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The methylation of peptide backbone amides is a hallmark of bioactive natural products, and it also greatly modifies the pharmacology of synthetic peptides. Usually, bioactive

Genetically-encoded calcium indicators (GECI) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of sub-optimal peak signal-to-baseline-ratio (SBR) with limited resolution for reporting subtle calcium transients. We report herein the development of a suite of calcium sensors, designated NEMO, with fast kinetics and wide dynamic ranges (>100-fold). NEMO indicators report Ca2+transients with peak SBRs ~20-fold larger than the top-of-the-range GCaMP series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared to GCaMPs, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times largerin vivo, thereby outperforming most existing state-of-the-art GECIs. Given their high sensitivity and resolution to report intracellular Ca2+signals, NEMO sensors may find broad applications in monitoring neuronal activities and other Ca2+-modulated physiological processes in both mammals and plants.

Although splicing is an integral part of the expression of many genes in our body, genetic syndromes with spliceosomal defects affect only specific tissues. To help understand the mechanism, we investigated the expression pattern of a core protein of the major spliceosome, SmB/B' (Small Nuclear Ribonucleoprotein Polypeptides B/B'), which is encoded by SNRPB. Loss-of-function mutations of SNRPB in humans cause cerebro-costo-mandibular syndrome (CCMS) characterized by rib gaps, micrognathia, cleft palate, and scoliosis. Our expression analysis focused on the affected structures as well as non-affected tissues, using chick and mouse embryos as model animals.Embryos at young stages (gastrula) showed ubiquitous expression of SmB/B'. However, the level and pattern of expression became tissue-specific as differentiation proceeded. The regions relating to CCMS phenotypes such as cartilages of ribs and vertebrae and palatal mesenchyme express SmB/B' in the nucleus sporadically. However, cartilages that are not affected in CCMS also showed similar expressions. Another spliceosomal gene, SNRNP200, which mutations cause retinitis pigmentosa, was also prominently expressed in cartilages in addition to the retina.The expression of SmB/B' is spatiotemporally regulated during embryogenesis despite the ubiquitous requirement of the spliceosome, however, the expression pattern is not strictly correlated with the phenotype presentation.

The System of Rice Intensification (SRI) seeks to increase both the sustainability and the productivity of smallholder rice farms, but the adoption of this methodology is constrained by local access to appropriate mechanical equipment. Valuable information about real difficulties in the adoption of SRI was collected through communications with five field partners. Two primary mechanization obstacles were identified, that is, the availability and performance of a roller component on a push seeder and a rotor component on a push weeder. The potential of additive manufacturing (AM), and especially material extrusion three-dimensional (3D) printing (ME3DP), to assist in overcoming the identified obstacles was assessed for two cases while considering both local prototyping and low-volume production. A simplified cost model was used to compare with the cost of manufacturing both in the United States and locally in the field. The acquired data suggests that in specific cases current ME3DP technology can more rapidly provide functional parts, accelerating the design cycle and lowering cost by about a factor of 10 relative to local fabrication routes. In the case where mechanical performance is critical and dimensional precision and surface finish are not, wire arc metal AM appears promising, but it is not as economical as fabrication by traditional means in the field.

Cyclic peptides are excellent drug candidates, placing macrocyclization reactions at the apex of drug development. PatG and related dual-action proteases from cyanobactin biosynthesis are responsible for cleaving off the C-terminal recognition sequence and macrocyclizing the substrate to provide cyclic peptides. This reaction has found use in the enzymatic synthesis of diverse macrocycles. However, these enzymes function best on substrates that terminate with the non-proteinogenic thiazole/thiazoline residue, complicating synthetic strategies. Here, we biochemically characterize a new class of PatG-like macrocyclases that natively use proline, obviating the necessity of additional chemical or biochemical steps. We experimentally define the biochemical steps involved in synthesizing the widespread prenylagaramide-like natural products, including macrocyclization and prenylation. Using saturation mutagenesis, we show that macrocyclase PagG and prenyltransferase PagF are highly promiscuous, producing a library of more than 100 cyclic peptides and their prenylated derivatives in vitro. By comparing our results to known cyanobactin macrocyclase enzymes, we catalog a series of enzymes that collectively should synthesize most small macrocycles. Collectively, these data reveal that, by selecting the right cyanobactin macrocyclase, a large array of enzymatically synthesized macrocycles are accessible.

