Engineering of NEMO as calcium indicators with large dynamics and high sensitivity.

Genetically encoded calcium indicators (GECIs) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of suboptimal 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 around 20-fold larger than the top-of-the-range GCaMP6 series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared with GCaMP6s, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times larger in 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.

CGI1746 targets σ1R to modulate ferroptosis through mitochondria-associated membranes.

Ferroptosis is iron-dependent oxidative cell death. Labile iron and polyunsaturated fatty acid (PUFA)-containing lipids are two critical factors for ferroptosis execution. Many processes regulating iron homeostasis and lipid synthesis are critically involved in ferroptosis. However, it remains unclear whether biological processes other than iron homeostasis and lipid synthesis are associated with ferroptosis. Using kinase inhibitor library screening, we discovered a small molecule named CGI1746 that potently blocks ferroptosis. Further studies demonstrate that CGI1746 acts through sigma-1 receptor (σ1R), a chaperone primarily located at mitochondria-associated membranes (MAMs), to inhibit ferroptosis. Suppression of σ1R protects mice from cisplatin-induced acute kidney injury hallmarked by ferroptosis. Mechanistically, CGI1746 treatment or genetic disruption of MAMs leads to defective Ca2+ transfer, mitochondrial reactive oxygen species (ROS) production and PUFA-containing triacylglycerol accumulation. Therefore, we propose a critical role for MAMs in ferroptosis execution.

Non-ubiquitous expression of core spliceosomal protein SmB/B' in chick and mouse embryos.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Recurrent allopolyploidizations diversify ecophysiological traits in marsh orchids (Dactylorhiza majalis s.l.).

Whole-genome duplication has shaped the evolution of angiosperms and other organisms, and is important for many crops. Structural reorganization of chromosomes and repatterning of gene expression are frequently observed in allopolyploids, with physiological and ecological consequences. Recurrent origins from different parental populations are widespread among polyploids, resulting in an array of lineages that provide excellent models to uncover mechanisms of adaptation to divergent environments in early phases of polyploid evolution. We integrate here transcriptomic and ecophysiological comparative studies to show that sibling allopolyploid marsh orchid species (Dactylorhiza, Orchidaceae) occur in different habitats (low nutrient fens vs. meadows with mesic soils) and are characterized by a complex suite of intertwined, pronounced ecophysiological differences between them. We uncover distinct features in leaf elemental chemistry, light-harvesting, photoprotection, nutrient transport and stomata activity of the two sibling allopolyploids, which appear to match their specific ecologies, in particular soil chemistry differences at their native sites. We argue that the phenotypic divergence between the sibling allopolyploids has a clear genetic basis, generating ecological barriers that maintain distinct, independent lineages, despite pervasive interspecific gene flow. This suggests that recurrent origins of polyploids bring about a long-term potential to trigger and maintain functional and ecological diversity in marsh orchids and other groups.

Association between oral health and cognitive function among Chinese older adults: the Taizhou imaging study.

BACKGROUND: We aimed to investigate the association between oral health and cognitive function in a sample of older adults from a Chinese rural community. METHODS: The cross-sectional cognitive function of 677 individuals were assessed by Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA). A comprehensive profile of the oral health status was evaluated by questionnaire and clinical examination. RESULTS: Multiple covariates-adjusted regression models demonstrated decayed teeth (DT) and decayed/missing/filled teeth (DMFT) were negatively associated with MoCA score (all p < 0.05). Calculus index (CI) and clinical attachment loss (CAL) were significantly associated with the lower MoCA, short-term memory and executive function score, respectively (all p < 0.05). Additionally, participants with missing teeth unrestored tend to get lower MMSE and MoCA scores (p < 0.05). The results also showed that increased DT and CI were modestly associated with higher odds of cognitive impairment (p < 0.05). CONCLUSIONS: There is an association between oral health and global cognition. Poor periodontal status was strongly associated with worse global cognition performance, especially in the short-term memory and executive domain for the aging population.

Dissolution at a Ductile Crack Tip.

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.

Photoautotrophic production of eicosapentaenoic acid.

