Spatiotemporal transcriptomic atlas of mouse organogenesis using DNA nanoball-patterned arrays.

Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development.

Strategies for the biological synthesis of D-glucuronic acid and its derivatives.

D-glucuronic acid is a kind of glucose derivative, which has excellent properties such as anti-oxidation, treatment of liver disease and hyperlipidemia, and has been widely used in medicine, cosmetics, food and other fields. The traditional production methods of D-glucuronic acid mainly include natural extraction and chemical synthesis, which can no longer meet the growing market demand. The production of D-glucuronic acid by biocatalysis has become a promising alternative method because of its high efficiency and environmental friendliness. This review describes different production methods of D-glucuronic acid, including single enzyme catalysis, multi-enzyme cascade, whole cell catalysis and co-culture, as well as the intervention of some special catalysts. In addition, some feasible enzyme engineering strategies are provided, including the application of enzyme immobilized scaffold, enzyme mutation and high-throughput screening, which provide good ideas for the research of D-glucuronic acid biocatalysis.

Strategies for the efficient biosynthesis of ß-carotene through microbial fermentation.

ß-Carotene is an orange fat-soluble compound, which has been widely used in fields such as food, medicine and cosmetics owing to its anticancer, antioxidant and cardiovascular disease prevention properties. Currently, natural ß-carotene is mainly extracted from plants and algae, which cannot meet the growing market demand, while chemical synthesis of ß-carotene cannot satisfy the pursuit for natural products of consumers. The ß-carotene production through microbial fermentation has become a promising alternative owing to its high efficiency and environmental friendliness. With the rapid development of synthetic biology and in-depth study on the synthesis pathway of ß-carotene, microbial fermentation has shown promising applications in the ß-carotene synthesis. Accordingly, this review aims to summarize the research progress and strategies of natural carotenoid producing strain and metabolic engineering strategies in the heterologous synthesis of ß-carotene by engineered microorganisms. Moreover, it also summarizes the adoption of inexpensive carbon sources to synthesize ß-carotene as well as proposes new strategies that can further improve the ß-carotene production.

Characterization of a novel esterase and construction of a Rhodococcus-Burkholderia consortium capable of catabolism bis (2-hydroxyethyl) terephthalate.

Bis (2-hydroxyethyl) terephthalate (BHET) is one of the main compounds produced by enzymatic hydrolysis or chemical depolymerization of polyethylene terephthalate (PET). However, the lack of understanding on BHET microbial metabolism is a main factor limiting the bio-upcycling of PET. In this study, BHET-degrading strains of Rhodococcus biphenylivorans GA1 and Burkholderia sp. EG1 were isolated and identified, which can grow with BHET as the sole carbon source. Furthermore, a novel esterase gene betH was cloned from strain GA1, which encodes a BHET hydrolyzing esterase with the highest activity at 30 °C and pH 7.0. In addition, the co-culture containing strain GA1 and strain EG1 could completely degrade high concentration of BHET, eliminating the inhibition on strain GA1 caused by the accumulation of intermediate metabolite ethylene glycol (EG). This work will provide potential strains and a feasible strategy for PET bio-upcycling.

Recent progress in unraveling the biosynthesis of natural sunscreens mycosporine-like amino acids.

Exposure to ultraviolet (UV) rays is a known risk factor for skin cancer, which can be notably mitigated through the application of sun care products. However, escalating concerns regarding the adverse health and environmental impacts of synthetic anti-UV chemicals underscore a pressing need for the development of biodegradable and eco-friendly sunscreen ingredients. Mycosporine-like amino acids (MAAs) represent a family of water-soluble anti-UV natural products synthesized by various organisms. These compounds can provide a two-pronged strategy for sun protection as they not only exhibit a superior UV absorption profile but also possess the potential to alleviate UV-induced oxidative stresses. Nevertheless, the widespread incorporation of MAAs in sun protection products is hindered by supply constraints. Delving into the biosynthetic pathways of MAAs can offer innovative strategies to overcome this limitation. Here, we review recent progress in MAA biosynthesis, with an emphasis on key biosynthetic enzymes, including the dehydroquinate synthase homolog MysA, the adenosine triphosphate (ATP)-grasp ligases MysC and MysD, and the nonribosomal peptide synthetase (NRPS)-like enzyme MysE. Additionally, we discuss recently discovered MAA tailoring enzymes. The enhanced understanding of the MAA biosynthesis paves the way for not only facilitating the supply of MAA analogs but also for exploring the evolution of this unique family of natural sunscreens. ONE-SENTENCE SUMMARY: This review discusses the role of mycosporine-like amino acids (MAAs) as potent natural sunscreens and delves into recent progress in their biosynthesis.

