Cross-linking Analysis Research Articles

Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes.

Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation.

A bibliometric and citation network analysis of crosslinking.

Purpose: Research on crosslinking has focused mainly on trying to slow the progression of keratoconus. The objective of this bibliometric analysis is to determine the relationships between the different publications and authors. As well as to identify the different areas of research on crosslinking. Methods: Web of Science (WOS) was the database for the search of publications for the period 1972 to December 2023, using the terms: crosslinking, cross-linking, ocular, vision, visual, cornea, ectasia AND Keratoconus. The analysis of the publication was performed using the CitNetExplorer, VOSviewer and CiteSpace software. Results: 7161 publications and 59415 citation networks were found. The year with the most publications is 2021, a total of 571 (7.97%) publications and 135 citation networks. The most cited publication was "Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus" published by Wollensak et al. in 2003, with a citation index of 1780. Using the Clustering function, five groups were found that cover the main research areas in this field: keratoconus, corneal transplantation, cataracts, corneal inflammation and axial growth. Conclusion: The treatment of keratoconus continues to be the topic of interest in this area of research. However, in recent years there has been an increase in research on how crosslinking helps to slow down axial growth.

BC-g-PAA: Characterization and Establishment of the IPN Hydrogel

A study on the manufacture of bacterial cellulose IPN hydrogel crosslinked poly acrylic acid using the MBA crosslinker. This study aims to characterize and observe the reaction to form bacterial cellulose-based hydrogel IPN. The BC-g-PAA was characterized by the degree of cross-linking, swelling ratio, and FTIR analysis. The results of this study found predictions of the reaction process for the formation of IPN Hydrogel in several process stages, namely 1) The polymerization process of bacterial cellulose chains that form cross-linked networks independently, 2) The process of cross-linking Acrylate monomers with MBA crosslinkers in three steps, i.e. initiation, propagation and termination, 3) Reaction process Formation of IPN network. The results of the characterization test for the degree of crosslinking were 54.55%; Swelling ratio of 1631.25%. FTIR analysis shows that there is a peak that identifies the occurrence of crosslinking

PRMT5-mediated arginine methylation of FXR1 is essential for RNA binding in cancer cells.

Emerging evidence indicates that arginine methylation promotes the stability of arginine-glycine-rich (RGG) motif-containing RNA-binding proteins (RBPs) and regulates gene expression. Here, we report that post-translational modification of FXR1 enhances the binding with mRNAs and is involved in cancer cell growth and proliferation. Independent point mutations in arginine residues of FXR1's nuclear export signal (R386 and R388) and RGG (R453, R455and R459) domains prevent it from binding to RNAs that form G-quadruplex (G4) RNA structures. Disruption of G4-RNA structures by lithium chloride failed to bind with FXR1, indicating its preference for G4-RNA structure containing mRNAs. Furthermore, loss-of-function of PRMT5 inhibited FXR1 methylation both in vivo and in vitro, affecting FXR1 protein stability, inhibiting RNA-binding activity and cancer cell growth and proliferation. Finally, the enhanced crosslinking and immunoprecipitation (eCLIP) analyses reveal that FXR1 binds with the G4-enriched mRNA targets such as AHNAK, MAP1B, AHNAK2, HUWE1, DYNC1H1 and UBR4 and controls its mRNA expression in cancer cells. Our findings suggest that PRMT5-mediated FXR1 methylation is required for RNA/G4-RNA binding, which promotes gene expression in cancer cells. Thus, FXR1's structural characteristics and affinity for RNAs preferentially G4 regions provide new insights into the molecular mechanism of FXR1 in oral cancer cells.

CLAUDIO: automated structural analysis of cross-linking data.