Whole-genome duplication, in particular allopolyploidy, has shaped the evolution of angiosperms and other organisms. Structural reorganization of chromosomes and repatterning of gene expression is frequently observed in early generation allopolyploids, with potential ecological consequences. The relative contributions of environmental and intrinsic drivers to these changes are unknown. We show here that in marsh orchids (Dactylorhiza, Orchidaceae), recurrently-formed allopolyploids are characterized by distinct eco-physiologies matching their respective environments, providing us with an excellent study system to address this question. Here we integrate eco-physiological and transcriptomic comparative studies to reveal a complex suite of intertwined, pronounced differences between sibling allopolyploids. We show that Dactylorhiza majalis that is distributed in Central and Southern Europe favors meadows with mesic soils. Its sibling allopolyploid D. traunsteineri occurs in fens, characterized by soils depleted by macro- and micronutrients, mainly in previously glaciated European areas. We further uncover distinct features in their nutrient transport, leaf elemental chemistry, light-harvesting, photoprotection, and stomata activity, that appear all linked to their distinct ecologies, in particular soil chemistry differences at their native sites. Recurrent polyploidization hence enriches biodiversity through eco-physiological diversification, providing the opportunity for sibling allopolyploids to evolve as distinct evolutionary units, despite pervasive interspecific gene flow.Significance StatementWhole-genome duplication resulting in polyploidy has underpinned the evolution of flowering plants and other organisms, and is important for many crops. However, the ecological implications of polyploidy remain little understood. Here, we demonstrate that two sibling allopolyploid marsh orchid species prefer distinct habitats, and have evolved a suite of distinctive ecophysiological characters (e.g. nutrient transport, energy harvesting and photoprotection). We argue that the divergence of these characters in the nascent polyploids drove adaptation into distinct ecological niches (low nutrient fens versus meadows with mesic soils), generating ecological barriers that maintains distinct, independent lineages, even in the presence of interspecific gene flow.

YcaO proteins are ubiquitous in living organisms, where they perform crucial post-translational modifications of peptides and proteins. They are used extensively in biotechnology in companies and academic settings around the world. They also underlie some of the most important natural products in pharmaceutical development, such as thiopeptides (thiostrepton, etc.). Here, we solve one of the major outstanding mysteries behind YcaO proteins: how they exert precision selectivity of the nucleophile. The resulting findings have major implications in understanding the >30,000 YcaO proteins currently in sequencing databases and will be used widely for precision synthetic biology applications.

The growth of cracks can be substantially influenced by the environment. Atomic modeling provides a means to isolate the action of individual mechanisms involved in such complex processes. Here, we utilize a newly implemented multiscale modeling approach to assess the role of material dissolution on long crack growth in a ductile material. While we find dissolution to be capable of freeing arrested fatigue cracks, the crack tip is always blunted under both static and cyclic loading, suggesting that dissolution has an overall crack arresting effect. Despite observations of plasticity-induced-dissolution and dissolution-induced-plasticity that are consistent with macroscale experiments, dissolution-induced-blunting is found to be independent of mechanical loading magnitude. This will simplify implementation of the dissolution-induced-blunting process into continuum crack growth models.

