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Lymphangioleiomyomatosis (LAM) is a progressive destructive neoplasm of the lung associated with inactivating mutations in the TSC1 or TSC2 tumor suppressor genes. Cell or animal models that accurately reflect the pathology of LAM have been challenging to develop. Here, we generated a robust human cell model of LAM by reprogramming TSC2 mutation–bearing fibroblasts from a patient with both tuberous sclerosis complex (TSC) and LAM (TSC-LAM) into induced pluripotent stem cells (iPSC), followed by selection of cells that resemble those found in LAM tumors by unbiased in vivo differentiation. We established expandable cell lines under smooth muscle cell (SMC) growth conditions that retained a patient-specific genomic TSC2+/− mutation and recapitulated the molecular and functional characteristics of pulmonary LAM cells. These include multiple indicators of hyperactive mTORC1 signaling, presence of specific neural crest and SMC markers, expression of VEGF-D and female sex hormone receptors, reduced autophagy, and metabolic reprogramming. Intriguingly, the LAM-like features of these cells suggest that haploinsufficiency at the TSC2 locus contributes to LAM pathology, and demonstrated that iPSC reprogramming and SMC lineage differentiation of somatic patient cells with germline mutations was a viable approach to generate LAM-like cells. The patient-derived SMC lines we have developed thus represent a novel cellular model of LAM that can advance our understanding of disease pathogenesis and develop therapeutic strategies against LAM. Cancer Res; 77(20); 5491–502. ©2017 AACR.

This file contains the full descriptive legends for Supplementary Figures S1-S7.

This file provides supplementary data pertaining to TSC2 mutation and protein expression analysis in human fibroblast cell lines (Figure S1); as well as Karyotypes (Figure S2), in vitro and in vivo pluripotency and differentiation potential (Figure S3), and genotyping mutation analysis (Figure S4) of human iPSC lines used in this study. Supplementary data regarding human-derived SMC lines, with and without TSC2 mRNA knock-down, specifically cell size analysis (Figure S5), gp100 and ER- expression (Figure S6) and cell death analysis (Figure S7), is also shown.

This file contains descriptions of materials and methods used in this study pertaining to cell and tissue staining, Western blot and mRNA analysis, and cell size and genotyping analyses. Corresponding references are also included.

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring constitutive mTORC1 signaling. Rapamycin is the only clinically approved disease-modifying treatment, but its action is cytostatic and disease progresses upon its withdrawal. There is a critical need to identify novel agents that prevent the invasive phenotype and/or eradicate the neoplastic LAM cells. Here, we employed novel cellular and extracellular models to screen for candidate therapeutics in a physiologically relevant setting. We observed that lung-mimetic hydrogel culture of pluripotent stem cell-derived diseased cells more faithfully recapitulates human LAM biology compared to conventional culture on two-dimensional tissue culture plastic. Leveraging our culture system, we conducted a three-dimensional drug screen using a custom 800-compound library, tracking cytotoxicity and invasion modulation phenotypes at the single cell level. We identified histone deacetylase (HDAC) inhibitors as a group of anti-invasive agents that are also selectively cytotoxic towards TSC2-/- cells. Unexpectedly, we observed that next generation ATP-competitive mTORC1/2 inhibitors potentiate invasion. We determined anti-invasive effects of HDAC inhibitors to be independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Drug performance was subsequently evaluated at the single cell level in zebrafish xenografts. We observed consistent therapeutic efficacy in vivo at equivalent concentrations to those used in vitro, substantiating HDAC inhibitors as potential therapeutic candidates for pursuit in patients with LAM.One Sentence SummaryWe performed a drug screen in 3D and discovered HDAC inhibitors exhibit therapeutic efficacy in models of the lung disease lymphangioleiomyomatosis.

