Multiple Promoters Research Articles

Vector design for enhancing expression level and assembly of knob-into-hole based FabscFv-Fc bispecific antibodies in CHO cells

Two-armed FabscFv-Fc is a favoured bispecific antibody (BsAb) format due to its advantages of the conventional IgG structure. Production of FabscFv-Fc requires expression of three polypeptide chains, one light chain (LC), one heavy chain (HC) and a scFv fused to the Fc (scFvFc) at optimal ratios. We designed a set of internal ribosome entry site (IRES)-mediated multi-cistronic vectors tailoring to various expression ratios of the three polypeptides to study how the chain ratios affect the FabscFv-Fc production. Expression of HC and scFvFc chains at 1:1 ratio and excess LC gave the highest yield of correctly assembled product. Compared to the use of IRES and multiple promoters, using 2A peptides for co-expression of the three polypeptides gave the highest titre and correctly assembled product. The results obtained in this work provide insights to the impacts of hetero-chain ratios on the BsAb production.

Tissue- and cell-specific whole-transcriptome meta-analysis from brain and retina reveals differential expression of dystrophin complexes and new dystrophin spliced isoforms.

The large DMD gene encodes a group of dystrophin proteins in brain and retina, produced from multiple promoters and alternative splicing events. Dystrophins are core components of different scaffolding complexes in distinct cell types. Their absence may thus alter several cellular pathways, which might explain the heterogeneous genotype-phenotype relationships underlying central comorbidities in Duchenne muscular dystrophy (DMD). However, the cell-specific expression of dystrophins and associated proteins (DAPs) is still largely unknown. The present study provides a first RNA-Seq-based reference showing tissue- and cell-specific differential expression of dystrophins, splice variants and DAPs in mouse brain and retina. We report that a cell type may express several dystrophin complexes, perhaps due to expression in separate cell subdomains and/or subpopulations, some of which with differential expression at different maturation stages. We also identified new splicing events in addition to the common exon-skipping events. These include a new exon within intron 51 (E51b) in frame with the flanking exons in retina, as well as inclusions of intronic sequences with stop codons leading to the presence of transcripts with elongated exons 40 and/or 41 (E40e, E41e) in both retina and brain. PCR validations revealed that the new exons may affect several dystrophins. Moreover, immunoblot experiments using a combination of specific antibodies and dystrophin-deficient mice unveiled that the transcripts with stop codons are translated into truncated proteins lacking their C-terminus, which we called N-Dp427 and N-Dp260. This study thus uncovers a range of new findings underlying the complex neurobiology of DMD.

Prolactin receptor gene transcriptional control, regulatory modalities relevant to breast cancer resistance and invasiveness.

The prolactin receptor (PRLR) is a member of the lactogen/cytokine receptor family, which mediates multiple actions of prolactin (PRL). PRL is a major hormone in the proliferation/differentiation of breast epithelium that is essential for lactation. It is also involved in breast cancer development, tumor growth and chemoresistance. Human PRLR expression is controlled at the transcriptional level by multiple promoters. Each promoter directs transcription/expression of a specific non-coding exon 1, a common non-coding exon 2 and coding exons E3-11. The identification of exon 11 of PRLR led to finding of alternative spliced products and two novel short forms (SF) that can inhibit the long form (LF) of PRLR activity with relevance in physiological regulation and breast cancer. Homo and heterodimers of LF and SF are formed in the absence of PRL that acts as a conformational modifier. Heterodimerization of SF with LF is a major mechanism through which SF inhibits some signaling pathways originating at the LF. Biochemical/molecular modeling approaches demonstrated that the human PRLR conformation stabilized by extracellular intramolecular S−S bonds and several amino acids in the extracellular D1 domain of PRLR SF are required for its inhibitory actions on PRLR LF-mediated functions. Studies in breast cancer cells demonstrated that the transcription of PRLR was directed by the preferentially utilized PIII promoter, which lacks an estrogen responsive element. Complex formation of non-DNA bound ERα dimer with Sp1 and C/EBPβ dimers bound to their sites at the PRLR promoter is required for basal activity. Estradiol induces transcriptional activation/expression of the PRLR gene, and subsequent studies revealed the essential role of autocrine PRL released by breast cancer cells and CDK7 in estradiol-induced PRLR promoter activation and upregulation. Other studies revealed stimulation of the PRLR promoter activity and PRLR LF protein by PRL in the absence of estrogen via the STAT5/phospho-ERα activation loop. Additionally, EGF/ERBB1 can induce the transcription of PRLR independent of estrogen and prolactin. The various regulatory modalities contributing to the upregulation of PRLR provide options for the development of therapeutic approaches to mitigate its participation in breast cancer progression and resistance.

