Search

This study aimed to reveal interactions of the stress response sigma subunits (factors) σD and σH of RNA polymerase and promoters in Gram-positive bacterium Corynebacterium glutamicum by combining wet-lab obtained data and in silico modeling. Computer modeling-guided point mutagenesis of C. glutamicum σH subunit led to the creation of a panel of σH variants. Their ability to initiate transcription from naturally occurring hybrid σD/σH-dependent promoter Pcg0441 and two control canonical promoters (σD-dependent PrsdA and σH-dependent PuvrD3) was measured and interpreted using molecular dynamics simulations of homology models of all complexes. The results led us to design the artificial hybrid promoter PD35H10 combining the −10 element of the PuvrD3 promoter and the −35 element of the PrsdA promoter. This artificial hybrid promoter PD35-rsdAH10-uvrD3 showed almost optimal properties needed for the bio-orthogonal transcription (not interfering with the native biological processes). [ABSTRACT FROM AUTHOR]

Gain‐of‐function variants in the WDR44 gene have recently been associated with an X‐linked ciliopathy‐related neurodevelopmental phenotype. Here, we report on a WDR44 loss‐of‐function (LOF) variant identified in the genome sequence from a male fetus enrolled in the Prenatal Genetic Diagnosis by Genomic Sequencing (PrenatalSEQ) multicenter study. The phenotype is consistent with the described X‐linked ciliopathy that includes developmental delay, microcephaly, congenital heart defects, kidney abnormalities, cryptorchidism, musculoskeletal abnormalities, craniofacial dysmorphism, and effusions. This is the first report of a WDR44 LOF variant in an affected individual with a prenatal presentation and supports LOF as a mechanism for the X‐linked WDR44 ciliopathy‐related phenotype. [ABSTRACT FROM AUTHOR]

Abstract Background Overactivated microglia are a key contributor to Parkinson’s disease (PD) by inducing neuroinflammation. CD200R1, a membrane glycoprotein mainly found on microglia, is crucial for maintaining quiescence with its dysregulation linked to microglia’s abnormal activation. We and other groups have reported a decline in CD200R1 levels in several neurological disorders including PD. However, the mechanism regulating CD200R1 expression and the specific reasons for its reduction in PD remain largely unexplored. Given the pivotal role of transcription factors in gene expression, this study aimed to elucidate the transcriptional regulation of CD200R1 and its implications in PD. Methods The CD200R1 promoter core region was identified via luciferase assays. Potential transcription factors were predicted using the UCSC ChIP-seq database and JASPAR. NFKB1 binding to the CD200R1 core promoter was substantiated through electrophoretic mobility shift and chromatin immunoprecipitation assays. Knocking-down or overexpressing NFKB1 validated its regulatory effect on CD200R1. Correlation between decreased CD200R1 and deficient NFKB1 was studied using Genotype-Tissue Expression database. The clinical samples of the peripheral blood mononuclear cells were acquired from 44 PD patients (mean age 64.13 ± 9.78, 43.2% male, median Hoehn-Yahr stage 1.77) and 45 controls (mean age 64.70 ± 9.41, 52.1% male). NFKB1 knockout mice were utilized to study the impact of NFKB1 on CD200R1 expression and to assess their roles in PD pathophysiology. Results The study identified the CD200R1 core promoter region, located 482 to 146 bp upstream of its translation initiation site, was directly regulated by NFKB1. Significant correlation between NFKB1 and CD200R1 expression was observed in human PMBCs. Both NFKB1 and CD200R1 were significantly decreased in PD patient samples. Furthermore, NFKB1-/- mice exhibited exacerbated microglia activation and dopaminergic neuron loss after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Conclusion Our study identified that NFKB1 served as a direct regulator of CD200R1. Reduced NFKB1 played a critical role in CD200R1 dysregulation and subsequent microglia overactivation in PD. These findings provide evidence that targeting the NFKB1-CD200R1 axis would be a novel therapeutic strategy for PD.

Abstract Background The dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes. Methods Based on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses. Results The use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35–75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies. Conclusions A DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established. Graphical Abstract

Abstract Maltotetraose amylase (Mta) catalyzes the hydrolysis of amylaceous polysaccharides into maltotetraose, which is an important functional sugar used in the food industry. However, the lack of efficient expression systems for recombinant Mta has hindered its scale-up production and application. In this study, a codon-optimized mta gene from Pseudomonas saccharophila was efficiently produced in Bacillus subtilis by optimizing the regulatory elements. First, a plasmid library containing 173 different signal peptide sequences placed upstream of mta gene was constructed, and transformed into B. subtilis strain WB800N(amyEΔ1) for high-throughput screening. The signal peptide yhcR was found to significantly enhance the secretion of Mta, reaching an activity of 75.4 U/mL in the culture medium. After optimization of the promoters, the Mta activity was further increased to 100.3 U/mL using a dual-promoter PHpaIIPamyE. Finally, the carbon sources and nitrogen sources for recombinant Mta production were optimized, yielding a highest Mta activity of 288.9 U/mL under the optimal culture conditions. The crude enzyme solution containing recombinant Mta produced a highest maltotetraose yield of 70.3% with 200 g/L of maltodextrin as the substrate. Therefore, the present study have demonstrated a high yield of Mta produced in B. subtilis, laying the foundation for large-scale Mta production and application.