Renal angiomyolipomas (R-AMLs) are hamartomatous kidney tumors which stain positively for markers of adipocytic, vascular, smooth muscle, and melanocytic lineages. These lesions possess loss of function mutations in either TSC1 or TSC2, which are canonical negative regulators of mTORC1 signaling. To date, there exists no in vitro or in vivo model which faithfully recapitulates the architectural and molecular complexity of R-AMLs. Considering these lesions can be detected very early in life- even congenitally- we hypothesized they arise as a consequence of aberrant tissue development. To test this hypothesis, we generated TSC1-/- and TSC2-/- mutants in four human pluripotent stem cell (hPSC) backgrounds using CRISPR/Cas9 genome engineering. Wild type hPSCs differentiated into renal organoids express markers of the glomerulus, proximal and distal tubules, in a topology that resembles human nephron patterning. Remarkably, wild type renal organoids downregulate mTORC1 signaling compared to adjacent undifferentiated cells. In contrast, both TSC1-/- and TSC2-/- hPSCs exhibit substantial lineage infidelity upon differentiation, staining positively for adipocytic and melanocytic markers which are absent in matched wild type controls. Additionally, knockout lines formed nodular growths with disorganized architecture, resembling the hamartomatous organization of R-AML lesions. These lesions exhibit hyperactive mTORC1 signaling, consistent with human pathology. Together, these data suggest three primary findings: loss of TSC1/2 drives lineage infidelity; TSC1/2 may be required for architectural organization of the kidney parenchyma; and a developmental approach to R-AML modelling may best recapitulate the human disease. Citation Format: Adam Pietrobon, Sean P. Delaney, Carole Doré, Julien Yockell-Lelievre, Lisa Julian, William L. Stanford. Loss of TSC1 or TSC2 drives lineage infidelity and hamartoma formation in a renal organoid model of angiomyolipoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2974.

Lymphangioleiomyomatosis (LAM) is a progressive destructive neoplasm of the lung associated with inactivating mutations in the TSC1 or TSC2 tumor suppressor genes. Cell or animal models that accurately reflect the pathology of LAM have been challenging to develop. Here, we generated a robust human cell model of LAM by reprogramming TSC2 mutation–bearing fibroblasts from a patient with both tuberous sclerosis complex (TSC) and LAM (TSC-LAM) into induced pluripotent stem cells (iPSC), followed by selection of cells that resemble those found in LAM tumors by unbiased in vivo differentiation. We established expandable cell lines under smooth muscle cell (SMC) growth conditions that retained a patient-specific genomic TSC2+/− mutation and recapitulated the molecular and functional characteristics of pulmonary LAM cells. These include multiple indicators of hyperactive mTORC1 signaling, presence of specific neural crest and SMC markers, expression of VEGF-D and female sex hormone receptors, reduced autophagy, and metabolic reprogramming. Intriguingly, the LAM-like features of these cells suggest that haploinsufficiency at the TSC2 locus contributes to LAM pathology, and demonstrated that iPSC reprogramming and SMC lineage differentiation of somatic patient cells with germline mutations was a viable approach to generate LAM-like cells. The patient-derived SMC lines we have developed thus represent a novel cellular model of LAM that can advance our understanding of disease pathogenesis and develop therapeutic strategies against LAM. Cancer Res; 77(20); 5491–502. ©2017 AACR.

Lignin depolymerization represents a promising approach to the sustainable production of aromatic molecules. One potential approach to the stepwise depolymerization of lignin involves oxidation of the benzylic alcohol group in β-O-4 and β-1 linkages, followed by Baeyer-Villiger oxidation (BVO) of the resulting ketones and subsequent ester hydrolysis. Towards this goal, BVO reactions were performed on 2-adamantanone, a series of acetophenone derivatives, and lignin model compounds using a tin beta zeolite/hydrogen peroxide biphasic system. XRD, 119Sn MAS NMR spectroscopy, DRUVS and XPS were used to determine tin speciation in the catalyst, the presence of both framework Sn and extra framework SnO2 being inferred. Conversion of ketones to BVO products was affected by electron donation as well as steric hindrance, 4′-methoxyacetophenone affording the highest yield of ester (81%). As the size and complexity of the ketone increased, excess hydrogen peroxide was typically needed for successful BVO. Yields of ester products derived from β-O-4 and β-1 lignin models were modest due to the formation of polymeric material stemming from direct ring hydroxylation.

Magnesium stearate is the salt of a complex mixture of fatty acids, with the majority being stearate and palmitate. It has multiple crystalline forms and, potentially, an amorphous form. Magnesium stearate is used in the pharmaceutical manufacturing industry as a powder lubricant, and typically is added at low levels (∼1%) during the manufacturing process and blended for a relatively short time (∼5 min). Proper levels and mixing times are needed, as too short a mixing time or too small a quantity will result in improper lubrication, and too much can negatively impact dissolution rates. The complex mixture of multiple fatty acids and crystalline forms in magnesium stearate leads to variability between commercial sources, and switching between sources can impact both the amount of lubricant and mixing time needed for proper lubrication. In order to better understand the complex nature of magnesium stearate, a variety of analytical techniques were used to characterize both synthesized and commercial magnesium stearate samples. The results show that correlation among differential scanning calorimetry, thermogravimetric analysis, solid-state NMR spectroscopy, and other techniques provides a unique insight into the forms of magnesium stearate. Finally, the ability to monitor form changes of magnesium stearate in an intact tablet using solid-state NMR spectroscopy is shown.

