Extraction of eutrophic and green ponds from segmentation of high-resolution imagery based on the EAF-Unet algorithm.

Inland ponds exhibit remarkable ubiquity across the globe, playing a vital role in the sustainability of global continental freshwater resources and contributing significantly to their biodiversity. Numerous ponds are eutrophic and experience recurrent seasonal or year-round algal blooms or persistent duckweed cover, conferring a characteristic green hue. Here, we denote these eutrophic and green ponds as EGPs. The excessive proliferation of algal blooms and duckweed within these EGPs poses a significant threat to the ecological functioning of these aquatic systems, which can lead to hypoxia or the release of microcystins. To identify these EGPs automatically, we constructed an Efficient Attention Fusion Unet (EAF-Unet) algorithm using Gaofen-2 (GF2) panchromatic and multispectral imagery. The attention mechanism was incorporated in Unet to help better detect EGPs. Using the first EGP labeled dataset, we determined the best input feature combination (RGB, NIR, NDVI, and Bright) and the most effective encoding (Rasnet50) for EAF-Unet for distinguishing EGPs from other ground cover types. The evaluation indices - Precision (0.81), Recall (0.79), F1-Score (0.80), and Intersection over Union (IoU, 0.67) - indicate that EAF-Unet can accurately and robustly extract EGPs from GF2 images without relying on pond water masks. Remote-sensing EGP products can assist in identifying ponds with severe eutrophication. Moreover, these products can serve as references for identifying high-risk areas prone to improper sewage discharge or inadequate sewer construction.

Manganese regulation of COPII condensation controls circulating lipid homeostasis.

Precise control of circulating lipids is instrumental in health and disease. Bulk lipids, carried by specialized lipoproteins, are secreted into the circulation, initially via the coat protein complex II (COPII). How the universal COPII machinery accommodates the abundant yet unconventional lipoproteins remains unclear, let alone its therapeutic translation. Here we report that COPII uses manganese-tuning, self-constrained condensation to selectively drive lipoprotein delivery and set lipid homeostasis in vivo. Serendipitously, adenovirus hijacks the condensation-based transport mechanism, thus enabling the identification of cytosolic manganese as an unexpected control signal. Manganese directly binds the inner COPII coat and enhances its condensation, thereby shifting the assembly-versus-dynamics balance of the transport machinery. Manganese can be mobilized from mitochondria stores to signal COPII, and selectively controls lipoprotein secretion with a distinctive, bell-shaped function. Consequently, dietary titration of manganese enables tailored lipid management that counters pathological dyslipidaemia and atherosclerosis, implicating a condensation-targeting strategy with broad therapeutic potential for cardio-metabolic health.

Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast.

Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential substitutes for fossil fuels, such as diesel and jet fuels, and have wide applications in various manufacturing processes. While today MCFOHs are mainly sourced from petrochemicals or plant oils, microbial biosynthesis represents a scalable, reliable, and sustainable alternative. Here, we aim to establish a Saccharomyces cerevisiae platform capable of selectively producing MCFOHs. This was enabled by tailoring the properties of a bacterial carboxylic acid reductase from Mycobacterium marinum (MmCAR). Extensive protein engineering, including directed evolution, structure-guided semirational design, and rational design, was implemented. MmCAR variants with enhanced activity were identified using a growth-coupled high-throughput screening assay relying on the detoxification of the enzyme's substrate, medium-chain fatty acids (MCFAs). Detailed characterization demonstrated that both the specificity and catalytic activity of MmCAR was successfully improved and a yeast strain harboring the best MmCAR variant generated 2.8-fold more MCFOHs than the strain expressing the unmodified enzyme. Through deletion of the native MCFA exporter gene TPO1, MCFOH production was further improved, resulting in a titer of 252 mg/L for the final strain, which represents a significant improvement in MCFOH production in minimal medium by S. cerevisiae.

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata.

Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.

Genome-Wide Characterization and Analysis of bHLH Transcription Factors Related to Crocin Biosynthesis in Gardenia jasminoides Ellis (Rubiaceae).

Crocins, enriched in Gardenia jasminoides fruits, have a pharmacological activity against central nervous system diseases, cardiovascular diseases, and cancer cell growth. The biosynthesis of crocins has been widely explored, but its regulatory mechanism remains unknown. Here, the basic helix-loop-helix (bHLH) transcription factors related to crocin biosynthesis were systematically identified on the basis of the genome of G. jasminoides. A total of 95 GjbHLH transcription factor genes were identified, and their phylogenetic analysis indicated that they could be classified into 23 subfamilies. The combination of gene-specific bHLH expression patterns, the coexpression analysis of biosynthesis genes, and the analysis of promoter sequences in crocin biosynthesis pathways suggested that nine bHLHs in G. jasminoides might negatively regulate crocin biosynthesis. This study laid a foundation for understanding the regulatory mechanism of crocin biosynthesis and the improvement and breeding of G. jasminoides varieties.

Chinese herbal medicine resources: Where we stand.

