Increased monoamine oxidase activity and imidazoline binding sites in insulin-resistant adipocytes from obese Zucker rats

BACKGROUND Despite overt insulin resistance,adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIM To investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide,since it is produced by monoamine oxidase(MAO)and semicarbazide-sensitive amine oxidase(SSAO)in adipocytes.METHODS 3H-2-deoxyglucose uptake(2-DG)was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine.14C-tyramine oxidation and binding of imidazolinic radioligands[3H-Idazoxan,3H-(2-benzofuranyl)-2-imidazoline]were studied in adipocytes,the liver,and muscle.The influence of in vivo administration of tyramine+vanadium on glucose handling was assessed in lean and obese rats.RESULTS 2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats,when compared to their lean littermates.Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited,while MAO was increased and SSAO decreased.These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat,when compared to the lean.In vitro,tyramine precluded the binding to I2 sites,while in vivo,its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese CONCLUSION The adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number.However,probably as a consequence of SSAO down-regulation,the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes.The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.

Low-affinity SPL binding sites contribute to subgenome expression divergence in allohexaploid wheat

Expression divergence caused by genetic variation and crosstalks among subgenomes of the allohexaploid bread wheat(Triticum aestivum.L.,BBAADD)is hypothesized to increase its adaptability and/or plasticity.However,the molecular basis of expression divergence remains unclear.Squamosa promoter-binding protein-like(SPL)transcription factors are critical for a wide array of biological processes.In this study,we constructed expression regulatory networks by combining DAP-seq for 40 SPLs,ATACseq,and RNA-seq.Our findings indicate that a group of low-affinity SPL binding regions(SBRs)were targeted by diverse SPLs and caused different sequence preferences around the core GTAC motif.The SBRs including the low-affinity ones are evolutionarily conserved,enriched GWAS signals related to important agricultural traits.However,those SBRs are highly diversified among the cis-regulatory regions(CREs)of syntenic genes,with less than 8%SBRs coexisting in triad genes,suggesting that CRE variations are critical for subgenome differentiations.Knocking out of Ta SPL7A/B/D and Ta SPL15A/B/D subfamily further proved that both high-and low-affinity SBRs played critical roles in the differential expression of genes regulating tiller number and spike sizes.Our results have provided baseline data for downstream networks of SPLs and wheat improvements and revealed that CRE variations are critical sources for subgenome divergence in the allohexaploid wheat.

Addition of heparin binding sites strongly increases the bone forming capabilities of BMP9 in vivo

Bone Morphogenetic proteins(BMPs)like BMP2 and BMP7 have shown great potential in the treatment of severe bone defects.In recent in vitro studies,BMP9 revealed the highest osteogenic potential compared to other BMPs,possibly due to its unique signaling pathways that differs from other osteogenic BMPs.However,in vivo the bone forming capacity of BMP9-adsorbed scaffolds is not superior to BMP2 or BMP7.In silico analysis of the BMP9 protein sequence revealed that BMP9,in contrast to other osteogenic BMPs such as BMP2,completely lacks so-called heparin binding motifs that enable extracellular matrix(ECM)interactions which in general might be essential for the BMPs’osteogenic function.Therefore,we genetically engineered a new BMP9 variant by adding BMP2-derived heparin binding motifs to the N-terminal segment of BMP9′s mature part.The resulting protein(BMP9 HB)showed higher heparin binding affinity than BMP2,similar osteogenic activity in vitro and comparable binding affinities to BMPR-II and ALK1 compared to BMP9.However,remarkable differences were observed when BMP9 HB was adsorbed to collagen scaffolds and implanted subcutaneously in the dorsum of rats,showing a consistent and significant increase in bone volume and density compared to BMP2 and BMP9.Even at 10-fold lower BMP9 HB doses bone tissue formation was observed.This innovative approach of significantly enhancing the osteogenic properties of BMP9 simply by addition of ECM binding motifs,could constitute a valuable replacement to the commonly used BMPs.The possibility to use lower protein doses demonstrates BMP9 HB’s high translational potential.

