Genome-wide association identifies a BAHD acyltransferase activity that assembles an ester of glucuronosylglycerol and phenylacetic acid.

Genome-wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD-family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl-CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP-sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate-starvation stress. GGES is a member of a well-supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant-specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism.

Biosynthesis of tetrahydropapaverine and semisynthesis of papaverine in yeast.

Tetrahydropapaverine (THP) and papaverine are plant natural products with clinically significant roles. THP is a precursor in the production of the drugs atracurium and cisatracurium, and papaverine is used as an antispasmodic during vascular surgery. In recent years, metabolic engineering advances have enabled the production of natural products through heterologous expression of pathway enzymes in yeast. Heterologous biosynthesis of THP and papaverine could play a role in ensuring a stable supply of these clinically significant products. Biosynthesis of THP and papaverine has not been achieved to date, in part because multiple pathway enzymes have not been elucidated. Here, we describe the development of an engineered yeast strain for de novo biosynthesis of THP. The production of THP is achieved through heterologous expression of two enzyme variants with activity on nonnative substrates. Through protein engineering, we developed a variant of N-methylcoclaurine hydroxylase with activity on coclaurine, enabling de novo norreticuline biosynthesis. Similarly, we developed a variant of scoulerine 9-O-methyltransferase capable of O-methylating 1-benzylisoquinoline alkaloids at the 3' position, enabling de novo THP biosynthesis. Flux through the heterologous pathway was improved by knocking out yeast multidrug resistance transporters and optimization of media conditions. Overall, strain engineering increased the concentration of biosynthesized THP 600-fold to 121 µg/L. Finally, we demonstrate a strategy for papaverine semisynthesis using hydrogen peroxide as an oxidizing agent. Through optimizing pH, temperature, reaction time, and oxidizing agent concentration, we demonstrated the ability to produce semisynthesized papaverine through oxidation of biosynthesized THP.

Mucin O-glycans are natural inhibitors of Candida albicans pathogenicity.

Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast-to-hyphal transition of Candida albicans, a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear despite their potential for therapeutic interventions. Here, we determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O-glycans. We isolated and cataloged >100 mucin O-glycans from three major mucosal surfaces and established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa. Using synthetic O-glycans, we identified three structures (core 1, core 1 + fucose and core 2 + galactose) that are sufficient to inhibit filamentation with potency comparable to the complex O-glycan pool. Overall, this work identifies mucin O-glycans as host molecules with untapped therapeutic potential to manage fungal pathogens.

Dry eye symptoms and signs in United States Gulf War era veterans with myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: To examine ocular symptoms and signs of veterans with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) diagnosis, ME/CFS symptoms, and controls. METHODS: This was a prospective, cross-sectional study of 124 South Florida veterans in active duty during the Gulf War era. Participants were recruited at an ophthalmology clinic at the Miami Veterans Affairs Hospital and evaluated for a diagnosis of ME/CFS, or symptoms of ME/CFS (intermediate fatigue, IF) using the Canadian Consensus criteria. Ocular symptoms were assessed via standardised questionnaires and signs via comprehensive slit lamp examination. Inflammatory blood markers were analysed and compared across groups. RESULTS: Mean age was 55.1 ± 4.7 years, 88.7% identified as male, 58.1% as White, and 39.5% as Hispanic. Ocular symptoms were more severe in the ME/CFS (n = 32) and IF (n = 48) groups compared to controls (n = 44) across dry eye (DE; Ocular Surface Disease Index [OSDI]: 48.9 ± 22.3 vs. 38.8 ± 23.3 vs. 19.1 ± 17.8, p < 0.001; 5 item Dry Eye Questionnaire [DEQ-5]: 10.8 ± 3.9 vs. 10.0 ± 4.6 vs. 6.6 ± 4.2, p < 0.001) and pain-specific questionnaires (Numerical Rating Scale 1-10 [NRS] right now: 2.4 ± 2.8 vs. 2.4 ± 2.9 vs 0.9 ± 1.5; p = 0.007; Neuropathic Pain Symptom Inventory modified for the Eye [NPSI-E]: 23.0 ± 18.6 vs. 19.8 ± 19.1 vs. 6.5 ± 9.0, p < 0.001). Ocular surface parameters and blood markers of inflammation were generally similar across groups. CONCLUSION: Individuals with ME/CFS report increased ocular pain but similar DE signs, suggesting that mechanisms beyond the ocular surface contribute to symptoms.

