Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.

The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component.

Beyond its role in cellular homeostasis, autophagy plays anti- and promicrobial roles in host-microbe interactions, both in animals and plants. One prominent role of antimicrobial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in the depletion of a bacterial virulence factor and unravel an unprecedented pathogen strategy to counteract defense-related autophagy in plant-bacteria interactions.

The p21CIP1-CDK4-DREAM axis is a master regulator of genotoxic stress-induced cellular senescence.

Cellular senescence, a major driver of aging, can be stimulated by DNA damage, and is counteracted by the DNA repair machinery. Here we show that in p16INK4a-deficient cells, senescence induction by the environmental genotoxin B[a]P or ionizing radiation (IR) completely depends on p21CIP1. Immunoprecipitation-based mass spectrometry interactomics data revealed that during senescence induction and maintenance, p21CIP1 specifically inhibits CDK4 and thereby activates the DREAM complex. Genome-wide transcriptomics revealed striking similarities in the response induced by B[a]P and IR. Among the top 100 repressed genes 78 were identical between B[a]P and IR and 76 were DREAM targets. The DREAM complex transcriptionally silences the main proliferation-associated transcription factors E2F1, FOXM1 and B-Myb as well as multiple DNA repair factors. Knockdown of p21CIP1, E2F4 or E2F5 diminished both, repression of these factors and senescence. The transcriptional profiles evoked by B[a]P and IR largely overlapped with the profile induced by pharmacological CDK4 inhibition, further illustrating the role of CDK4 inhibition in genotoxic stress-induced senescence. Moreover, data obtained by live-cell time-lapse microscopy suggest the inhibition of CDK4 by p21CIP1 is especially important for arresting cells which slip through mitosis. Overall, we identified the p21CIP1/CDK4/DREAM axis as a master regulator of genotoxic stress-induced senescence.

SPIRO - the automated Petri plate imaging platform designed by biologists, for biologists.

Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photographs of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists' needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO's excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared with manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing, and photoblastic response.

Loss of the mitochondrial protein Abcb10 results in altered arginine metabolism in MEL and K562 cells and nutrient stress signaling through ATF4.

Abcb10 is a mitochondrial membrane protein involved in hemoglobinization of red cells. Abcb10 topology and ATPase domain localization suggest it exports a substrate, likely biliverdin, out of mitochondria that is necessary for hemoglobinization. In this study, we generated Abcb10 deletion cell lines in both mouse murine erythroleukemia and human erythroid precursor human myelogenous leukemia (K562) cells to better understand the consequences of Abcb10 loss. Loss of Abcb10 resulted in an inability to hemoglobinize upon differentiation in both K562 and mouse murine erythroleukemia cells with reduced heme and intermediate porphyrins and decreased levels of aminolevulinic acid synthase 2 activity. Metabolomic and transcriptional analyses revealed that Abcb10 loss gave rise to decreased cellular arginine levels, increased transcripts for cationic and neutral amino acid transporters with reduced levels of the citrulline to arginine converting enzymes argininosuccinate synthetase and argininosuccinate lyase. The reduced arginine levels in Abcb10-null cells gave rise to decreased proliferative capacity. Arginine supplementation improved both Abcb10-null proliferation and hemoglobinization upon differentiation. Abcb10-null cells showed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha, increased expression of nutrient sensing transcription factor ATF4 and downstream targets DNA damage inducible transcript 3 (Chop), ChaC glutathione specific gamma-glutamylcyclotransferase 1 (Chac1), and arginyl-tRNA synthetase 1 (Rars). These results suggest that when the Abcb10 substrate is trapped in the mitochondria, the nutrient sensing machinery is turned on remodeling transcription to block protein synthesis necessary for proliferation and hemoglobin biosynthesis in erythroid models.

Organic Cocrystal Alloys: From Three Primary Colors to Continuously Tunable Emission and Applications on Optical Waveguides and Displays.