Systematic identification and annotation of multiple-variant compound effects at transcription factor binding sites in human genome

Understanding the functional effects of genetic variants is crucial in modern genomics and genetics.Transcription factor binding sites(TFBSs) are one of the most important cis-regulatory elements. While multiple tools have been developed to assess functional effects of genetic variants at TFBSs, they usually assume that each variant works in isolation and neglect the potential "interference" among multiple variants within the same TFBS. In this study, we presented COPE-TFBS(Context-Oriented Predictor for variant Effect on Transcription Factor Binding Site), a novel method that considers sequence context to accurately predict variant effects on TFBSs. We systematically re-analyzed the sequencing data from both the 1000 Genomes Project and the Genotype-Tissue Expression(GTEx) Project via COPE-TFBS, and identified numbers of novel TFBSs, transformed TFBSs and discordantly annotated TFBSs resulting from multiple variants, further highlighting the necessity of sequence context in accurately annotating genetic variants. COPE-TFBS is freely available for academic use at http://cope.cbi.pku.edu.cn/.

基于3A组装的多拷贝基因策略优化重组水蛭素变体Ⅲ的生产

水蛭素变体Ⅲ(Hirudin varidantⅢ,Hv3)是一种从水蛭中提取的活性成分,在预防和治疗白内障方面具有潜在作用。为了制备足量Hv3用于进一步的临床前应用研究,该研究开发了一种在谷氨酸棒杆菌(Corynebacterium glutamicum)中高效、稳定表达重组水蛭素变体Ⅲ(recombinant Hirudin varidantⅢ,Rhv3)的办法。首先,在C.glutamicum中构建含Ptac启动子和CspA信号肽的Rhv3分泌表达菌株,比较其在不同培养基中的表达情况。结果表明在BHI培养基中Rhv3活性最高,达到3.02×10^(3) ATU/L。为进一步提升Rhv3产量,通过核糖体结合位点(ribosome binding site,RBS)筛选,选用RBS1应用于Rhv3多拷贝菌株构建和表达。然后利用3A组装技术构建含不同信号肽和不同拷贝数的Rhv3分泌表达菌株进一步优化其产量。通过放大发酵培养,Rhv3产量达到1.89 g/L,活性达到10.91×10^(3) ATU/L。最后利用镍柱对Rhv3进行简单纯化,其纯度达到90%。该异源表达策略有效提高Rhv3表达量,为Rhv3的重组生产提供了一种质量可靠、低成本的表达体系。

Flumazenil-insensitive benzodiazepine effects in vitro and in vivo

OBJECTIVE To identify benzodi⁃azepine(BZD)effects that are insensitive to the classical BZD binding site antagonist,flumazenil.Whether the flumazenil-insensitive BZD effects have selectivity on different GABAA receptor sub⁃types was also investigated.METHODS The high-concentration effects of BZDs and their sensitivity to flumazenil were determined on recombi⁃nant synaptic(α1β2γ2,α2β2γ2,α5β2γ2)and extra-synaptic(α4β2δ)GABAA receptors using the voltage-clamp electrophysiology technique.The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice loss of right reflex(LORR)test.RESULTS Diazepam induced a biphasic potentiation for theα1β2γ2,α2β2γ2 andα5β2γ2 receptor channels,but did not affect theα4β2δreceptor.In contrast to the nanomolar com⁃ponent of potentiation,the second potentiation elicited by micromolar diazepam was insensitive to flumazenil.Midazolam,clonazepam,and loraz⁃epam at 200μmol·L-1 exhibited similar flumaze⁃nil-insensitive effects onα1β2γ2,α2β2γ2 andα5β2γ2 receptors,whereas the potentiation induced by 200μmol·L-1 zolpidem or triazolam was abol⁃ished by flumazenil.Consistent with the in vitro results,flumazenil antagonized the zolpidem(50 mg·kg-1)-induced LORR,but not those induced by 50 mg·kg-1 diazepam or 100 mg·kg-1 midazolam.CONCLUSION The existence of non-classical BZD binding sites on certain GABAA receptor subtypes and the flumazenil-insensitive effects depend on the chemical structures of the allosteric modulators.