Cross-linking mass spectrometry has made remarkable advancements in the high-throughput characterization of protein structures and interactions. The resulting pairs of cross-linked peptides typically require geometric assessment and validation, given the availability of their corresponding structures. CLAUDIO (Cross-linking Analysis Using Distances and Overlaps) is an open-source software tool designed for the automated analysis and validation of different varieties of large-scale cross-linking experiments. Many of the otherwise manual processes for structural validation (i.e. structure retrieval and mapping) are performed fully automatically to simplify and accelerate the data interpretation process. In addition, CLAUDIO has the ability to remap intra-protein links as inter-protein links and discover evidence for homo-multimers. CLAUDIO is available as open-source software under the MIT license at https://github.com/KohlbacherLab/CLAUDIO.

Cytoplasmic and nuclear DROSHA in human villous trophoblasts

In villous trophoblasts, DROSHA is a key ribonuclease III enzyme that processes pri-microRNAs (pri-miRNAs) into pre-miRNAs at the placenta-specific, chromosome 19 miRNA cluster (C19MC) locus. However, little is known of its other functions. We performed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq) analysis of terminal chorionic villi to identify DROSHA-binding RNAs in villous trophoblasts. In villous trophoblasts, DROSHA predominantly generated placenta-specific C19MC pre-miRNAs, including antiviral C19MC pre-miRNAs. The fCLIP-seq analysis also identified non-miRNA transcripts with hairpin structures potentially capable of binding to DROSHA (e.g., SNORD100 and VTRNA1–1). Moreover, in vivo immunohistochemical analysis revealed DROSHA in the cytoplasm of villous trophoblasts. DROSHA was abundant in the cytoplasm of villous trophoblasts, particularly in the apical region of syncytiotrophoblast, in the full-term placenta. Furthermore, in BeWo trophoblasts infected with Sindbis virus (SINV), DROSHA translocated to the cytoplasm and recognized the genomic RNA of SINV. Therefore, in trophoblasts, DROSHA not only regulates RNA metabolism, including the biogenesis of placenta-specific miRNAs, but also recognizes viral RNAs. After SINV infection, BeWo DROSHA-binding VTRNA1–1 was significantly upregulated, and cellular VTRNA1–1 was significantly downregulated, suggesting that DROSHA soaks up VTRNA1–1 in response to viral infection. These results suggest that the DROSHA-mediated recognition of RNAs defends against viral infection in villous trophoblasts. Our data provide insight into the antiviral functions of DROSHA in villous trophoblasts of the human placenta.

Efficacy of Forced Choice Testing in Detecting a Secret Cell Hiding Within a Group

The purpose of this project was to test whether a previously validated method of detecting deception known as forced choice testing would be effective at finding a “cell” of deceptive individuals hiding in a larger group. 64 men and women enrolled in a State Trooper Academy were the participants of this study; 57 participants were randomized to the truthful group and 7 were randomized to the deceptive group. Each participant was given an FCT by a member of the cadre who was blind to the status of the participants. The distribution of responses deviated significantly below the binomial prediction for 14 individuals; a cross-link analysis indicated 7 of these 14 did not select each other. These were the members of the deceptive group. Consistent with the hypothesis underpinning traditional FCT methods, deceptive individuals motivated to hide what they knew (i.e., the identity of the “cell” of deceptive individuals) avoided picking any of the other deceptive participants who appeared on the FCT. This avoidance behavior resulted in fewer number of “selections” than predicted by the binomial calculations. These data suggest it may be fruitful to explore how well the principles of FCT can be used in law enforcement or national security to detect potentially dangerous “cells” of individuals hiding within larger groups.

