The MdNAC72-MdABI5 module acts as an interface integrating jasmonic acid and gibberellin signals and undergoes ubiquitination-dependent degradation regulated by MdSINA2 in apple.

Jasmonic acid (JA) and gibberellin (GA) coordinately regulate plant developmental programs and environmental cue responses. However, the fine regulatory network of the cross-interaction between JA and GA remains largely elusive. In this study, we demonstrate that MdNAC72 together with MdABI5 positively regulates anthocyanin biosynthesis through an exquisite MdNAC72-MdABI5-MdbHLH3 transcriptional cascade in apple. MdNAC72 interacts with MdABI5 to promote the transcriptional activation of MdABI5 on its target gene MdbHLH3 and directly activates the transcription of MdABI5. The MdNAC72-MdABI5 module regulates the integration of JA and GA signals in anthocyanin biosynthesis by combining with JA repressor MdJAZ2 and GA repressor MdRGL2a. MdJAZ2 disrupts the MdNAC72-MdABI5 interaction and attenuates the transcriptional activation of MdABI5 by MdNAC72. MdRGL2a sequesters MdJAZ2 from the MdJAZ2-MdNAC72 protein complex, leading to the release of MdNAC72. The E3 ubiquitin ligase MdSINA2 is responsive to JA and GA signals and promotes ubiquitination-dependent degradation of MdNAC72. The MdNAC72-MdABI5 interface fine-regulates the integration of JA and GA signals at the transcriptional and posttranslational levels by combining MdJAZ2, MdRGL2a, and MdSINA2. In summary, our findings elucidate the fine regulatory network connecting JA and GA signals with MdNAC72-MdABI5 as the core in apple.

Enhancing the expression of chondroitin 4-O-sulfotransferase for one-pot enzymatic synthesis of chondroitin sulfate A.

Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4-O-sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5' untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.

Enzymatic construction of a library of even- and odd-numbered heparosan oligosaccharides and their N-sulfonated derivatives.

Low-molecular-weight heparins (LMWHs), especially the specific-sized heparin oligosaccharides, are attractive for the therapeutic applications, while their synthesis remains challenging. In the present study, unsaturated even-numbered heparosan oligosaccharides were firstly prepared by cleaving high-molecular-weight heparosan using recombinant heparinase III (HepIII). The conversion rates of the unsaturated disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides were 33.9 %, 47.9 %, 78.7 %, 71.8 %, and 53.4 %, respectively. After processing the aforementioned heparosan oligosaccharides with the Δ4,5 unsaturated glycuronidase, saturated odd-numbered heparosan trisaccharides, pentasaccharides, heptasaccharides, and nonasaccharides were produced. It was observed that among them, the pentasaccharides were the smallest units of saturated odd-numbered oligosaccharides recognized by HepIII. These oligosaccharides were further catalyzed with bifunctional heparan sulfate N-deacetylase/N-sulfotransferase (NDST) under optimized reaction conditions. It was found that the tetrasaccharide was defined as the smallest recognition unit for NDST, obtaining the N-sulfonated heparosan tetrasaccharides, pentasaccharides, and hexasaccharides with a single sulfonate group, as well as N-sulfonated heparosan heptasaccharides, octasaccharides, and nonasaccharides with multiple sulfonate groups. These results provide an easy pathway for constructing a library of specific-sized N-sulfonated heparosan oligosaccharides that can be used as the substrates for the enzymatic synthesis of LMWHs and heparin oligosaccharides, shedding new light on the substrate preference of NDST.

MdbHLH162 connects the gibberellin and jasmonic acid signals to regulate anthocyanin biosynthesis in apple.