CW198 acts as a genetic insulator to block enhancer-promoter interaction in plants.

Insulators in vertebrates play a role in genome architecture and orchestrate temporo-spatial enhancer-promoter interactions. In plants, insulators and their associated binding factors have not been documented as of yet, largely as a result of a lack of characterized insulators. In this study, we took a comprehensive strategy to identify and validate the enhancer-blocking insulator CW198. We show that a 1.08-kb CW198 fragment from Arabidopsis can, when interposed between an enhancer and a promoter, efficiently abrogate the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and tobacco plants. In plants, both transcriptional crosstalk and spreading of histone modifications were rarely detectable across CW198, which resembles the insulation property observed across the CTCF insulator in the mammalian genome. Taken together, our findings support that CW198 acts as an enhancer-blocking insulator in both Arabidopsis and tobacco. The significance of the present findings and their relevance to the mitigation of mutual interference between enhancers and promoters, as well as multiple promoters in transgenes, is discussed.

Transcriptome and carotenoids profiling of flowers in different Osmanthus fragrans cultivars provide insight into transcriptional control network of carotenoid-related genes expression

• The difference in the content and composition of carotenoids in four Osmanthus fragrans cultivars was investigated. • The critical genes contributing to carotenoids accumulation were identified. • Potential TF candidates participating in expression regulation of critical genes were predicated. • Nine TFs were identified having a similar expression pattern with OfCCD4 in two cultivars. • Established a possible regulatory network between transcription factors and critical genes. Carotenoids are important natural pigments and essential for the coloration formation in plant petals. The difference in the accumulation of carotenoids always leads to the difference in flower coloration in O. fragrans . In this study, flower petals from different O. fragrans groups with different coloration were used. The critical genes and corresponding transcription factor (TF) candidates playing important role in carotenoid accumulation in petals were investigated through the integrated carotenoid profiles and transcriptomic analysis. We found the difference in the contents and compositions of carotenoids endow flower petal with diversified coloration, and expression levels of OfNCED3, OfDXS2, OfCCD1, OfGGPPS2, OfDXR3, OfBCH2 , and OfCCD4 were critical factors modulating carotenoid accumulation. Further analysis showed that 61, 19, 21, 47, 16, 21, and 29 potential TF candidates with high Pearson correlation coefficients (|r|> 0.80) were but also for the first time speculated to interact with the promoter of OfNCED3, OfDXS2, OfCCD1, OfGGPPS2, OfDXR3, OfBCH2 , and OfCCD4 , respectively. In which, 17 TF candidates might bind to multiple gene promoters (>2) for affecting the carotenoid accumulation in O. fragrans petals. Furthermore, nine and one of OfCCD4 potential TFs presented similar and opposite expression pattern with OfCCD4 , respectively, further indicating they might potentially participate in OfCCD4 expression regulation. Collectively, our data established the possible regulatory network between the critical genes and potential TF candidates and provided useful information for studying the underlying transcriptional mechanism of carotenoid accumulation in O. fragrans .