Enhanced ß-carotene production in Yarrowia lipolytica through the metabolic and fermentation engineering.

ß-Carotene is a kind of high-value tetraterpene compound, which shows various applications in medical, agricultural, and industrial areas owing to its antioxidant, antitumor, and anti-inflammatory activities. In this study, Yarrowia lipolytica was successfully metabolically modified through the construction and optimization of ß-carotene biosynthetic pathway for ß-carotene production. The ß-carotene titer in the engineered strain Yli-C with the introduction of the carotenogenesis genes crtI, crtE, and crtYB can reach 34.5 mg/L. With the overexpression of key gene in the mevalonate pathway and the enhanced expression of the fatty acid synthesis pathway, the ß-carotene titer of the engineered strain Yli-CAH reached 87 mg/L, which was 152% higher than that of the strain Yli-C. Through the further expression of the rate-limiting enzyme tHMGR and the copy number of ß-carotene synthesis related genes, the ß-carotene production of Yli-C2AH2 strain reached 117.5 mg/L. The final strain Yli-C2AH2 produced 2.7 g/L ß-carotene titer by fed-batch fermentation in a 5.0-L fermenter. This research will greatly speed up the process of developing microbial cell factories for the commercial production of ß-carotene. ONE-SENTENCE SUMMARY: In this study, the ß-carotene synthesis pathway in engineered Yarrowia lipolytica was enhanced, and the fermentation conditions were optimized for high ß-carotene production.

Total Syntheses of Calyciphylline A-Type Alkaloids (-)-10-Deoxydaphnipaxianine A, (+)-Daphlongamine E and (+)-Calyciphylline R via Late-Stage Divinyl Carbinol Rearrangements.

Among the famous Daphniphyllum alkaloids family, the calyciphylline A-type subfamily has triggered particular interest from the organic synthesis community in recent years. Here, we report divergent total syntheses of three calyciphylline A-type alkaloids, namely, (-)-10-deoxydaphnipaxianine A, (+)-daphlongamine E, and (+)-calyciphylline R. Our work highlights an efficient, divergent strategy via late-stage divinyl carbinol rearrangements, including an unprecedented oxidative Nazarov electrocyclization using an unfunctionalized tertiary divinyl carbinol and an unusual allylic alcohol rearrangement. A highly efficient "donor-acceptor" platinum catalyst was used for a critical nitrile hydration step. Moreover, the power of selective amide reductions has also been showcased by novel and classic tactics.

Indoleamine 2,3-dioxygenase 1 regulates breast cancer tamoxifen resistance through interleukin-6/signal transducer and activator of transcription.

Breast cancer is the primary cause of cancer-related deaths in women. Tamoxifen (TAM) is the preferred drug for treating premenopausal luminal-type breast cancer, but TAM resistance restricts its ability to benefit patients. To date, the mechanism of this resistance remains unclear, and there is currently no effective treatment for reversing it. The expression of indoleamine 2,3-dioxygenase 1 (IDO1) has been shown to be elevated in various malignancies. Here, we aimed to investigate the role of IDO1 in TAM-resistant breast cancer. We confirmed that IDO1 is strongly expressed in TAM-resistant breast cancer, and it mediates drug-resistant cell proliferation, metastasis, and TAM resistance in vivo and in vitro through interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3). We also found that the mechanism by which TAM upregulates IDO1 is dependent on STAT1 activation. In summary, IDO1 regulates TAM resistance and can serve as a novel target for treatment of TAM-resistant breast cancer.

Autonomic behavioral impairment induced by simazine exposure during early life of male mouse is mediated by Lmx1a/Wnt1 pathway.

Simazine is a widely used herbicide and known as an environmental estrogen. Multiple studies have proved simazine can induced the degeneration of dopaminergic neuron resulting in a degenerative disease-like syndrome. Herein, we explored the neurotoxicity of simazine on the dopaminergic nervous system of embryos and weaned offspring during the maternal gestation period or the maternal gestation and lactation periods. We found that simazine disturbed the crucial components expression involved in Lmx1a/Wnt1 pathway of dopaminergic neuron in embryonic and weaned offspring. Furthermore, morphological and behavioral tests performed on weaned male offspring treated by simazine suggested that the grip strength, autonomic exploring, and the space sense ability were weakened, as well as the pathological damage of dopaminergic neuron was clearly observed. But, the same neurotoxicity of simazine is less significantly observed in female offspring. Our findings will provide reliable reference for the determination of environmental limits and new insight into the pathogenesis of nonfamilial neurodegenerative diseases related to environmental risk factors.