Abstract Background Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers. Results In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter. Conclusion Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts. Graphical Abstract

Introduction: Human genome consists of the three billion base pairs that has about one percent of genetic variation from one person to another، which determines physical، psychological، and susceptibility to diseases. Among the types of genetic diversity, single nucleotide polymorphisms are one of the most important genetic differences between two people. Single nucleotide polymorphism variation is located in the promoter region, exons، introns، untranslated regions and other Deoxyribonucleic acid (DNA) regions. While variation in the exon region can change susceptibility to diseases depending on whether it changes the protein structure or affects translation kinetics. Diversity in the promoter region can affect the interaction of genetic and epigenetic elements. Also، variation in the promoter region can affect the DNA methylation status. Polymorphic variation in the intron region can affect Messenger Ribonucleic acid splicing and the function of cis-regulatory elements. Polymorphic variation in the 5' Untranslated region، region causes a change in translation efficiency,، while a change in the 3' Untranslated region binds micro Ribonucleic acids to their position then affects the effects. In some cases، variations in Transfer Ribonucleic acid (tRNA) and Ribosomal ribonucleic acid (rRNA) affect the function of these regulatory cis elements. Conclusion: From a clinical point of view, a deep knowledge of this type of genetic variation can help the treatment process, manage patients and understand the prognosis based on these SNPs. Private or personalized medicine is also fundamentally based on genetic diversity. In this article, it was reviewed the types of single nucleotide genetic variation and presented examples of types of cancer, neurological and immunological diseases.

DNA guanine (G)-quadruplexes (G4s) are unique secondary structures formed by two or more stacked G-tetrads in G-rich DNA sequences. These structures have been found to play a crucial role in highly transcribed genes, especially in cancer-related oncogenes, making them attractive targets for cancer therapeutics. Significantly, targeting oncogene promoter G4 structures has emerged as a promising strategy to address the challenge of undruggable and drug-resistant proteins, such as MYC, BCL2, KRAS, and EGFR. Natural products have long been an important source of drug discovery, particularly in the fields of cancer and infectious diseases. Noteworthy progress has recently been made in the discovery of naturally occurring DNA G4-targeting drugs. Numerous DNA G4s, such as MYC-G4, BCL2-G4, KRAS-G4, PDGFR-β-G4, VEGF-G4, and telomeric-G4, have been identified as potential targets of natural products, including berberine, telomestatin, quindoline, sanguinarine, isaindigotone, and many others. Herein, we summarize and evaluate recent advancements in natural and nature-derived DNA G4 binders, focusing on understanding the structural recognition of DNA G4s by small molecules derived from nature. We also discuss the challenges and opportunities associated with developing drugs that target DNA G4s.

Abstract High Mobility Group A2 (HMGA2) oncofetal proteins are a distinct category of Transcription Factors (TFs) known as “architectural factors” due to their lack of direct transcriptional activity. Instead, they modulate the three-dimensional structure of chromatin by binding to AT-rich regions in the minor grooves of DNA through their AT-hooks. This binding allows HMGA2 to interact with other proteins and different regions of DNA, thereby regulating the expression of numerous genes involved in carcinogenesis. Consequently, multiple mechanisms exist to finely control HMGA2 protein expression at various transcriptional levels, ensuring precise concentration adjustments to maintain cellular homeostasis. During embryonic development, HMGA2 protein is highly expressed but becomes absent in adult tissues. However, recent studies have revealed its re-elevation in various cancer types. Extensive research has demonstrated the involvement of HMGA2 protein in carcinogenesis at multiple levels. It intervenes in crucial processes such as cell cycle regulation, apoptosis, angiogenesis, epithelial-to-mesenchymal transition, cancer cell stemness, and DNA damage repair mechanisms, ultimately promoting cancer cell survival. This comprehensive review provides insights into the HMGA2 protein, spanning from the genetic regulation to functional protein behavior. It highlights the significant mechanisms governing HMGA2 gene expression and elucidates the molecular roles of HMGA2 in the carcinogenesis process. Graphical Abstract

Abstract Background White lupin (Lupinus albus L.) is a high-protein Old World grain legume with remarkable food and feed production interest. It is sown in autumn or early spring, depending on the local agroclimatic conditions. This study aimed to identify allelic variants associated with vernalization responsiveness, in order to improve our knowledge of legume flowering regulatory pathways and develop molecular selection tools for the desired phenology as required for current breeding and adaptation to the changing climate. Results Some 120 white lupin accessions originating from a wide range of environments of Europe, Africa, and Asia were phenotyped under field conditions in three environments with different intensities of vernalization, namely, a Mediterranean and a subcontinental climate sites of Italy under autumn sowing, and a suboceanic climate site of France under spring sowing. Two hundred sixty-two individual genotypes extracted from them were phenotyped in a greenhouse under long-day photoperiod without vernalization. Phenology data, and marker data generated by Diversity Arrays Technology sequencing (DArT-seq) and by PCR-based screening targeting published quantitative trait loci (QTLs) from linkage map and newly identified insertion/deletion polymorphisms in the promoter region of the FLOWERING LOCUS T homolog, LalbFTc1 gene (Lalb_Chr14g0364281), were subjected to a genome-wide association study (GWAS). Population structure followed differences in phenology and isolation by distance pattern. The GWAS highlighted numerous loci significantly associated with flowering time, including four LalbFTc1 gene promoter deletions: 2388 bp and 2126 bp deletions at the 5’ end, a 264 bp deletion in the middle and a 28 bp deletion at the 3’ end of the promoter. Besides LalbFTc1 deletions, this set contained DArT-seq markers that matched previously published major QTLs in chromosomes Lalb_Chr02, Lalb_Chr13 and Lalb_Chr16, and newly discovered QTLs in other chromosomes. Conclusions This study highlighted novel QTLs for flowering time and validated those already published, thereby providing novel evidence on the convergence of FTc1 gene functional evolution into the vernalization pathway in Old World lupin species. Moreover, this research provided the set of loci specific for extreme phenotypes (the earliest or the latest) awaiting further implementation in marker-assisted selection for spring- or winter sowing.