Antioxidant water-resistant fish gelatin nanofibers: A comparative analysis of fructose and citric acid crosslinking and investigation of chlorogenic acid release kinetics

The use of bio-based and biodegradable electrospun mats encapsulating antioxidants is a promising strategy for creating innovative multi-layered food packaging materials, since the nanometric size of the fibers forming the mat maximizes the availability of the active compound. However, the development of water-resistant active electrospun mats using only non-toxic natural components remains a challenge. In this paper, water-resistant fish gelatin (FG) nanofibers incorporating chlorogenic acid (CGA), a natural antioxidant, were developed using either citric acid (CA) or fructose (F) as crosslinking agents. The CA-crosslinked fibers had a mean diameter of (200 ± 50) nm and a solubility in cold water of (28 ± 2)%, while F-crosslinked fibers were obtained with a mean diameter of (300 ± 50) nm and a solubility of (5 ± 3)%. CGA was quickly released when the mats were immersed in several food simulants, achieving equilibrium in less than 30 min for both materials. ABTS scavenging activity did not show significant differences (p > 0.05) regardless of the crosslinker, but DPPH activity was significantly higher (p < 0.05) in those samples crosslinked with CA. Physico-chemical properties of the developed materials were studied by FTIR, DSC, and TGA tests. Results demonstrate the release kinetics and antioxidant activity of electrospinning-fabricated water-resistant FG nanofibers.

The Saccharomyces cerevisiae Spo7 basic tail is required for Nem1–Spo7/Pah1 phosphatase cascade function in lipid synthesis

The Saccharomyces cerevisiae Nem1-Spo7 protein phosphatase complex dephosphorylates and thereby activates Pah1 at the nuclear/endoplasmic reticulum membrane. Pah1, a phosphatidate phosphatase catalyzing the dephosphorylation of phosphatidate to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The diacylglycerol produced in the lipid phosphatase reaction is utilized for the synthesis of triacylglycerol that is stored in lipid droplets. Disruptions of the Nem1-Spo7/Pah1 phosphatase cascade cause a plethora of physiological defects. Spo7, the regulatory subunit of the Nem1-Spo7 complex, is required for the Nem1 catalytic function and interacts with the acidic tail of Pah1. Spo7 contains three conserved homology regions (CR1-3) that are important for the interaction with Nem1, but its region for the interaction with Pah1 is unknown. Here, by deletion and site-specific mutational analyses of Spo7, we revealed that the C-terminal basic tail (residues 240-259) containing five arginine and two lysine residues is important for the Nem1-Spo7 complex-mediated dephosphorylation of Pah1 and its cellular function (triacylglycerol synthesis, lipid droplet formation, maintenance of nuclear/endoplasmic reticulum membrane morphology, and cell growth at elevated temperatures). The glutaraldehyde cross-linking analysis of synthetic peptides indicated that the Spo7 basic tail interacts with the Pah1 acidic tail. This work advances our understanding of the Spo7 function and the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.

Alzheimer’s-specific brain amyloid interactome: Neural-network analysis of intra-aggregate crosslinking identifies novel drug targets

Alzheimer's disease (AD) is characterized by peri-neuronal amyloid plaque and intra-neuronal neurofibrillary tangles. These aggregates are identified by the immunodetection of "seed" proteins (Aβ1-42 and hyperphosphorylated tau, respectively), but include many other proteins incorporated nonrandomly. Using click-chemistry intra-aggregate crosslinking, we previously modeled amyloid "contactomes" in SY5Y-APPSw neuroblastoma cells, revealing that aspirin impedes aggregate growth and complexity. By an analogous strategy, we now construct amyloid-specific aggregate interactomes of AD and age-matched-controlhippocampi. Comparing these interactomes reveals AD-specific interactions, from which neural-network (NN) analyses predict proteins with the highest impact on pathogenic aggregate formation and/or stability. RNAi knockdowns of implicated proteins, in C.elegans and human-cell-culture models of AD, validated those predictions. Gene-Ontology meta-analysis of AD-enriched influential proteins highlighted the involvement of mitochondrial and cytoplasmic compartments in AD-specific aggregation. This approach derives dynamic consensus models of aggregate growth and architecture, implicating highly influential proteins as new targets to disrupt amyloid accrual in the AD brain.