Anthocyanins are secondary metabolites induced by environmental stimuli and developmental signals. The positive regulators of anthocyanin biosynthesis have been reported, whereas the anthocyanin repressors have been neglected. Although the signal transduction pathways of gibberellin (GA) and jasmonic acid (JA) and their regulation of anthocyanin biosynthesis have been investigated, the cross-talk between GA and JA and the antagonistic mechanism of regulating anthocyanin biosynthesis remain to be investigated. In this study, we identified the anthocyanin repressor MdbHLH162 in apple and revealed its molecular mechanism of regulating anthocyanin biosynthesis by integrating the GA and JA signals. MdbHLH162 exerted passive repression by interacting with MdbHLH3 and MdbHLH33, which are two recognized positive regulators of anthocyanin biosynthesis. MdbHLH162 negatively regulated anthocyanin biosynthesis by disrupting the formation of the anthocyanin-activated MdMYB1-MdbHLH3/33 complexes and weakening transcriptional activation of the anthocyanin biosynthetic genes MdDFR and MdUF3GT by MdbHLH3 and MdbHLH33. The GA repressor MdRGL2a antagonized MdbHLH162-mediated inhibition of anthocyanins by sequestering MdbHLH162 from the MdbHLH162-MdbHLH3/33 complex. The JA repressors MdJAZ1 and MdJAZ2 interfered with the antagonistic regulation of MdbHLH162 by MdRGL2a by titrating the formation of the MdRGL2a-MdbHLH162 complex. Our findings reveal that MdbHLH162 integrates the GA and JA signals to negatively regulate anthocyanin biosynthesis. This study provides new information for discovering more anthocyanin biosynthesis repressors and explores the cross-talk between hormone signals.

Manipulation of Giant Multipole Resonances via Vortex γ Photons.

Traditional photonuclear reactions primarily excite giant dipole resonances, making the measurement of isovector giant resonances with higher multipolarities a great challenge. In this Letter, the manipulation of collective excitations of different multipole transitions in even-even nuclei via vortex γ photons is investigated. We develop the calculation method for photonuclear cross sections induced by the vortex γ photon beam using the fully self-consistent random-phase approximation plus particle-vibration coupling (RPA+PVC) model based on Skyrme density functional. We find that the electromagnetic transitions with multipolarity J<|m_{γ}| are forbidden for vortex γ photons due to the angular momentum conservation, with m_{γ} being the projection of total angular momentum of γ photon on its propagation direction. For instance, this allows for probing the isovector giant quadrupole resonance without interference from dipole transitions using vortex γ photons with m_{γ}=2. Furthermore, the electromagnetic transition with J=|m_{γ}|+1 vanishes at a specific polar angle. Therefore, the giant resonances with specific multipolarity can be extracted via vortex γ photons. Moreover, the vortex properties of γ photons can be meticulously diagnosed by measuring the nuclear photon-absorption cross section. Our method opens new avenues for photonuclear excitations, generation of coherent γ photon laser and precise detection of vortex particles, and consequently, has significant impact on nuclear physics, nuclear astrophysics and strong laser physics.

Engineering sulfonate group donor regeneration systems to boost biosynthesis of sulfated compounds.

Sulfonation as one of the most important modification reactions in nature is essential for many biological macromolecules to function. Development of green sulfonate group donor regeneration systems to efficiently sulfonate compounds of interest is always attractive. Here, we design and engineer two different sulfonate group donor regeneration systems to boost the biosynthesis of sulfated compounds. First, we assemble three modules to construct a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) regeneration system and demonstrate its applicability for living cells. After discovering adenosine 5'-phosphosulfate (APS) as another active sulfonate group donor, we engineer a more simplified APS regeneration system that couples specific sulfotransferase. Next, we develop a rapid indicating system for characterizing the activity of APS-mediated sulfotransferase to rapidly screen sulfotransferase variants with increased activity towards APS. Eventually, the active sulfonate group equivalent values of the APS regeneration systems towards trehalose and p-coumaric acid reach 3.26 and 4.03, respectively. The present PAPS and APS regeneration systems are environmentally friendly and applicable for scaling up the biomanufacturing of sulfated products.

Advances and challenges in biotechnological production of chondroitin sulfate and its oligosaccharides.

