Rice calcium/calmodulin-dependent protein kinase directly phosphorylates a mitogen-activated protein kinase kinase to regulate abscisic acid responses.

Calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of abscisic acid (ABA) and abiotic stress signaling in plants and is believed to act upstream of mitogen-activated protein kinase (MAPK) in ABA signaling. However, it is unclear how CCaMK activates MAPK in ABA signaling. Here, we show that OsDMI3, a rice (Oryza sativa) CCaMK, directly interacts with and phosphorylates OsMKK1, a MAPK kinase (MKK) in rice, in vitro and in vivo. OsDMI3 was found to directly phosphorylate Thr-25 in the N-terminus of OsMKK1, and this Thr-25 phosphorylation is OsDMI3-specific in ABA signaling. The activation of OsMKK1 and its downstream kinase OsMPK1 is dependent on Thr-25 phosphorylation of OsMKK1 in ABA signaling. Moreover, ABA treatment induces phosphorylation in the activation loop of OsMKK1, and the two phosphorylations, in the N-terminus and in the activation loop, are independent. Further analyses revealed that OsDMI3-mediated phosphorylation of OsMKK1 positively regulates ABA responses in seed germination, root growth, and tolerance to both water stress and oxidative stress. Our results indicate that OsMKK1 is a direct target of OsDMI3, and OsDMI3-mediated phosphorylation of OsMKK1 plays an important role in activating the MAPK cascade and ABA signaling.

Synthesis of Fluorine-Containing Multisubstituted Oxa-Spiro[4,5]cyclohexadienones via a Fluorinated Alcohol-Catalyzed One-Pot Sequential Cascade Strategy.

In recent years, the application of fluorinated alcohols as solvents, cosolvents, or additives has become important in modern organic synthesis. However, their potential as efficient catalysts in organic synthesis has not been well-explored. In this article, we report on the development of a one-pot sequential cascade reaction of p-quinone methides with difluoroenoxysilanes using hexafluoroisopropanol as catalyst. This reaction allows for the preparation of fluorinated multisubstituted oxa-spiro[4,5]cyclohexadienones. By using 50 mol % 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP), the reaction proceeds smoothly to yield 1,6-conjugated products, which are then subjected to oxidative dearomatization/hemiacetalization using PhI(OAc)2. The overall process affords moderate to high yields and excellent diastereoselectivities.

Origins of Diastereoselectivity in the Addition of Enoxysilanes to Vinyl Diazonium Salts.

The addition of enoxysilanes to vinyl diazonium ions occurs with varying levels of diastereoselectivity. To understand the origins of the stereoselectivity, we studied these transformations using density functional theory (DFT) calculations. The selectivity stems from a stabilizing cation-π interaction that orients the nucleophile and the diazonium ion.

Organocatalyzed Photoelectrochemistry for the Generation of Acyl and Phosphoryl Radicals through Hydrogen Atom-Transfer Process.

An organocatalyzed photoelectrochemical method for the generation of acyl and phosphoryl radicals from formamides, aldehydes, and phosphine oxides has been developed. This protocol utilizes 9,10-phenanthrenequinone (PQ) as both a molecular catalyst and a hydrogen atom-transfer (HAT) reagent, eliminating the requirement for external metal-based reagents, HAT reagents, and oxidants. The generated acyl radicals can be applied to a range of radical-mediated transformation reactions, including C-H carbamoylation of heteroarenes, intermolecular tandem radical cyclization of CF3-substituted N-arylacrylamides, as well as intramolecular cyclization reactions. The use of acyl radicals in these transformations offers an efficient and sustainable approach to accessing structurally diverse carbonyl compounds.

La(OTf)3-Catalyzed [3+2] Cycloaddition Reactions for the Synthesis of Benzo[d]oxazoles/Benzofurans.