Factors Affecting University Students’ Sleep Quality during the Normalisation of COVID-19 Epidemic Prevention and Control in China: A Cross-Sectional Study

Background: Insufficient and poor-quality sleep have significant negative health consequences for university students in China. In this study, we aimed to assess the subjective sleep quality of university students during the normalisation of COVID-19 epidemic prevention and control in China and to identify key factors affecting their sleep quality. Materials and Methods: A cross-sectional survey was conducted among 1326 university students from Hubei Province, China. Latent profile analysis was conducted on the results of class-difference tests of sleep patterns. Multinomial logistic regression was used to explore the relationship between the influencing factors and three classes of sleep quality. Results: The overall score of sleep quality (9.18 ± 3.22) among university students was assessed by using the PSQI scale, and 427 (32.20%) students reported poor sleep quality. Three distinct classes of sleep patterns were identified, namely, good sleepers (Class 1, 70.44%), poor sleep quality with less medication use (Class 2, 26.55%) and poor sleepers (Class 3, 3.01%). Conclusions: Compared with ‘good sleepers’, students having ‘poor sleep quality with less hypnotic drug use’ were influenced by their education stage, smoking habits, physical activity, depression and anxiety. Meanwhile, ‘poor sleepers’ may be affected by their age, origins, smoking habits, mental stress, depression and anxiety. Significant heterogeneity was confirmed in the sleep patterns of university students. Their behavioural lifestyles and mental health-related factors demonstrated different relationship patterns with sleep quality. Multiple sleep promotion interventions, including moderate aerobic exercises, psychological counselling and mindfulness training, should be regularly performed in groups to improve their sleep quality.

Characterization of transcriptional activities at a divergent promoter of the type VI secretion system in enterohemorrhagic Escherichia coli O157:H7.

The type VI secretion system (T6SS) is a novel secretion system found in many Gram-negative bacteria that plays a role in bacterial competition, virulence, and host immune evasion. The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 has a single functional T6SS gene cluster. In this study, we attempted to characterize the transcriptional pattern of the T6SS effector gene Z0264 in EDL933. Transcriptional analyses showed that Z0264 and other T6SS genes were transcribed in vitro in a growth-phase-dependent manner, but Z0264 was not secreted in the rich medium. Using adapter- and radioactivity-free transcription start site analysis, we identified an overlapping divergent promoter between Z0264 and Z0265. A β-galactosidase assay with truncated promoter regions showed that the divergent promoter is functional. In addition, we demonstrated the role of H-NS as a repressor in the transcription of Z0264. Notably, the cDNA PCR assay showed that the mRNA transcript from the Z0264 promoter did not include the entire main T6SS cluster, suggesting segmented gene expression by multiple promoters in the T6SS cluster. In conclusion, we identified a divergent promoter for Z0264 located in the T6SS cluster of EDL933 and characterized its in vitro transcriptional activity during growth. Our findings provide insights and a preliminary understanding of the regulatory mechanisms underlying T6SS transcription.

Characterization of BbKlf1 as a novel transcription factor vital for asexual and infection cycles of Beauveria bassiana.

The large family of C2H2-type zinc finger transcription factors (TFs) comprise the Kruppel-like factors (KLFs) that evolved relatively late in eukaryotes but remain unexplored in filamentous fungi. Here, we report that an orthologue (BbKlf1) of yeast Klf1 mediating cell wall integrity (CWI) is a wide-spectrum TF evidently localized in nucleus and cytoplasm in Beauveria bassiana. BbKlf1 features conserved domains and multiple DNA-binding motifs predicted to bind multiple promoter DNA fragments of target genes across asexual developmental and stress-responsive pathways. Despite limited impact on normal colony growth, deletion of Bbklf1 resulted in impaired CWI and hypersensitivity to Congo red-induced cell wall stress. Also, the deletion mutant was severely compromised in tolerance to oxidative and osmotic stresses, hyphal septation and differentiation, conidiation capacity (reduced by 95%), conidial quality (viability and hydrocarbon epitope pattern) and virulence. Importantly, these phenotypes correlated well with sharply repressed or nearly abolished expressions of those genes required for or involved in chitin biosynthesis, antioxidant activity, cell division and differentiation, aerial conidiation and conidial maturation. These findings indicate an essentiality of BbKlf1 for the asexual and insect-pathogenic lifecycles of B. bassiana and a novel scenario much beyond the yeast orthologue-mediated CWI, suggesting important roles of its orthologues in filamentous fungi.