Chondroitin sulfate (CS) is a member of glycosaminoglycans (GAGs) and has critical physiological functions. CS is widely applied in medical and clinical fields. Currently, the supply of CS relies on traditional animal tissue extraction methods. From the perspective of medical applications, the biggest drawback of animal-derived CS is its uncontrollable molecular weight and sulfonated patterns, which are key factors affecting CS activities. The advances of cell-free enzyme catalyzed systems and de novo biosynthesis strategies have paved the way to rationally regulate CS sulfonated pattern and molecular weight. In this review, we first present a general overview of biosynthesized CS and its oligosaccharides. Then, the advances in chondroitin biosynthesis, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) synthesis and regeneration, and CS biosynthesis catalyzed by sulfotransferases are discussed. Moreover, the progress of mining and expression of chondroitin depolymerizing enzymes for preparation of CS oligosaccharides is also summarized. Finally, we analyze and discuss the challenges faced in synthesizing CS and its oligosaccharides using microbial and enzymatic methods. In summary, the biotechnological production of CS and its oligosaccharides is a promising method in addressing the drawbacks associated with animal-derived CS and enabling the production of CS oligosaccharides with defined structures.

Production of different molecular weight glycosaminoglycans with microbial cell factories.

Glycosaminoglycans (GAGs) are naturally occurring acidic polysaccharides with wide applications in pharmaceuticals, cosmetics, and health foods. The diverse biological activities and physiological functions of GAGs are closely associated with their molecular weights and sulfation patterns. Except for the non-sulfated hyaluronan which can be synthesized naturally by group A Streptococcus, all the other GAGs such as heparin and chondroitin sulfate are mainly acquired from animal tissues. Microbial cell factories provide a more effective platform for the production of structurally homogeneous GAGs. Enhancing the production efficiency of polysaccharides, accurately regulating the GAGs molecular weight, and effectively controlling the sulfation degree of GAGs represent the major challenges of developing GAGs microbial cell factories. Several enzymatic, metabolic engineering, and synthetic biology strategies have been developed to tackle these obstacles and push forward the industrialization of biotechnologically produced GAGs. This review summarizes the recent advances in the construction of GAGs synthesis cell factories, regulation of GAG molecular weight, and modification of GAGs chains. Furthermore, the challenges and prospects for future research in this field are also discussed.

The Spliceosome Factor EFTUD2 Promotes IFN Anti-HBV Effect through mRNA Splicing.

Methods: Using a CRISPR/Cas9 gene-editing system, EFTUD2 single allele knockout HepG2.2.15 cells were constructed. Subsequently, the HBV biomarkers in EFTUD2+/- HepG2.2.15 cells and wild-type (WT) cells with or without IFN-α treatment were detected. And the EFTUD2-regulated genes were then identified using mRNA sequence. Selected gene mRNA variants and their proteins were examined by qRT-PCR and Western blotting. To confirm the effects of EFTUD2 on HBV replication and IFN-stimulated gene (ISG) expression, a rescue experiment in EFTUD2+/- HepG2.2.15 cells was performed by EFTUD2 overexpression. Results: IFN-induced anti-HBV activity was found to be restricted in EFTUD2+/- HepG2.2.15 cells. The mRNA sequence showed that EFTUD2 could regulate classical IFN and virus response genes. Mechanistically, EFTUD2 single allele knockout decreased the expression of ISG-encoded proteins, comprising Mx1, OAS1, and PKR (EIF2AK2), through mediated gene splicing. However, EFTUD2 did not affect the expression of Jak-STAT pathway genes. Furthermore, EFTUD2 overexpression could restore the attenuation of IFN anti-HBV activity and the reduction of ISG resulting from EFTUD2 single allele knockout. Conclusion: EFTUD2, the spliceosome factor, is not IFN-inducible but is an IFN effector gene. EFTUD2 mediates IFN anti-HBV effect through regulation of gene splicing for certain ISGs, including Mx1, OAS1, and PKR. EFTUD2 does not affect IFN receptors or canonical signal transduction components. Therefore, it can be concluded that EFTUD2 regulates ISGs using a novel, nonclassical mechanism.

