Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Molecular cloning,characterization and promoter analysis of LbgCWIN1 and its expression profiles in response to exogenous sucrose during in vitro bulblet initiation in lily

Lily(Lilium spp.) is an important ornamental flower, which is mainly propagated by bulbs. Cell wall invertases(CWINs), which catalyze the irreversibly conversion of sucrose into glucose and fructose in the extracellular space, are key enzymes participating in sucrose allocation in higher plants. Previous studies have shown that CWINs play an essential role in bulblet initiation process in bulbous crops, but the underlying molecular mechanism remains unclear. Here, a CWIN gene of Lilium brownii var. giganteum(Lbg) was identified and amplified from genomic DNA. Quantitative RT-PCR assays revealed that the expression level of LbgCWIN1 was highly upregulated exactly when the endogenous starch degraded in non-sucrose medium during in vitro bulblet initiation in Lbg. Phylogenetic relationship, motif, and domain analysis of LbgCWIN1 protein and CWINs in other plant species showed that all sequences of these CWIN proteins were highly conserved. The promoter sequence of LbgCWIN1 possessed a number of alpha-amylase-, phytohormone-, light-and stress-responsive cis-elements. Meanwhile, β-glucuronidase(GUS) assay showed that the 459 bp upstream fragment from the translational start site displayed maximal promoter activity. These results revealed that LbgCWIN1 might function in the process of in vitro bulblet initiation and be in the response to degradation of endogenous starch.

Lineage-Specific Genome Architecture Links Enhancers and Non-coding Disease Variants to Target Gene Promoters

Long-range interactions between regulatory elements and gene promoters play key roles in transcriptional regulation.The vast majority of interactions are uncharted,constituting a major missing link in understanding genome control.Here,we use promoter capture Hi-C to identify interacting regions of 31,253 promoters in 17 human primary hematopoietic cell types.We show that promoter interactions are highly cell type specific and enriched for links between active promoters and epigenetically marked enhancers.Promoter interactomes reflect lineage relationships of the hematopoietic tree,consistent with dynamic remodeling of nuclear architecture during differentiation.Interacting regions are enriched in genetic variants linked with altered expression of genes they contact,highlighting their functional role.We exploit this rich resource to connect non-coding disease variants to putative target promoters,prioritizing thousands of disease-candidate genes and implicating disease pathways.Our results demonstrate the power of primary cell promoter interactomes to reveal insights into genomic regulatory mechanisms underlying common diseases.

Promoter and coding sequence diversity of CsCCD1 may contribute to the differential accumulation of floralβ-ionone in fresh tea leaves

The carotenoid-derived volatileβ-ionone makes an important contribution to tea fragrance.Here,we qualitatively and quantitatively analysed 15 carotenoids in tea leaves of 13 cultivars by UHPLC-APCI-MS/MS.The 13 cultivars were divided into two groups by PCA(Principal Component Analysis)clustering analysis of their carotenoid content,and OPLS-DA(Orthogonal projections to latent structures)indicated that the levels ofβ-carotene(VIP=2.89)and lutein(VIP=2.30)were responsible for much of the variation between the two groups.Interestingly,theβ-carotene toβ-ionone conversion rates in Group 1 were higher than in Group 2,while theβ-carotene content was significantly lower in Group 1 than in Group 2.Theβ-ionone content was significantly higher in Group 1.Pearson Correlation Coefficient calculation between the transcription level of candidate genes(CsCCD1 and CsCCD4)and the accumulation ofβ-ionone indicated that CsCCD1 may involve in the formation ofβ-ionone in 13 cultivars.Prokaryotic expression and in vitro enzyme activity assays showed that‘Chuanhuang 1’had an amino acid mutation in carotenoid cleavage dioxygenases 1(CsCCD1)compared with‘Shuchazao’,resulting in a significantly higherβ-ionone content in‘Chuanhuang 1’.Sequence analysis showed that‘Chuanhuang 1’and‘Huangdan’had different CsCCD1 promoter sequences,leading to significantly higher CsCCD1 expression andβ-ionone accumulation in‘Chuanhuang 1’.These results indicated that the promoter and coding sequence diversity of CsCCD1 might contribute to the differential accumulation ofβ-ionone in different tea cultivars.