Genetic and methylation profiles distinguish benign, malignant and spitzoid melanocytic tumors.

Accurate classification of melanocytic tumors is important for prognostic evaluation, treatment and follow-up protocols of patients. The majority of melanocytic proliferations can be classified solely based on clinical and pathological criteria, however in select cases a definitive diagnostic assessment remains challenging and additional diagnostic biomarkers would be advantageous. We analyzed melanomas, nevi, Spitz nevi and atypical spitzoid tumors using parallel sequencing (exons of 611 genes and 507 gene translocation analysis) and methylation arrays (850k Illumina EPIC). By combining detailed genetic and epigenetic analysis with reference-based and reference-free DNA methylome deconvolution we compared Spitz nevi to nevi and melanoma and assessed the potential for these methods in classifying challenging spitzoid tumors. Results were correlated with clinical and histologic features. Spitz nevi were found to cluster independently of nevi and melanoma and demonstrated a different mutation profile. Multiple copy number alterations and TERT promoter mutations were identified only in melanomas. Genome-wide methylation in Spitz nevi was comparable to benign nevi while the Leukocytes UnMethylation for Purity (LUMP) algorithm in Spitz nevi was comparable to melanoma. Histologically difficult to classify Spitz tumor cases were assessed which, based on methylation arrays, clustered between Spitz nevi and melanoma and in terms of genetic profile or copy number variations demonstrated worrisome features suggesting a malignant neoplasm. Comprehensive sequencing and methylation analysis verify Spitz nevi as an independent melanocytic entity distinct from both nevi and melanoma. Combined genetic and methylation assays can offer additional insights in diagnosing difficult to classify Spitzoid tumors.

Association between eight hypermethylation-related genes and gastric cancer: a systematic review and meta-analysis.

BackgroundAlthough multiple gene promoter hypermethylation has been associated with gastric carcinogenesis, data on their specific relationship remains scant. We aimed to investigate the correlation between the status of multiple gene promoter methylation and gastric cancer (GC).MethodsWe searched PubMed, EMBASE, CNKI, Wanfang, Cqvip and Cochrane Library up to May 2021. We systematically assessed the association between methylation status of the CpG islands and the risk of GC. We compared the incidence of DNA methylation between tumor and non-tumor tissues, and evaluated the clinicopathological significance of the DNA methylation in gastric carcinoma. The data was presented by an odds ratio (OR) with an accompanying 95% confidence interval (CI). We then generated forest plots calculated by fixed-effects or random-effects model.ResultsThis study enrolled a total of 201 studies (140 papers). Our analysis showed a higher frequency of methylation of the CpG islands in GC tissues compared to non-neoplastic tissues. Besides, the data demonstrated that polygene’s aberrant promoter methylation might be linked to the initial development and progression of GC.DiscussionThe genes with altered DNA methylation might serve as epigenetic biomarkers, providing a promising molecular diagnostic and prognostic tool for human GC. However, our findings need further evaluation in large randomized controlled trials.

Abstract 5593: DNMT inhibitor induces NSCLC inflammasome signaling and mitochondrial dysfunction, producing to DSB repair defects and PARP inhibitor sensitivity