The La(OTf)3-catalyzed [3+2] cycloaddition reactions for the synthesis of benzo[d]oxazoles/benzofurans via quinones and 1,2-di-tert-butyl-3-(cyanimino)diaziridine (1,3-di-tert-butyl-2-cyanoguanidine)/vinyl azides have been explored. A series of 5-hydroxybenzofuran-4-carboxylic acid derivatives and 5-hydroxybenzo[d]oxazole-4-carboxylic acid derivatives were conveniently obtained with high yields and good stereoselectivities, which could be used for further transformations to valuable compounds.

High-throughput process development from gene cloning to protein production.

In the post-genomic era, the demand for faster and more efficient protein production has increased, both in public laboratories and industry. In addition, with the expansion of protein sequences in databases, the range of possible enzymes of interest for a given application is also increasing. Faced with peer competition, budgetary, and time constraints, companies and laboratories must find ways to develop a robust manufacturing process for recombinant protein production. In this review, we explore high-throughput technologies for recombinant protein expression and present a holistic high-throughput process development strategy that spans from genes to proteins. We discuss the challenges that come with this task, the limitations of previous studies, and future research directions.

Palladium-Catalyzed C-H Allylation/Annulation Reaction of Amides and Allylic Alcohols: Regioselective Construction of Vinyl-Substituted 3,4-Dihydroisoquinolones.

A palladium-catalyzed highly regioselective C-H allylation/annulation reaction of N-sulfonyl amides with secondary or tertiary allylic alcohols has been developed to construct 3,4-dihydroisoquinolones bearing a synthetically valuable vinyl substituent. This cascade cyclization approach of allylic alcohols involving C-H allylation has not been reported previously. The commercially available allylic alcohol substrates, the only by-product of water, and the used terminal oxidant of O2 provide environmentally benign advantages.

Lobosteroids A-F: Six New Highly Oxidized Steroids from the Chinese Soft Coral Lobophytum sp.

To explore the steroidal constituents of the soft coral Lobophytum sp. at the coast of Xuwen County, Guangdong Province, China, a chemical investigation of the above-mentioned soft coral was carried out. After repeated column chromatography over silica gel, Sephadex LH-20, and reversed-phase HPLC, six new steroids, namely lobosteroids A-F (1-6), along with four known compounds 7-10, were obtained. Their structures were determined by extensive spectroscopic analysis and comparison with the spectral data reported in the literature. Among them, the absolute configuration of 1 was determined by X-ray diffraction analysis using Cu Kα radiation. These steroids were characterized by either the presence of an α,ß-α',ß'-unsaturated carbonyl, or an α,ß-unsaturated carbonyl moiety in ring A, or the existence of a 5α,8α-epidioxy system in ring B, as well as diverse oxidation of side chains. The antibacterial bioassays showed that all isolated steroids exhibited significant inhibitory activities against the fish pathogenic bacteria Streptococcus parauberis FP KSP28, Phoyobacterium damselae FP2244, and Streptococcus parauberis SPOF3K, with IC90 values ranging from 0.1 to 11.0 µM. Meanwhile, compounds 2 and 6-10 displayed potent inhibitory effects against the vancomycin-resistant Enterococcus faecium bacterium G7 with IC90 values ranging from 4.4 to 18.3 µM. Therefore, ten highly oxidized steroids with strong antibacterial activities were isolated from the Chinese soft coral Lobophytum sp., which could be developed as new chemotypes of antibacterial drug leads.

Genome-Wide Identification and Expression Analysis of the NAC Gene Family in Kandelia obovata, a Typical Mangrove Plant.