In the current investigation, the role of drug-polymer hydrogen bonding (H-bonding) with respect to the phase behavior of amorphous solid dispersions (ASDs) is studied in depth on a nanometer level. Melt-quenched dispersions of felodipine (FEL) with poly(vinylpyrrolidone), or PVP, poly(vinylpyrrolidone-co-vinylacetate), or PVP/VA, and poly(vinylacetate), or PVAc, were prepared at drug loadings of 50-90% w/w. Modulated differential scanning calorimetry (MDSC) was used to detect microscopic homogeneity for each set of ASDs. A single composition dependent glass transition temperature (Tg) was observed over the entire composition range in MDSC data for each set of ASDs; however some samples within each set of ASDs showed a crystallization exotherm and corresponding melting endotherm in the first heating scan. Solid-state nuclear magnetic resonance spectroscopy (SSNMR) was further employed to understand phase homogeneity in these systems. The proton spin-lattice relaxation times in the laboratory and rotating frame (1H T1 and T1ρ) for the drug and individual polymer for each set of ASDs were measured to evaluate phase homogeneity. On the basis of proton relaxation measurements, it was revealed that FEL:PVP and FEL:PVP/VA ASDs exhibited better compositional homogeneity than FEL:PVAc ASDs. The strength and the extent of H-bonding were studied by using 13C SSNMR spectra. In addition, deconvolution of the carbonyl region of amorphous FEL revealed that 40% of amorphous FEL molecules were hydrogen bonded (H-bonded) through dimers and the remaining 60% were free/non H-bonded. The dimer fraction decreased as the polymer content increased for each set of ASDs, while the free fraction increased. This indicated that the polymers containing hydrogen bond acceptor groups disrupted dimers and formed intermolecular H-bonding interactions with FEL. The strength and extent of FEL:polymer H-bonding was rank ordered as PVP > PVP/VA > PVAc. These findings were also confirmed through DFT calculations on these systems. Our results suggest that drug-polymer H-bonding interaction may impact the phase homogeneity in ASDs formulated by a specific method. The data from the current study further demonstrate that SSNMR is a powerful tool for characterizing phase homogeneity in ASDs with sub-50 nm resolution. In addition, SSNMR can provide insights into drug-polymer interactions and speciation in ASDs.

SUMMARY TSC2 inactivating mutations elicit mTORC1 hyperactivation and underlie neural and mesenchymal tumorigenesis in tuberous sclerosis complex (TSC). Given the paucity of humanized and lineage-relevant experimental systems relevant to TSC we have developed the first multi-system TSC model, based on CRISPR/Cas9-mediated knockout of TSC2 in human pluripotent stem cells (hPSCs) and their differentiation into putative cell types driving TSC tumors and neurological dysfunction. Mesenchymal neural crest cell (NCC) differentiation recapitulates critical features of lung and kidney tumors, while neural lineage induction of TSC2−/− hPSCs progressively models aberrant neuronal, glial and neural precursor cells (NPCs) that constitute TSC brain tumors. We additionally provide an RNA-sequencing (RNA-seq) resource in which multiple time-points during induction of WT and TSC2−/− cells into neural and neural crest lineages were profiled. Temporal RNA-seq analyses exposed a massive proteostatic stress response activated acutely during neuroepithelial induction in TSC2-deficient cells. Remarkably, this is resolved upon NCC specification but leads to endosomal and metabolic reprogramming in NPCs. Furthermore, TSC2−/− NPCs, but not NCCs, are uniquely vulnerable to death via proteasome inhibition independent of mTORC1 regulation, highlighting cell type-specific proteostatic states. Thus, our data reveal that mechanisms regulating proteostasis stress are strikingly lineage-specific in TSC2−/−cells, and these differences are acquired developmentally. This drives long-term catabolic adaptations specifically in neural cells that counter typical mTORC1 paradigms, resulting in previously unrecognized tumor/lineage-specific therapeutic sensitivities. Overall, our work exemplifies the complexity of elucidating underlying biological mechanisms and therapeutic approaches for multisystem diseases, illustrating the power of utilizing hPSC disease models with tissue-specific relevance.