Lipids from ocular meibum and tears may serve as biomarkers for depression and post-traumatic stress disorder.

BACKGROUND: There is a need to develop biomarkers for diagnosis and prediction of treatment responses in depression and post-traumatic stress disorder (PTSD). METHODS: Cross-sectional study examining correlations between tear inflammatory proteins, meibum and tear sphingolipids, and symptoms of depression and PTSD-associated anxiety. Ninety individuals filled depression (Patient Health Questionnaire 9, PHQ-9) and PTSD-associated anxiety (PTSD Checklist-Military Version, PCL-M) questionnaires. In 40 patients, a multiplex assay system was used to quantify 23 inflammatory proteins in tears. In a separate group of 50 individuals, liquid chromatography-mass spectrometry was performed on meibum and tears to quantify 34 species of sphingolipids, encompassing ceramides, monohexosyl ceramides and sphingomyelins. RESULTS: The mean age of the population was 59.4 ± 11.0 years; 89.0% self-identified as male, 34.4% as White, 64.4% as Black, and 16.7% as Hispanic. The mean PHQ-9 score was 11.1 ± 7.6, and the mean PCL-M score was 44.3 ± 19.1. Symptoms of depression and PTSD-associated anxiety were highly correlated (ρ =0.75, p < 0.001). Both PHQ9 and PCL-M scores negatively correlated with multiple sphingolipid species in meibum and tears. In multivariable models, meibum Monohexosyl Ceramide 26:0 (pmol), tear Ceramide 16:0 (mol%), meibum Monohexosyl Ceramide 16:0 (mol%), and tear Ceramide 26:1 (mol%) remained associated with depression and meibum Monohexosyl Ceramide 16:0 (mol%), meibum Monohexosyl Ceramide 26:0 (pmol), tear Sphingomyelin 20:0 (mol%), and tear Sphingosine-1-Phosphate (mol%) remained associated with PTSD-associated anxiety. CONCLUSIONS: Certain meibum and tear sphingolipid species were related to mental health indices. These interactions present opportunities for innovative diagnostic and therapeutic approaches for mental health disorders.

Improved single-swab sample preparation for recovering bacterial and phage DNA from human skin and wound microbiomes.

BACKGROUND: Characterization of the skin and wound microbiome is of high biomedical interest, but is hampered by the low biomass of typical samples. While sample preparation from other microbiomes (e.g., gut) has been the subject of extensive optimization, procedures for skin and wound microbiomes have received relatively little attention. Here we describe an improved method for obtaining both phage and microbial DNA from a single skin or wound swab, characterize the yield of DNA in model samples, and demonstrate the utility of this approach with samples collected from a wound clinic. RESULTS: We find a substantial improvement when processing wound samples in particular; while only one-quarter of wound samples processed by a traditional method yielded sufficient DNA for downstream analysis, all samples processed using the improved method yielded sufficient DNA. Moreover, for both skin and wound samples, community analysis and viral reads obtained through deep sequencing of clinical swab samples showed significant improvement with the use of the improved method. CONCLUSION: Use of this method may increase the efficiency and data quality of microbiome studies from low-biomass samples.

Single molecule FRET investigation of pressure-driven unfolding of cold shock protein A.

We demonstrate that fused silica capillaries are suitable for single molecule fluorescence resonance energy transfer (smFRET) measurements at high pressure with an optical quality comparable to the measurement on microscope coverslips. Therefore, we optimized the imaging conditions in a standard square fused silica capillary with an adapted arrangement and evaluated the performance by imaging the focal volume, fluorescence correlation spectroscopy benchmarks, and FRET measurements. We demonstrate single molecule FRET measurements of cold shock protein A unfolding at a pressure up to 2000 bars and show that the unfolded state exhibits an expansion almost independent of pressure.

To do or not to do: Large-dose steroid treatment for severe vision loss secondary to compressive inflammatory optic neuropathy in the setting of invasive fungal sinusitis.