4D-diaXLMS: Proteome-wide Four-Dimensional Data-Independent Acquisition Workflow for Cross-Linking Mass Spectrometry.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for examining protein structures and interactions. Nevertheless, analysis of low-abundance cross-linked peptides is often limited in the data-dependent acquisition (DDA) mode due to its semistochastic nature. To address this issue, we introduced a workflow called 4D-diaXLMS, representing the first-ever application of four-dimensional data-independent acquisition for proteome-wide cross-linking analysis. Cross-linking studies of the HeLa cell proteome were evaluated using the classical cross-linker disuccinimidyl suberate as an example. Compared with the DDA analysis, 4D-diaXLMS exhibited marked improvement in the identification coverage of cross-linked peptides, with a total increase of 36% in single-shot analysis across all 16 SCX fractions. This advantage was further amplified when reducing the fraction number to 8 and 4, resulting in 125 and 149% improvements, respectively. Using 4D-diaXLMS, up to 83% of the cross-linked peptides were repeatedly identified in three replicates, more than twice the 38% in the DDA mode. Furthermore, 4D-diaXLMS showed good performance in the quantitative analysis of yeast cross-linked peptides even in a 15-fold excess amount of HeLa cell matrix, with a low coefficient of variation and high quantitative accuracies in all concentrations. Overall, 4D-diaXLMS was proven to have high coverage, good reproducibility, and accurate quantification for in-depth XL-MS analysis in complex samples, demonstrating its immense potential for advances in the field.

Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

Chemical cross-linking with mass spectrometry provides low-resolution structural information on proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells or young and old mice. A difference can originate from protein conformational changes that alter the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications of the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein with the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences in cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.

Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides.

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.

Dimerization of European Robin Cryptochrome 4a.

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerization of avian Crys and the role it could play in the mechanism of magnetic sensing in migratory birds. Here, we present a combined experimental and computational investigation of the dimerization of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulfide bonds, chemical cross-linking, and photometric measurements show that disulfide-linked dimers are routinely formed, that their formation is promoted by exposure to blue light, and that the most likely cysteines are C317 and C412. Computational modeling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.

Extracellular matrix remodelling and stiffening contributes to tumorigenesis of salivary carcinoma ex pleomorphic adenoma——A study based on patient-derived organoids

BackgroundSalivary carcinoma ex pleomorphic adenoma (CXPA) is defined as a carcinoma that develops from benign pleomorphic adenoma (PA). Abnormally activated Androgen signaling pathway and amplification of HER-2/neu(ERBB-2) gene are known to be involved in CXPA tumorigenesis. Recent progress in tumour microenvironment research has led to identification that extracellular matrix (ECM) remodelling and increased stiffness act as critical contributing role in tumour carcinogenesis. This study examined ECM modifications to elucidate the mechanism underlying CXPA tumorigenesis.ResultsPA and CXPA organoids were successfully established. Histological observation, immunohistochemistry (IHC), and whole-exome sequencing demonstrated that organoids recapitulated phenotypic and molecular characteristics of their parental tumours. RNA-sequencing and bioinformatic analysis of organoids showed that differentially expressed genes are highly enriched in ECM-associated terms, implying that ECM alternations may be involved in carcinogenesis. Microscopical examination for surgical samples revealed that excessive hyalinized tissues were deposited in tumour during CXPA tumorigenesis. Transmission electron microscopy confirmed that these hyalinized tissues were tumour ECM in nature. Subsequently, examination by picrosirius red staining, liquid chromatography with tandem mass spectrometry, and cross-linking analysis indicated that tumour ECM was predominantly composed of type I collagen fibers, with dense collagen alignment and an increased level of collagen cross-linking. IHC revealed the overexpression of COL1A1 protein and collagen-synthesis-related genes, DCN and IGFBP5 (p < 0.05). Higher stiffness of CXPA than PA was demonstrated by atomic force microscopy and elastic imaging analysis. We utilized hydrogels to mimic ECM with varying stiffness degrees in vitro. Compared with softer matrices (5Kpa), CXPA cell line and PA primary cells exhibited more proliferative and invasive phenotypes in stiffer matrices (50Kpa, p < 0.01). Protein-protein interaction (PPI) analysis of RNA-sequencing data revealed that AR and ERBB-2 expression was associated with TWIST1. Moreover, surgical specimens demonstrated a higher TWIST1 expression in CXPA over PA. After knocking down TWIST1 in CXPA cells, cell proliferation, migration, and invasiveness were significantly inhibited (p < 0.01).ConclusionDeveloping CXPA organoids provides a useful model for cancer biology research and drug screening. ECM remodelling, attributed to overproduction of collagen, alternation of collagen alignment, and increased cross-linking, leads to increased ECM stiffness. ECM modification is an important contributor in CXPA tumorigenesis.