This study quantifies the lubricating efficiency of two grades of crystalline vegetable-derived magnesium stearate (MgSt-V) using the DM3 approach, which utilizes design of experiments (D) and multivariate analysis techniques (M3) to evaluate the effect of a material's (M1) molecular and macroscopic properties and manufacturing factors (M2) on critical product attributes. A 23 factorial design (2 continuous variables plus 1 categorical factor) with three center points for each categorical factor was used to evaluate the effect of MgSt-V fraction and blend time on running powder basic flow energy (BFE), tablet mechanical strength (TMS), disintegration time (DT), and running powder lubricant sensitivity ratio (LSR). Molecular characterization of MgSt-V employed moisture sorption–desorption analysis, 13C nuclear magnetic resonance spectroscopy, thermal analysis, and powder X-ray diffraction. MgSt-V macroscopic analysis included mean particle size, specific surface area, particle morphology, and BFE. Principal component analysis and partial least squares multivariate analysis techniques were used to develop predictive qualitative and quantitative relationships between the molecular and macroscopic properties of MgSt-V grades, design variables, and resulting tablet formulation properties. MgSt-V fraction and blending time and their square effects showed statistical significant effects. Significant variation in the molecular and macroscopic properties of MgSt-V did not have a statistically significant impact on the studied product quality attributes (BFE, TMS, DT, and LSR). In setting excipient release specifications, functional testing may be appropriate in certain cases to assess the effect of statistically significant different molecular and macroscopic properties on product quality attributes.

Lymphangioleiomyomatosis (LAM) is a rare lung cancer characterized by immature smooth muscle-like cell invasion of the lung parenchyma, leading to cystonodular destruction and respiratory decline. Interestingly, LAM is a monogetic disease caused by loss of TSC2, conferring constitutive mTORC1 activation. Currently, there are no approved therapeutics which exhibit LAM cell-specific cytotoxicity. Here, we describe a novel 3D drug screening platform which couples a rationally designed, synthetic, lung-mimetic hydrogel to high content imaging. We have designed and synthesized a hyaluronic acid-based hydrogel which mimics endogenous lung tissue. Human LAM cell models maintain mTORC1 activation in the hydrogel compared to matched controls, reflective of human LAM lesions. We observe LAM cells to exhibit both protease-dependent and independent modes of invasion: the former imparted by gel crosslinking with an MMP-cleavable peptide, the latter enabled by methylcellulose conjugation. Proliferation is inhibited as LAM cells actively invade through the gel. We coupled our culture system to high content confocal microscopy, permitting measurements of cell viability and invasion depth in response to therapeutic screening. We subsequently tested an 800-drug library of Health Canada-approved small molecule cancer therapeutics on LAM cells and matched controls. Surprisingly, LAM cells exhibited pan-therapeutic resistance as measured by both invasion and viability metrics. Enrichment analysis revealed categories of therapeutics which demonstrated LAM-selective cytotoxicity and/or anti-invasiveness. We performed a refinement screen on select therapeutics and identified AURA inhibition as an effective LAM cell-specific therapeutic avenue. Work is ongoing to establish the most efficacious small molecule inhibitor. In conclusion, we describe a novel drug screening platform which enables high content measurements of viability and invasion in response to drug screening in a lung-mimetic system. We have identified AURA inhibition as a potential therapeutic avenue for LAM patients using this screening platform. Future work will involve target validation, elucidation of the mechanism of action, and testing in a pre-clinical mouse model. As these small molecules are Health Canada-approved, we anticipate rapid clinical translation of our preferred candidate. Citation Format: Adam Pietrobon, Julien Yockell-Lelievre, Carole Doré, Roger Y. Tam, Sean P. Delaney, Molly Shoichet, William L. Stanford. High throughput small molecule screening with synthetic 3D lung-mimetic hydrogels in the rare lung cancer lymphangioleiomyomatosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6316.