Exploring the proteins and metabolites associated with male antennae responses to female exposure of Antheraea pernyi (Lepidoptera: Saturniidae) moths.

Detection of sex pheromones of insects relies on the antennae. The female pheromone signal transmission in the male antennae ultimately initiates the courtship and mating behaviors of males. To investigate the proteins and metabolites involved in this neural transduction, integrative proteomics and metabolomics analysis including tandem mass tag (TMT) proteomic quantification and liquid chromatography tandem mass spectrometry (LC/MS)-based metabolomics was adopted for comparing proteomic and metabolic changes between the antennae of male moths following stimulation by females and the non-stimulated males of Antheraea pernyi (Guérin-Méneville, Lepidoptera: Saturniidae) in this study. A total of 92 differentially expressed proteins (DEPs) containing 52 upregulated and 40 downregulated proteins and 545 differentially expressed metabolites (DEMs) including 218 upregulated and 327 downregulated metabolites were identified from the antennae of female-stimulated male moths based on the proteome and metabolome data, respectively. Bioinformatics analysis was performed for the 45 DEPs and 160 DEMs, including Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encylopaedia of Genes and Genomes (KEGG) enrichment analysis and Human Metabolome Database (HMDB) annotation. A number of DEPs and DEMs related to neural transmission of female pheromone signals in the male antennae of A. pernyi were screened, including tyrosine hydroxylase, cryptochrome-1, tachykinin, arylalkylamine N-acetyltransferase, cadherin-23, glutathione S-transferase delta 3, tyramine, tryptamine, n-oleoyl dopamine, n-stearoyl dopamine, and n-stearoyl tyrosine. The altered expression levels of those proteins or metabolites were speculated involved in regulating the neuron activity for enhanced transmission of neural impulses and continuous perception, reception, and transduction of female pheromone signals. Our findings yielded novel insights into the potential mechanisms in the antennae of male A. pernyi responding to female attraction.

MdVQ10 promotes wound-triggered leaf senescence in association with MdWRKY75 and undergoes antagonistic modulation of MdCML15 and MdJAZs in apple.

Wounding stress leads to leaf senescence. However, the underlying molecular mechanism has not been elucidated. In this study, we investigated the role of the MdVQ10-MdWRKY75 module in wound-induced leaf senescence. MdWRKY75 was identified as a key positive modulator of wound-induced leaf senescence by activating the expression of the senescence-associated genes MdSAG12 and MdSAG18. MdVQ10 interacted with MdWRKY75 to enhance MdWRKY75-activated transcription of MdSAG12 and MdSAG18, thereby promoting leaf senescence triggered by wounding. In addition, the calmodulin-like protein MdCML15 promoted MdVQ10-mediated leaf senescence by stimulating the interaction between MdVQ10 and MdWRKY75. Moreover, the jasmonic acid signaling repressors MdJAZ12 and MdJAZ14 antagonized MdVQ10-mediated leaf senescence by weakening the MdVQ10-MdWRKY75 interaction. Our results demonstrate that the MdVQ10-MdWRKY75 module is a key modulator of wound-induced leaf senescence and provides insights into the mechanism of leaf senescence caused by wounding.

Improvement of the stability and catalytic efficiency of heparan sulfate N-sulfotransferase for preparing N-sulfated heparosan.

The chemo-enzymatic and enzymatic synthesis of heparan sulfate and heparin are considered as an attractive alternative to the extraction of heparin from animal tissues. Sulfation of the hydroxyl group at position 2 of the deacetylated glucosamine is a prerequisite for subsequent enzymatic modifications. In this study, multiple strategies, including truncation mutagenesis based on B-factor values, site-directed mutagenesis guided by multiple sequence alignment, and structural analysis were performed to improve the stability and activity of human N-sulfotransferase. Eventually, a combined variant Mut02 (MBP-hNST-NΔ599-602/S637P/S741P/E839P/L842P/K779N/R782V) was successfully constructed, whose half-life at 37°C and catalytic activity were increased by 105-fold and 1.35-fold, respectively. After efficient overexpression using the Escherichia coli expression system, the variant Mut02 was applied to N-sulfation of the chemically deacetylated heparosan. The N-sulfation content reached around 82.87% which was nearly 1.88-fold higher than that of the wild-type. The variant Mut02 with high stability and catalytic efficiency has great potential for heparin biomanufacturing.