Multicolor luminescence of organic fluorescent materials is an essential part of lighting and optical communication. However, the conventional construction of a multicolor luminescence system based on integrating multiple organic fluorescent materials of a single emission band remains complicated and to be improved. Herein, organic alloys (OAs) capable of full-color emission are synthesized based on charge transfer (CT) cocrystals. By adjusting the molar ratio of electron donors, the emission color of the OAs can be conveniently and continuously regulated in a wide visible range from blue (CIE: 0.187, 0.277), to green (CIE: 0.301, 0.550), and to red (CIE: 0.561, 0.435). The OAs show analogous 1D morphology with smooth surface, allowing for full-color waveguides with low optical-loss coefficient. Impressively, full-color optical displays are easily achieved through the OAs system with continuous emission, which shows promising applications in the field of optical display and promotes the development of organic photonics.

A model of hybrid speciation process drawn from three new poplar species originating from distant hybridization between sections.

Although numerous studies have been conducted on hybrid speciation, our understanding of this process remains limited. Through an 18-year systematic investigation of all taxa of Populus on the Qinghai-Tibet Plateau, we discovered three new taxa with clear characteristics of sect. Leucoides. Further evidence was gathered from morphology, whole-genome bioinformatics, biogeography, and breeding to demonstrate synthetically that they all originated from distant hybridization between sect. Leucoides and sect. Tacamahaca. P. gonggaensis originated from the hybridization of P. lasiocarpa with P. cathayana, P. butuoensis from the hybridization of P. wilsonii with P. szechuanica, and P. dafengensis from the hybridization of P. lasiocarpa with P. szechuanica. Due to heterosis, the three hybrid taxa possess greater ecological adaptability than their ancestral species. We propose a hybrid speciation process model that incorporates orthogonal, reverse, and backcrossing events. This model can adequately explain some crucial evolutionary concerns, such as the nuclear-cytoplasmic conflict on phylogeny and the extinction of ancestral species within the distribution range of hybrid species.

A new species of Populus and the extensive hybrid speciation arising from it on the Qinghai-Tibet Plateau.

While the diversity of species formation is broadly acknowledged, significant debate exists regarding the universal nature of hybrid species formation. Through an 18-year comprehensive study of all Populus species on the Qinghai-Tibet Plateau, 23 previously recorded species and 8 new species were identified. Based on morphological characteristics, these can be classified into three groups: species in section Leucoides, species with large leaves, and species with small leaves in section Tacamahaca. By conducting whole-genome re-sequencing of 150 genotypes from these 31 species, 2.28 million single nucleotide polymorphisms (SNPs) were identified. Phylogenetic analysis utilizing these SNPs not only revealed a highly intricate evolutionary network within the large-leaf species of section Tacamahaca but also confirmed that a new species, P. curviserrata, naturally hybridized with P. cathayana, P. szechuanica, and P. ciliata, resulting in 11 hybrid species. These findings indicate the widespread occurrence of hybrid species formation within this genus, with hybridization serving as a key evolutionary mechanism for Populus on the plateau. A novel hypothesis, "Hybrid Species Exterminating Their Ancestral Species (HSEAS)," is introduced to explain the mechanisms of hybrid species formation at three different scales: the entire plateau, the southeastern mountain region, and individual river valleys.

Strategies for the Construction of Benzobicyclo[3.2.1]octane in Natural Product Synthesis.

Benzobicyclo[3.2.1]octane is a cage-like unique motif containing a bicyclo[3.2.1]octane structure fused with at least one benzene ring. It is found in various natural products that exhibit structural complexities and important biological activities. The total synthesis of natural products possessing this challenging structure has received considerable attention, and great advances have been made in this field during the past 15 years. This review summarizes thus far achieved chemical syntheses and synthetic studies of natural compounds featuring the benzobicyclo[3.2.1]octane core. It focuses on strategic approaches constructing the bridged structure, aiming to provide a useful reference for inspiring further advancements in strategies and total syntheses of natural products with such a framework.

Improvement of electrothermal and photothermal properties of ultra-thin graphite film on oxygen plasma-treated polyimide substrate.