© The Author(s) 2020., The causes and consequences of genome reduction in animals are unclear because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8-megabase genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene-dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes., This study was supported by Sars Centre core budget and the European Research Council (ERC) grant agreement no. 648861 to A.H. J.M.M.-D. was additionally supported by the ERC grant agreement no. 801669, and B.C.V. by an EMBO Long-Term Fellowship (ALTF 74-2018). J.L.G.-S. received funding from the ERC (grant agreement no. 740041) and the Spanish Ministerio de Economía y Competitividad (grant no. BFU2016-74961-P) and the institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687). We dedicate this manuscript to the loving memory of José Luis Gómez-Skarmeta.

© 2021 The Authors. There is an increasing need for sustainable sources of omega-3 fatty acids; this challenge can be addressed through large-scale production of eicosapentaenoic acid (EPA) using microalgae. Identification of suitable strains is key in successful process scale-up. However, it is not certain whether conclusions from small-scale systems (i.e. flasks and well plates) can be translated to larger photobioreactors. To examine this issue the productivity and fatty acid composition of eleven different microalgal strains was quantified using both flask and photobioreactor cultures, generating a significant experimental dataset. Results from the flask screening offered relatively poor predictions of performance in photobioreactors, suggesting a need for improved screening tools. Of the species examined, Phaeodactylum tricornutum was found to be the most promising when grown in photobioreactors. To further guide scale-up the effect of environmental conditions (temperature and salinity) on EPA production was examined. It was found that the EPA content was ~5% of the dry biomass and this was approximately constant for the range of temperatures (13–27 °C) and salinities (35–50 g L−1) examined. Finally, detailed nutritional information about the biomass is presented, which can serve as a starting point for its formulation into food products. Australian Government Research Training Program Scholarship.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are ubiquitous natural products. Bioactive RiPPs are produced from a precursor peptide, which is modified by enzymes. Usually, a single product is encoded in a precursor peptide. However, in cyanobactins and several other RiPP pathways, a single precursor peptide encodes multiple bioactive products flanking with recognition sequences known as “cassettes”. The role of multiple cassettes in one peptide is mysterious, but in general their presence is a marker of biosynthetic plasticity. Here, we show that in cyanobactin biosynthesis the presence of multiple cassettes confers distributive enzyme processing to multiple steps of the pathway, a feature we propose to be a hallmark of multi-cassette RiPPs. TruD heterocyclase is stochastic and distributive. Although a canonical biosynthetic route is favored with certain substrates, every conceivable biosynthetic route is accepted. Together, these factors afford greater plasticity to the biosynthetic pathway by equalizing the processing of each cassette, enabling access to chemical diversity.

Natural products are significant therapeutic agents and valuable drug leads. This is likely owing to their three-dimensional structural complexity, which enables them to form complex interactions with biological targets. Enzymes from natural product biosynthetic pathways show great potential to generate natural product-like compounds and libraries. Many challenges still remain in biosynthesis, such as how to rationally synthesize small molecules with novel structures, and how to generate maximum chemical diversity. In this account, we describe recent advances from our laboratory in the synthesis of natural product-like libraries using natural biosynthetic machinery. Our work has focused on the pat and tru biosynthetic pathways to patellamides, trunkamide, and related compounds from cyanobacterial symbionts in marine tunicates. These belong to the cyanobactin class of natural products, which are part of the larger group of Ribosomally synthesized and Posttranslationally modified Peptides (RiPPs). These results have enabled the synthesis of rationally designed small molecules and libraries covering more than 1 million estimated derivatives. Because the RiPPs are translated on the ribosome and then enzymatically modified, they are highly compatible with recombinant technologies. This is important because it means that the resulting natural products, their derivatives, and wholly new compounds can be synthesized using the tools of genetic engineering. The RiPPs also represent possibly the most widespread group of bioactive natural products, although this is in part because of the broad definition of what constitutes a RiPP. In addition, the underlying ideas may form the basis for broad-substrate biosynthetic pathways beyond the RiPPs. For example, some of the ideas about kinetic ordering of broad-substrate pathways may apply to polyketide or nonribosomal peptide biosynthesis as well. While making these products, we have sought to understand what makes biosynthetic pathways plastic, and whether or not there are any rules that might generally apply to plastic biosynthetic pathways. We present three principles of diversity-generating biosynthesis: 1) Substrate evolution, in which the substrates change while enzymes remain constant; 2) Pairing of recognition sequences on substrates with biosynthetic enzymes; 3) An inverse metabolic flux in comparison to canonical pathways. If these principles are general, they may enable the design of unimagined derivatives using biosynthetic engineering. For example, it is possible to discover substrate evolution directly by examining sequencing data. By shuffling appropriate recognition sequences and biosynthetic enzymes, it has already been possible to make new hybrid products of multiple pathways. While cases so far have been limited, if this is more general designed synthesis will become routine. Finally, biosynthesis of natural products is regulated in elaborate ways that are just beginning to be understood. If the inverse metabolic flux model is widespread, it potentially informs on what the timing and relative production level of each enzyme in a designer pathway should be in order to optimize the synthesis of new compounds in vivo. [Image: see text]