Bioinformatics Analysis of the Human TCF7L2 Gene Promoter Region

[Objectives]This study was conducted to investigate characteristics of the human TCF7 L2 gene promoter.[Methods]The 2000 bp sequence of the 5’regulatory region of the human TCF7 L2 gene was obtained from the UCSC genome database.The promoter,transcription factor binding sites,CpG islands,SNPs and so on were analyzed by a variety of online softwares.[Results]The bioinformatics analysis results showed there were at least 5 potential promoters in the positive-sense strand of the 2000 bp sequence,among which-242--192 bp,-853--803 bp might contain core promoters.A TATA box and a CpG island with a length of 499 bp were found.241,944 and 1035(positive-sense strand)transcription factor binding sites were predicted by the AliBaba2.1,PROMO and JASPAR softwares,respectively.207 common transcription factor binding sites in the conserved region of human and mouse homologous TCF7 L2 gene promoter were identified with CONREAL program,involving 66 kinds of transcription factors.Two SNPs were found in the promoter region.[Conclusions]The promoter of the human TCF7 L2 gene was analyzed by bioinformatics,and the promoter characteristics were obtained.

Current progress in application of 1-Methylcyclopropene to improve postharvest quality of cut flowers

Floral senescence or petal abscission that determine cut flower quality and longevity are primarily caused by the regulation of endogenous ethylene and perception of endogenous and exogenous ethylene in floral organs(such as the petals and gynoecium),in ethylene-sensitive species.Several attempts have been made to prolong flower quality and longevity using silver ions and other chemicals that inhibit ethylene production and perception in floral organs.Among the available compounds,1-methylcyclopropene(1-MCP)has been increasingly used as ethylene-action/perception inhibitor because of its ability to inhibit ethylene action/perception and it is environmentally safe.Although 1-MCP has been used in several cut flower crops for about 25 years,there has been no review yet that critically emphasizes the benefits or difficulties of using this compound for the prolongation of cut flower quality and longevity.Here,we outline the role of 1-MCP in inhibiting ethylene action/perception and petal senescence in cut flowers through the blocking of receptor binding sites,and we discuss how various factors affecting efficacy of 1-MCP(such as concentration,treatment time and temperature,genotype,and flower stage)are involved in the achievement of flower longevity.Moreover,we highlight the advantages of applying nonvolatile and liquid 1-MCP formulation types,as opposed to using the conventional 1-MCP treatment(powder formulation type).We expect that this review will provide useful information for the future utilization of 1-MCP for the maintenance of cut flower longevity.

3-Arylisothiazoloquinols as potent ligands for the benzodiazepine site of GABA_(A) receptors

3-Arylisothiazolo[5,4-b]quinolin-4(9H)-ones and 3-arylisoxazolo[5,4-b]quinolin-4(9H)-ones were synthesized and assayed for affinity for the benzodiazepine binding site of the GABAA receptors. While the 3-arylisothiazoloquinolin-4-ones were found to be potent ligands, with affinities (expressed as the affinity Ki value) down to 1 nM, the 3-arylisoxazoloquinolin-4-ones are less potent. This is suggested to depend on sterical repulsive interaction of the 3-arylisoxazoloquinolin-4-ones with the receptor essential volume of the binding site, and a higher electron density at the nitrogen in the azole ring (N-2) as well as the carbonyl oxygen in the isothiazoloquinolin-4-ones enabling them to interact stronger with hydrogen bond donor sites at the binding site.

A cost-effective ts CUT&Tag method for profiling transcription factor binding landscape

Knowledge of the transcription factor binding landscape(TFBL)is necessary to analyze gene regulatory networks for important agronomic traits.However,a low-cost and high-throughput in vivo chromatin profiling method is still lacking in plants.Here,we developed a transient and simplified cleavage under targets and tagmentation(tsCUT&Tag)that combines transient expression of transcription factor proteins in protoplasts with a simplified CUT&Tag without nucleus extraction.Our tsCUT&Tag method provided higher data quality and signal resolution with lower sequencing depth compared with traditional ChIP-seq.Furthermore,we developed a strategy combining tsCUT&Tag with machine learning,which has great potential for profiling the TFBL across plant development.