Eicosapentaenoic acid (EPA) is an omega-3 fatty acid which is an essential nutrient for both humans and animals. This review examines the global need for EPA, both in human nutrition and aquaculture. The potential shortfall in supply of this important nutrient as well as sustainability issues with wild-caught fish have generated increased interest into alternative sources of EPA. Various approaches are summarized, including heterotrophic production and the use of genetically modified microorganisms and plants. Studies on photoautotrophic production of EPA are extensively reviewed. Widely used species for large-scale production of EPA includes Phaeodactylum tricornutum and Nannochloropsis due to their robustness and relatively high growth rates and EPA content (typically 5% of dry biomass). Approaches for large-scale production have also been reviewed. Closed reactors like flat panels, tubular reactors and bubble columns may be the most suitable due to their high productivity. However, there is no agreement in the literature as to which design generates the lowest cost of production. The economics of the process has also been examined. The best estimates for large-scale (100 hectare) plants give EPA prices of the order 39-90 USD per kilogram. This is approximately ten times higher than the price of EPA derived from fish oil. Potential avenues for lowering the cost are highlighted, along with the need to better understand the advantages and disadvantages of different EPA production methods from a more holistic perspective.

Trend on dental caries status and its risk indicators in children aged 12 years in China: a multilevel analysis based on the repeated national cross-sectional surveys in 2005 and 2015.

BACKGROUND: This study aimed to explore the trend and risk indicators for dental caries of children aged 12 years in China based on national oral health survey data in 2005 and 2015. METHODS: Research data were from the two latest national oral health surveys conducted in mainland China, including 30 and 31 provinces, autonomous regions, and municipalities in 2005 and 2015, respectively. Children aged 12 years were clinically examined for dental caries and dental fluorosis according to the World Health Organization criteria. Sociodemographic characteristics and oral health-related behaviours were collected using questionnaires. Multilevel zero-inflated negative binomial regression model was used to investigate the association between dental caries severity and dental fluorosis, sociodemographic characteristics, and oral health-related behaviours. RESULTS: The final analyses included 12,350 and 27,818 children surveyed in 2005 and 2015, respectively. The standardized prevalence of dental caries increased from 27.05% (95% confidence interval [CI], 24.25-28.85) in 2005 to 37.92% (95% CI, 34.94-40.90) in 2015, and the respective standardized mean decayed, missing, filled teeth (DMFT) index scores increased from 0.50 (standard deviation [SD], 1.04) to 0.83 (SD, 1.45) (P < 0.001). Fujian province had the highest increase in dental caries, followed by Liaoning, Heilongjiang, Hainan, and Yunnan. Results revealed that children who were girls, more frequently experienced dental pain, and had more recent dental visits, had significantly higher DMFT scores after adjusting for the survey year and other variables (all P < 0.05). CONCLUSIONS: Dental caries of 12-year-old children in China deteriorated from 2005 to 2015, particularly in the northeast and southwest regions. Dental caries was associated with sex, dental pain, and dental service utilization.

Roads to Rome: Role of Multiple Cassettes in Cyanobactin RiPP Biosynthesis.

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 multicassette 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.

Three Principles of Diversity-Generating Biosynthesis.

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 post-translationally 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 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.

Copper/silver-mediated direct ortho-ethynylation of unactivated (hetero)aryl C-H bonds with terminal alkyne.

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.

Atomic mechanism of near threshold fatigue crack growth in vacuum.

Structural failures resulting from prolonged low-amplitude loading are particularly problematic. Over the past century a succession of mechanisms have been hypothesized, as experimental validation has remained out of reach. Here we show by atomistic modeling that sustained fatigue crack growth in vacuum requires emitted dislocations to change slip planes prior to their reabsorption into the crack on the opposite side of the loading cycle. By harnessing a new implementation of a concurrent multiscale method we (1) assess the validity of long-hypothesized material separation mechanisms thought to control near-threshold fatigue crack growth in vacuum, and (2) reconcile reports of crack growth in atomistic simulations at loading amplitudes below experimental crack growth thresholds. Our results provide a mechanistic foundation to relate fatigue crack growth tendency to fundamental material properties, e.g. stacking fault energies and elastic moduli, opening the door for improved prognosis and the design of novel fatigue resistance alloys.

Applying Promiscuous RiPP Enzymes to Peptide Backbone N-Methylation Chemistry.