OsAMT1.1 Expression by Nitrate-Inducible Promoter of OsNAR2.1 Increases Nitrogen Use Efficiency and Rice Yield

Nitrate(NO_(3)^(-))and ammonium(NH_(4)^(+))are two main inorganic nitrogen(N)sources during crop growth.Here,we enhanced the expression of OsAMT1.1,which encodes a NH_(4)^(+)transporter,using the NO_(3)^(-)-inducible promoter of OsNAR2.1 and an ubiquitin promoter in transgenic rice plants.Under field condition of 120 kg/hm2 N,agronomic N use efficiency,N recovery efficiency and N transport efficiency,and grain yield of the pOsNAR2.1:OsAMT1.1 transgenic lines were increased compared with those of the wild type(WT)and the pUbi:OsAMT1.1 transgenic plants.Under 2.0 mmol/L NO_(3)^(-)+0.5 mmol/L NH_(4)^(+)and 0.5 mmol/L NO_(3)^(-)+2.0 mmol/L NH_(4)^(+)conditions of hydroponic culture,compared with the WT,both biomass and total N content were increased in the pOsNAR2.1:OsAMT1.1 transgenic lines.However,biomass was significantly reduced in pUbi:OsAMT1.1 transgenic plants under 0.5 mmol/L NO_(3)^(-)+2.0 mmol/L NH_(4)^(+)condition.The lines expressing pOsNAR2.1:OsAMT1.1 exhibited increased OsAMT1.1 expression and 15NH_(4)^(+)influx in roots under both 2.0 mmol/L NO_(3)^(-)+0.5 mmol/L NH_(4)^(+)and 0.5 mmol/L NO_(3)^(-)+2.0 mmol/L NH_(4)^(+)conditions.Our study showed that expression of OsAMT1.1 can be promoted when driven by the OsNAR2.1 promoter,especially under high-level nitrate condition,leading to enhancement of NH_(4)^(+)uptake,N use efficiency and grain yield.

Methyltransferase 3A-mediated promoter methylation represses retinoic acid receptor responder 3 expression in basal-like breast cancer

Retinoic acid receptor responder 3(RARRES3)has been characterized as a tumor suppressor in multiple types of cancer.This study aimed to examine the expression profile of RARRES3 across the PAM50 subtypes of breast cancer.The DNA methylation status of RARRES3 was checked in the basal-like subtype,and the underlying mechanisms of its dysregulation were explored.RNA-sequencing(seq)and methylation data from The Cancer Genome Atlas were used for in-silico analysis.Basal-like representative SUM149 and MDA-MB-468 cell lines were used for in vitro and in vivo studies.Compared to tumor-adjacent normal tissues,only the basal-like tumor tissues had significantly downregulated RARRES3 expression.The methylation level of four CpG sites in the promoter region showed a strong negative correlation with RARRES3 expression.The gene coding for DNA methyltransferase 3A(DNMT3A)had consistent positive correlations with the methylation of the CpG sites.Chromatin Immunoprecipitation-quantitative polymerase chain-reaction and bisulfite sequencing PCR showed that DNMT3A could bind to the promoter region of RARRES3 and promote methylation of the CpG sites within the region.DNMT3A knockdown significantly restored RARRES3 expression at the mRNA and protein level in the two cell lines.CCK-8,colony formation,and flow cytometric analysis showed that RARRES3 overexpression attenuated the growth-promoting effects of DNMT3A overexpression and also weakened the DNMT3A overexpression-induced activation of ERK1/2 and PI3K/AKT signaling.In summary,this study revealed that DNMT3A enhances promoter methylation of the RARRES3 gene and suppresses its transcription in basal-like breast cancer.The DNMT3A-RARRES3 signaling pathway might be a potential target for the treatment of this tumor subtype.

Theoretical study on the morphology of cobalt nanoparticles modulated by alkali metal promoters

Cobalt nanoparticles(NPs)catalysts are extensively used in heterogeneous catalytic reactions,and the addition of alkali metal promoters is a common method to modulate the catalytic performance because the catalyst's surface structures and morphologies are sensitive to the addition of promoters.However,the underlying modulation trend remains unclear.Herein,the adsorption of alkali metal promoters(Na and K)on the surfaces of face-centered-cubic(FCC)and hexagonal-closest packed(HCP)polymorphous cobalt was systematically investigated using density functional theory.Furthermore,the effect of alkali promoters on surface energies and nanoparticle morphologies was revealed on the basis of Wulff theory.For FCC-Co,the exposed area of the(111)facet in the nanoparticle increases with the adsorption coverage of alkali metal oxide.Meanwhile,the(311),(110),and(100)facets would disappear under the higher adsorption coverage of alkali metals.For HCPCo,the Wulff morphology is dominated by the(0001)and(1011)facets and is independent of the alkali metal adsorption coverage.This work provides insights into morphology modulation by alkali metal promoters for the rational design and synthesis of cobalt-based nanomaterials with desired facets and morphologies.