Flufenamic acid is the most polymorphic rich molecular solid currently known. Here, the origins of the two most common polymorphs of flufenamic acid (Form I and Form III) were investigated using terahertz spectroscopy and solid-state density functional theory. Terahertz spectroscopy probes the low-frequency vibrations that are sensitive to both the intermolecular and intramolecular interactions of molecules in the solid-state. This ability makes terahertz spectroscopy a particularly useful tool for characterizing polymorphic systems, where slight changes in packing arrangements distinguish different polymorphs. Solid-state density functional theory provides a means to achieve better understanding of the chemical origins of the low-frequency vibrations, while also revealing the energetic details of the polymorph relative stabilities. The exploration of these flufenamic acid polymorphs revealed that molecular conformation drives the formation of these particular forms, specifically the magnitude and orientation of the molecular dipoles dictate the preferred solid-state packing arrangements.

Two series of thermosensitive hydrogels were synthesized by copolymerizing N-isopropylacrylamide (NIPAAm) with various contents of novel hydrophobic crosslinkers, curcumin multiacrylate (CMA) and quercetin multiacrylate (QMA). The compositions of the resulting hydrogels were characterized using solid state-NMR (ss-NMR), and the temperature dependent swelling behavior and lower critical solution temperature (LCST) were characterized using swelling studies and differential scanning calorimetry (DSC). Increasing the crosslinker content resulted in a significant decrease in the LCST and swelling ratio of hydrogels, which could be attributed to the increased hydrophobicity introduced by CMA or QMA. All of the hydrogels demonstrated temperature responsive swelling with the extent of swelling decreasing with increasing crosslinker content. The lower crosslinker content gels displayed sharper phase transitions, while the high crosslinker content gels had broader phase transitions.

A new crystalline polymorph (Form VIII) of the antidepressant drug aripiprazole has been discovered, resulting from a previously unknown enantiotropic phase transition from Form II at 225 K. This finding makes aripiprazole one of the most polymorphic flexible organic solids currently known, equaling flufenamic acid in number of solved forms (eight polymorphs). Enantiotropic solid–solid phase transitions are relatively uncommon for pharmaceuticals; however, for the aripiprazole system, such phase transitions play a central role in several of its polymorphic transformations. A combination of solid-state density functional theory and single-crystal X-ray diffraction has been used to investigate the energies involved in the formation of Form VIII. This work reveals that Form VIII is stable despite containing molecules with unfavorable conformations. The stability of this polymorph originates from improved intermolecular binding energy due to enhanced London dispersion forces at low temperatures.

The previously unknown crystal structure of α,α,α′,α′-tetrabromo- p -xylene has been determined using single-crystal X-ray diffraction, and it was discovered that the molecular packing primarily involves electrostatic interactions between neighboring molecules. Due to the unusual nature of the packing in this system, the vibrational motions were investigated. The low energy lattice and molecular vibrations unique to molecular crystals were measured in the terahertz (THz) region (10–100 cm −1 ). These vibrational motions were then reproduced using solid-state density functional theory (DFT) as implemented in CRYSTAL09, and it was found that the majority of the motions in this region involve either whole molecular translation or molecular scissoring.

Polymorphism in molecular solids is driven by the competition between internal molecular conformational strain and external binding forces. The pharmaceutical gabapentin exhibits three polymorphic forms each differing in molecular conformation and intermolecular interactions. To improve the understanding of the polymorphic formation, terahertz spectroscopy and solid-state density functional theory have been applied in the analysis of the factors leading to these particular crystalline arrangements. It was found that the most stable solid-state polymorph contained gabapentin molecules under considerable internal strain. The origins of this unexpected stability are described in terms of intramolecular conformational changes and the amount of cohesive binding energy present in each polymorph. Finally, the temperature dependence of the polymorph stabilities is investigated by calculation of the free–energy curves of each form and determining the solid–solid transition points connecting the various phases.

The relative stabilities of crystalline polymorphs are an important aspect of the manufacturing and effective utilization of pharmaceuticals. These stabilities are driven by both molecular conformational energy within the solid-state components and cohesive binding energy of the crystalline arrangement. The combined approach of experimental vibrational terahertz spectroscopy with solid-state density functional theory provides a powerful tool to study such properties and is applied here in the analysis of conformational polymorphism in crystalline aripiprazole. The low-frequency (

The characterization of crystalline polymorphs of drug molecules is an area of great interest since these variations in solid-state structure directly influence the physical properties of such substances. Terahertz spectroscopy provides a powerful analytical tool for these investigations and has been used here to study tautomeric polymorphism and conformational disorder in crystallized irbesartan, an antihypertensive medication. The low-frequency (

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin‐1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems‐level analyses, we compiled a MKRN1‐centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA‐binding proteins, and ensuing RIP‐chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule‐resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems‐level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.