Biological production of xylitol by using nonconventional microbial strains.

Xylitol (C5H12O5), an amorphous sugar alcohol of crystalline texture has received great interest on the global market due to its numerous applications in different industries. In addition to its high anticariogenic and sweetening properties, characteristics such as high solubility, stability and low glycemic index confer xylitol its fame in the food and odontological industries. Moreover, it also serves as a building-block in the production of polymers. As a result of the harmful effects of the chemical production of xylitol, the biotechnological means of producing this polyol have evolved over the decades. In contrast to the high consumption of energy, long periods of purification, specialized equipment and high production cost encountered during its chemical synthesis, the biotechnological production of xylitol offers advantages both to the economy and the environment. Non-Saccharomyces yeast strains, also termed as nonconventional, possess the inherent capacity to utilize D-xylose as a sole carbon source, unlike Saccharomyces species.

An engineered azurin with a lanthanide binding site capable of copper sensing.

Proteins with hetero-bimetallic metal centers can catalyze important reactions and are challenging to design. Azurin is a mononuclear copper center that has been extensively studied for electron transfer. Here we inserted the lanthanide binding tag (LBT), which binds lanthanide with sub µM affinity, into the copper binding loop of azurin, while keeping the type 1 copper center unperturbed. The resulting protein, Az-LBT, which has two metal bonding centers, shows strong luminescence upon coordination with Tb3+ and luminescence quenching upon Cu2+ binding. The in vitro luminescence quenching has high metal specificity and a limit-of-detection of 0.65 µM for Cu2+. With the low background from lanthanide's long luminescence lifetime, bacterial cells expressing Az-LBT in the periplasm also shows sensitivity for metal sensing.

Quantitative proteomic analysis to reveal expression differences for butanol production from glycerol and glucose by Clostridium sp. strain CT7.

Clostridium sp. strain CT7 is a new emerging microbial cell factory with high butanol production ratio owing to its non-traditional butanol fermentation mode with uncoupled acetone and 1,3-propanediol formation. Significant changes of metabolic products profile were shown in glycerol- and glucose-fed strain CT7, especially higher butanol and lower volatile fatty acids (VFAs) production occurred from glycerol-fed one. However, the mechanism of this interesting phenomenon was still unclear. To better elaborate the bacterial response towards glycerol and glucose, the quantitative proteomic analysis through iTRAQ strategy was performed to reveal the regulated proteomic expression levels under different substrates. Proteomics data showed that proteomic expression levels related with carbon metabolism and solvent generation under glycerol media were highly increased. In addition, the up-regulation of hydrogenases, ferredoxins and electron-transferring proteins may attribute to the internal redox balance, while the earlier triggered sporulation response in glycerol-fed media may be associated with the higher butanol production. This study will pave the way for metabolic engineering of other industrial microorganisms to obtain efficient butanol production from glycerol.

Microbial application of thermophilic Thermoanaerobacterium species in lignocellulosic biorefinery.

Recently, thermophilic Thermoanaerobacterium species have attracted increasing attentions in consolidated bioprocessing (CBP), which can directly utilize lignocellulosic materials for biofuels production. Compared to the mesophilic process, thermophilic process shows greater prospects in CBP due to its relatively highly efficiency of lignocellulose degradation. In addition, thermophilic conditions can avoid microbial contamination, reduce the cooling costs, and further facilitate the downstream product recovery. However, only few reviews specifically focused on the microbial applications of thermophilic Thermoanaerobacterium species in lignocellulosic biorefinery. Accordingly, this review will comprehensively summarize the recent advances of Thermoanaerobacterium species in lignocellulosic biorefinery, including their secreted xylanases and bioenergy production. Furthermore, the co-culture can significantly reduce the metabolic burden and achieve the more complex work, which will be discussed as the further perspectives. KEY POINTS: • Thermoanaerobacterium species, promising chassis for lignocellulosic biorefinery. • Potential capability of hemicellulose degradation for Thermoanaerobacterium species. • Efficient bioenergy production by Thermoanaerobacterium species through metabolic engineering.