Functional characterization of the NF-kB binding site in the human NOD2 promoter

Nucleotide-binding and oligomerization domain 2(NOD2),a member of the NOD protein family,plays an important role in innate immunity.In response to pathogen attack,NOD2 stimulates cytokine and defensin production by activating nuclear factor(NF)-kB,a key transcription factor responsible for mediating downstream reactions.However,the mechanism linking NOD2 regulation and NF-kB activation is poorly understood.Using bioinformatics,we found a completely preserved canonical NF-kB binding site in the NOD2 core promoter(216 to 225 bp)in both humans and chimpanzees.The functional role of this NF-kB binding site was investigated using the enhanced green fluorescent protein(EGFP)reporter system,site-directed mutagenesis,the NF-kB activation inhibitor(JSH-23)and the chromatin immunoprecipitation(ChIP)assay.The results show that the NF-kB binding site is critical for regulation of the NOD2 gene.Either deletion of the NF-kB binding elements within the NOD2 promoter or treatment with an NF-kB activation inhibitor could lead to a significant loss of NOD2 promoter activity as detected by reporter gene assay.The canonical NF-kB binding site was bound by NF-kB as determined by the ChIP method.Based on these results,we suggest a positive feedback regulation between NF-kB and NOD2,which may represent an efficient mechanism in response to pathogen invasion.

Computational prediction and validation of specific EmbR binding site on PknH

Tuberculosis drug resistance continues to threaten global health but the underline molecular mechanisms are not clear.Ethambutol(EMB),one of the well-known first-line drugs in tuberculosis treatment is,unfortunately,not free from drug resistance problems.Genomic studies have shown that some genetic mutations in Mycobacterium tuberculosis(Mtb)EmbR,and EmbC/A/B genes cause EMB resistance.EmbR-PknH pair controls embC/A/B operon,which encodes EmbC/A/B genes,and EMB interacts with EmbA/B proteins.However,the EmbR binding site on PknH was unknown.We conducted molecular simulation on the EmbR-peptides binding structures and discovered phosphorylated PknH 273-280(N′-HEALS^(P)DPD-C′)makesβstrand with the EmbR FHA domain,asβ-MoRF(MoRF;molecular recognition feature)does at its binding site.Hydrogen bond number analysis also supported the peptides’β-MoRF forming activity at the EmbR FHA domain.Also,we discovered that previously known phosphorylation residues might have their chronological order according to the phosphorylation status.The discovery validated that Mtb PknH 273-280(N′-HEALSDPD-C′)has reliable EmbR binding affinity.This approach is revolutionary in the computer-aided drug discovery field,because it is the first trial to discover the protein-protein interaction site,and find binding partner in nature from this site.

Mechanisms and biotechnological applications of transcription factors

Transcription factors play an indispensable role in maintaining cellular viability and finely regulating complex internal metabolic networks.These crucial bioactive functions rely on their ability to respond to effectors and concurrently interact with binding sites.Recent advancements have brought innovative insights into the understanding of transcription factors.In this review,we comprehensively summarize the mechanisms by which transcription factors carry out their functions,along with calculation and experimental-based methods employed in their identification.Additionally,we highlight recent achievements in the application of transcription factors in various biotechnological fields,including cell engineering,human health,and biomanufacturing.Finally,the current limitations of research and provide prospects for future investigations are discussed.This review will provide enlightening theoretical guidance for transcription factors engineering.

SARS-CoV-2 spike variants differ in their allosteric responses to linoleic acid

The SARS-CoV-2 spike protein contains a functionally important fatty acid(FA)binding site,which is also found in some other coronaviruses,e.g.SARS-CoV and MERS-CoV.The occupancy of the FA site by linoleic acid(LA)reduces infectivity by‘locking’the spike in a less infectious conformation.Here,we use dynamical-nonequilibrium molecular dynamics(D-NEMD)simulations to compare the allosteric responses of spike variants to LA removal.D-NEMD simulations show that the FA site is coupled to other functional regions of the protein,e.g.the receptor-binding motif(RBM),N-terminal domain(NTD),furin cleavage site,and regions surrounding the fusion peptide.D-NEMD simulations also identify the allosteric networks connecting the FA site to these functional regions.The comparison between the wild-type spike and four variants(Alpha,Delta,Delta plus,and Omicron BA.1)shows that the variants differ significantly in their responses to LA removal.The allosteric connections to the FA site on Alpha are generally similar to those on the wild-type protein,with the exception of the RBM and the S71–R78 region,which show a weaker link to the FA site.In contrast,Omicron is the most different variant,exhibiting significant differences in the RBM,NTD,V622–L629,and furin cleavage site.These differences in the allosteric modulation may be of functional relevance,potentially affecting transmissibility and virulence.Experimental comparison of the effects of LA on SARS-CoV-2 variants,including emerging variants,is warranted.