Construction of a Protein Crystalline Inclusion-Based Enzyme Immobilization System for Biosynthesis of PAPS from ATP and Sulfate.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the bioactive form of sulfate and is involved in all biological sulfation reactions. The enzymatic transformation method for PAPS is promising, but the low efficiency and high cost of enzyme purification and storage restrict its practical applications. Here, we reported PAPS biosynthesis with a protein crystalline inclusion (PCI)-based enzyme immobilization system. First, the in vivo crystalline inclusion protein CipA was identified as an efficient auto-assembly tag for immobilizing the bifunctional PAPS synthase (ASAK). After characterizing the pyrophosphokinase activity of a polyphosphate exonuclease PaPPX from Pseudomonas aeruginosa, and optimizing the linker fragment, auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA were constructed. Then, the auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA with high stability were co-expressed and immobilized for constructing a transformation system. The highest transformation rate of PAPS from ATP and sulfate reached 90%, and the immobilized enzyme can be reused 10 times. The present work provided a convenient, efficient, and easy to be enlarged auto-immobilization system for PAPS biosynthesis from ATP and sulfate. The immobilization system also represented a new approach to reduce the production cost of PAPS by facilitating the purification, storage, and reuse of related enzymes, and it would boost the studies on biotechnological production of glycosaminoglycans and sulfur-containing natural compounds.

Plerixafor and resatorvid inhibit hepatitis B virus in vitro by upregulating elongation factor Tu GTP-binding domain containing 2.

Background: An increase in the demand for a functional cure has accelerated research on new methods of therapy for chronic hepatitis B, which is mainly focused on restoring antiviral immunity for controlling viral infections. Previously, we had described elongation factor Tu GTP-binding domain containing 2 (EFTUD2) as an innate immune regulator and suggested that it might be an antiviral target. Methods: In this study, we generated the Epro-LUC-HepG2 cell model for screening compounds that target EFTUD2. Plerixafor and resatorvid were screened from 261 immunity and inflammation-related compounds due to their ability to highly upregulate EFTUD2. The effects of plerixafor and resatorvid on hepatitis B virus (HBV) were examined in HepAD38 cells and HBV-infected HepG2-NTCP cells. Results: The dual-luciferase reporter assays showed that the EFTUD2 promoter hEFTUD2pro-0.5 kb had the strongest activity. In Epro-LUC-HepG2 cells, plerixafor and resatorvid significantly upregulated the activity of the EFTUD2 promoter and the expression of the gene and protein. In HepAD38 cells and HBV-infected HepG2-NTCP cells, treatment with plerixafor and resatorvid strongly inhibited HBsAg, HBV DNA, HBV RNAs, and cccDNA in a dose-dependent manner. Furthermore, the anti-HBV effect was enhanced when entecavir was administered along with either of the previous two compounds, and the effect could be blocked by knocking down EFTUD2. Conclusion: We established a convenient model for screening compounds that target EFTUD2 and further identified plerixafor and resatorvid as novel HBV inhibitors in vitro. Our findings provided information on the development of a new class of anti-HBV agents that act on host factors rather than viral enzymes.

Morphology and location of sensilla in the antennae and ovipositor of Sirex noctilio (Hymenoptera: Siricidae).