Tunable, thiol-ene, interpenetrating network hydrogels of norbornene-modified carboxymethyl cellulose and cellulose nanofibrils

Carboxymethyl cellulose modified with norbornene groups (NorCMC) and cellulose nanofibrils (CNFs) produced through mechanical refining without chemical pretreatment formed interpenetrating network hydrogels through a UV-light initiated thiol-ene reaction. The molar ratio of thiols in crosslinkers to norbornene groups off the NorCMC (T:N), total polymer weight percent in the hydrogel, and weight percent of CNFs of the total polymer content of the hydrogels were varied to control hydrogel properties. This method enabled orders of magnitude changes to behavior. Swelling in aqueous environments could be significant (>150 %) without CNFs to minimal (<15 %) with the use of 50 % CNFs. NorCMC and CNF networks interacted synergistically to create hydrogels with compression modulus values spanning 1 to 150 kPa - the values of most biological tissues. T:N and total polymer weight percent could be varied to create hydrogels with different CNF content, but the same compression modulus, targeting 10 and 100 kPa hydrogels and providing a system that can independently vary fibrillar content and bulk modulus. Analysis of the effective crosslinks, thiol-ene network mesh size, and burst release of the polymer indicated synergistic interactions of the NorCMC thiol-ene and CNFs networks. These interactions enhanced modulus and degradation control of the network under physiological conditions.

Fibrinogen BOE II: dysfibrinogenemia with bleeding and defective thrombin binding

BackgroundVariants of fibrinogen sequences that bind to thrombin’s catalytic sites are mostly associated with bleeding phenotypes, while variants with fibrinogen nonsubstrate-thrombin–binding sites are commonly believed to cause thrombosis. AαGlu39 and BβAla68 play important roles in fibrin(ogen)-thrombin–nonsubstrate binding. The BβAla68Thr variant has been described in several unrelated families with apparent thrombotic phenotypes. ObjectivesHomozygous AαGlu39Lys variant (fibrinogen BOE II) was identified in a boy with dysfibrinogenemia who had multiple cerebral hemorrhages. A series of analyses were performed to assess the variant’s functions and elucidate underlying bleeding mechanisms. MethodsAbnormal fibrinogen was purified from plasma and subjected to Western blot, fibrinogen and fibrin monomer polymerization, clottability, fibrinopeptides release, activated factor (F)XIII (FXIIIa) cross-linking, fibrinolysis, and scanning electron microscopy analyses. ResultsFibrinogen BOE II weakened the binding capacity of thrombin to fibrinogen and delayed the formation of fibrin clots. The release of fibrinopeptides, polymerization of fibrinogen catalyzed by thrombin, and cross-linking of FXIIIa of fibrinogen BOE II were impaired. In contrast, batroxobin-catalyzed fibrinogen polymerization and desA/desAB fibrin monomer polymerization did not differ from those in normal controls. Fibrin clots formed by fibrinogen BOE II were composed of thicker fibrin fibers and showed a faster fibrinolysis rate. ConclusionDefective fibrin(ogen)-thrombin–nonsubstrate binding is not necessarily associated with thrombotic disorders. When the hypercoagulable state created by increased circulating free thrombin is insufficient to compensate for defective hemostasis caused by slowly formed but rapidly lysed clots, the primary concern of thrombin-binding deficiency dysfibrinogenemia appears to be hemorrhage rather than thrombosis.