Graphene and its derivatives are widely used in the field of energy conversion and management due to their excellent physical and chemical properties. In this paper, ultra-thin graphite film (GF) with thickness of 100-150 nm prepared by chemical vapor deposition was transferred to oxygen plasma-treated polyimide (PI) substrate as flexible heating film. The electrothermal and photothermal properties of GF on PI substrates with different treatment time were studied. The experimental results show that the PI substrate pretreated by oxygen plasma can change the surface morphology of GF, increase its electrical conductivity and light absorption capacity, and significantly improve the electrothermal and photothermal properties of GF heater. Under the low applied voltage of 5 V (power density of 0.81 W cm-2), the surface temperature of GF on 40 min plasma-treated PI substrate can rise to 250 °C, which is nearly 50 °C higher than that of GF on untreated PI substrate. When 100 nm thick commercial multilayer graphene film (MLG) is used, plasma-treated PI substrate can increase the electric heating temperature of MLG by 70 °C. In terms of photothermal performance, the surface temperature of GF on 50 min plasma-treated PI substrate can reach 73 °C under one Sun irradiation, which is 8 °C higher than that on untreated substrate. The experimental results are in good agreement with the simulation research. Our strategy has important implications for the development of efficient and energy-saving graphene/graphite-based heating films for advanced electrothermal and photothermal conversion devices.

ABCB10 Loss Reduces CD4+ T Cell Activation and Memory Formation.

T cells must shift their metabolism to respond to infections and tumors and to undergo memory formation. The ATP-binding cassette transporter ABCB10 localizes to the mitochondrial inner membrane, where it is thought to export a substrate important in heme biosynthesis and metabolism, but its role in T cell development and activation is unknown. In this article, we use a combination of methods to study the effect of ABCB10 loss in primary and malignantly transformed T cells. Although Abcb10 is dispensable for development of both CD4+ and CD8+ T cells, it is required for expression of specific cytokines in CD4+, but not CD8+, T cells activated in vitro. These defects in cytokine expression are magnified on repeated stimulation. In vivo, CD8+ cells lacking ABCB10 expand more in response to viral infection than their control counterparts, while CD4+ cells show reductions in both number and percentage. CD4+ cells lacking ABCB10 show impairment in Ag-specific memory formation and recall responses that become more severe with time. In malignant human CD4+ Jurkat T cells, we find that CRISPR-mediated ABCB10 disruption recapitulates the same cytokine expression defects upon activation as observed in primary mouse T cells. Mechanistically, ABCB10 deletion in Jurkat T cells disrupts the ability to switch to aerobic glycolysis upon activation. Cumulatively, these results show that ABCB10 is selectively required for specific cytokine responses and memory formation in CD4+ T cells, suggesting that targeting this molecule could be used to mitigate aberrant T cell activation.

Kinase-controlled phase transition of membraneless organelles in mitosis.

Liquid-liquid phase separation has been shown to underlie the formation and disassembly of membraneless organelles in cells, but the cellular mechanisms that control this phenomenon are poorly understood. A prominent example of regulated and reversible segregation of liquid phases may occur during mitosis, when membraneless organelles disappear upon nuclear-envelope breakdown and reappear as mitosis is completed. Here we show that the dual-specificity kinase DYRK3 acts as a central dissolvase of several types of membraneless organelle during mitosis. DYRK3 kinase activity is essential to prevent the unmixing of the mitotic cytoplasm into aberrant liquid-like hybrid organelles and the over-nucleation of spindle bodies. Our work supports a mechanism in which the dilution of phase-separating proteins during nuclear-envelope breakdown and the DYRK3-dependent degree of their solubility combine to allow cells to dissolve and condense several membraneless organelles during mitosis.

Analysis of factors influencing hospitalization cost of patients with distal radius fractures: an empirical study based on public traditional Chinese medicine hospitals in two cities, China.

BACKGROUND: Distal radius fractures (DRFs) have become a public health problem for all countries, bringing a heavier economic burden of disease globally, with China's disease economic burden being even more acute due to the trend of an aging population. This study aimed to explore the influencing factors of hospitalization cost of patients with DRFs in traditional Chinese medicine (TCMa) hospitals to provide a scientific basis for controlling hospitalization cost. METHODS: With 1306 cases of DRFs patients hospitalized in 15 public TCMa hospitals in two cities of Gansu Province in China from January 2017 to 2022 as the study object, the influencing factors of hospitalization cost were studied in depth gradually through univariate analysis, multiple linear regression, and path model. RESULTS: Hospitalization cost of patients with DRFs is mainly affected by the length of stay, surgery and operation, hospital levels, payment methods of medical insurance, use of TCMa preparations, complications and comorbidities, and clinical pathways. The length of stay is the most critical factor influencing the hospitalization cost, and the longer the length of stay, the higher the hospitalization cost. CONCLUSIONS: TCMa hospitals should actively take advantage of TCMb diagnostic modalities and therapeutic methods to ensure the efficacy of treatment and effectively reduce the length of stay at the same time, to lower hospitalization cost. It is also necessary to further deepen the reform of the medical insurance payment methods and strengthen the construction of the hierarchical diagnosis and treatment system, to make the patients receive reasonable reimbursement for medical expenses, thus effectively alleviating the economic burden of the disease in the patients with DRFs.