Abstract DNA methyltransferase inhibitors (DNMTis) modulate genome-wide methylation patterns, previously linked by our group to downregulation of DNA double strand break repair (DSBR) that sensitizes non-small cell lung cancer (NSCLC) to poly(ADP-ribose) polymerase inhibitors (PARPis). DNMTi hypomethylation of endogenous retroviral (ERV) elements produces cytosolic aggregation of double-stranded RNA (dsRNA) that activates antiviral interferon (IFN) and inflammasome signaling. Accordingly, DNMTi treatment of NSCLC cell lines (A549, H460) increases expression of 1) multiple ERV transcripts; 2) dsRNA sensors RIG-I and MDA5; 3) downstream mitochondrial antiviral signaling protein (MAVS); 4) MAVS-activated antiviral kinases (TBK1, IKKα/β) and transcriptional regulators (IRF3/7, NFκB); and 5) IFNβ and IFN-stimulated genes (ISGs). A second mechanism of cellular antiviral signaling is STING (stimulator of interferon genes) activation triggered by cytosolic dsDNA. Following DNMTi treatment, we observe mitochondrial dysfunction including impairment of oxidative phosphorylation, reactive oxygen species generation, and reduced mitochondrial membrane potential. This dysfunction is associated with increased mitochondrial DNA (mtDNA) damage and accumulation of cytosolic mtDNA, leading to increased STING expression and IFN activation that can be abrogated by siSTING or IKK inhibition. Treatment with PARPi, which activates STING via cytosolic accumulation of damaged nuclear DNA, further enhances DNMTi-induced IFN signaling. We previously linked IFN activation to DSBR downregulation in breast and ovarian cancer, and now present TCGA analysis in NSCLC indicating a similar negative correlation, corroborated by reduced DSBR in NSCLC cell lines after IFNβ treatment. Computational interrogation indicates multiple DSBR promoters contain IFN-stimulated response elements (ISREs), including potential negative regulators, that may account for these findings. Notably, we find that precise patterns of immune activation may be TP53 dependent: NSCLC cell lines with missense mutations (H23, H441, H1115), as observed in ~30% patients, exhibit increased NFκB inflammasome signaling and decreased IFN activation compared to wildtype (A549, H460). TP53 mutation is associated with significantly reduced baseline STING expression, potentially affording a more robust DNMTi treatment response; accordingly, TP53-mutant NSCLC cells exhibit significantly greater sensitivity to DNMTi/PARPi. Our new data expands on our published work to further understanding of novel mechanisms of 1) inflammasome induction and 2) mitochondrial dysfunction through which DNMTis activate cellular antiviral signaling and downregulate DSBR to induce PARPi sensitivity in NSCLC. We also identify TP53 as a possible biomarker for future personalization of DNMTi therapy. Citation Format: Rachel Abbotts, Lora Stojanovic, Michael J. Topper, Brian M. Polster, Stephen B. Baylin, Feyruz V. Rassool. DNMT inhibitor induces NSCLC inflammasome signaling and mitochondrial dysfunction, producing to DSB repair defects and PARP inhibitor sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5593.

Identification and expression analysis of capsaicin biosynthesis pathway genes at genome level in Capsicum chinense

Capsicum chinense is one of the best ingredients for making chili sauces and extracting capsaicin. In this study, we identified 59 genes associated with shikimic acid, phenylpropane, unsaturated fatty acids, phenylalanine, valine, leucine, isoleucine and other metabolic pathways based on pepper genome. In the present study, 58 of the 59 genes involved in capsaicin synthesis were found to be unevenly distributed on 11 chromosomes, and several genes with the same number of exons and high homology were found to be clustered in a duplication region of the chromosome. The promoter regions of these genes were predicted and analyzed, and multiple gene promoters were found to contain MYB binding sites, which provided a reference for the subsequent exploration of MYB target genes. At the same time, the expression levels of genes related to capsaicin synthesis were analyzed based on different transcriptomes, and it was found that these genes showed significant differences in different plant development stages and different tissues. The results of this study will be helpful to further study the synthesis mechanism of capsaicin in Capsicum chinense and the functional relationship between genes.