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 N-methylated peptides are cyclic. However, there is very limited knowledge about how post-translational enzymes can be applied to the synthesis of designed N-methylated peptides or peptide libraries. Here, driven by the established ability of some RiPP enzymes to process diverse substrates, we sought to define catalysts for the in vivo and in vitro macrocyclization of backbone-methylated peptides. We developed efficient methods in which short, synthetic N-methylated peptides could be modified using side chain and mainchain macrocyclases, PsnB and PCY1 from plesiocin and orbitide biosynthetic pathways, respectively. Most significantly, a strategy for PsnB cyclase was designed enabling simple in vitro methods compatible with solid-phase peptide synthesis. We show that cyanobactin N-terminal protease PatA is a broadly useful catalyst that is also compatible with N-methylation chemistry, but that cyanobactin macrocyclase PatG is strongly biased against N-methylated substrates. Finally, we sought to marry these macrocyclase tools with an enzyme that N-methylates its core peptide: OphMA from the omphalotin pathway. However, instead, we reveal some limitations of OphMA and demonstrate that it unexpectedly and extensively modified the enzyme itself in vivo. Together, these results demonstrate proof-of-concept for enzymatic synthesis of N-methylated peptide macrocycles.

A scalable model for EPA and fatty acid production by Phaeodactylum tricornutum.

Large-scale photoautotrophic production of microalgae has the potential to provide a sustainable supply of omega-3 fatty acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) for human and animal nutrition. This study presents a kinetic model for the EPA-producing microalga Phaeodactylum tricornutum in photoautotrophic conditions, with light and nitrogen being the growth limiting factors. The model was developed using a dataset obtained from bench-scale (5 L) cultures and was successfully validated against pilot-scale (50 L) cultures. This model is the first to predict the biomass and total fatty acid accumulation along with the EPA concentrations in the biomass and total fatty acid fraction for microalgae. The model was used to develop an optimized repeated-batch strategy; implementation of this led to increases in the biomass and EPA productivities of 50 and 20% respectively. This clearly indicates the potential of the model to be used as a tool in the design, optimization and scale-up of microalgal systems for EPA production.

Control of Nucleophile Chemoselectivity in Cyanobactin YcaO Heterocyclases PatD and TruD.

Members of the YcaO superfamily are among the most common post-translational modification enzymes in natural product biosynthesis, with wide usage in biotechnology and synthetic biology applications. Here, we use domain-swapped chimeras and discovered unstructured regions in cyanobactin YcaOs that guide interactions with the substrates, governing access to interior amino acids in the substrates and explaining the chemoselectivity between PatD and TruD. These results define how the cyanobactin heterocyclases modify exceptionally sequence diverse substrates, yet with a high degree of positional and nucleophile selectivity.

Conservative route to genome compaction in a miniature annelid.

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.

Assessment of Additive Manufacturing for Increasing Sustainability and Productivity of Smallholder Agriculture.

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.

Expanding the chemical space of synthetic cyclic peptides using a promiscuous macrocyclase from prenylagaramide biosynthesis.

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 macrocyclases, we catalog a series of enzymes from this family that 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.

Independent associations of body-size adjusted fat mass and fat-free mass with the metabolic syndrome in Chinese.

BACKGROUND: Excess fat leads to adverse health outcomes. Most previous studies investigating body fatness using BMI or fat percentage, which contain both fat mass and fat-free mass, were not able to differentiate the exposure. AIM: The present study assessed the independent association of fat and fat-free mass with metabolic syndrome (MetS) in Chinese. SUBJECTS AND METHODS: A population-based study of 1144 subjects aged 50-70 from urban and rural areas of Shanghai in 2005-2006 was employed. Body composition was measured with DEXA. Fat mass index (FMI) and fat-free mass index (FFMI) were calculated. MetS was defined using the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria without waist circumference for its high correlation with body composition. RESULTS: Both FMI and FFMI were significantly related with higher odds of MetS (OR 3.97, 95% CI 2.58-6.09 for FMI; OR 2.67, 95% CI 1.70-4.18 for FFMI, the highest quartile vs the lowest group) after adjusting for age, residence, sex, smoking, drinking, physical activity, medication, family history of chronic diseases, and fat-free mass (for FMI) or fat mass (for FFMI). CONCLUSION: Both FMI and FFMI are independently associated with increased MetS risks. Proper expression of body composition is essential in assessing body composition and disease risk association.