Abstract Background Identifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agent Phytophthora infestans, which is a tractable model for studying life-stage differentiation within the group. Results Mining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestans life cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families within P. infestans often discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genes in vivo. Conclusions We established a large dataset of binding specificities for P. infestans TFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms.

5-Hydroxytryptophan (5-HTP), a precursor of the neurotransmitter serotonin in mammals, has demonstrated efficacy in treating various diseases such as depression, fibromyalgia and obesity. However, conventional biosynthesis methods of 5-HTP are limited by low yield and high reagent and process costs. In this study, the strain C1T7-S337A/F318Y with optimized promoter distribution was obtained, and the 5-HTP yield was 60.30 % higher than that of the initial strain. An efficient fermentation process for 5-HTP synthesis was developed using strain C1T7-S337A/F318Y with whey powder as a substrate for cell growth and inducer production. Shake flask fermentation experiments yielded 1.302 g/L 5-HTP from 2.0 g/L L-tryptophan (L-Trp), surpassing the whole-cell biocatalysis by 42.86 %. Scale-up to a 5 L fermenter further increased the yield to 1.649 g/L. This fermentation strategy substantially slashed reagent cost by 95.39 %, providing a more economically viable and environmentally sustainable route for industrial biosynthesis of 5-HTP. Moreover, it contributes to the broader utilization of whey powder in various industries. [Display omitted] • An efficient 5-HTP fermentation process was realized by condition optimization. • Whey powder was used as a substrate for cell growth and inducer production. • The strain C1T7 increased the 5-HTP yield by 60.30 % through promoter adjustment. • The 5-HTP yield of the new fermentation process was increased by 42.86 %. • The total reagent cost of the new fermentation process was reduced by 95.39 %. [ABSTRACT FROM AUTHOR]

AbstractPurposeMethodsResultsConclusionThe purpose of the present study was to construct a substantial theory that can systematically deal with barriers, promotors, and adaptive actions when older adults with chronic conditions move in a community.The present study is qualitative research based on grounded theory methodology. We recruited participants in 2 day-service facilities and one nursing station, and selected participants according to principle of theoretical sampling.We included sixteen participants. We have constructed a theory including eight categories. The core category of the theory was “the set of primary indicators in community mobility” This core category is related to difficulty, comfortability, and safety when persons move in a community, and consists of four elements: difficulty of general actions, degree of psychological burden, degree of accumulated fatigue, and probability of accidents.The theory integrated around the core category could provide a way of thinking for clinical practice. [ABSTRACT FROM AUTHOR]

Background: Overactivated microglia are a key contributor to Parkinson's disease (PD) by inducing neuroinflammation. CD200R1, a membrane glycoprotein mainly found on microglia, is crucial for maintaining quiescence with its dysregulation linked to microglia's abnormal activation. We and other groups have reported a decline in CD200R1 levels in several neurological disorders including PD. However, the mechanism regulating CD200R1 expression and the specific reasons for its reduction in PD remain largely unexplored. Given the pivotal role of transcription factors in gene expression, this study aimed to elucidate the transcriptional regulation of CD200R1 and its implications in PD. Methods: The CD200R1 promoter core region was identified via luciferase assays. Potential transcription factors were predicted using the UCSC ChIP-seq database and JASPAR. NFKB1 binding to the CD200R1 core promoter was substantiated through electrophoretic mobility shift and chromatin immunoprecipitation assays. Knocking-down or overexpressing NFKB1 validated its regulatory effect on CD200R1. Correlation between decreased CD200R1 and deficient NFKB1 was studied using Genotype-Tissue Expression database. The clinical samples of the peripheral blood mononuclear cells were acquired from 44 PD patients (mean age 64.13 ± 9.78, 43.2% male, median Hoehn-Yahr stage 1.77) and 45 controls (mean age 64.70 ± 9.41, 52.1% male). NFKB1 knockout mice were utilized to study the impact of NFKB1 on CD200R1 expression and to assess their roles in PD pathophysiology. Results: The study identified the CD200R1 core promoter region, located 482 to 146 bp upstream of its translation initiation site, was directly regulated by NFKB1. Significant correlation between NFKB1 and CD200R1 expression was observed in human PMBCs. Both NFKB1 and CD200R1 were significantly decreased in PD patient samples. Furthermore, NFKB1-/- mice exhibited exacerbated microglia activation and dopaminergic neuron loss after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Conclusion: Our study identified that NFKB1 served as a direct regulator of CD200R1. Reduced NFKB1 played a critical role in CD200R1 dysregulation and subsequent microglia overactivation in PD. These findings provide evidence that targeting the NFKB1-CD200R1 axis would be a novel therapeutic strategy for PD. [ABSTRACT FROM AUTHOR]