Spatiotemporal variations,influencing factors,and configurational promotion paths of energy efficiency in China’s cities

The promotion of energy efficiency(EE)helps address energy constraints and promote environmental sustainability.This study comprehensively explores the spatiotemporal variations,influencing factors,and configuration promotion paths of EE in 284 Chinese cities during 2003‒2019 using the global super-efficiency minimum distance to strong efficient frontier(G-S-MinDS),exploratory spatial data analysis(ESDA),multiscale geographically weighted regression(MGWR),and fuzzy set qualitative comparative analysis(fsQCA)methods.The findings are:①China’s cities have an annual average EE of 0.658 with a growth rate of 0.53%,showing considerable promotion potential.②Industrial structure optimization,population agglomeration,economic development,and increased green coverage contribute positively,while government intervention and openness hinder China’s urban EE.③Four configurational promotion paths for enhancing China’s urban EE are identified,where among those paths population density is a core condition,while government intervention is not.This study provides valuable insights into substantially improving urban EE,emphasizing the need for targeted policies to address energy and environmental crises in China.

Pueraria isoflavones inhibit XOD and GLUT9 to decrease uric acid production and promote uric acid excretion, respectively

Objective: To analyze the possible mechanism of Pueraria isoflavones inhibiting XOD and GLUT9 to reduce uric acid production and promote uric acid excretion. Methods: August 2021-April 2022, a total of forty SPF male Kunming mice were divided into the healthy group (carboxymethylcellulose sodium at a dose of 250 mg/kg), the model group (HUA mice were given carboxymethylcellulose sodium at a dose of 250 mg/kg), the low group (HUA mice were given pueraria isoflavone at a dose of 125 mg/kg), HUA mice were given pueraria isoflavones at a dose of 250 mg/kg once d frequency)and the high group (HUA mice were given pueraria isoflavones at a dose of 500 mg/kg once d frequency) dosage groups, with 8 mice in each group. The contents of uric acid (SUA), urea nitrogen (BUN) and creatinine (SCr) in serum and urine of each group were compared before and after intervention (30 d). Statistical differences of xanthine oxidase (XOD) and human glucose transporter 9(GLUT9), cy- clooxygenase- 2(COX-2), tumor necrosis factor (TNF-α) and interleukin-1 (IL-1β) contents in renal tissues of each group after intervention (30 d) were compared. Results: After intervention, kidney inflammatory factors (COX-2, TNF-α and IL-1β) in the model group were compared. Blood and urine indexes (SUA, BUN, SCr);The contents of XOD and GLUT9 were higher than those of healthy group(P<0.05). Renal inflammatory cytokines (COX-2, TNF-α and IL-1β) in low, medium and high dose groups;Blood and urine indexes (SUA, BUN, SCr);The contents of XOD and GLUT9 were lower than those of model group, and there were low > medium > high dose groups, the comparison between the two groups had statistical significance(P< 0.05). After intervention, the contents of 3 indicators in blood or urine(COX-2, TNF-α and IL-1β) all decreased compared with before intervention, and the differences in intra-group comparison were statistically significant (P<0.05). Conclusion: Pueraria isoflavones can treat HUA mice by inhibiting the expression of XOD and GLUT9, and then play a role in reducing uric acid pro- duction and promoting uric acid excretion, as well as alleviating the degree of disease inflammation.

From Inside Out:Internal Marketing as an Engine for Service Promotion-A Secondary Publication

Focusing on“from inside out”means understanding that the driving force behind a successful service promotion campaign lies in a deep connection with the internal team,turning it into the engine that propels a positive brand perception.The University of Panama faces challenges in the effective implementation of strategies involving its internal staff,impacting the coherence and strength of its promotional messages.The research aimed to determine internal marketing strategies as an engine for service promotion at the Central Campus and CRUSAM.The applied study was descriptive,non-experimental field research,and cross-sectional.The sample consisted of 80 individuals using a quota sampling method to measure and quantify the expression of a construct or variable that is not directly observable.A selfadministered questionnaire was used to collect information,measuring variables in their various dimensions.Content validity of the questionnaire was ensured through the evaluation of three experts,and the reliability of the Likert-type instrument was assessed using Cronbach’s α coefficient,which was found to be very high(0.918).The results led to the conclusion that establishing internal marketing strategies is necessary for the transformation and innovation process in the university through service promotion.