The woodwasp Sirex noctilio Fabricius is a major quarantine pest that was reported in China in 2013 and mostly damages Pinus sylvestris var. mongolica. Reverse chemical ecology, which uses chemical lures to catch or block insects from mating is the classic way to control forestry pests. This indicates that insect sensilla play a crucial role in detecting external chemical and physical stimuli. Nonetheless, the categorization and distribution of sensilla on the antennae and ovipositor of S. noctilio are insufficiently specific. In this paper, scanning electron microscopy (SEM) was used to observe the ultrastructure of the sensilla of S. noctilio on the antenna and ovipositor. It was found that the types and distribution of sensilla on the antennae of S. noctilio male and female are consistent, and six types of sensilla are found: sensilla trichodea (ST), sensilla chaetica (SC), Böhm bristles (BB), sensilla basiconica (SB), sensilla ampullacea (SA), and contact chemoreceptors (CC). Besides, there are five types of sensilla on the female ovipositor. In addition to ST, SC and BB, two more types of sensilla are also found: sensilla cavity (SCa) and sensilla coeloconica (SCo). Through identification of the morphology and distribution of the sensilla, the functions of different sensilla in the mating and host selection mechanisms of S. noctilio are proposed, thereby establishing a foundation for S. noctilio chemical communication research.

Apple U-box-type E3 ubiquitin ligase MdPUB23 reduces cold-stress tolerance by degrading the cold-stress regulatory protein MdICE1.

Cold stress limits plant growth, geographical distribution, and crop yield. The MYC-type bHLH transcription factor ICE1 is recognized as the core positive regulator of the cold-stress response. However, how ICE1 protein levels are regulated remains to be further studied. In this study, we observed that a U-box-type E3 ubiquitin ligase, MdPUB23, positively regulated the cold-stress response in apple. The expression of MdPUB23 increased at both the transcriptional and post-translational levels in response to cold stress. Overexpression of MdPUB23 in transgenic apple enhanced sensitivity to cold stress. Further study showed that MdPUB23 directly interacted with MdICE1, promoting the ubiquitination-mediated degradation of the MdICE1 protein through the 26S-proteasome pathway and reducing the MdICE1-improved cold-stress tolerance in apple. Our results reveal that MdPUB23 regulates the cold-stress response by directly mediating the stability of the positive regulator MdICE1. The PUB23-ICE1 ubiquitination module may play a role in maintaining ICE1 protein homeostasis and preventing overreactions from causing damage to plants. The discovery of the ubiquitination regulatory pathway of ICE1 provides insights for the further exploration of plant cold-stress-response mechanisms.

Enzymatic Production of Chondroitin Oligosaccharides and Its Sulfate Derivatives.

Chondroitin sulfate (CS) has a wide range of physiological functions and clinical applications. However, the biosynthesis of chondroitin oligosaccharides (o-CHs) and sulfate derivatives with specific length is always challenging. Herein, we report enzymatic strategies for producing homogeneous o-CHs and its sulfate derivatives from microbial sourced chondroitin. Chondroitin disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides with defined structure were produced by controllably depolymerizing microbial sourced chondroitin with an engineered chondroitinase ABC I. The highest conversion rates of the above corresponding o-CHs were 65.5%, 32.1%, 12.7%, 7.2%, and 16.3%, respectively. A new efficient enzymatic sulfation system that directly initiates from adenosine 5'-triphosphate (ATP) and sulfate was developed and improved the sulfation of chondroitin from 8.3% to 85.8% by optimizing the temperature, sulfate and ATP concentration. o-CHs decasaccharide, octasaccharide, hexasaccharide, tetrasaccharide and disaccharide were modified and the corresponding sulfate derivatives with one sulfate group were prepared. The enzymatic approaches constructed here for preparing o-CHs and its sulfate derivatives pave the way for the study of structure-activity relationship and applications.

[One-pot synthesis of chondroitin sulfate A by engineered Pichia pastoris].