Potential biocontrol and plant growth promotion of an endophytic bacteria isolated from Glycyrrhiza uralensis seeds

BackgroundEndophytic bacteria have been demonstrated to be one of the most potentially important biocontrol agents for their efficiently improved plant growth and protection of host plants from infection by phytopathogens.ResultsNine strains of endophytic bacteria isolated from Glycyrrhiza uralensis Fisch. (G. uralensis) seeds were screened for the inhibitory effect against 5 fungal phytopathogens by using dual culture method. Among which, the isolate FT2 showed the highest percentage of inhibition to all the test fungal phytopathogens. Detached root assay showed that FT2 significantly reduced disease index and rotted area on Angelica sinensis root slices caused by Fusarium acuminatum. FT2 also exhibited multiple plant growth promotion traits by qualitative analytical method. Based on phenotypic, physiological and biochemical characterization and genotypic characterization, the FT2 strain was identified as Stenotrophomonas rhizophila. Application of strain FT2 reduced the disease index of Cucumber Fusarium Wilt, and the biocontrol effect was equal to chemical fungicide, carbendazim. Moreover, strain FT2 increased the plant height, stem diameter, leaf number and fresh weight of cucumber seedlings and the activities of the defense-related enzymes including phenylalanine ammonia-lyase, polyphenol oxidase and peroxidase. In addition, strain FT2 could promote seed germination and seedling growth of G. uralensis and increase enzyme activities, available nutrient contents and bacterial count in G. uralensis rhizosphere soil.ConclusionsThe results demonstrated that the strain FT2 could be used to develop environmentally friendly microbial products to improve the activity against pathogenic and promote plant growth.

Developing a recombinant expression system for human serine protease PRSS23

The PRSS23 gene, encoding putative serine protease 23, has been associated with several diseases by correlative gene expression studies and genetic‐based comparative models. Our group has recently developed data implicating the expression of this gene as a potential driver of ovarian cancer malignancy. However, there have been no studies defining PRSS23 protein structure or demonstrating its hypothesized proteolytic function. We aim to elucidate PRSS23 protein structure and enzymatic activity. The first step to achieve this goal is to develop a robust expression system and purification protocol for recombinant PRSS23 protein. We hypothesize that developing a methodology to produce PRSS23 recombinant protein will enable us to determine its crystal structure, evaluate its biological and enzyme function, and ultimately target the enzyme therapeutically. We strategized to maximize chances of producing PRSS23 in its native form and with high yield by testing multiple systems and host species: human, yeast, and bacterial. We anticipate, based on the protein sequence, that PRSS23 is endogenously secreted and may be activated in the secretory pathway by furin, releasing the C‐terminal catalytic domain from a smaller N‐terminal domain. The protein is also predicted to be glycosylated. We generated expression plasmids for full‐length PRSS23, codon‐optimized for expression in human and Pichia pastoris expression systems, incorporating a His‐tag for detection and purification. In the human HEK 293F and HEK 293T cell systems, despite optimizing transfection protocols and testing multiple expression constructs, secretion signals, and promoters, the expression level remained very low and was judged unlikely to produce sufficient protein for structural and functional studies. Integration of codon‐optimized PRSS23 into the genome of P. pastoris produced integrants showing a spectrum of expression levels from very low to moderate. Experiments are ongoing to optimize expression conditions of the best clone and develop a purification workflow involving ammonium sulfate precipitation, immobilized nickel ion chromatography, and additional chromatographic steps as needed. In parallel, we cloned the catalytic domain of PRSS23, appropriately codon‐optimized and also containing a C‐terminal His‐tag, into an expression vector for Escherichia coli. High levels of protein were produced as insoluble inclusion bodies, and a protocol was developed for nickel ion affinity purification under denaturing conditions. Experiments are ongoing to identify suitable conditions for in vitro refolding of the PRSS23 catalytic domain. Additionally, we are evaluating a maltose binding protein fusion with the PRSS23 catalytic domain as an alternative strategy to improve soluble protein production in E. coli. Significant quantities of pure protein will be needed to enable crystal structure determination and enzymatic activity elucidation, to unravel the biological mechanism of action and identify endogenous substrates, and potentially lead to new therapeutic strategies targeting PRSS23. The expression systems and purification workflows developed here will be a step forward toward these long‐term goals.