Background Genome-wide association studies have identified several nonfunctional tag single-nucleotide polymorphisms (SNPs) associated with severe malaria. We hypothesized that causal SNPs could play a significant role in severe malaria by altering promoter or enhancer activity. Here, we sought to identify such regulatory SNPs. Methods SNPs in linkage disequilibrium with tagSNPs associated with severe malaria were identified and were further annotated using FUMA. Then, SNPs were prioritized using the integrative weighted scoring method to identify regulatory ones. Gene reporter assays were performed to assess the regulatory effect of a region containing candidates. The association between SNPs and severe malaria was assessed using logistic regression models in a Senegalese cohort. Results Among 418 SNPs, the best candidates were rs116525449 and rs79644959, which were in full disequilibrium between them, and located within the ARL14 promoter. Our gene reporter assay results revealed that the region containing the SNPs exhibited cell-specific promoter or enhancer activity, while the SNPs influenced promoter activity. We detected an association between severe malaria and those 2 SNPs using the overdominance model and we replicated the association of severe malaria with the tagSNP rs116423146. Conclusions We suggest that these SNPs regulate ARL14 expression in immune cells and the presentation of antigens to T lymphocytes, thus influencing severe malaria development. [ABSTRACT FROM AUTHOR]

Tumour necrosis factor (TNF) superfamily member 11 (TNFSF11), also known as RANKL, plays a crucial role in regulating several physiological and pathological activities. Additionally, it is a vital factor in bone physiology, and the sex hormone progesterone regulates the expansion of stem cells and the proliferation of mammary epithelial cells. It is essential for animal growth and reproductive physiological processes. This study aimed to evaluate the tissue-specific expression characteristics and promoter activity of the TNFSF11 gene in pigs. As a result, the study examined the presence of TNFSF11 expression in the tissues of Xiangsu pigs at 0.6 and 12 months of age. Moreover, the core promoter region of TNFSF11 was also identified by utilizing a combination of bioinformatic prediction and dual-luciferase activity tests. Finally, the effect of transcription factors on the transcriptional activity of the core promoter region was determined using site-directed mutagenesis. TNFSF11 was uniformly expressed in all tissues; however, its expression in muscles was comparatively low. The core promoter region of TNFSF11 was located in the −555 to −1 region. The prediction of the transcription start site of TNFSF11 gene-2000 ∼ + 500bp showed that there was a CpG site in 17 ∼ + 487bp. Analysis of mutations in the transcription factor binding sites revealed that mutations in the Stat5b , Myog , Trl , and EN1 binding sites had significant effects on the transcriptional activity of the TNFSF11 gene, particularly following the EN1 binding site mutation (P < 0.001). This study provides insights into both the tissue-specific expression patterns of TNFSF11 in the tissues of Xiangsu pigs and the potential regulatory effects of transcription factors on its promoter activity. These results may be helpful for future research aimed at clarifying the expression and role of the porcine TNFSF11 gene. [ABSTRACT FROM AUTHOR]

Background and purpose: Women are considered more vulnerable than men due to various biological, genetic, and physical factors, including conditions such as pregnancy and breastfeeding. Additionally, societal structures contribute to this vulnerability. Factors such as the lack of social justice, gender biases, unemployment, unequal job opportunities, insecurity, limited access to education, cultural problems, traditional gender roles, and a lack of recognition for women's capacity and potential all exacerbate the vulnerability of women. Over recent decades, the population of vulnerable women has increased significantly. On the other hand, health-promoting behaviors are major determinants of public health. Therefore, this study was designed and conducted to assess health-enhancing lifestyles among women who visited the Behavioral Diseases Counseling Center in Sari city, as well as the related demographic and social factors. Materials and methods: This descriptive-analytical cross-sectional study included 174 vulnerable women who visited the Behavioral Diseases Counseling Center in Sari city. Participants were selected using a convenience sampling method. Data were collected using a questionnaire that measured sociodemographic factors and health-promoting lifestyles. The questionnaire comprised 52 items in six areas: nutrition (9 questions), physical activity (8 questions), health responsibility (9 questions), stress management (8 questions), interpersonal relationships (9 questions), and spiritual growth (9 questions). Data were analyzed using SPSS software (version 20). Descriptive statistics, such as frequency, mean, median, and standard deviation, were used to summarize demographic characteristics. For inferential analysis, the normality of variables was assessed using the Shapiro-Wilk test. Independent t-tests and one-way analysis of variance were used to compare health-promoting lifestyle scores across two-level and multi-level variables, respectively. Spearman's correlation coefficient and linear regression were also employed for further analysis. Results: The mean and standard deviation (SD) scores for the components of the health-promoting lifestyle questionnaire were as follows: self-actualization (mean ± SD: 28.12±6.77), health responsibility (31.90±6.18), interpersonal support (19.25±4.65), stress management (11.13±3.35), physical activity (14.83±5.62), and nutrition (19.81±4.96). The highest and lowest scores were observed in the components of health responsibility and stress management, respectively. Overall, the mean score for health-promoting lifestyles was 127.02±23.23, with 43.3% of participants having a favorable health-promoting lifestyle. Logistic regression analysis revealed that socio-demographic variables such as age, education, occupation, monthly income sufficiency, and history or current infection with sexually transmitted diseases were significantly associated with the desirability of health-promoting lifestyles (P=0.001). The coefficient of determination (R²=0.34) indicated that these variables accounted for 34% of the variance in health-promoting lifestyle scores. Conclusion: Given the importance of health-promoting behaviors in improving public health, particularly among women, it is essential to identify and enhance these behaviors through applied and interventional studies. Increasing awareness and addressing socio-demographic factors can play a crucial role in improving women's health. [ABSTRACT FROM AUTHOR]