Soybean isoflavones protect SH-SY5Y neurons from atrazine-induced toxicity by activating mitophagy through stimulation of the BEX2/BNIP3/NIX pathway.

Atrazine (ATR) is a widely used herbicide that can induce the degeneration of dopaminergic (DAergic) neurons in the substantia nigra, resulting in a Parkinson's disease-like syndrome. Despite the high risk of environmental exposure, few studies have investigated strategies for the prevention of ATR neurotoxicity. Our previous studies demonstrated that ATR can impair mitochondrial function, leading to metabolic failure. Cells maintain mitochondrial quality through selective autophagic elimination, termed mitophagy. Soybean isoflavones (SI) possess multiple beneficial bioactivities, including preservation of mitochondria function, so it was hypothesized that SI can protect neurons against ATR toxicity by promoting mitophagy. Pretreatment of SH-SY5Y neurons with SI prevented ATR-induced metabolic failure and cytotoxicity as assessed by intracellular ATP, Na+-K+-ATPase activity, mitochondrial membrane potential, and cell viability assays. The neuroprotective efficacy of SI was superior to the major individual components genistein, daidzein, and glycitein. Ultrastructural analyses revealed that ATR induced mitochondrial damage, while SI promoted the sequestration of damaged mitochondria into autophagic vesicles. Soybean isoflavones also induced mitophagy as evidenced by upregulated expression of BNIP3/NIX, BEX2, and LC3-II, while co-treatment with the mitophagy inhibitor Mdivi-1 blocked SI-mediated neuroprotection and prevented SI from reversing ATR-induced BEX2 downregulation. Furthermore, BEX2 knockdown inhibited SI-induced activation of the BNIP3/NIX pathway, mitophagy, and neuroprotection. These findings suggest that SI protects against ATR-induced mitochondrial dysfunction and neurotoxicity by activating the BEX2/BNIP3/NIX pathway.

Challenges and Future Perspectives of Promising Biotechnologies for Lignocellulosic Biorefinery.

Lignocellulose is a kind of renewable bioresource containing abundant polysaccharides, which can be used for biochemicals and biofuels production. However, the complex structure hinders the final efficiency of lignocellulosic biorefinery. This review comprehensively summarizes the hydrolases and typical microorganisms for lignocellulosic degradation. Moreover, the commonly used bioprocesses for lignocellulosic biorefinery are also discussed, including separated hydrolysis and fermentation, simultaneous saccharification and fermentation and consolidated bioprocessing. Among these methods, construction of microbial co-culturing systems via consolidated bioprocessing is regarded as a potential strategy to efficiently produce biochemicals and biofuels, providing theoretical direction for constructing efficient and stable biorefinery process system in the future.

Consolidated bioprocessing performance of a two-species microbial consortium for butanol production from lignocellulosic biomass.

Consolidated bioprocessing (CBP) by using microbial consortium was considered as a promising approach to achieve direct biofuel production from lignocellulose. In this study, the interaction mechanism of microbial consortium consisting of Thermoanaerobacterium thermosaccharolyticum M5 and Clostridium acetobutylicum NJ4 was analyzed, which could achieve efficient butanol production from xylan through CBP. Strain M5 possesses efficient xylan degradation capability, as 19.73 g/L of xylose was accumulated within 50 hr. The efficient xylose utilization capability of partner strain NJ4 could relieve the substrate inhibition to hydrolytic enzymes of xylanase and xylosidase secreted by strain M5. In addition, the earlier solventogenesis of strain NJ4 was observed due to the existence of butyrate generated by strain M5. The mutual interaction of these two strains finally gave 13.28 g/L of butanol from 70 g/L of xylan after process optimization, representing a relatively high butanol production from hemicellulose. Moreover, 7.61 g/L of butanol was generated from untreated corncob via CBP. This successfully constructed microbial consortium exhibits efficient cooperation performance on butanol production from lignocellulose, which could provide a platform for the emerging butanol production from lignocellulose.

Pediatric Malignant Phyllodes Tumors of the Breast: Characteristics and Outcomes Based on the Surveillance Epidemiology and End Results Database.

