bims-supasi 2022-09-18 papers

bims-supasi

Biomed News

on Sulfation pathways and signalling

Issue of 2022–09–18
twelve papers selected by
Jonathan Wolf Mueller, University of Birmingham

Insights into the reaction of chondroitin sulfate with glycidyl methacrylate: 1D and 2D NMR investigation.
Glycosaminoglycan linkage region of urinary bikunin as a potentially useful biomarker for β3GalT6-deficient spondylodysplastic Ehlers-Danlos syndrome.
Analysis of Prediagnostic Circulating Levels of Gonadotropins and Androgens with Risk of Epithelial Ovarian Cancer.
Trajectory of plasma syndecan-1 and heparan sulfate during major surgery: A retrospective observational study.
Salt-Wasting Form of Congenital Adrenal Hyperplasia: A Case Report.
Biochemical characterization of a disease-causing human osteoprotegerin variant.
Presence of chondroitin sulphate and requirement for heparan sulphate biosynthesis in the developing zebrafish inner ear.
6-O-desulfated heparin attenuates myocardial ischemia/reperfusion injury in mice through the regulation of miR-199a-5p/klotho axis.
Parity History and Later Life Sex Hormone Levels in the Multi-Ethnic Study of Atherosclerosis (MESA).
Pharmacogenetics of human sulfotransferases and impact of amino acid exchange on Phase II drug metabolism.
Use of heparin to rescue immunosuppressive monocyte reprogramming by glioblastoma-derived extracellular vesicles.
Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation.

Carbohydr Polym. 2022 Nov 15. pii: S0144-8617(22)00821-9. [Epub ahead of print]296 119916

Insights into the reaction of chondroitin sulfate with glycidyl methacrylate: 1D and 2D NMR investigation.

Laura Di Muzio, Patrizia Paolicelli, Jordan Trilli, Stefania Petralito, Vito Cosimo Carriero, Chiara Brandelli, Mattia Spano, Anatoly Petrovich Sobolev, Luisa Mannina, Maria Antonietta Casadei.

  Chondroitin sulfate methacrylate (CS-MA) is a semisynthetic biopolymer increasingly used for the fabrication of chemical hydrogels. In this study, the methacrylation reaction of native CS was carried out with glycidyl methacrylate in dimethyl sulfoxide and optimized to obtain tunable and reproducible methacrylation degrees in a short reaction time. The methacrylation reaction was deeply characterized by mono- and bi-dimensional (1D, 2D) NMR spectroscopy of CS-MA derivatives with different methacrylation degrees. In contrast to what previously reported in the literature, HSQC, HMBC and TOCSY analyses revealed that the methacrylation reaction proceeds via both epoxy ring-opening and transesterification, involving predominantly the primary hydroxyl groups of CS, while preserving sulfate and carboxyl groups of the biopolymer. These findings are of fundamental importance for appropriate and rational design of CS-MA-based biomaterials.

Keywords:  2D-NMR; Chondroitin sulfate methacrylate; Epoxy ring-opening; Glycosaminoglycans; Transesterification

DOI:  https://doi.org/10.1016/j.carbpol.2022.119916
JIMD Rep. 2022 Sep;63(5): 462-467

Glycosaminoglycan linkage region of urinary bikunin as a potentially useful biomarker for β3GalT6-deficient spondylodysplastic Ehlers-Danlos syndrome.

Mahnaz Nikpour, Fredrik Noborn, Jonas Nilsson, Tim Van Damme, Olivier Kaye, Delfien Syx, Fransiska Malfait, Göran Larson.

  The spondylodysplastic type of Ehlers-Danlos syndrome (spEDS) is caused by genetic defects in the B4GALT7 or B3GALT6 genes both deranging the biosynthesis of the glycosaminoglycan linkage region of chondroitin/dermatan sulfate and heparan sulfate proteoglycans. In this study, we have analyzed the linkage regions of urinary chondroitin sulfate proteoglycans of three siblings, diagnosed with spEDS and carrying biallelic pathogenic variants of the B3GALT6 gene. Proteoglycans were digested with trypsin, glycopeptides enriched on anion-exchange columns, depolymerized with chondroitinase ABC, and analyzed by nLC-MS/MS. In urine of the unaffected mother, the dominating glycopeptide of bikunin/protein AMBP appeared as only one dominating (99.9%) peak with the canonical tetrasaccharide linkage region modification. In contrast, the samples of the three affected siblings contained two different glycopeptide peaks, corresponding to the canonical tetrasaccharide and to the non-canonical trisaccharide linkage region modifications in individual ratios of 61/38, 73/27, and 59/41. We propose that the relative distribution of glycosaminoglycan linkage regions of urinary bikunin glycopeptides may serve as a phenotypic biomarker in a diagnostic test but also as a biomarker to follow the effect of future therapies in affected individuals.