Nonadherence to health promotion depending on chronic obstructive pulmonary disease severity

BackgroundNonadherence to recommended health behaviors reportedly correlates with the prevalence and health outcomes of COPD, but the factors contributing to multiple health promotions are not well understood. ObjectiveTo examine the factors associated with nonadherence to health behaviors, specifically focusing on COPD severity. MethodsWe conducted a secondary data analysis with data from the 2016–2019 Korea National Health and Nutrition Examination Survey. The analyzed data were from 1,627 individuals aged over 40 years who had a FEV1/FVC ratio < 70% measured via spirometer. In this study, nonadherence to health behaviors is the sum of seven health-promoting behaviors in terms of smoking cessation, alcohol abstinence, regular exercise, weight control, recommended sleep time, influenza vaccination, and medical check-up. ResultsCOPD patients’ nonadherence to health behaviors was significantly associated with comorbidity (p=0.017), stress (p=0.002), and COPD severity (moderate, severe; p=0.010, p=0.039, respectively). In addition, the moderate and severe COPD groups showed a higher nonadherence rate than the mild COPD group. Specifically, nonadherence to weight control was the most significantly different behavior according to COPD severity levels, based on the GOLD classification of airflow limitation. ConclusionOur study findings support that those with higher levels of stress, comorbidities, and severe disease conditions are less likely to adhere to health behaviors. Thus, healthcare providers and policymakers should provide tailored approaches according to COPD severity to educate and support appropriate health behaviors for individuals with COPD.

Research on regional differences of China's new energy vehicles promotion policies: A perspective of sales volume forecasting

After breaking through the technical bottleneck in the early stage of development, China's NEVs (New Energy Vehicles) industry has ushered in breakthrough growth under the joint action of multiple promotion policies. To improve the market penetration of NEVs, verify whether the market penetration of NEVs can reach the target of 20% in 2025. Taking the sales volume of NEVs as a decision reference, this study proposes a multi-factor prediction model integrating DWT (Discrete Wavelet Transform) and BiLSTM (Bidirectional Long Short Term Memory). The optimal model is obtained through comparative experiments, the MAE, MAPE, and RMSE values of the DWT-BiLSTM model are 0.811, 5.671, and 1.001. Through the analysis of the average impact value of three representative cities, the relative importance of each input index is discussed quantitatively. From 2020 to 2025, the sales volume of NEVs in China will show growth trends in varying degrees under three scenarios, but none of them can achieve the goal of 20% market penetration of NEVs in 2025; Corresponding policy suggestions are put forward for the three types of cities, including adjusting the license quota policy, accelerating the construction of charging infrastructure and delaying the decline of subsidies for NEV.

Establishment of an Efficient Polyethylene Glycol (PEG)-Mediated Transformation System in Pleurotus eryngii var. ferulae Using Comprehensive Optimization and Multiple Endogenous Promoters

Pleurotus eryngii var. ferulae, a fungus of the genus Pleurotus, efficiently degrades lignin, especially during co-cultivation with other fungi. However, low transformation efficiency and heterologous gene expression restrict systematic studies of the molecular mechanisms and metabolic control of natural products in this mushroom. In this study, the homologous resistance marker carboxin (cbx) was used to establish a polyethylene glycol-mediated transformation (PMT) system in P. eryngii var. ferulae. Optimization of the transformation process greatly improved the number of positive transformants. In particular, we optimized: (i) protoplast preparation and regeneration; (ii) screening methods; and (iii) transformation-promoting factors. The optimized transformation efficiency reached 72.7 CFU/μg, which is higher than the average level of Pleurotus sp. (10–40 CFU/μg). Moreover, three endogenous promoters (Ppfgpd1, Ppfgpd2, and Ppfsar1) were screened and evaluated for different transcription initiation characteristics. A controllable overexpression system was established using these three promoters that satisfied various heterologous gene expression requirements, such as strong or weak, varied, or stable expression levels. This study lays the foundation for recombinant protein expression in P. eryngii var. ferulae and provides a method to investigate the underlying molecular mechanisms and secondary metabolic pathway modifications.