Chrysanthemum is the most popular ornamental plant, after roses and lilies. The cauliflower mosaic virus (CaMV) promoter, which remains the most widely used promoter in dicotyledons, is a very strong promoter with sufficient effects in most crops. However, weak expression has often been reported in Chrysanthemum. Therefore, we searched for constitutive promoters available in Chrysanthemum. Based on the transcriptome analysis data of Chrysanthemum, nine constitutively expressed genes were selected, and each promoter region (1.0–3.0 kb) was isolated by genome walking. Only two of the nine promoters expressed GUS in tobacco and chrysanthemums. The major motif of the CmERF promoter (U41, 2060 bp) was related to the regulation of ethylene (ERELEE4) or gibberellin (PYRIMIDINEBOXOSRAMY1 and WRKY71OS). Similarly, the motif of the CmGA2 ox promoter (U47, 1060 bp) also contained gibberellin signaling factors, such as PRIMIDINEBOXHVEPB1 and WRKY71OS. Both promoters showed strong systemic expression in tobacco using GUS staining. Although weaker than in tobacco, significant expression was confirmed in the flowers and stems in chrysanthemum. The results of the GUS activity assay using chrysanthemum transformants showed that the transgenic line (#12) containing the U47 promoter had higher expression in all tissues than that containing the 35S-CaMV promoter. The U41 promoter was found to have a higher expression than the 35S-CaMV promoter in the stem. [ABSTRACT FROM AUTHOR]

Methanogens are the main biological producers of methane on Earth. Methanosarcina acetivorans is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter--RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the β-glucuronidase reporter gene in M. acetivorans cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter--RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter--RBS combinations. Our promoter--RBS library is effective in enabling the fine-tuning of gene expression in M. acetivorans for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds. [ABSTRACT FROM AUTHOR]

Local gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. To optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. AAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. A novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. The combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies. • A novel AAV capsid termed AAVDJK2 was developed and was superior to existing AAVs in terms of gene transduction efficiency and specificity for keratinocytes in vitro and in vivo. • A novel tissue-specific promotor termed K14 SCP3 promotor was developed and was superior to existing promoters in terms of gene transduction efficiency and specificity for keratinocytes in vitro and in vivo. • The novel AAV system benefits experimental research and development of new epidermis-targeted gene therapies. [ABSTRACT FROM AUTHOR]

Amylin promoter and transcriptional factors are well-established, inducible factors in the production of the main amyloidogenic pancreatic hormone, human islet amyloid peptide (hIAPP) or amylin. However, posttranscriptional mechanisms driving hIAPP expression in pancreas remain enigmatic, and hence were explored here. The translational assay revealed that both 5′ and 3′ untranslated regions (UTRs) of hIAPP restricted expression of the luciferase constructs only in constructs driven by the hIAPP promoter. Bioinformatics analysis revealed several putative seed sequences for a dozen micro RNAs (miRNAs) in hIAPP's 3′ UTR. miR-182, miR-335, and miR-495 were the most downregulated miRNAs in stressed human islets exposed to endoplasmic reticulum (ER) or metabolic stressors, thapsigargin (TG) or high glucose (HG). Correspondingly, miR-335 mimics alone or in combination with miR-495 and miR-182 mimics significantly and potently (>3-fold) reduced hIAPP protein expression in HG-treated cultured human islets. siRNA-mediated silencing of Ago2 but not Ago1 significantly stimulated hIAPP expression and secretion from transfected, HG-treated human islets. Conversely, ectopic expression of Ago2 in hIAPP-expressing RIN-m5F cell line driven by CMV promoter reduced hIAPP intracellular protein levels. Collectively, the results point to a novel and synergistic role for hIAPP promoter, 5/3′ UTRs and Ago-2/miR-335 complex in post-transcriptional regulation of hIAPP gene expression in normal and metabolically active β-cells. [ABSTRACT FROM AUTHOR]

Simple Summary: This research explores the role of the Cs-g2e3, a type of E3 ubiquitin ligase, in the gametogenesis of the Chinese Tongue Sole (Cynoglossus semilaevis), a marine fish known for its significant sexual dimorphism due to its unique chromosome system. E3 ubiquitin ligases are enzymes that help tag proteins for degradation or other regulatory functions and are involved in various biological processes, including gametogenesis. Our study focused on understanding how Cs-g2e3 influences gametogenesis—the process by which gametes or germ cells are produced. We found that Cs-g2e3 was highly expressed in the gonadal tissues of C. semilaevis, with its expression peaking at 8 months of age. By using techniques such as RNA interference, we discovered that the knockdown of Cs-g2e3 in ovarian and testicular germ cell lines significantly downregulated the expression of spermatogenesis-related and oogenesis-related genes. Additionally, we looked at how different transcription factors, which help turn genes on or off, affect the activity of the Cs-g2e3 promoter. Our findings highlight the crucial role of Cs-g2e3 in gametogenesis, suggesting its potential as a novel genetic tool that could significantly advance artificial reproduction technologies in aquaculture. Gametogenesis, the intricate developmental process responsible for the generation of germ cells (gametes), serves as a fundamental prerequisite for the perpetuation of the reproductive cycle across diverse organisms. The g2e3 enzyme is a putative ubiquitin E3 ligase implicated in the intricate regulatory mechanisms underlying cellular proliferation and division processes. The present study delves into the function of G2/M phase-specific E3 ubiquitin protein ligase (Cs-g2e3) in gametogenesis in Chinese Tongue Sole (Cynoglossus semilaevis). Sequence analysis shows that the Cs-g2e3 mRNA spans 6479 bp, encoding a 733 amino acid protein characterized by three conserved structural domains: PHD, RING, and HECT—typical of HECT E3 ubiquitin ligases. The predominant expression of Cs-g2e3 in the gonad tissues is further verified by qPCR. The expression profile of Cs-g2e3 in the gonads of the Chinese Tongue Sole is analyzed at different ages, and the results show that its expression peaks at 8 months of age and then begins to decline and stabilize. It is noteworthy that the expression level remains significantly elevated compared to that observed during the juvenile period. In situ hybridization shows that the mRNA of Cs-g2e3 is mainly localized in the germ cells of the ovary and the testis. RNA interference experiments show that the knockdown of Cs-g2e3 in ovarian and testicular germ cell lines significantly downregulates the expression of key genes involved in oogenesis (e.g., sox9 and cyp19a) and spermatogenesis (e.g., tesk1 and piwil2), respectively. Furthermore, the analysis of mutations in the transcription factor binding sites reveals that mutations within the Myogenin, YY1, and JunB binding sites significantly impact the transcriptional activity of the Cs-g2e3 gene, with the mutation in the YY1 binding site exhibiting the most pronounced effect (p < 0.001). This study contributes to a deeper understanding of the tissue-specific expression patterns of Cs-g2e3 across various tissues in Cynoglossus semilaevis, as well as the potential regulatory influences of transcription factors on its promoter activity. These findings may facilitate future research endeavors aimed at elucidating the expression and functional roles of the Cs-g2e3 gene. [ABSTRACT FROM AUTHOR]