Keywords:  Ehlers–Danlos syndrome; bikunin; glycopeptides; glycoproteomics; glycosaminoglycan linkage region; linkeropathies; liquid chromatography; tandem mass spectrometry; β3GalT6

DOI:  https://doi.org/10.1002/jmd2.12311
J Lab Physicians. 2022 Mar;14(1): 47-56

Analysis of Prediagnostic Circulating Levels of Gonadotropins and Androgens with Risk of Epithelial Ovarian Cancer.

Deepak Parchwani, Sagar Jayantilal Dholariya, Sohil Takodara, Ragini Singh, Vivek Kumar Sharma, Alpana Saxena, Digishaben D Patel, Madhuri Radadiya.

  Background Prevailing experimental and epidemiological evidence supports the role of circulating endogenous sex steroid hormones in the pathogenesis of ovarian carcinogenesis by dysregulation of cell differentiation, proliferation, and apoptosis but is scarce and inconclusive. Objectives This article evaluates the role of circulating levels of gonadotropins (follicle-stimulating hormone [FSH], luteinizing hormone [LH]) and androgens (testosterone, dehydroepiandrosterone-sulfate [DHEA-S]) for the risk of epithelial ovarian cancer in a case-control approach using samples collected in advance of clinical diagnosis. Materials and Methods A total of 100 epithelial ovarian cancer (EOC) patients and 100 healthy female controls were consequently enrolled in this hospital-based case-control study. Serum FSH, LH, testosterone, and DHEA-S were measured based on the principle of electrochemiluminescence immunoassay. Suitable descriptive statistics were used for different variables. Results Median values of FSH (58.9 vs. 45.5 IU/L, p = 0.02) and DHEA-S (163.43 vs. 142.2 ug/dL, p = 0.03) were significantly high in EOC patients compared with controls. Conditional logistic regression was used to estimate the odds ratio (OR) across increasing thirds of FSH and DHEA-S concentrations, and the results revealed that the highest third tertile of FSH (> 72.6 IU/L; OR = 3.0, confidence interval [CI] = 1.24-7.29, p trend = 0.04) and DHEA-S (> 194.2 ug/dL; OR = 3.8, CI = 1.26-11.61, p trend = 0.03) were significantly associated with increased risk of ovarian cancer in postmenopausal and premenopausal women, respectively. The statistically significant trend observed for FSH in postmenopausal women, remained only for the subgroup with menopause duration greater than 10 years (OR = 5.9, CI = 1.33-26.66, p trend = 0.04). FSH and DHEA-S concentrations and ovarian cancer risk were internally consistent with groups defined by oral contraceptive pill use, hormone replacement therapy, and smoking. However, no evidence was found for the association between serum LH and testosterone level with the occurrence of ovarian tumorigenesis. Conclusion Prediagnostic circulating concentration of FSH and DHEA-S unveiled a significant positive association with augmented risk of EOC, thus might serve as a predictive marker for the susceptibility to ovarian carcinogenesis and should be added in the screening profile of EOC for early recognition and scheduling necessary interventions/management strategies.

Keywords:  dehydroepiandrosterone-sulfate; epithelial ovarian cancer; follicle-stimulating hormone

DOI:  https://doi.org/10.1055/s-0041-1741443
Acta Anaesthesiol Scand. 2022 Sep 16.

Trajectory of plasma syndecan-1 and heparan sulfate during major surgery: A retrospective observational study.

Laurence Weinberg, Fumitaka Yanase, Shervin Tosif, Bernhard Riedel, Rinaldo Bellomo, Robert G Hahn.

