between the novel RNA-binding compound F1 and a structured region in the 5' untranslated region of quiescin sulfhydryl oxidase 1 isoform a (QSOX1-a), not present in isoform b. Additional studies show that F1 specifically bound RNA over DNA and protein; that is, we studied the entire DNA, RNA, and protein interactome. This interaction was used to design a ribonuclease targeting chimera (RIBOTAC) to locally recruit Ribonuclease L to degrade QSOX1 mRNA in an isoform-specific manner, as QSOX1-a, but not QSOX1-b, mRNA and protein levels were reduced. The RIBOTAC alleviated QSOX1-mediated phenotypes in cancer cells. This approach can be broadly applied to discover ligands that bind RNA in cells, which could be bioactive themselves or augmented with functionality such as targeted degradation.

Comparison of aerosol mitigation strategies and aerosol persistence in dental environments.

OBJECTIVE: To determine the impact of various aerosol mitigation interventions and to establish duration of aerosol persistence in a variety of dental clinic configurations. METHODS: We performed aerosol measurement studies in endodontic, orthodontic, periodontic, pediatric, and general dentistry clinics. We used an optical aerosol spectrometer and wearable particulate matter sensors to measure real-time aerosol concentration from the vantage point of the dentist during routine care in a variety of clinic configurations (eg, open bay, single room, partitioned operatories). We compared the impact of aerosol mitigation strategies (eg, ventilation and high-volume evacuation (HVE), and prevalence of particulate matter) in the dental clinic environment before, during, and after high-speed drilling, slow-speed drilling, and ultrasonic scaling procedures. RESULTS: Conical and ISOVAC HVE were superior to standard-tip evacuation for aerosol-generating procedures. When aerosols were detected in the environment, they were rapidly dispersed within minutes of completing the aerosol-generating procedure. Few aerosols were detected in dental clinics, regardless of configuration, when conical and ISOVAC HVE were used. CONCLUSIONS: Dentists should consider using conical or ISOVAC HVE rather than standard-tip evacuators to reduce aerosols generated during routine clinical practice. Furthermore, when such effective aerosol mitigation strategies are employed, dentists need not leave dental chairs fallow between patients because aerosols are rapidly dispersed.

Bioinformatic Searching for Optimal RNA Targets of Dimeric Compounds Informs Design of a MicroRNA-27a Inhibitor.

Various studies have shown that selective molecular recognition of RNA targets by small molecules in cells, although challenging, is indeed possible. One facile strategy to enhance selectivity and potency is binding two or more sites within an RNA simultaneously with a single molecule. To simplify the identification of targets amenable to such a strategy, we informatically mined all human microRNA (miRNA) precursors to identify those with two proximal noncanonically paired sites. We selected oncogenic microRNA-27a (miR-27a) for further study as a lead molecule binds its Drosha site and a nearby internal loop, affording a homodimer that potently and specifically inhibits miR-27a processing in both breast cancer and prostate cancer cells. This reduction of mature miR-27a ameliorates an oncogenic cellular phenotype with nanomolar activity. Collectively, these studies demonstrate that synergistic bioinformatic and experimental approaches can define targets that may be more amenable to small molecule targeting than others.

Machine Learning for Health: Algorithm Auditing & Quality Control.

Developers proposing new machine learning for health (ML4H) tools often pledge to match or even surpass the performance of existing tools, yet the reality is usually more complicated. Reliable deployment of ML4H to the real world is challenging as examples from diabetic retinopathy or Covid-19 screening show. We envision an integrated framework of algorithm auditing and quality control that provides a path towards the effective and reliable application of ML systems in healthcare. In this editorial, we give a summary of ongoing work towards that vision and announce a call for participation to the special issue  Machine Learning for Health: Algorithm Auditing & Quality Control in this journal to advance the practice of ML4H auditing.

A Small Molecule Exploits Hidden Structural Features within the RNA Repeat Expansion That Causes c9ALS/FTD and Rescues Pathological Hallmarks.

The hexanucleotide repeat expansion GGGGCC [r(G4C2)exp] within intron 1 of C9orf72 causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD. , the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r(G4C2)exp RNA gain- and loss-of-function mechanisms. The r(G4C2)exp RNA folds into both a hairpin structure with repeating 1 × 1 nucleotide GG internal loops and a G-quadruplex structure. Here, we report the identification of a small molecule (CB253) that selectively binds the hairpin form of r(G4C2)exp. Interestingly, the small molecule binds to a previously unobserved conformation in which the RNA forms 2 × 2 nucleotide GG internal loops, as revealed by a series of binding and structural studies. NMR and molecular dynamics simulations suggest that the r(G4C2)exp hairpin interconverts between 1 × 1 and 2 × 2 internal loops through the process of strand slippage. We provide experimental evidence that CB253 binding indeed shifts the equilibrium toward the 2 × 2 GG internal loop conformation, inhibiting mechanisms that drive c9ALS/FTD pathobiology, such as repeat-associated non-ATG translation formation of stress granules and defective nucleocytoplasmic transport in various cellular models of c9ALS/FTD.