Supplemental material, Supplementary_materials for Species identification of felis and vulpes by a novel loop-mediated isothermal amplification assay in fur products by Shunji Yu, Wenjia Gu, Yi Yu, Qinfeng Qu and Yi’nan Zhang in Journal of Engineered Fibers and Fabrics

A Correction to this paper has been published: https://doi.org/10.1038/s41559-020-01327-6.

Cyanobactin biosynthetic enzymes have exceptional versatility in the synthesis of natural and unnatural products. Cyanobactins are ribosomally synthesized and posttranslationally modified peptides synthesized by multistep pathways involving a broad suite of enzymes, including heterocyclases/cyclodehydratases, macrocyclases, proteases, prenyltransferases, methyltransferases, and others. Here, we describe the enzymology and structural biology of cyanobactin biosynthetic enzymes, aiming at the twin goals of understanding biochemical mechanisms and biosynthetic plasticity. We highlight how this common suite of enzymes may be utilized to generate a large array or structurally and chemically diverse compounds.

A copper(II)-catalyzed methoxylation of unactivated (hetero)aryl C–H bonds directed by our newly developed PIP directing group with methanol has been achieved. This protocol occurs efficiently with a catalytic amount of copper (5 mol%), is a simple and environmentally benign reaction system, has a broad substrate scope and high functional group tolerance.

Subcritical crack growth can occur in a glass when the stress intensity factor is less than the fracture toughness if water molecules are present. A novel bi-material beam specimen is proposed to investigate environmentally assisted crack growth (EACG). Two materials with different coefficients of thermal expansion are diffusion bonded at high temperature and cooled to the room temperature which introduces residual stress in the beam. A Finite element (FE) model is developed and initially validated with an analytical model. Steady-state crack (SSC) depth at which mode II stress intensity factor (KII) is zero and the corresponding mode I stress intensity factor (KI) value are obtained for different material pairs and thickness ratios of the top and bottom materials using the FE model. Crack propagation path is also predicted. We finally modify the geometry of the specimen to generate non-constant KI values as the crack propagates.

A copper/silver-mediated oxidative ortho-ethynylation of unactivated aryl C-H bonds with terminal alkyne has been developed. The reaction uses the removable PIP directing group and features broad substrate scope, high functional-group tolerance, and compatibility with a wide range of heterocycles, providing an efficient synthesis of aryl alkynes. This procedure highlights the potential of copper catalysts to promote unique, synthetically enabling C-H functionalization reactions that lie outside of the current scope of precious metal catalysis.

The regulation of citrate on amorphous calcium phosphate (ACP)-mediated crystallization of hydroxyapatite (HAP) is revealed in this work. The surface associated citrate on ACP plays the key role in controlling the nucleation of HAP by inhibiting the reaction of surface nucleation, and the effect of embedded citrate inside ACP or citrate in solution is weak.