   BACKGROUND: Surgical trauma-induced inflammation during major surgery may disrupt endothelial integrity and affect plasma concentrations of glycocalyx constituents, such as syndecan-1 and heparan sulfate. To date, no studies have focused on their perioperative temporal changes.
METHODS: As part of a trial, we obtained plasma and urine specimens sampled during the perioperative period in 72 patients undergoing major abdominal surgery. The plasma concentration of syndecan-1 and heparan sulfate was measured on five occasions, from baseline to the second postoperative day. Plasma and urinary creatinine and urinary syndecan-1 concentrations were measured before surgery and on the first postoperative morning.
RESULTS: We observed three different temporal patterns of plasma syndecan-1 concentration. Group 1 "low" (64% of patients) showed only minor changes from baseline despite a median heparan sulfate increase of 67% (P < 0.005). Group 2 "increase" (21% of patients) showed a marked increase in median plasma syndecan-1 from 27 μg/L to 118 μg/L during the first postoperative day (P < 0.001) with a substantial (+670%; P < 0.005) increase in median plasma heparan sulfate from 279 to 2,196 μg/L. Group 3 "high" (14% of patients) showed a constant elevation of plasma syndecan-1 to > 100 μg/L, but low heparan sulfate levels. The plasma C-reactive protein concentration did not differ across the three groups and 90% of colon surgeries occurred in Group 1. Treatment with dexamethasone was similar across the three groups. Surgical blood loss, duration of surgery, and liver resection were greatest in Group 2.
CONCLUSION: Changes in syndecan-1 and heparan sulfate after surgery appear to show three different patterns, with greatest increases in those patients with greater blood loss, more liver surgery, and longer operations. These observations suggest that increases in syndecan-1 and heparan sulfate reflect the degree of surgical injury.

Keywords:  Fluids; Glycocalyx; Inflammation; Surgery; anaesthesia

DOI:  https://doi.org/10.1111/aas.14150
Cureus. 2022 Aug;14(8): e27807

Salt-Wasting Form of Congenital Adrenal Hyperplasia: A Case Report.

Anu R Twayana, Neela Sunuwar, Sulav Deo, Wasiq B Tariq, Azwar Anjum, Sushil Rayamajhi, Bishayeeta Shrestha.

  Congenital adrenal hyperplasia (CAH) is a set of autosomal recessive disorders characterized by enzyme abnormalities in the adrenal steroidogenesis pathway, which cause impaired cortisol biosynthesis. Glucocorticoid, mineralocorticoid, and sex steroid production can all be altered in individuals, necessitating hormone replacement therapy. The symptoms might range from prenatal salt loss and abnormal genitalia to adult hirsutism and irregular menses. We present the case of a girl who presented with a seizure initially at the age of three months. Laboratory results revealed hypoglycemia, hyponatremia, and hyperkalemia with increased renin activity, increased adrenocorticotropic hormone (ACTH), low aldosterone, low cortisol, high dehydroepiandrosterone sulfate (DHEAS), and high 17 hydroxyprogesterone levels. Imaging findings were normal. The patient was managed with hydrocortisone and fludrocortisone. She is currently on regular follow-up and is doing well with dexamethasone therapy.

Keywords:  17 hydroxyprogesterone; 21-hydroxylase deficiency; congenital adrenal hyperplasia; dehydroepiandrosterone sulfate; virilization

DOI:  https://doi.org/10.7759/cureus.27807
Sci Rep. 2022 Sep 10. 12(1): 15279

Biochemical characterization of a disease-causing human osteoprotegerin variant.

Yin Luo, Miaomiao Li, Ding Xu.

Recently, a human mutation of OPG was identified to be associated with familial forms of osteoarthritis. This missense mutation (c.1205A = > T; p.Stop402Leu) occurs on the stop codon of OPG, which results in a 19-residue appendage to the C-terminus (OPG+19). The biochemical consequence of this unusual sequence alteration remains unknown. Here we expressed OPG+19 in 293 cells and the mutant OPG was purified to homogeneity by heparin affinity chromatography and size exclusion chromatography. We found that in sharp contrast to wildtype OPG, which mainly exists in dimeric form, OPG+19 had a strong tendency to form higher-order oligomers. To our surprise, the hyper-oligomerization of OPG+19 had no impact on how it binds cell surface heparan sulfate, how it inhibits RANKL-induced osteoclastogenesis and TRAIL-induced chondrocytes apoptosis. Our data suggest that in biological contexts where OPG is known to play a role, OPG+19 functions equivalently as wildtype OPG. The disease-causing mechanism of OPG+19 likely involves an unknown function of OPG in cartilage homeostasis and mineralization. By demonstrating the biochemical nature of this disease-causing OPG mutant, our study will likely help elucidating the biological roles of OPG in cartilage biology.