The second messenger guanosine 3',5'‐bis(diphosphate)/guanosine tetraphosphate (ppGpp) and guanosine 3'‐diphosphate 5'‐triphosphate/guanosine pentaphosphate (pppGpp) ((p)ppGpp) has been shown to be crucial for the survival of mycobacteria under hostile conditions. Unexpectedly, deletion of primary (p)ppGpp synthetase‐Rel did not completely diminish (p)ppGpp levels leading to the discovery of novel bifunctional enzyme‐RelZ, which displayed guanosine 5'‐monophosphate,3'‐diphosphate (pGpp), ppGpp, and pppGpp ((pp)pGpp) synthesis and RNAseHII activity. What conditions does it express itself under, and does it work in concert with Rel? The regulation of its transcription and whether the Rel enzyme plays a role in such regulation remain unclear. In this article, we have studied relZ promoter and compared its activity with rel promoter in different growth conditions. We observed that the promoter activity of relZ was constitutive; it is weaker than rel promoter, lies within 200 bp upstream of translation‐start site, and it increased under carbon starvation. Furthermore, the promoter activity of relZ was compromised in the rel‐knockout strain in the stationary phase. Our study unveils the dynamic regulation of relZ promoter activity by SigA and SigB sigma factors in different growth phases in mycobacteria. Importantly, elucidating the regulatory network of RelZ would enable the development of the targeted interventions for treating mycobacterial infections. [ABSTRACT FROM AUTHOR]

Burkholderia is the second largest source of natural product bacteria after Actinomyces and can produce many secondary metabolites including pyrrolnitrin (PRN). Natural products of microbial origin are usually found in trace amounts, so in metabolic engineering, promoter engineering is often used to regulate gene expression to increase yield. In this study, an endogenous strong promoter was identified based on RNA-seq to overexpress biosynthetic genes to increase the production of PRN. By analyzing the transcriptomic data of the antagonistic bacterium Burkholderia sp. JP2-270 in three different development periods, we screened 50 endogenous promoters with high transcriptional activity, nine of which were verified by an obvious fluorescent signal via fluorescence observation. Then, combined with RT-qPCR analysis, Php, the promoter of a hypothetical protein, was found to be significantly expressed in all three periods. In order to increase the suitability of endogenous promoters, the promoter Php was shortened at different lengths, and the results show that a sequence length of 173 bp was necessary for its activity. Moreover, this promoter was used to overexpress the PRN biosynthesis genes (prnA, prnB, prnC and prnD) in JP2-270, resulting in a successful increase in gene expression levels by 40–80 times. Only the overexpression of the prnB gene successfully increased PRN production to 1.46 times that of the wild type. Overall, the endogenous strong promoters screened in this study can improve gene expression and increase the production of secondary metabolites in JP2-270 and other strains. [ABSTRACT FROM AUTHOR]

The expansin genes are commonly expressed in plant cells, and the encoded proteins influence plant growth and stress resistance by loosening the structure and increasing the flexibility of the cell wall. The objective of this study was to characterize expansin gene promoters in Populus trichocarpa to clarify the regulatory mechanisms underlying gene expression and evolution. Sequence alignments revealed that the similarity among 36 poplar expansin genes was greater for the coding sequences than for the promoter sequences, which suggested these promoter sequences evolved asynchronously. The bases flanking the start codon exhibited a usage bias, with sites +3, +4, and +5 biased toward GC, whereas the other sites were biased toward AT. The flanking sites were significantly correlated with gene expression, especially sites −10 and −17, in which C and G are the bases positively associated with gene expression. A total of 435 regulatory elements (61 types) were identified on the promoters of the poplar expansin genes; Skn-1 was the most common element in 23 promoters. Some expansin genes had more regulatory elements on their promoters (e.g., PtrEXPA4, PtrEXPA3, PtrEXPB3, and PtrEXPB1), whereas some others had less (e.g., PtrEXLA2, PtrEXLB1, and PtrEXPA23). Furthermore, 26 types of elements were involved in expansin gene expression, 25 of which positively affected expression in all analyzed samples. The exception was the endosperm expression-related element Skn-1, which negatively regulated expression in four tissues or treatments. Expression analysis showed that the expansin genes in Populus trichocarpa performed much differently under regular and abiotic stress conditions, which well matched the diversity of their promoter sequences. The results show that expansin genes play an important role in plant growth and development and stress resistance through expression adjustment. [ABSTRACT FROM AUTHOR]