BACKGROUND: Malignant phyllodes tumors of the breast are uncommon in women and rare in children. This study aimed to assess the differences in survival among five specific pathologic groups of breast malignancies and the differences between pediatric and adult breast phyllodes malignancy. MATERIALS AND METHODS: Using the Surveillance, Epidemiology, and End Results database, we collected data on 270 pediatric (aged ≤21 y) female breast malignant tumor patients and 2773 female malignant phyllodes tumor patients between 1976 and 2015. We evaluated survival differences among younger patients with breast malignancy and compared the pediatric and adult groups based on characteristics, treatment patterns, and survival months. Finally, we identified the risk and protective factors for breast phyllodes cases using a multivariable Cox analysis. RESULTS: We collected and analyzed 270 malignant breast cancer patients aged ≤21 y and 2773 malignant phyllodes tumor patients. Pediatric patients with malignant phyllodes tumors (22.2%, n = 60) exhibited better overall survival (OS; log-rank, P = 0.012) and cancer-specific survival (CSS; log-rank, P = 0.005) among the younger patients with malignant breast tumors. Furthermore, pediatric patients with malignant phyllodes tumors showed better OS (log-rank, P = 0.004), and similar CSS (log-rank, P = 0.105), compared with older patients. After adjustments for potential confounding factors, age >21 y, Black race, tumor size of >100 mm, high grade, wider invasion, positive nodal status, larger scope surgery, and no surgery were found to be associated with worse OS. All these factors, except for race, were found to be independent risk factors for CSS. CONCLUSIONS: The prognosis of malignant phyllodes tumors in children is better than that of adults. Appropriate surgical scope and risk of overtreatment should be considered when treating pediatric malignant phyllodes tumor patients.

Heterologous expression of cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and its application in 2-O-α-D-glucopyranosyl-L-ascorbic acid production.

BACKGROUND: Cyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries. RESULTS: In this study, the cgt gene encoding α-CGTase from Paenibacillus macerans was codon-optimized (opt-cgt) and cloned into pET-28a (+) for intracellular expression in E. coli BL21 (DE3). The Opt-CGT was purified by Ni2+-NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75 U·mg- 1. In addition, the enzyme was adopted to transform L-AA into 9.1 g/L of AA-2G. Finally, more economic substrates, including ß-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5 g/L of AA-2G were obtained, respectively. CONCLUSIONS: N-terminal amino acid is critical to the activity of CGTase suggested by its truncation study. Furthermore, when the Opt-CGT was flanked by His6-tags on the C- and N-terminal, the recovery of purification by Ni2+-NTA resin is appreciably enhanced. α-cyclodextrin was the ideal glycosyl donor for AA-2G production. In addition, the selection of low cost glycosyl donors would make the process of AA-2G production more economically competitive.

Recent advances on conversion and co-production of acetone-butanol-ethanol into high value-added bioproducts.

Butanol is an important bulk chemical and has been regarded as an advanced biofuel. Large-scale production of butanol has been applied for more than 100 years, but its production through acetone-butanol-ethanol (ABE) fermentation process by solventogenic Clostridium species is still not economically viable due to the low butanol titer and yield caused by the toxicity of butanol and a by-product, such as acetone. Renewed interest in biobutanol as a biofuel has spurred technological advances to strain modification and fermentation process design. Especially, with the development of interdisciplinary processes, the sole product or even the mixture of ABE produced through ABE fermentation process can be further used as platform chemicals for high value added product production through enzymatic or chemical catalysis. This review aims to comprehensively summarize the most recent advances on the conversion of acetone, butanol and ABE mixture into various products, such as isopropanol, butyl-butyrate and higher-molecular mass alkanes. Additionally, co-production of other value added products with ABE was also discussed.

Microbial co-culturing systems: butanol production from organic wastes through consolidated bioprocessing.

Biobutanol can be indigenously synthesized by solventogenic Clostridium species; however, these microorganisms possess inferior capability of utilizing abundant and renewable organic wastes, such as starch, lignocellulose, and even syngas. The common strategy to achieve direct butanol production from these organic wastes is through genetic modification of wild-type strains. However, due to the complex of butanol synthetic and hydrolytic enzymes expression systems, the recombinants show unsatisfactory results. Recently, setting up microbial co-culturing systems became more attractive, as they could not only perform more complicated tasks, but also endure changeable environments. Hence, this mini-review comprehensively summarized the state-of-the-art biobutanol production from different substrates by using microbial co-culturing systems. Furthermore, strategies regarding establishment principles of microbial co-culturing systems were also analyzed and compared.