Study on the Promotion of Digital Trade to Northeast Asian Regional Economy

Society is currently transitioning from the era of informatization and digitization to the era of intelligence.Advanced technologies such as artificial intelligence,the Internet of Things,and big data are permeating every aspect of production and life.This penetration presents a rare opportunity for the development of digital trade and catalyzes innovation in traditional trade patterns.This paper first analyzes the inevitability of digital trade development in Northeast Asia.Secondly,it delves into the challenges faced by digital trade development and explores in-depth the promotion strategy for digital trade’s impact on the economic development of the Northeast Asia region,providing insights from various perspectives for reference.

A Multimodal Critical Discourse Analysis of Lingnan Cultural Promotional Videos on Social Media

In recent years,more and more directors of culture and tourism have taken part in the promotion of local cultural tourism by cross-dressing,talent shows,and pushing their limits on self-media platforms.This study investigates short videos of Lingnan culture promoted by directors general and deputy directors general of the Culture,Radio,Television,Tourism,and Sports Bureau of counties and cities in Guangdong Province on social media by the method of multimodal critical discourse analysis.The analysis of 33 videos shows that Lingnan culture is a domineering and confident culture,historical culture,graceful and elegant culture,and vibrant and active culture.Domineering and confident culture is embedded in the utterances and behaviors of the directors general or deputy directors general in the video.Historical culture is realized through the conversation with historical figures through time travel.Graceful and elegant culture is constructed in the depiction of sceneries and the depiction of characters’manners.Vibrant and active culture is represented in the depiction of the characters’actional process and analytical process.

Marketing Strategies for Reading Promotion Activities in Academic Libraries:A Case Study of the University of Shanghai for Science and Technology

In the contemporary digital age,traditional reading habits have been compromised by digital media,a situation that has forced academic libraries to adopt proactive marketing approaches to promote reading among students and faculty.This article investigates the implementation of marketing strategies in promoting reading activities within academic libraries,taking the University of Shanghai for Science and Technology as a case study.The paper explores various marketing strategies employed by academic libraries,including market segmentation,utilization of new media platforms,and the creation of personalized content for readers.Drawing from an extensive literature review and real-world examples,the study establishes a framework for marketing reading promotion activities tailored to academic libraries.These findings underscore how effective marketing strategies can enhance user participation,expand library influence,and optimize resource utilization.

The Discourse-Historical Approach of Identity Construction in Corporate Promotional Videos for External Publicity Image

Corporate identity construction of external publicity image is an important part of the development of enterprises.Based on Wodak’s discourse-historical approach,this study takes the text of COFCO’s English promotional video as the research object,analyzes the corporate brand image,media image,organizational image,and environmental image constructed by the enterprises from three steps:linguistic expression,discourse strategy,and theme to provide references for Chinese enterprises to enhance their international influence.

Economic Analysis and Research of New Energy Vehicle Promotion Model

New energy vehicles represent the inevitable trend of future development.Compared to traditional fuel vehicles,they are more energy-saving and environmentally friendly,effectively reducing air pollution and mitigating excessive exploitation of oil resources,a stance strongly supported by governments.However,new energy vehicles possess certain drawbacks in terms of price and usability compared to traditional counterparts.Therefore,external support is imperative for their development.This paper delineates four main sections:the background of new energy vehicle promotion and application,a comparative analysis of domestic and foreign promotion models,specific promotion suggestions,and future development prospects.By leveraging insights from economic analysis,the optimal promotion model for new energy vehicles is elucidated.

牙鲆GHR基因Promoter区微卫星序列多态性与生长性状关系的初步研究

采用牙鲆GHR基因5’端Promoter区的1个微卫星标记,对胶南和日照2个牙鲆养殖群体进行了群体遗传多样性的研究,并探索该基因多态性位点与牙鲆生长性状之间的相关性。结果表明,2个群体在该座位的等位基因数为12和9个,有效等位基因数为6.26和5.04个,多态信息含量为0.84和0.80。2个群体该座位的Hardy-Weinberg遗传偏离指数均为正值,并没有显示出杂合子缺失,但各基因型分布频率都在一定程度上偏离Hardy-Weinberg平衡(P<0.01)。连锁分析中发现,在胶南群体中,IM基因型对应的个体在全重、全长、体长、头长、体高和眼径形态学数据中均是最大的;在日照群体中,BC基因型对应的个体在全重、全长、体高、尾柄高、尾柄长和眼径数据中均是最大的;而CJ基因型对应的个体在体长和头长这两组数据中是最大的。该结果为研究GHR基因的变异对鱼类生长发育的影响及探索将该基因作为生长遗传标记的可行性奠定了实验基础。