The NAC (NAM, ATAF1/2, and CUC2) gene family, one of the largest transcription factor families in plants, acts as positive or negative regulators in plant response and adaption to various environmental stresses, including cold stress. Multiple reports on the functional characterization of NAC genes in Arabidopsis thaliana and other plants are available. However, the function of the NAC genes in the typical woody mangrove (Kandelia obovata) remains poorly understood. Here, a comprehensive analysis of NAC genes in K. obovata was performed with a pluri-disciplinary approach including bioinformatic and molecular analyses. We retrieved a contracted NAC family with 68 genes from the K. obovata genome, which were unevenly distributed in the chromosomes and classified into ten classes. These KoNAC genes were differentially and preferentially expressed in different organs, among which, twelve up-regulated and one down-regulated KoNAC genes were identified. Several stress-related cis-regulatory elements, such as LTR (low-temperature response), STRE (stress response element), ABRE (abscisic acid response element), and WUN (wound-responsive element), were identified in the promoter regions of these 13 KoNAC genes. The expression patterns of five selected KoNAC genes (KoNAC6, KoNAC15, KoNAC20, KoNAC38, and KoNAC51) were confirmed by qRT-PCR under cold treatment. These results strongly implied the putative important roles of KoNAC genes in response to chilling and other stresses. Collectively, our findings provide valuable information for further investigations on the function of KoNAC genes.

Abscisic Acid Inhibits Rice Protein Phosphatase PP45 via H2O2 and Relieves Repression of the Ca2+/CaM-Dependent Protein Kinase DMI3.

In plants, Ca2+/calmodulin-dependent protein kinase (CCaMK) is a positive regulator of abscisic acid (ABA) responses, including root growth, antioxidant defense, and tolerance of both water stress and oxidative stress. However, the underlying molecular mechanisms are poorly understood. Here, we show a direct interaction between DMI3 (Doesn't Make Infections 3), a rice (Oryza sativa) CCaMK and PP45, a type 2C protein phosphatase in rice (PP2C). This interaction involves the CaM binding domain of DMI3 and the PP2C domain of PP45. In the absence of ABA, PP45 directly inactivates DMI3 by dephosphorylating Thr-263 in DMI3. However, in the presence of ABA, ABA-induced H2O2 production by the NADPH oxidases RbohB/E inhibits the activity of PP45 not only by inhibiting the expression of PP45 but also by oxidizing Cys-350 and Cys-428 residues to form PP45 intermolecular dimers. ABA-induced oxidation of Cys-350 and Cys-428 in PP45 blocked the interaction between PP45 and DMI3 and substantially prevented PP45-mediated inhibition in DMI3 activity. Genetic analysis indicated that PP45 is an important negative regulator of ABA signaling. These results reveal important pathways for the inhibition of DMI3 under the basal state and for its ABA-induced activation in rice.

Hypersecretion of OmlA antigen in Corynebacterium glutamicum through high-throughput based development process.

Outer membrane lipoprotein A (OmlA) is a vaccine antigen against porcine contagious pleuropneumonia (PCP), a disease severely affecting the swine industry. Here, we aimed to systematically potentiate the secretory production of OmlA in Corynebacterium glutamicum (C. glutamicum), a widely used microorganism in the food industry, by establishing a holistic development process based on our high-throughput culture platform. The expression patterns, expression element combinations, medium composition, and induction conditions were comprehensively screened or optimized in microwell plates (MWPs), followed by fermentation parameter optimization in a 4 × 1 L parallel fermentation system (CUBER4). An unprecedented yield of 1.01 g/L OmlA was ultimately achieved in a 5-L bioreactor following the scaling-up strategy of fixed oxygen mass transfer coefficient (kLa), and the produced OmlA antigen showed well-protective immunity against Actinobacillus pleuropneumoniae challenge. This result provides a rapid and reliable pipeline to achieve the hyper-production of OmlA, and possibly other recombinant vaccines, in C. glutamicum. KEY POINTS: • Established a holistic development process and applied it to potentiate the secretion of OmlA. • The secretion of OmlA reached an unprecedented yield of 1.01 g/L. • The recombinant OmlA antigen induced efficient protective immunity.

Palladium-Catalyzed Regioselective C-H Functionalization/Annulation Reaction of Amides and Allylbenzenes for the Synthesis of Isoquinolinones and Pyridinones.

A regioselective C-H functionalization/annulation reaction of N-sulfonyl amides and allylbenzenes through a palladium-catalyzed C(sp2)-H allylation/aminopalladation/ß-H elimination/isomerization sequence has been reported. Various aryl and alkenyl carboxamides are found to be efficient substrates to construct isoquinolinones and pyridinones in up to 96% yield. Using ambient air as the terminal oxidant is another advantage regarding environmental friendliness and operational simplicity.