Ascorbate peroxidases (APXs) are essential for plants as they act as hydrogen peroxide-scavenging enzymes, providing protection against oxidative damage. Using bioinformatic methods, five APX genes were discovered in the genome of Citrus sinensis in this study. APX genes of C. sinensis (CsAPXs) encode polypeptides between 250 and 436 residues in length, with molecular weights that range from 27.56 to 47.34 kDa. Additionally, the isoelectric point of CsAPXs varies from 5.64 to 8.63. The predicted locations of CsAPXs are peroxisome, chloroplast, and mitochondrion, with an uneven distribution across four chromosomes and eight orthologous gene pairs with Arabidopsis thaliana. A phylogenetic analysis revealed that the CsAPXs were divided into three clades. The CsAPXs all contained a conserved APX domain and six common motifs. Upon promoter analysis, it was found that CsAPXs could respond to abscisic acid, auxin, ethylene, gibberellin, methyl jasmonate, salicylic acid, and wounding stress treatments. In addition, the analysis of expression patterns revealed that the presence of Candidatus Liberibacter asiaticus (CLas) has a dynamic impact on the expression of CsAPXs, with CsAPX2 showing significant inhibition in response to CLas infection. These findings will provide novel insights for the forthcoming functional investigations of CsAPXs within the process of citrus-CLas interactions. [ABSTRACT FROM AUTHOR]

β-glucosidase is a key component of cellulase for its function in hydrolyzing cellobiose to glucose in the final step of cellulose degradation. The high-level expression of β-glucosidase is essential for cellulose conversion. Aspergillus niger ATCC 20611 has the potential for efficient protein expression because of its ability to secret enzymes for the industrial production of fructooligosaccharides, but it lacks robust promoters for high-level protein expression. Here, the development of A. niger 20611 as a powerful protein expression system exploited the conserved constitutive promoter Pgpd1 of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene from Trichoerma reesei to drive the expression of the enhanced green fluorescent protein in A. niger ATCC 20611. The mycelium of the transformant AGE9 exhibited intense fluorescence. Then, the promotor Pgpd1 was used to drive the expression of β-glucosidase and the enzyme activity of transformants AGB1 and AGB33 were 1.02 and 0.51 U/mL, respectively. These results demonstrate that the promotor Pgpd1 from T. reesei was applicable for A. niger ATCC 20611. Furthermore, the T. reesei-specific robust promoter Pcdna1 was used to drive the expression of β-glucosidase. The β-glucosidase exhibited a high-level expression with a yield of 15.2 U/mL, which was over 13.9 times higher than that driven by the promoter Pgpd1. The β-glucosidase was thermally stable and accounted for 85% of the total extracellular proteins. Subsequently, the fermentation broth including β-glucosidase was directly added to the cellulase mixture of T. reesei for saccharification of the acid-treated corncob residues and the delignified corncob residues, which increased the saccharification efficiency by 26.21% and 29.51%, respectively. Thus, β-glucosidase exhibited a high level of expression in A. niger ATCC 20611 and enhanced cellulose degradation by addition in vitro. In addition, the robust promoter Pcdna1 of T. reesei could drive the high-level expression of protein in A. niger ATCC 20611. These results demonstrate that the promoters in filamentous fungi could be employed across species in A. niger ATCC 20611 and further facilitated the efficient expression of β-glucosidase to optimize cellulases for efficient cellulose transformation. [ABSTRACT FROM AUTHOR]

Variability in expression levels in response to random genomic mutations varies among genes, influencing both the facilitation and constraint of phenotypic evolution in organisms. Despite its importance, both the underlying mechanisms and evolutionary origins of this variability remain largely unknown due to the mixed contributions of cis - and trans -acting elements. To address this issue, we focused on the mutational variability of cis -acting elements, that is, promoter regions, in Escherichia coli. Random mutations were introduced into the natural and synthetic promoters to generate mutant promoter libraries. By comparing the variance in promoter activity of these mutant libraries, we found no significant difference in mutational variability in promoter activity between promoter groups, suggesting the absence of a signature of natural selection for mutational robustness. In contrast, the promoters controlling essential genes exhibited a remarkable bias in mutational variability, with mutants displaying higher activities than the wild types being relatively rare compared to those with lower activities. Our evolutionary simulation on a rugged fitness landscape provided a rationale for this vulnerability. These findings suggest that past selection created nonuniform mutational variability in promoters biased toward lower activities of random mutants, which now constrains the future evolution of downstream essential genes toward higher expression levels. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Maltotetraose amylase (Mta) catalyzes the hydrolysis of amylaceous polysaccharides into maltotetraose, which is an important functional sugar used in the food industry. However, the lack of efficient expression systems for recombinant Mta has hindered its scale-up production and application. In this study, a codon-optimized mta gene from Pseudomonas saccharophila was efficiently produced in Bacillus subtilis by optimizing the regulatory elements. First, a plasmid library containing 173 different signal peptide sequences placed upstream of mta gene was constructed, and transformed into B. subtilis strain WB800N(amyEΔ1) for high-throughput screening. The signal peptide yhcR was found to significantly enhance the secretion of Mta, reaching an activity of 75.4 U/mL in the culture medium. After optimization of the promoters, the Mta activity was further increased to 100.3 U/mL using a dual-promoter PHpaIIPamyE. Finally, the carbon sources and nitrogen sources for recombinant Mta production were optimized, yielding a highest Mta activity of 288.9 U/mL under the optimal culture conditions. The crude enzyme solution containing recombinant Mta produced a highest maltotetraose yield of 70.3% with 200 g/L of maltodextrin as the substrate. Therefore, the present study have demonstrated a high yield of Mta produced in B. subtilis, laying the foundation for large-scale Mta production and application. [ABSTRACT FROM AUTHOR]