A rapid on-site detection of exogenous proteins without the need for equipped laboratories or skilled personnel would benefit many areas. We built a rapid protein detection platform based on aptamer-induced inner-membrane scaffolds dimerization by virtue of bacterial ghost system. When the detection platform was coincubated with two kinds of aptamers targeting two different sites of thrombin, green fluorescence or β-lactamase activity were yielded with two different designs. The latter was detected by commercially available testing strips.

Background: Few studies have investigated differences in bone health and associated lifestyle factors between urban and rural populations in countries, such as China, undergoing rapid nutrition transition. Such a study may help to identify risk factors of osteoporosis and provide evidence for future preventive strategies. Objective: To determine primarily whether differences in bone mineral content (BMC) and bone mineral density (BMD) exist between urban and rural Chinese men and women and secondly whether any urban–rural differences could be explained by body size or lifestyle factors. Methods: In total, 490 men and 689 women aged 50–70 were included in the study. 535 of them were from urban Shanghai and 644 from surrounding rural areas. Anthropometric measurements were conducted and spine lumber 1–4 BMC measurements were determined by dualenergy X-ray absorptiometry (DEXA). Information on socioeconomic status, medical history, smoking and drinking habits and physical activity were collected. Results: Urban men and women had significantly higher spine BMC, BMD and bone area than their rural counterparts (Pb0.01). After controlling body size, the differences between urban–rural spine BMC and BMD remained in women (Pb0.001), but were no longer significant in men. The urban and rural differences of BMC and BMD in women could not be explained by including the lifestyle factors such as income, intake of milk, vitamin D and calcium, total physical activity level, walking and social activity. Conclusion: This study found the significant differences in both spine BMC and BMD between urban and rural men and women in Shanghai, China. This difference could be explained by the body size in men; however, it remained unexplained in women after adjusting for body size and certain lifestyle risk factors.

An efficient synthesis of aryl alkynes using the removable PIP as directing group is developed.

A copper-mediated C–H hydroxylation of arenes and heteroarenes using our newly developed PIP directing group has been developed. This procedure is scalable and compatible with a wide range of functional groups and heteroarenes, providing an operationally simple protocol for the synthesis of o-hydroxybenzamides. The hydroxylation of nicotinamides gave 4-oxo-1,4-dihydropyridine-3-carboxamides selectively. Preliminary mechanistic studies implicate that a basic ligand-enabled, irreversible, rate-determining CMD step is most likely involved in this process.

Cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. These compounds discourage most pests on the plant and have also been shown to have antitumor properties. With genomics and biochemistry, we identified nine cucumber genes in the pathway for biosynthesis of cucurbitacin C and elucidated four catalytic steps. We discovered transcription factors Bl (Bitter leaf) and Bt (Bitter fruit) that regulate this pathway in leaves and fruits, respectively. Traces in genomic signatures indicated that selection imposed on Bt during domestication led to derivation of nonbitter cucurbits from their bitter ancestors.

A copper-mediated C–H hydroxylation of arenes and heteroarenes using our newly developed PIP directing group has been developed. This procedure is scalable and compatible with a wide range of functional groups and heteroarenes, providing an operationally simple protocol for the synthesis of o-hydroxybenzamides. The hydroxylation of nicotinamides gave 4-oxo-1,4-dihydropyridine-3-carboxamides selectively. Preliminary mechanistic studies implicate that a basic ligand-enabled, irreversible, rate-determining CMD step is most likely involved in this process.

Cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. These compounds discourage most pests on the plant and have also been shown to have antitumor properties. With genomics and biochemistry, we identified nine cucumber genes in the pathway for biosynthesis of cucurbitacin C and elucidated four catalytic steps. We discovered transcription factors Bl ( Bitter leaf ) and Bt ( Bitter fruit ) that regulate this pathway in leaves and fruits, respectively. Traces in genomic signatures indicated that selection imposed on Bt during domestication led to derivation of nonbitter cucurbits from their bitter ancestors.