In recent years, the development of Chinese herbal medicine (CHM) has been challenged by shortages of CHM resources and drug safety concerns related to end products. There have been significant efforts by Chinese scholars to tackle these challenges, which are revealed by analyzing the research trend of CHM resources via surveying Chinese Traditional and Herbal Drugs (Zhong Cao Yao), a representative journal in CHM. Our study focused on 781 articles in CHM resources from 2013 to 2018 and included four subject areas: germplasm resources, quality analysis and evaluation, cultivation, and bioengineering of CHM. Discussion and prospective for future investigations were also presented, including: construct the core germplasm of medicinal plants and expand germplasms; combine molecular research with field experiments and promote the deeper study of cultivation of CHM plants; improve the quality evaluation method of CHM and strengthen the identification of Chinese patented medicines; promote the sustainable development of CHM resources by utilizing bioengineering and synthetic biology. This study helps international scholars understand the status quo of CHM research and provides theoretical support for the healthy, modern, and international development of CHM, and it will facilitate the sustainable development of the traditional Chinese medicine industry.

Metabolic engineering of Saccharomyces cerevisiae for production of germacrene A, a precursor of beta-elemene.

Beta-elemene, a sesquiterpene and the major component of the medicinal herb Curcuma wenyujin, has antitumor activity against various types of cancer and could potentially serve as a potent antineoplastic drug. However, its current mode of production through extraction from plants has been inefficient and suffers from limited natural resources. Here, we engineered a yeast cell factory for the sustainable production of germacrene A, which can be transformed to beta-elemene by a one-step chemical reaction in vitro. Two heterologous germacrene A synthases (GASs) converting farnesyl pyrophosphate (FPP) to germacrene A were evaluated in yeast for their ability to produce germacrene A. Thereafter, several metabolic engineering strategies were used to improve the production level. Overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase and fusion of FPP synthase with GAS, led to a sixfold increase in germacrene A production in shake-flask culture. Finally, 190.7 mg/l of germacrene A was achieved. The results reported in this study represent the highest titer of germacrene A reported to date. These results provide a basis for creating an efficient route for further industrial application re-placing the traditional extraction of beta-elemene from plant sources.

Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway.

Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes.

[Cloning and bioinformatics analysis of ent-kaurene oxidase synthase gene in Salvia miltiorrhiza].

Based on the transcriptome database of Salvia miltiorrhiza, specific primers were designed to clone a full-length cDNA of ent-kaurene oxidase synthase (SmKOL) using the RACE strategy. ORF Finder was used to find the open reading frame of SmKOL cDNA, and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA 5.1. The transcription level of SmKOL from the hairy roots induced by elicitor methyl jasmonate (MeJA) was qualifiedby real-time quantitative PCR. The full length of SmKOL cDNA was of 1 884 bp nucleotides encoding 519 amino acids. The molecular weight of the SmKOL protein was about 58.88 kDa with isoelectric point (pI) of 7.62. Results of real-time quantitative PCR analyses indicated that the level of SmKOL mRNA expression in hairy roots was increased by elicitor oMeJA, and reached maximum in 36 h. The full-length cDNA of SmKOL was cloned from S. miltiorrhiza hairy root, which provides a target gene for further studies of its function, gibberellin biosynthesis and regulation of secondary metabolites.

Adiponectin inhibits KISS1 gene transcription through AMPK and specificity protein-1 in the hypothalamic GT1-7 neurons.

Adiponectin secreted from adipose tissues plays a role in the regulation of energy homeostasis, food intake, and reproduction in the hypothalamus. We have previously demonstrated that adiponectin significantly inhibited GNRH secretion from GT1-7 hypothalamic GNRH neuron cells. In this study, we further investigated the effect of adiponectin on hypothalamic KISS1 gene transcription, which is the upstream signal of GNRH. We found that globular adiponectin (gAd) or AICAR, an artificial AMPK activator, decreased KISS1 mRNA transcription and promoter activity. Conversely, inhibition of AMPK by Compound C or AMPKα1-SiRNA augmented KISS1 mRNA transcription and promoter activity. Additionally, gAd and AICAR decreased the translocation of specificity protein-1 (SP1) from cytoplasm to nucleus; however, Compound C and AMPKα1-siRNA played an inverse role. Our experiments in vivo demonstrated that the expression of Kiss1 mRNA was stimulated twofold in the Compound C-treated rats and decreased about 60-70% in gAd- or AICAR-treated rats compared with control group. The numbers of kisspeptin immunopositive neurons in the arcuate nucleus region of Sprague Dawley rats mimicked the same trend seen in Kiss1 mRNA levels in animal groups with different treatments. In conclusion, our results provide the first evidence that adiponectin reduces Kiss1 gene transcription in GT1-7 cells through activation of AMPK and subsequently decreased translocation of SP1.

Globular adiponectin regulates energy homeostasis through AMP-activated protein kinase-acetyl-CoA carboxylase (AMPK/ACC) pathway in the hypothalamus.

Adiponectin is a newly researched adipokine which participates in the regulation of energy homeostasis. AMP-activated protein kinase (AMPK) represents an energy sensor that responds to hormone and nutrition status in vivo and exerts a regulatory effect in the hypothalamus and multiple peripheral tissues. We investigated the possible mechanisms involved in appetite regulation by adiponectin in vitro with GT1-7 cells, a mouse immortalized hypothalamic neuron. The results showed that adiponectin increased the phosphorylation of AMPK, activated AMPK phosphorylated and inactivated acetyl-CoA carboxylase (ACC), and subsequently increased expression of agouti-related peptide (AgRP) mRNA. Our results also indicated that adiponectin had no effect on signal transducer and activator of transcription (STAT3). Together these findings suggest that adiponectin regulated energy homeostasis through the AMPK/ACC pathway but not the JAK/STAT3 pathway in the hypothalamus.