Protection levels of N95-level respirator substitutes proposed during the COVID-19 pandemic: safety concerns and quantitative evaluation procedures.

OBJECTIVE: The COVID-19 pandemic has precipitated widespread shortages of filtering facepiece respirators (FFRs) and the creation and sharing of proposed substitutes (novel designs, repurposed materials) with limited testing against regulatory standards. We aimed to categorically test the efficacy and fit of potential N95 respirator substitutes using protocols that can be replicated in university laboratories. SETTING: Academic medical centre with occupational health-supervised fit testing along with laboratory studies. PARTICIPANTS: Seven adult volunteers who passed quantitative fit testing for small-sized (n=2) and regular-sized (n=5) commercial N95 respirators. METHODS: Five open-source potential N95 respirator substitutes were evaluated and compared with commercial National Institute for Occupational Safety and Health (NIOSH)-approved N95 respirators as controls. Fit testing using the 7-minute standardised Occupational Safety and Health Administration fit test was performed. In addition, protocols that can be performed in university laboratories for materials testing (filtration efficiency, air resistance and fluid resistance) were developed to evaluate alternate filtration materials. RESULTS: Among five open-source, improvised substitutes evaluated in this study, only one (which included a commercial elastomeric mask and commercial HEPA filter) passed a standard quantitative fit test. The four alternative materials evaluated for filtration efficiency (67%-89%) failed to meet the 95% threshold at a face velocity (7.6 cm/s) equivalent to that of a NIOSH particle filtration test for the control N95 FFR. In addition, for all but one material, the small surface area of two 3D-printed substitutes resulted in air resistance that was above the maximum in the NIOSH standard. CONCLUSIONS: Testing protocols such as those described here are essential to evaluate proposed improvised respiratory protection substitutes, and our testing platform could be replicated by teams with similar cross-disciplinary research capacity. Healthcare professionals should be cautious of claims associated with improvised respirators when suggested as FFR substitutes.

Massively Parallel Optimization of the Linker Domain in Small Molecule Dimers Targeting a Toxic r(CUG) Repeat Expansion.

RNA contributes to disease pathobiology and is an important therapeutic target. The downstream biology of disease-causing RNAs can be short-circuited with small molecules that recognize structured regions. The discovery and optimization of small molecules interacting with RNA is, however, challenging. Herein, we demonstrate a massively parallel one-bead-one-compound methodology, employed to optimize the linker region of a dimeric compound that binds the toxic r(CUG) repeat expansion [r(CUG)exp] causative of myotonic dystrophy type 1 (DM1). Indeed, affinity selection on a 331,776-member library allowed the discovery of a compound with enhanced potency both in vitro (10-fold) and in DM1-patient-derived myotubes (5-fold). Molecular dynamics simulations revealed additional interactions between the optimized linker and the RNA, resulting in ca. 10 kcal/mol lower binding free energy. The compound was conjugated to a cleavage module, which directly cleaved the transcript harboring the r(CUG)exp and alleviated disease-associated defects.

Genome-wide transcriptional analysis unveils the molecular basis of organ-specific expression of isosteroidal alkaloids biosynthesis in critically endangered Fritillaria roylei Hook.

Fritillaria roylei Hook. is a critically endangered high altitude Himalayan medicinal plant species with rich source of pharmaceutically active structurally diverse steroidal alkaloids. Nevertheless, except few marker compounds, the chemistry of the plant remains unexplored. Therefore, in the current study, transcriptome sequencing efforts were made to elucidate isosteroidal alkaloids biosynthesis by creating first organ-specific genomic resource using bulb, stem, and leaf tissues derived from natural populations of Indian Himalayan region. Overall, 349.9 million high quality paired-end reads obtained using NovaSeq 6000 platform were assembled (de novo) into 82,848 unigenes and 31,061 isoforms. Functional annotation and organ specific differential expression (DE) analysis identified 2488 significant DE transcripts, grouped into three potential sub-clusters (sub-cluster I: 728 transcripts; sub-cluster II: 446 transcripts and Sub-cluster III: 1314 transcripts). Subsequently, pathway enrichment (GO, KEGG) and protein-protein network analysis revealed significantly higher enrichment of phenyl-propanoid and steroid backbone including terpenoid, sesquiterpenoid and triterpenoid biosynthesis in bulb. Additionally, upregulated expression of cytochrome P450, UDP-dependent Glucuronosyltransferase families and key transcription factor families (FAR1, bHLH, GRAS, C2H2, TCP and MYB) suggests 'bulb' as a primary site of MVA mediated isosteroidal alkaloids biosynthesis. The comprehensive elucidation of molecular insights in this study is a first step towards the understanding of isosteroidal alkaloid biosynthesis pathway in F. roylei. Furthermore, key genes and regulators identified here can facilitate metabolic engineering of potential bioactive compounds at industrial scale.