Bicistronic design as recombinant expression enhancer: characteristics, applications, and structural optimization.

The bicistronic design (BCD) is characterized by a short fore-cistron sequence and a second Shine-Dalgarno (SD2) sequence upstream of the target gene. The outstanding performance of this expression cassette in promoting recombinant protein production has attracted attention. Recently, the application of the BCD has been further extended to gene expression control, protein translation monitoring, and membrane protein production. In this review, we summarize the characteristics, molecular mechanisms, applications, and structural optimization of the BCD expression cassette. We also specifically discuss the challenges that the BCD system still faces. This is the first review of the BCD expression strategy, and it is believed that an in-depth understanding of the BCD will help researchers to better utilize and develop it. KEY POINTS: • Summary of the characteristics and molecular mechanisms of the BCD system. • Review of the actual applications of the BCD expression cassette. • Summary of the structural optimization of the BCD system.

Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system.

BACKGROUND: Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. RESULTS: In this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L. CONCLUSION: In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Construction of a 3A system from BioBrick parts for expression of recombinant hirudin variants III in Corynebacterium glutamicum.

Standardized parts can be efficiently assembled into novel biological systems using the three antibiotic (3A) system, ensuring the reusability of components and repeatability of experiments. In this study, we created the 3A expression system for easy construction of gene expression cassettes in Corynebacterium glutamicum (C. glutamicum), which was applied to screen combinations of promoters and signal peptides for improved secreted rhv3 production. We first obtained three strong promoters P2252, Podhi, and PyweA from all of promoters, which drive the highest expression of green fluorescent protein (egfp). The three promoters were then assembled with different signal peptides to generate a series of constructs using the 3A expression system developed in this study, from which the highest activity of rhv3 reached 3187.5 ATU/L of PyweA-CspA-rhv3. Further increased production of rhv3 achieved large-scale fermentation using 5-L jar bioreactor, with the highest rhv3 accumulation 1.21 g/L obtained after 40 h of cultivation, which is higher than 0.95 g/L reported in E. coli. To the best of our knowledge, this is the first report of rhv3 secretory expression in C. glutamicum, which could be applied for the production of other recombinant proteins with significant applications.Key points• We have exploited a 3A system for the genetic manipulation in C. glutamicum.• We constructed element libraries for assembling standard sequence in C. glutamicum.• The secreted expression of rhv3 was realized by 3A system in C. glutamicum.

MTD: a mammalian transcriptomic database to explore gene expression and regulation.

A systematic transcriptome survey is essential for the characterization and comprehension of the molecular basis underlying phenotypic variations. Recently developed RNA-seq methodology has facilitated efficient data acquisition and information mining of transcriptomes in multiple tissues/cell lines. Current mammalian transcriptomic databases are either tissue-specific or species-specific, and they lack in-depth comparative features across tissues and species. Here, we present a mammalian transcriptomic database (MTD) that is focused on mammalian transcriptomes, and the current version contains data from humans, mice, rats and pigs. Regarding the core features, the MTD browses genes based on their neighboring genomic coordinates or joint KEGG pathway and provides expression information on exons, transcripts and genes by integrating them into a genome browser. We developed a novel nomenclature for each transcript that considers its genomic position and transcriptional features. The MTD allows a flexible search of genes or isoforms with user-defined transcriptional characteristics and provides both table-based descriptions and associated visualizations. To elucidate the dynamics of gene expression regulation, the MTD also enables comparative transcriptomic analysis in both intraspecies and interspecies manner. The MTD thus constitutes a valuable resource for transcriptomic and evolutionary studies. The MTD is freely accessible at http://mtd.cbi.ac.cn.

Asymmetric Palladium-Catalyzed C-H Functionalization Cascade for Synthesis of Chiral 3,4-Dihydroisoquinolones.