Crystal engineering of porous coordination networks for C3 hydrocarbon separation

C3 hydrocarbons(HCs),especially propylene and propane,are high‐volume products of the chemical industry as they are utilized for the production of fuels,polymers,and chemical commodities.Demand for C3 HCs as chemical building blocks is increasing but obtaining them in sufficient purity(>99.95%)for polymer and chemical processes requires economically and energetically costly methods such as cryogenic distillation.Adsorptive separations using porous coordination networks(PCNs)could offer an energy‐efficient alternative to current technolo-gies for C3 HC purification because of the lower energy footprint of sorbent separations for recycling versus alternatives such as distillation,solvent extraction,and chemical transformation.In this review,we address how the structural modularity of porous PCNs makes them amenable to crystal engineering that in turn enables control over pore size,shape,and chemistry.We detail how control over pore structure has enabled PCN sorbents to offer benchmark performance for C3 separations thanks to several distinct mechanisms,each of which is highlighted.We also discuss the major challenges and opportunities that remain to be addressed before the commercial development of PCNs as advanced sorbents for C3 separation becomes viable.

Minor fibrillar collagens,variable regions alternative splicing,intrinsic disorder,and tyrosine sulfation

Minor fibrillar collagen types V and XI,are those less abundant than the fibrillar collagen types I,II and III.The alpha chains share a high degree of similarity with respect to protein sequence in all domains except the variable region.Genomic variation and,in some cases,extensive alternative splicing contribute to the unique sequence characteristics of the variable region.While unique expression patterns in tissues exist,the functions and biological relevance of the variable regions have not been elucidated.In this review,we summarize the existing knowledge about expression patterns and biological functions of the collagen types V and XI alpha chains.Analysis of biochemical similarities among the peptides encoded by each exon of the variable region suggests the potential for a shared function.The alternative splicing,conservation of biochemical characteristics in light of low sequence conservation,and evidence for intrinsic disorder,suggest modulation of binding events between the surface of collagen fibrils and surrounding extracellular molecules as a shared function.

Cascade biocatalysis for production of enantiopure(S)-2-hydroxybutyric acid using recombinant Escherichia coli with a tunable multi-enzyme-coordinate expression system

Racemize 2-hydroxybutyric acid is usually synthesized by organic methods and needs additional deracemization to obtain optically pure enantiomers for industrial application.Here we present a cascade biocatalysis system in Escherichia coli BL21 which employed L-threonine deaminase(TD),NAD-dependent L-lactate dehydrogenase(LDH)and alcohol dehydrogenase(ADH)for producing optically pure(S)-2-hydroxybutyric acid((S)-2-HBA)from bulk chemical L-threonine.To solve the mismatch in the conversion rate and the consumption rate of intermediate 2-oxobutyric acid(2-OBA)formed in the multi-enzyme catalysis reaction,ribosome binding site regulation strategy was explored to control TD expression levels,achieving an eightfold alteration in the conversion rate of 2-OBA.With the optimized activity ratio of the three enzymes and using ADH for NADH regeneration,the recombinant strain ADH-r53 showed increased production of(S)-2-HBA with the highest titer of 129 g/L and molar yield of 93%within 24 h,which is approximately 1.65 times that of the highest yield reported so far.Moreover,(S)-2-HBA could easily be purified by distillation,making it have great potential for industrial application.Additionally,our results indicated that constructing a tunable multi-enzyme-coordinate expression system in single cell had great significance in biocatalysis of hydroxyl acids.

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12 mol/L-1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1×10-9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recog-nition properties previously determined for the RTRES domain. These operators containing the con-sensus sequence GTAAGAAARNTTACN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA inter-actions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.

Observation of oxygen evolution over a {Ni12}-cluster-based metal-organic framework

The development of efficient electrocatalysts based on non-noble metals for oxygen evolution reaction(OER) remains an important and challenging task. Multinuclear transition-metal clusters with high structural stability are promising OER catalysts but their catalytic role is poorly understood. Here we report the crystallographic observation of OER activity over robust {Ni12}-clusters immobilised in a porous metal-organic framework, NKU-100, by single-crystal X-ray diffraction as a function of external applied potential. We observed the aggregation of confined oxygen species around the {Ni12}-cluster as a function of applied potential during the electrocatalytic process. The refined occupancy of these oxygen species shows a strong correlation with the variation of current density. This study demonstrates that the enrichment of oxygen species in the secondary coordination sphere of multinuclear transition-metal clusters can promote the OER activity.