Antagonistic fungal enterotoxins intersect at multiple levels with host innate immune defences.

Animals and plants need to defend themselves from pathogen attack. Their defences drive innovation in virulence mechanisms, leading to never-ending cycles of co-evolution in both hosts and pathogens. A full understanding of host immunity therefore requires examination of pathogen virulence strategies. Here, we take advantage of the well-studied innate immune system of Caenorhabditis elegans to dissect the action of two virulence factors from its natural fungal pathogen Drechmeria coniospora. We show that these two enterotoxins have strikingly different effects when expressed individually in the nematode epidermis. One is able to interfere with diverse aspects of host cell biology, altering vesicle trafficking and preventing the key STAT-like transcription factor STA-2 from activating defensive antimicrobial peptide gene expression. The second increases STA-2 levels in the nucleus, modifies the nucleolus, and, potentially as a consequence of a host surveillance mechanism, causes increased defence gene expression. Our results highlight the remarkably complex and potentially antagonistic mechanisms that come into play in the interaction between co-evolved hosts and pathogens.

Diabetes and osteoporosis: a two-sample mendelian randomization study.

BACKGROUND: The effects on bone mineral density (BMD)/fracture between type 1 (T1D) and type 2 (T2D) diabetes are unknown. Therefore, we aimed to investigate the causal relationship between the two types of diabetes and BMD/fracture using a Mendelian randomization (MR) design. METHODS: A two-sample MR study was conducted to examine the causal relationship between diabetes and BMD/fracture, with three phenotypes (T1D, T2D, and glycosylated hemoglobin [HbA1c]) of diabetes as exposures and five phenotypes (femoral neck BMD [FN-BMD], lumbar spine BMD [LS-BMD], heel-BMD, total body BMD [TB-BMD], and fracture) as outcomes, combining MR-Egger, weighted median, simple mode, and inverse variance weighted (IVW) sensitivity assessments. Additionally, horizontal pleiotropy was evaluated and corrected using the residual sum and outlier approaches. RESULTS: The IVW method showed that genetically predicted T1D was negatively associated with TB-BMD (ß = -0.018, 95% CI: -0.030, -0.006), while T2D was positively associated with FN-BMD (ß = 0.033, 95% CI: 0.003, 0.062), heel-BMD (ß = 0.018, 95% CI: 0.006, 0.031), and TB-BMD (ß = 0.050, 95% CI: 0.022, 0.079). Further, HbA1c was not associated with the five outcomes (ß ranged from - 0.012 to 0.075). CONCLUSIONS: Our results showed that T1D and T2D have different effects on BMD at the genetic level. BMD decreased in patients with T1D and increased in those with T2D. These findings highlight the complex interplay between diabetes and bone health, suggesting potential age-specific effects and genetic influences. To better understand the mechanisms of bone metabolism in patients with diabetes, further longitudinal studies are required to explain BMD changes in different types of diabetes.

Adolescents and Children Age Estimation Using Machine Learning Based on Pulp and Tooth Volumes on CBCT Images.