A palladium-catalyzed C-H functionalization/intramolecular asymmetric allylation cascade of N-sulfonyl benzamides with 1,3-dienes has been developed. In the presence of a chiral pyridine-oxazoline ligand, this protocol enables the synthesis of chiral 3,4-dihydroisoquinolones in yields of up to 83% with enantioselectivities of up to 96%, using environmentally friendly air as the terminal oxidant.

Highly Diastereoselective [3 + 2] Cycloaddition of Aziridines with Difluorinated Silyl Enol Ethers: Divergent Synthesis of 4,4-Difluoropyrrolidines and 4-Fluoropyrroles.

A highly diastereoselective [3 + 2] cycloaddition of aziridines with difluorinated silyl enol ethers has been developed. This approach provides a facile methodology for highly functionalized gem-difluorinated pyrrolidines in good to excellent yields with good functional group tolerance. A one-pot, two-step approach for synthesis of structurally interesting fluorinated pyrroles has also been developed through a cycloaddition/aromatization/desulfonation sequence. Moreover, readily available substrates, mild reaction conditions, and easy scale-up synthesis show practical advantages.

HFIP-Promoted Divergent Cycloadditions of Difluoroenoxysilanes with 2-Indolylmethanols: Synthesis of Fluoro 2H-Pyrano[3,4-b]indoles and gem-Difluoro Cyclopenta[b]indoles.

An oxa-6π-electrocyclization of difluoroenoxysilanes with diaryl 2-indolylmethanols has been developed. In addition, a rarely reported C3-nucleophilic [3+2] cycloaddition of difluoroenoxysilanes with dialkyl 2-indolylmethanols has been disclosed. This divergent cycloaddition approach affording readily available difluoroenoxysilanes as three-atom and C2 synthons provides rapid access to fluoro 2H-pyrano[3,4-b]indoles and gem-difluoro cyclopenta[b]indoles in good to excellent yields with good functional group tolerance. The metal-free and mild conditions using only HFIP as the solvent without any external acid catalyst illuminate practical and environmentally benign advantages.

Synergistic synthesis of gold nanoflowers as upconversion near-infrared nanoprobe energy acceptor and recognition unit for improved hydrogen sulfide sensing.

A highly sensitive and selective upconversion near-infrared (NIR) fluorescence and colorimetric dual readout hydrogen sulfide (H2S) nanoprobe was constructed based on the excellent NIR fluorescence emission performance of upconversion nanomaterials (UCNPs), the specific recognition effect of synergistically synthesized gold nanoflowers (trypsin-stabled AuNFs (Try-AuNFs)) and the effective NIR fluorescence quenching capability. In this assay, the sensing strategy included three processes. First of all, the synthesized UCNPs can emit 803 nm NIR fluorescence when they were excited by 980 nm excitation light. Secondly, as a result of the principle of fluorescence resonance energy transfer (FRET), Try-AuNFs can effectively quench the NIR fluorescence of UCNPs at 803 nm, which can effectively improve the signal-to-background ratio of nanoprobes, thereby improving the sensitivity of the probes. Thirdly, in the presence of H2S, the Try protective layer on the surface of Try-AuNFs was specifically penetrated, which will subsequently cleave Try-AuNFs via the strong S-Au bond. As such, the NIR fluorescence of UCNPs will be restored, achieving high selectivity and sensitivity detection of H2S. Under optimized conditions, the linear response range of H2S was 0.1-300 µM, and the detection limit was 53 nM. It is worth noting that the Try on the surface of Try-AuNFs via the synergistic effect can increase the steric hindrance of the probe, and this can effectively prevent the interaction between the probe with biothiols (cysteine (Cys), homocysteine (Hcy)) and other natural amino acids (non-thiol-containing) with resultant in the high selectivity regarding the detection of H2S in human serum, which is unlikely to be achieved by AuNFs synthesized by the gold seed method (Se-AuNFs). This work not only provided a new type of UCNPs fluorescence quencher and recognition unit, but also exemplified that the use of the physical properties (steric hindrance) of protein ligands on the surface of nanoflowers can improve the specificity of the probe. This will provide new ideas for the design of other nanoprobes.