DOI: https://doi.org/10.1038/s41598-022-19522-9
Front Cell Dev Biol. 2022 ;10 959624

Presence of chondroitin sulphate and requirement for heparan sulphate biosynthesis in the developing zebrafish inner ear.

Ana A Jones, Elvira Diamantopoulou, Sarah Baxendale, Tanya T Whitfield.

  Epithelial morphogenesis to form the semicircular canal ducts of the zebrafish inner ear depends on the production of the large glycosaminoglycan hyaluronan, which is thought to contribute to the driving force that pushes projections of epithelium into the lumen of the otic vesicle. Proteoglycans are also implicated in otic morphogenesis: several of the genes coding for proteoglycan core proteins, together with enzymes that synthesise and modify their polysaccharide chains, are expressed in the developing zebrafish inner ear. In this study, we demonstrate the highly specific localisation of chondroitin sulphate to the sites of epithelial projection outgrowth in the ear, present before any morphological deformation of the epithelium. Staining for chondroitin sulphate is also present in the otolithic membrane, whereas the otoliths are strongly positive for keratan sulphate. We show that heparan sulphate biosynthesis is critical for normal epithelial projection outgrowth, otolith growth and tethering. In the ext2 mutant ear, which has reduced heparan sulphate levels, but continues to produce hyaluronan, epithelial projections are rudimentary, and do not grow sufficiently to meet and fuse to form the pillars of tissue that normally span the otic lumen. Staining for chondroitin sulphate and expression of versican b, a chondroitin sulphate proteoglycan core protein gene, persist abnormally at high levels in the unfused projections of the ext2 mutant ear. We propose a model for wild-type epithelial projection outgrowth in which hyaluronan and proteoglycans are linked to form a hydrated gel that fills the projection core, with both classes of molecule playing essential roles in zebrafish semicircular canal morphogenesis.

Keywords:  Ext2; chondroitin sulphate proteoglycan; epithelial morphogenesis; extracellular matrix; heparan sulphate proteoglycan; otoliths; semicircular canals; zebrafish

DOI:  https://doi.org/10.3389/fcell.2022.959624
Glycoconj J. 2022 Sep 15.

6-O-desulfated heparin attenuates myocardial ischemia/reperfusion injury in mice through the regulation of miR-199a-5p/klotho axis.

Yujie Wang, Ting Li, Niansheng Li, Chuyi Huang, Xiaoming Xiong, Xu Xie, Meiting Wu, Lianchun Wang, Junlin Jiang.

  Heparin has been documented to reduce myocardial injury caused by ischemia/reperfusion (I/R), but its clinical application is limited due to its strong intrinsic anticoagulant property. Some desulfated derivatives of heparin display low anticoagulant activity and may have potential value as therapeutic agents for myocardial I/R injury. In this study, we observed that 6-O-desulfated heparin, a desulfated derivative of heparin, shortened the activated partial thromboplastin time and exhibited lower anticoagulant activity compared with heparin or 2-O-desulfated heparin (another desulfated derivative of heparin). Then, we explored whether 6-O-desulfated heparin could protect against myocardial I/R injury, and elucidated its possible mechanisms. Administration of 6-O-desulfated heparin significantly reduced creatine kinase activity, myocardial infarct size and cell apoptosis in mice subjected to 30 min of myocardial ischemia following 2 h of reperfusion, accompanied by a reverse in miR-199a-5p elevation, klotho downregulation and reactive oxygen species (ROS) accumulation. In cultured H9c2 cells, the mechanism of 6-O-desulfated heparin against myocardial I/R injury was further explored. Consistent with the results in vivo, 6-O-desulfated heparin significantly ameliorated hypoxia/reoxygenation-induced injury, upregulated klotho and decreased miR-199a-5p levels and ROS accumulation, and these effects were reversed by miR-199a-5p mimics. In conclusion, these results suggested that 6-O-desulfated heparin with lower anticoagulant activity attenuated myocardial I/R injury through miR-199a-5p/klotho and ROS signaling. Our study may also indicate that 6-O-desulfated heparin, as an excellent heparin derivative, is a potential therapeutic agent for myocardial I/R injury.