Improved Cross-Linking Coverage for Protein Complexes Containing Low Levels of Lysine by Using an Enrichable Photo-Cross-Linker.

Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.

Micronutrient optimization for tissue engineered articular cartilage production of type II collagen.

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Gaussia Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen promoter-driven Gaussia luciferase expression. While the target of this study was to establish a combination of all micronutrients, alpha-linolenic acid, copper, cobalt, chromium, manganese, molybdenum, vitamins A, E, D and B7 were all found to have a significant effect on type II collagen promoter activity. Five conditions containing all micronutrients predicted to produce the greatest luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen promoter activity. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Young's tensile modulus compared to engineered cartilage in basal media alone.Collagen cross-linking analysis confirmed formation of type II-type II collagen and type II-type IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.

Structure Elucidation of Secondary Metabolites: Current Frontiers and Lingering Pitfalls.

Analytical methods allow for the structure determination of submilligram quantities of complex secondary metabolites. This has been driven in large part by advances in NMR spectroscopic capabilities, including access to high-field magnets equipped with cryogenic probes. Experimental NMR spectroscopy may now be complemented by remarkably accurate carbon-13 NMR calculations using state-of-the-art DFT software packages. Additionally, microED analysis stands to have a profound effect on structure elucidation by providing X-ray-like images of microcrystalline samples of analytes. Nonetheless, lingering pitfalls in structure elucidation remain, particularly for isolates that are unstable or highly oxidized. In this Account, we discuss three projects from our laboratory that highlight nonoverlapping challenges to the field, with implications for chemical, synthetic, and mechanism of action studies. We first discuss the lomaiviticins, complex unsaturated polyketide natural products disclosed in 2001. The original structures were derived from NMR, HRMS, UV-vis, and IR analysis. Owing to the synthetic challenges presented by their structures and the absence of X-ray crystallographic data, the structure assignments remained untested for nearly two decades. In 2021, the Nelson group at Caltech carried out microED analysis of (-)-lomaiviticin C, leading to the startling discovery that the original structure assignment of the lomaiviticins was incorrect. Acquisition of higher-field (800 MHz 1H, cold probe) NMR data as well as DFT calculations provided insights into the basis for the original misassignment and lent further support to the new structure identified by microED. Reanalysis of the 2001 data set reveals that the two structure assignments are nearly indistinguishable, underscoring the limitations of NMR-based characterization. We then discuss the structure elucidation of colibactin, a complex, nonisolable microbiome metabolite implicated in colorectal cancer. The colibactin biosynthetic gene cluster was detected in 2006, but owing to colibactin's instability and low levels of production, it could not be isolated or characterized. We used a combination of chemical synthesis, mechanism of action studies, and biosynthetic analysis to identify the substructures in colibactin. These studies, coupled with isotope labeling and tandem MS analysis of colibactin-derived DNA interstrand cross-links, ultimately led to a structure assignment for the metabolite. We then discuss the ocimicides, plant secondary metabolites that were studied as agents against drug-resistant P. falciparum. We synthesized the core structure of the ocimicides and found significant discrepancies between our experimental NMR spectroscopic data and that reported for the natural products. We determined the theoretical carbon-13 NMR shifts for 32 diastereomers of the ocimicides. These studies indicated that a revision of the connectivity of the metabolites is likely needed. We end with some thoughts on the frontiers of secondary metabolite structure determination. As modern NMR computational methods are straightforward to execute, we advocate for their systematic use in validating the assignments of novel secondary metabolites.