OBJECTIVES: To estimate adolescents and children age using stepwise regression and machine learning methods based on the pulp and tooth volumes of the left maxillary central incisor and cuspid on cone beam computed tomography (CBCT) images, and to compare and analyze the estimation results. METHODS: A total of 498 Shanghai Han adolescents and children CBCT images of the oral and maxillofacial regions were collected. The pulp and tooth volumes of the left maxillary central incisor and cuspid were measured and calculated. Three machine learning algorithms (K-nearest neighbor, ridge regression, and decision tree) and stepwise regression were used to establish four age estimation models. The coefficient of determination, mean error, root mean square error, mean square error and mean absolute error were computed and compared. A correlation heatmap was drawn to visualize and the monotonic relationship between parameters was visually analyzed. RESULTS: The K-nearest neighbor model (R2=0.779) and the ridge regression model (R2=0.729) outperformed stepwise regression (R2=0.617), while the decision tree model (R2=0.494) showed poor fitting. The correlation heatmap demonstrated a monotonically negative correlation between age and the parameters including pulp volume, the ratio of pulp volume to hard tissue volume, and the ratio of pulp volume to tooth volume. CONCLUSIONS: Pulp volume and pulp volume proportion are closely related to age. The application of CBCT-based machine learning methods can provide more accurate age estimation results, which lays a foundation for further CBCT-based deep learning dental age estimation research.

Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan.

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

Posttranslational regulation of mitochondrial frataxin and identification of compounds that increase frataxin levels in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a degenerative disease caused by a decrease in the mitochondrial protein frataxin (Fxn), which is involved in iron-sulfur cluster (ISC) synthesis. Diminutions in Fxn result in decreased ISC synthesis, increased mitochondrial iron accumulation, and impaired mitochondrial function. Here, we show that conditions that result in increased mitochondrial reactive oxygen species in yeast or mammalian cell culture give rise to increased turnover of Fxn but not of other ISC synthesis proteins. We demonstrate that the mitochondrial Lon protease is involved in Fxn degradation and that iron export through the mitochondrial metal transporter Mmt1 protects yeast Fxn from degradation. We also determined that when FRDA fibroblasts were grown in media containing elevated iron, mitochondrial reactive oxygen species increased and Fxn decreased compared to WT fibroblasts. Furthermore, we screened a library of FDA-approved compounds and identified 38 compounds that increased yeast Fxn levels, including the azole bifonazole, antiparasitic fipronil, antitumor compound dibenzoylmethane, antihypertensive 4-hydroxychalcone, and a nonspecific anion channel inhibitor 4,4-diisothiocyanostilbene-2,2-sulfonic acid. We show that top hits 4-hydroxychalcone and dibenzoylmethane increased mRNA levels of transcription factor nuclear factor erythroid 2-related factor 2 in FRDA patient-derived fibroblasts, as well as downstream antioxidant targets thioredoxin, glutathione reductase, and superoxide dismutase 2. Taken together, these findings reveal that FRDA progression may be in part due to oxidant-mediated decreases in Fxn and that some approved compounds may be effective in increasing mitochondrial Fxn in FRDA, delaying disease progression.

Metabolomics and Genomics Enable the Discovery of a New Class of Nonribosomal Peptidic Metallophores from a Marine Micromonospora.

Although microbial genomes harbor an abundance of biosynthetic gene clusters, there remain substantial technological gaps that impair the direct correlation of newly discovered gene clusters and their corresponding secondary metabolite products. As an example of one approach designed to minimize or bridge such gaps, we employed hierarchical clustering analysis and principal component analysis (hcapca, whose sole input is MS data) to prioritize 109 marine Micromonospora strains and ultimately identify novel strain WMMB482 as a candidate for in-depth "metabologenomics" analysis following its prioritization. Highlighting the power of current MS-based technologies, not only did hcapca enable the discovery of one new, nonribosomal peptide bearing an incredible diversity of unique functional groups, but metabolomics for WMMB482 unveiled 16 additional congeners via the application of Global Natural Product Social molecular networking (GNPS), herein named ecteinamines A-Q (1-17). The ecteinamines possess an unprecedented skeleton housing a host of uncommon functionalities including a menaquinone pathway-derived 2-naphthoate moiety, 4-methyloxazoline, the first example of a naturally occurring Ψ[CH2NH] "reduced amide", a methylsulfinyl moiety, and a d-cysteinyl residue that appears to derive from a unique noncanonical epimerase domain. Extensive in silico analysis of the ecteinamine (ect) biosynthetic gene cluster and stable isotope-feeding experiments helped illuminate the novel enzymology driving ecteinamine assembly as well the role of cluster collaborations or "duets" in producing such structurally complex agents. Finally, ecteinamines were found to bind nickel, cobalt, zinc, and copper, suggesting a possible biological role as broad-spectrum metallophores.