Keywords:  6-O-desulfated heparin; Klotho; Myocardial ischemia/reperfusion; miR-199a-5p

DOI:  https://doi.org/10.1007/s10719-022-10081-9
Can J Cardiol. 2022 Sep 07. pii: S0828-282X(22)00790-5. [Epub ahead of print]

Parity History and Later Life Sex Hormone Levels in the Multi-Ethnic Study of Atherosclerosis (MESA).

Brigitte Kazzi, Oluseye Ogunmoroti, Carla P Rodriguez, Di Zhao, Anum S Minhas, Olatokunbo Osibogun, Vinita Subramanya, Matthew A Allison, Pamela Ouyang, Erin D Michos.

   BACKGROUND: Multiparity is a risk factor for cardiovascular disease (CVD). A more androgenic sex hormone profile, with a higher testosterone/estradiol (T/E2) ratio, is associated with worse CVD outcomes in women and may be one mechanism linking multiparity to increased CVD risk. We investigated the relationship between parity and sex hormones at mid-to-older age.
METHODS: We performed a cross-sectional analysis of 2,979 women with data on parity and endogenous sex hormone levels from MESA, a community-based cohort. Parity and gravidity (our exposures) were categorized as 0 (reference), 1-2, 3-4, or ≥5. Our outcome measures were testosterone (T), estradiol (E2), sex-hormone binding globulin (SHBG), dehydroepiandrosterone (DHEA), and T/E2 ratio. Progressively-adjusted linear regression was used to evaluate the association of parity/gravidity with sex hormones.
RESULTS: In multivariable-adjusted models, there were no significant associations of parity with E2, DHEA, and SHBG. Compared to nulliparity, after adjustment for CVD risk factors, women with 1-2 and 3-4 live births had higher T, but this was not significant for grand multiparity (≥5 live births). However, grand multigravidity (≥5 pregnancies) was associated with 10% (95% CI: 1%, 20%) higher T and 14% (1%, 29%) higher T/E2, compared to null-gravidity. Grand multiparity was associated with an 18% (4%, 34%) higher T/E2 ratio compared to nulliparity, after adjustment for CVD risk factors.
CONCLUSIONS: In this multiethnic cohort, women with grand multigravidity and grand multiparity had higher T/E2 levels, reflecting a more androgenic sex hormone profile. Longitudinal studies evaluating sex hormones' influence on the relationship between multiparity and CVD are warranted.

Keywords:  cardiovascular; gravidity; parity; prevention (6/6); sex hormones; women

DOI:  https://doi.org/10.1016/j.cjca.2022.09.004
Drug Discov Today. 2022 Sep 09. pii: S1359-6446(22)00342-7. [Epub ahead of print] 103349

Pharmacogenetics of human sulfotransferases and impact of amino acid exchange on Phase II drug metabolism.

Adriana Isvoran, Yunhui Peng, Silvana Ceauranu, Leon Schmidt, Arnaud B Nicot, Maria A Miteva.

  Sulfotransferases (SULTs) are Phase II drug-metabolizing enzymes (DMEs) catalyzing the sulfation of a variety of endogenous compounds, natural products, and drugs. Various drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDS) can inhibit SULTs, affecting drug-drug interactions. Several polymorphisms have been identified for SULTs that might be crucial for interindividual variability in drug response and toxicity or for increased disease risk. Here, we review current knowledge on non-synonymous single nucleotide polymorphisms (nsSNPs) of human SULTs, focusing on the coded SULT allozymes and molecular mechanisms explaining their variable activity, which is essential for personalized medicine. We discuss the structural and dynamic bases of key amino acid (AA) variants implicated in the impacts on drug metabolism in the case of SULT1A1, as revealed by molecular modeling approaches. Teaser: Sulfotransferases (SULTs) are drug-metabolizing enzymes catalyzing the sulfation of many endogenous compounds and drugs. We review current knowledge on polymorphisms of SULTs, focusing on coded SULT allozymes that might be crucial for interindividual variability in drug response and toxicity.