Chondroitin sulfate (CS) is a linear polysaccharide, which is widely used in medical, health care and other fields. Compared with the traditional animal tissue extraction method, microbial synthesis of CS has the advantages of controllability and easiness of scaling-up. In order to achieve an efficient synthesis of chondroitin sulfate A (CSA), we constructed a recombinant Pichia pastoris GS115 strain capable of synthesizing chondroitin (Ch) from glycerol by introducing the Ch synthase coding genes kfoC, kfoA and UDP-glucose dehydrogenase coding gene tuaD into the P. pastoris chromosome. The titer of Ch reached 2.6 g/L in fed-batch cultures upon optimizing the synthesis pathway of Ch. After further expressing the chondroitin-4-O-sulfotransferase (C4ST), we developed a one-pot biosynthesis system for CSA production by directly adding 3'-adenosine-5'-phosphoryl sulfate and C4ST into the high-pressure homogenized recombinant P. pastoris cells. Eventually, controllable synthesis of 0-40% CSA with different sulfation degrees were achieved by optimizing the catalytic conditions. The one-pot biosynthesis system constructed here is easy to operate and easy to scale up for industrial production of CSA. The idea of the present study may also facilitate the biosynthesis of other glycosaminoglycan, for instance, heparin.

[Preparation of tanshinone â ¡_A-glycyrrhetinic acid solid lipid nanoparticles and its inhibitory effect on acne].

The optimal prescription of tanshinone Ⅱ_A(TSN)-glycyrrhetinic acid(GA) solid lipid nanoparticles(GT-SLNs) was explored and evaluated in vivo and in vitro, and its effect on acne after oral administration was investigated. The preparation processing and prescription were optimized and verified by single factor and response surface methodology. The in vitro release of GA and TSN in GT-SLNs was determined by ultra-performance liquid chromatography(UPLC). The effect of GT-SLNs on acne was investigated by the levels of sex hormones in mice, ear swelling model, and tissue changes in sebaceous glands, and the pharmacokinetics was evaluated. The 24-hour cumulative release rates of GA and TSN in SLNs were 65.87%±5.63% and 36.13%±2.31% respectively. After oral administration of GT-SLNs and the mixture of GA and TSN(GT-Mix), the AUC_(0-t) and AUC_(0-∞) of TSN in GT-SLNs were 1.98 times and 4.77 times those in the GT-Mix group, respectively, and the peak concentration of TSN in the GT-SLNs group was 17.2 times that in the GT-Mix group. After intragastric administration of GT-SLNs, the serum levels of testosterone(T) and the ratio of testosterone to estradiol(T/E2) in the GT-SLNs group significantly declined, and the sebaceous glands of mice were atrophied to a certain extent. The results demonstrated that obtained GT-SLNs with good encapsulation efficiency and uniform particle size could promote the release of GA and TSN. GT-SLNs displayed therapeutic efficacy on acne manifested by androgen increase, abnormal sebaceous gland secretion, and inflammatory damage.

Abscisic acid insensitive 4 interacts with ICE1 and JAZ proteins to regulate ABA signaling-mediated cold tolerance in apple.

Abscisic acid is involved in the regulation of cold stress response, but its molecular mechanism remains to be elucidated. In this study, we demonstrated that the APETALA2/ethylene responsive factor (AP2/ERF) family protein MdABI4 positively regulates abscisic acid-mediated cold tolerance in apple. We found that MdABI4 interacts with MdICE1, a key regulatory protein involved in the cold stress response, and enhances the transcriptional regulatory function of MdICE1 on its downstream target gene MdCBF1, thus improving abscisic acid-mediated cold tolerance. The jasmonate-ZIM domain (JAZ) proteins MdJAZ1 and MdJAZ2 negatively modulate MdABI4-improved cold tolerance in apple by interacting with the MdABI4 protein. Further investigation showed that MdJAZ1 and MdJAZ2 interfere with the interaction between the MdABI4 and MdICE1 proteins. Together, our data revealed that MdABI4 integrates jasmonic acid and abscisic acid signals to precisely modulate cold tolerance in apple through the JAZ-ABI4-ICE1-CBF regulatory cascade. These findings provide insights into the crosstalk between jasmonic acid and abscisic acid signals in response to cold stress.