Mesenchymal Stem Cells and Selenium Nanoparticles Synergize with Low Dose of Gamma Radiation to Suppress Mammary Gland Carcinogenesis via Regulation of Tumor Microenvironment

Breast cancer is one of the most prevalent and deadliest cancers among women in the world because of its aggressive behavior and inadequate response to conventional therapies. Mesenchymal stem cells (MSCs) combined with green nanomaterials could be an efficient tool in cell cancer therapy. This study examined the curative effects of bone marrow–derived mesenchymal stem cells (BM-MSCs) with selenium nanoparticles (SeNPs) coated with fermented soymilk and a low dose of gamma radiation (LDR) in DMBA-induced mammary gland carcinoma in female rats. DMBA-induced mammary gland carcinoma as marked by an elevation of mRNA level of cancer promoter genes (Serpin and MIF, LOX-1, and COL1A1) and serum level of VEGF, TNF-α, TGF-β, CA15-3, and caspase-3 with the reduction in mRNA level of suppressor gene (FST and ADRP). These deleterious effects were hampered after treatment with BM-MSCs (1 × 106 cells/rat) once and daily administration of SeNPs (20 mg/kg body weight) and exposure once to (0.25 Gy) LDR. Finally, MSCs, SeNPs, and LDR notably modulated the expression of multiple tumor promoters and suppressor genes playing a role in breast cancer induction and suppression.

Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research.

The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolites or therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitative characterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used N. benthamiana leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expression of firefly/Renilla luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporter protein and mRNA levels. In addition, we investigated the effects of terminator-promoter combinations on gene expression and found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest and weakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterized promoters to engineer the betalain pathway in N. benthamiana. Seven selected plant promoters with different expression strengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0-362.4 μg/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequences in N. benthamiana and BY2 cells but also adds to the toolbox of plant promoters for plant engineering.

Environmental Tuning of Homologs of the Orange Carotenoid Protein-Encoding Gene in the Cyanobacterium Fremyella diplosiphon.

The orange carotenoid protein (OCP) family of proteins are light-activated proteins that function in dissipating excess energy absorbed by accessory light-harvesting complexes, i.e., phycobilisomes (PBSs), in cyanobacteria. Some cyanobacteria contain multiple homologs of the OCP-encoding gene (ocp). Fremyella diplosiphon, a cyanobacterium studied for light-dependent regulation of PBSs during complementary chromatic acclimation (CCA), contains several OCP homologs – two full-length OCPs, three Helical Carotenoid Proteins (HCPs) with homology to the N-terminus of OCP, and one C-terminal domain-like carotenoid protein (CCP) with homology to the C-terminus of OCP. We examined whether these homologs are distinctly regulated in response to different environmental factors, which could indicate distinct functions. We observed distinct patterns of expression for some OCP, HCP, and CCP encoding genes, and have evidence that light-dependent aspects of ocp homolog expression are regulated by photoreceptor RcaE which controls CCA. RcaE-dependent transcriptional regulator RcaC is also involved in the photoregulation of some hcp genes. Apart from light, additional environmental factors associated with cellular redox regulation impact the mRNA levels of ocp homologs, including salt, cold, and disruption of electron transport. Analyses of conserved sequences in the promoters of ocp homologs were conducted to gain additional insight into regulation of these genes. Several conserved regulatory elements were found across multiple ocp homolog promoters that potentially control differential transcriptional regulation in response to a range of environmental cues. The impact of distinct environmental cues on differential accumulation of ocp homolog transcripts indicates potential functional diversification of this gene family in cyanobacteria. These genes likely enable dynamic cellular protection in response to diverse environmental stress conditions in F. diplosiphon.