Keywords:  drug metabolism; drug–drug interactions; in silico modeling; pharmacogenomics; sulfotransferase

DOI:  https://doi.org/10.1016/j.drudis.2022.103349
J Neurosurg. 2022 Sep 16. 1-11

Use of heparin to rescue immunosuppressive monocyte reprogramming by glioblastoma-derived extracellular vesicles.

Benjamin T Himes, Cori E Fain, Zachariah P Tritz, Cody L Nesvick, Helen J Jin-Lee, Philipp A Geiger, Timothy E Peterson, Mi-Yeon Jung, Ian F Parney.

   OBJECTIVE: The profound immunosuppression found in glioblastoma (GBM) patients is a critical barrier to effective immunotherapy. Multiple mechanisms of tumor-mediated immune suppression exist, and the induction of immunosuppressive monocytes such as myeloid-derived suppressor cells (MDSCs) is increasingly appreciated as a key part of this pathology. GBM-derived extracellular vesicles (EVs) can induce the formation of MDSCs. The authors sought to identify the molecular consequences of these interactions in myeloid cells in order to identify potential targets that could pharmacologically disrupt GBM EV-monocyte interaction as a means to ameliorate tumor-mediated immune suppression. Heparin-sulfate proteoglycans (HSPGs) are a general mechanism by which EVs come into association with their target cells, and soluble heparin has been shown to interfere with EV-HSPG interactions. The authors sought to assess the efficacy of heparin treatment for mitigating the effects of GBM EVs on the formation of MDSCs.
METHODS: GBM EVs were collected from patient-derived cell line cultures via staged ultracentrifugation and cocultured with monocytes collected from apheresis cones from healthy blood donors. RNA was isolated from EV-conditioned and unconditioned monocytes after 72 hours of coculture, and RNA-sequencing analysis performed. For the heparin treatment studies, soluble heparin was added at the time of EV-monocyte coculture and flow cytometry analysis was performed 72 hours later. After the initial EV-monocyte coculture period, donor-matched T-cell coculture studies were performed by adding fluorescently labeled and stimulated T cells for 5 days of coculture.
RESULTS: Transcriptomic analysis of GBM EV-treated monocytes demonstrated downregulation of several important immunological and metabolic pathways, with upregulation of the pathways associated with synthesis of cholesterol and HSPG. Heparin treatment inhibited association between GBM EVs and monocytes in a dose-dependent fashion, which resulted in a concomitant reduction in MDSC formation (p < 0.01). The authors further demonstrated that reduced MDSC formation resulted in a partial rescue of immune suppression, as measured by effects on activated donor-matched T cells (p < 0.05).
CONCLUSIONS: The authors demonstrated that GBM EVs induce broad but reproducible reprogramming in monocytes, with enrichment of pathways that may portend an immunosuppressive phenotype. The authors further demonstrated that GBM EV-monocyte interactions are potentially druggable targets for overcoming tumor-mediated immune suppression, with heparin inhibition of EV-monocyte interactions demonstrating proof of principle.

Keywords:  MDSCs; extracellular vesicles; glioblastoma; heparin; immunosuppression; oncology

DOI:  https://doi.org/10.3171/2022.6.JNS2274
Nat Commun. 2022 Sep 16. 13(1): 5434

Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation.

Yuda Chen, Shikai Jin, Mengxi Zhang, Yu Hu, Kuan-Lin Wu, Anna Chung, Shichao Wang, Zeru Tian, Yixian Wang, Peter G Wolynes, Han Xiao.

Despite the great promise of genetic code expansion technology to modulate structures and functions of proteins, external addition of ncAAs is required in most cases and it often limits the utility of genetic code expansion technology, especially to noncanonical amino acids (ncAAs) with poor membrane internalization. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. These engineered cells can produce site-specifically sulfated proteins at a higher yield than cells fed exogenously with the highest level of sTyr reported in the literature. We use these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.

DOI: https://doi.org/10.1038/s41467-022-33111-4