Purpose: Invasive fungal sinusitis (IFS) is associated with high rates of morbidity and mortality and often presents with orbital apex syndrome. Prompt diagnosis and management are crucial to prevent irreversible visual loss. We report a case of an immunosuppressed patient with rapidly progressive severe visual loss associated with frontal lobe cerebritis and leptomeningitis related to IFS, causing an adjacent compressive inflammatory optic neuropathy, which was treated successfully by large-dose corticosteroids. Observations: A 29-year-old woman with acute myeloid leukemia status post chemotherapy presented with right-sided headaches and periorbital swelling. Her examination was significant for subjective red desaturation and trace right eyelid edema and ptosis. The remainder of her initial ocular examination was normal. Her labs demonstrated neutropenia and thrombocytopenia. Imaging of the brain and orbits was concerning for extensive sinus disease with intracranial extension. An urgent multi-sinus and optic nerve decompression was performed given concern for compressive optic neuropathy, and the biopsy was consistent with invasive fungal infection. Despite aggressive antifungal treatment, vision in her right eye decreased rapidly to counting fingers. No optic nerve abnormalities were observed on serial MRIs, but adjacent inferior frontal lobe enhancement was present. After a vigorous debate in a multidisciplinary meeting, her severe vision loss was attributed to cerebritis causing an adjacent compressive inflammatory optic neuropathy, and large-dose intravenous (IV) steroid treatment was initiated while maintaining systemic antifungal therapy. Remarkably, she had a full recovery of her vision. Conclusions and importance: Severe vision loss in IFS can occur due to compressive inflammatory optic neuropathy without direct fungal invasion as a contributing factor. Timely and effective intervention is crucial in preventing vision loss. Large-dose steroid therapy may be a potential treatment option for immunocompromised patients with invasive fungal sinusitis and intracranial invasion, provided strict fungal infection control measures are in place.

Comparative Analysis of the Intrinsic Disorder Within the Layers of the Human Cornea.

PURPOSE: The human cornea is essential for vision, providing structural integrity and refractive power to the eye. Recent advancements have deepened our understanding of the corneal molecular composition, yet the role of intrinsically disordered proteins within the cornea is unexplored. METHODS: We analyzed 3,250 corneal proteins identified by Dyrlund et al, focusing on the epithelium, stroma, and endothelium layers. We performed a bioinformatics analysis to characterize the amino acid composition, the propensity for intrinsic protein disorder, and the distribution of protein types in 3 corneal layer proteome. RESULTS: Our study demonstrates that each corneal layer exhibited unique patterns in amino acid composition related to protein disorder. Order-promoting amino acids were generally depleted except for leucine, whereas disorder-promoting amino acids like arginine and glutamic acid were enriched across all layers. Significant variations were observed in the levels of intrinsic disorder among the different corneal layers, with substantial proportions of highly disordered proteins present in each. Analysis of protein class type in each layers revealed that no significant differences were detected in the distribution of protein classifications across the layers, suggesting a consistent population of the protein types across all corneal layers. CONCLUSIONS: Our findings reveal a sophisticated landscape of protein structures where intrinsic disorder varies across layers, suggesting an adaptation of the corneal proteome to the unique physiological demands of each layer. These structural variations may reflect the intricate requirements for corneal transparency, biomechanical stability, and environmental responsiveness.

Effects of Aging on Intrinsic Protein Disorder in Human Lenses and Zonules.

This study aims to compare the levels of intrinsic protein disorder within the human lens and zonule proteomes and investigate the role of aging as a potential influencing factor on disorder levels. A cross-sectional proteomic analysis was employed, utilizing a dataset of 1466 proteins derived from the lens and zonule proteomes previously published by Wang et al. and De Maria et al. Bioinformatics tools, including a composition profiler and a rapid intrinsic disorder analysis online tool, were used to conduct a comparative analysis of protein disorder. Statistical tests such as ANOVA, Tukey's HSD, and chi-squared tests were applied to evaluate differences between groups. The study revealed distinct amino acid compositions for each proteome, showing a direct correlation between aging and increased protein disorder in the zonular proteomes, whereas the lens proteomes exhibited the opposite trend. Findings suggest that age-related changes in intrinsic protein disorder within the lens and zonule proteomes may be linked to structural transformations in these tissues. Understanding how protein disorder evolves with age could enhance knowledge of the molecular basis for age-related conditions such as cataracts and pseudoexfoliation, potentially leading to better therapeutic strategies.

Is Meibomian Gland Dysfunction One Disease? Heterogeneity Among Phenotypes.