种子特异性启动子(napinB promoter)分离、表达载体构建及转基因植物获得

利用PCR技术从油菜BrassicanapusH1 65基因组DNA中分离了napinB启动子。序列分析表明 ,扩增片段 (nap30 0 )与文献报道的napinB启动子相应区域的同源性为 97%。将其与gus连接构建种子特异性表达载体 ,农杆菌介导转化烟草。PCR、Southern结果显示 ,nap30 0已整合到烟草基因组DNA中 ,获得了转基因植株。

pGL3-Basic-COX-2-promoter报告基因重组质粒的构建及其功能鉴定

目的:构建COX-2基因启动子荧光素酶报告基因重组质粒pGL3-Basic-COX-2-promoter,并进行功能鉴定.方法:根据已知人的COX-2基因启动子序列设计两端引物,扩增人基因组DNA中的COX-2启动子.载体质粒pGL3-Basic和COX-2启动子分别用限制性内切酶HindⅢ和BglⅡ双酶切,将COX-2基因启动子插入到pGL3-Basic报告载体中.构建的pGL3-Basic-COX-2-promoter重组质粒和内参质粒pRL-SV40瞬时共转染胃癌MKN45细胞,加入100倍细胞数量的Hpylori共培养不同时间后,检测双荧光素酶的活性.结果:成功构建COX-2基因启动子重组质粒pGL3-Basic-COX-2-promoter,质粒测序及酶切结果完全正确.瞬时转染实验显示,COX-2启动子在MKN45细胞中的转录表达随时间的变化而升高,转染后40h的双报告基因活性是转染后8h的3.5倍(P<0.01);加入Hpylori共培养后,同时间点的荧光素酶活性明显升高(P<0.05或0.01),转染后40h的活性是转染后8h的5倍(P<0.01).结论:pGL3-Basic-COX-2-promoter在胃癌MKN45细胞中能被转录激活,加入Hpylori刺激后COX-2活性显著增强,pGL3-Basic-COX-2-promoter可以用来鉴定和检测细胞中COX-2的水平.

Core promoter: A critical region where the hepatitis B virus makes decisions

The core promoter(CP)of the viral genome plays an important role for hepatitis B virus(HBV)replication as it directs initiation of transcription for the synthesis of both the precore and pregenomic(pg)RNAs.The CP consists of the upper regulatory region and the basal core promoter(BCP).The CP overlaps with the 3’-end of the X open reading frames and the 5’-end of the precore region,and contains cis-acting elements that can independently direct transcription of the precore mRNA and pgRNA.Its transcription regulation is under strict control of viral and cellular factors.Even though this regulatory region exhibits high sequence conservation,when variations appear,they may contribute to the persistence of HBV within the host,leading to chronic infection and cirrhosis,and eventually,hepatocellular carcinoma.Among CP sequence variations,those occurring at BCP may dysregulate viral gene expression with emphasis in the hepatitis B e antigen,and contribute to disease progression.In this review these molecular aspects and pathologic topics of core promoter are deeply evaluated.

Cloning and expression analysis of the FvNCED3 gene and its promoter from ash(Fraxinus velutina)

The 9-cis-epoxycarotenoid dioxygenase(NCED)gene is rate-limiting in abscisic acid(ABA) biosynthesis.In this study, an NCED gene, designated FvNCED3(KY008746), was cloned from velvet ash(Fraxinus velutina Torr.) with a RACE method. The full length c DNA of FvNCED3 encodes a 573-amino acid polypeptide.Sequencing analysis showed that the FvNCED3 protein was highly homologous to other NCED proteins. The expression patterns of FvNCED3 in different ash organs were analyzed by real-time PCR which revealed that FvNCED3 expression levels were highest in leaves and lowest in roots. The gene expression patterns of FvNCED3 under abiotic stress indicated that its expression increased under drought, salt and ABA stress and decreased due to high and low temperatures. There were no obvious changes under ultraviolet light. The 1094-bp upstream sequence 5' flank regulation region of the FvNCED3 gene was also cloned from ash using the Genome Walking method. To assess the activity of the FvNCED3 promoter, a p FvNCED3 p::GUS plant expression vector was constructed for tobacco transformation. GUS expression of the FvNCED3 GUS enzyme activity was detected in almost all transgenic tobacco tissues, especially in the young leaves,stigma, anther, ovule and ovary. After treating the transgenic tobacco with NaCl and placing it under drought stress, GUS staining of tobacco leaves increased compared with that under normal growth conditions. This result indicates that gene expression driven by the FvNCED3 promoter can be induced by salt and drought stress.