Terahertz spectroscopy probes the low-frequency vibrations that are sensitive to both the intermolecular and intramolecular interactions of molecules in the solid state. Thus, terahertz spectroscopy can be a useful tool in the investigation of crystalline pharmaceutical compounds, where slight changes in the packing arrangement can modify the overall effectiveness of a drug formulation. This is especially true for cases of polymorphic systems, hydrates/solvates, and cocrystals. In this work, the cocrystal of flufenamic acid with nicotinamide was investigated using terahertz spectroscopy and solid-state density functional theory. The solid-state simulations enable understanding of the low-frequency vibrations seen in the terahertz spectra, while also providing insight into the energetics involved in the formation of the cocrystal. The comparison of the cocrystal to the pure forms of the molecular components reveals that the cocrystal has better overall binding energy, driven by increased intermolecular hydrogen bond strength and greater London dispersion forces and that the trifluoromethyl torsional potential is significantly different between the studied solids.

A new high-temperature polymorph of 5-(4-pyridyl)tetrazole has been discovered and characterized using X-ray crystallography and terahertz (THz) spectroscopy. The THz spectrum of the new polymorph was compared to the previously published form and was replicated by means of solid-state density functional theory. Terahertz spectroscopy was used to determine the influence of the different packing motifs on the molecular and low energy lattice vibrations displayed in the region from 10 to 100 cm(-1). It was found that there is only a ∼2 cm(-1) difference in the primary peak location, caused by a whole molecule rotation along the principal a axis, between the two polymorphic forms. In addition, the energy of formation was determined, and it was found that the previously known polymorphic form is more stable by ∼0.25 kJ/mol, compared to the newly discovered form.

Terahertz spectroscopy is sensitive to the interactions between molecules in the solid-state and recently has emerged as a new analytical tool for investigating polymorphism. Here, this technique is applied for the first time to the phenomenon of tautomeric polymorphism where the crystal structures of anthranilic acid (2-aminobenzoic acid) have been investigated. Three polymorphs of anthranilic acid (denoted Forms I, II and III) were studied using terahertz spectroscopy and the vibrational modes and relative polymorph stabilities analyzed using solid-state density functional theory calculations augmented with London dispersion force corrections. Form I consists of both neutral and zwitterionic molecules and was found to be the most stable polymorph as compared to Forms II and III (both containing only neutral molecules). The simulations suggest that a balance between steric interactions and electrostatic forces is responsible for the favoring of the mixed neutral/zwitterion solid over the all neutral or all zwitterion crystalline arrangements.

Arabidopsis thaliana contains two salicylic acid (SA) glucosyltransferase enzymes designated UGT74F1 and UGT74F2. UGT74F1 forms only SA 2-O-beta-D-glucose (SAG), while UGT74F2 forms both SAG and the SA glucose ester (SGE). In an attempt to determine the in vivo role of each SA glucosyltransferase (SAGT), the metabolism of SA in ugt74f1 and ugt74f2 mutants was examined and compared with that of the wild-type. The three major metabolites formed in wild-type Arabidopsis included SAG, SGE, and 2,5-dihydroxbenzoic acid 2-O-beta-D-glucose (DHB2G). This is the first description of DHB2G as a major metabolite of SA in plants. The major metabolites of SA formed in ugt74f1 mutants were SGE, SAG and 2,5-dihydroxybenzoic acid 5-O-beta-D-glucose (DHB5G). DHB5G was not formed in the wild-type plants. SAG and DHB2G were the main metabolites of SA in ugt74f2 mutants. The ugt74f2 mutant was unable to form SGE. Only SGE could be detected during in vitro SAGT assays of untreated wild-type and ugt74f1 mutants. This activity was because of constitutive UGT74F2 activity. Both SGE and SAG could be formed during in vitro assays of SA-pretreated wild-type and ugt74f1 leaves. Neither SAG nor SGE could be detected during the in vitro SAGT assays of untreated ugt74f2 leaves. Only SAG was formed during the in vitro SAGT assays of SA-pretreated ugt74f2 leaves. The SAG formation was a result of the UGT74F1 activity. This work demonstrates that changes in the activity of either SAGT enzyme can have a dramatic effect on the metabolism of exogenously supplied SA in Arabidopsis.