PURPOSE: The purpose of this study was to examine ocular surface symptoms, tear metrics, and tear cytokines by Meibomian gland dysfunction (MGD) features. METHODS: Symptom questionnaires and an ocular surface evaluation were performed on 40 individuals with varied MGD signs (Meibomian gland [MG] plugging, eyelid vascularity, meibum quality, and MG dropout). Tear proteins were extracted off Schirmer strips and analyzed for 23 human inflammation-related proteins. Statistical analysis was performed to examine associations between dry eye metrics inflammatory proteins and MGD features. RESULTS: The study involved 40 South Florida veterans with a mean age of 61 ± 13 years; most individuals were male (95%), White (31%), and non-Hispanic (85%). MGD features differentially related to dry eye signs. Eyelid vascularity, meibum quality, and MG dropout, but not MG plugging, correlated with higher corneal staining and lower tear production. MGD features also differentially related to tear cytokines. Eyelid vascularity most closely related to inflammation with significant correlations for interferon-gamma-γ (r = 0.36, P = 0.02), interleukin-4 (IL-4) (r = 0.43, P = 0.006), IL-17A (r = 0.42, P = 0.007), matrix metalloproteinase-2 (r = 0.39, P = 0.01), C-X-C motif chemokine ligand 5 (Regulated upon Activation, Normal T-Cell Expressed and presumably Secreted [RANTES]) (r = 0.32, P = 0.04), and tumor necrosis factor α (r = 0.36, P = 0.02). The other 3 MGD signs were less related to inflammation. Multivariable models revealed IL-4 to be most closely related to eyelid vascularity (standardized ß = 0.39, P < 0.0001). CONCLUSIONS: Eyelid vascularity was the MGD sign most closely related to inflammatory cytokines, suggesting that different MGD features may be driven by different pathophysiological mechanisms.

A genetically encoded fluorescent reporter for polyamines.

Polyamines are abundant and evolutionarily conserved metabolites that are essential for life. Dietary polyamine supplementation extends life-span and health-span. Dysregulation of polyamine homeostasis is linked to Parkinson's disease and cancer, driving interest in therapeutically targeting this pathway. However, measuring cellular polyamine levels, which vary across cell types and states, remains challenging. We introduce a first-in-class genetically encoded polyamine reporter for real-time measurement of polyamine concentrations in single living cells. This reporter utilizes the polyamine-responsive ribosomal frameshift motif from the OAZ1 gene. We demonstrate broad applicability of this approach and reveal dynamic changes in polyamine levels in response to genetic and pharmacological perturbations. Using this reporter, we conducted a genome-wide CRISPR screen and uncovered an unexpected link between mitochondrial respiration and polyamine import, which are both risk factors for genetic Parkinson's disease. By offering a new lens to examine polyamine biology, this reporter may advance our understanding of these ubiquitous metabolites and accelerate therapy development.

Emergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY.

Plants contain rapidly evolving specialized enzymes that support the biosynthesis of functionally diverse natural products. In coumarin biosynthesis, a BAHD acyltransferase-family enzyme COSY was recently discovered to accelerate coumarin formation as the only known BAHD enzyme to catalyze an intramolecular acyl transfer reaction. Here we investigate the structural and mechanistic basis for COSY's coumarin synthase activity. Our structural analyses reveal an unconventional active-site configuration adapted to COSY's specialized activity. Through mutagenesis studies and deuterium exchange experiments, we identify a unique proton exchange mechanism at the α-carbon of the o-hydroxylated trans-hydroxycinnamoyl-CoA substrates during the catalytic cycle of COSY. Quantum mechanical cluster modeling and molecular dynamics further support this key mechanism for lowering the activation energy of the rate-limiting trans-to-cis isomerization step in coumarin production. This study unveils an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on COSY's evolutionary origin and its recruitment to coumarin biosynthesis in eudicots.

Genomics and biochemical analyses reveal a metabolon key to ß-L-ODAP biosynthesis in Lathyrus sativus.

Grass pea (Lathyrus sativus L.) is a rich source of protein cultivated as an insurance crop in Ethiopia, Eritrea, India, Bangladesh, and Nepal. Its resilience to both drought and flooding makes it a promising crop for ensuring food security in a changing climate. The lack of genetic resources and the crop's association with the disease neurolathyrism have limited the cultivation of grass pea. Here, we present an annotated, long read-based assembly of the 6.5 Gbp L. sativus genome. Using this genome sequence, we have elucidated the biosynthetic pathway leading to the formation of the neurotoxin, ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP). The final reaction of the pathway depends on an interaction between L. sativus acyl-activating enzyme 3 (LsAAE3) and a BAHD-acyltransferase (LsBOS) that form a metabolon activated by CoA to produce ß-L-ODAP. This provides valuable insight into the best approaches for developing varieties which produce substantially less toxin.

The chloroalkaloid (-)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants.

Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)- and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (-)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (-)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.