Background: The dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes. Methods: Based on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses. Results: The use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35–75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies. Conclusions: A DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established. [ABSTRACT FROM AUTHOR]

Background: Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers. Results: In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter. Conclusion: Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts. Highlights: The broad-spectrum promoters enable broad cross-species functionality. Five Kluyveromyces marxianus promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) can all express genes in Yarrowia lipolytica. Five Y. lipolytica promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) can all express genes in K. marxianus. The Kluyveromyces marxianus promoter km.TEF1 can strongly express RFP in bacteria E. coli and C. glutamicum. [ABSTRACT FROM AUTHOR]

CO 2 methanation over Ni-based catalysts provides a promising way to address the energy crisis and the environmental problems caused by massive CO 2 emissions. Nevertheless, Ni-based catalysts still face the challenges of poor activity at low temperatures. Herein, a series of MgO-promoted Ni-based catalysts supported by HNTs (halloysite nanotubes) were successfully prepared to investigate the role of MgO in CO 2 methanation. Various characterization results demonstrated that introducing MgO can enhance metal-support interaction, resulting in generating finer and more stable metal particles. Meanwhile, MgO can also offer sufficient alkaline sites and oxygen vacancies for CO 2 activation. NiMg1.0/Na-HNTs exhibited the maximum CO 2 conversion (79.0%) and CH 4 selectivity (97.5%) even at 275 °C as well as outstanding long-term stability over 100 h reaction. Additionally, in situ DRIFTS suggested that the mechanism for CO 2 methanation in this work mainly followed the formate pathway, and introducing magnesium can accelerate to convert intermediates. Briefly, this study provides an efficient low-temperature catalyst for CO 2 methanation with potential industrial applications. [Display omitted] • The metal-support interaction is strengthened through introducing MgO. • MgO-modified Ni-based catalysts possess more basic sites and oxygen vacancies. • NiMg1.0/Na-HNTs exhibits 79.0% CO 2 conversion even at 275 °C. • NiMg1.0/Na-HNTs shows outstanding long-term stability over 100 h reaction. • The formates are the main intermediates over NiMg1.0/Na-HNTs. [ABSTRACT FROM AUTHOR]

Oestrogen receptor alpha (ER; gene symbol ESR1) is the most important prognostic and treatment‐predictive biomarker in breast cancer. Drugs targeting oestrogen and ER for endocrine therapy of breast cancer include aromatase inhibitors, the selective ER modulator tamoxifen and the selective ER degrader fulvestrant. Tumours can develop resistance to endocrine therapy through several mechanisms, which is often linked to altered expression of ER. To investigate the role of promoter methylation in the regulation of ESR1 expression, we used bisulfite sequencing to measure methylation at CpG sites in alternative ER promoter regions for six cell line models of fulvestrant resistance. Both CpG methylation and expression of alternative first exons changed dynamically, with striking differences between cell lines that had stable or unstable resistance upon fulvestrant withdrawal. Methylation at some CpG sites was strongly negatively correlated with expression of specific first exons. In a breast tumour cohort, higher relative expression of upstream alternative first exons was associated with worse prognosis in post‐menopausal women with ER‐positive tumours who received endocrine therapy. [ABSTRACT FROM AUTHOR]

Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterization of genetic elements would make heterologous gene expression more predictable in this test bed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S × 2) provided the highest yield of proteins, although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia genes for ETHYLENE RESPONSE FACTOR 1 and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER protein drove expression to higher levels across all tissues without a growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed RUBY , a polycistronic betalain synthesis cassette linked by P2A sequences, to demonstrate coordinated expression of metabolic enzymes. A heat-shock-inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing tool kit for gene expression in Marchantia and provided new resources for the Marchantia research community. [ABSTRACT FROM AUTHOR]

The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG. Translation initiation regions (TIRs) of both genes of the operon (hol and endo) were further analyzed using chimeric constructs, in which parts of the hol/endo regulatory regions were fused with the gene of a reporter protein (EGFP). It was found that TIR of hol was 20 times less effective than that of endo. As it turned out, the level of EGFP production was influenced by the composition of the constructs and the type of the hol start codon. Apparently, the translational suppression of holin's accumulation and posttranslational activation of endolysin by Ca2+ are the main factors ensuring the proper timing of the host cell lysis by bacteriophage T5. The approach based on the use of chimeric constructs proposed in the paper can be recommended for studying other native or artificial operons of any complexity: analyzing the impacts of separate DNA regions, as well as their coupled effect, on the processes of transcription and translation of recombinant protein(s). [ABSTRACT FROM AUTHOR]

Bacillus licheniformis is recognised as an exceptional expression platform in biomanufacturing due to its ability to produce high-value products. Consequently, metabolic engineering of B. licheniformis is increasingly pursued to enhance its utility as a biomanufacturing vehicle. Effective B. licheniformis cell factories require promoters that enable regulated expression of target genes. This review discusses recent advancements in the characterisation, synthesis, and engineering of B. licheniformis promoters. We highlight the application of constitutive promoters, quorum sensing promoters, and inducible promoters in protein and chemical synthesis. Additionally, we summarise efforts to expand the promoter toolbox through hybrid promoter engineering, transcription factor-based inducible promoter engineering, and ribosome binding site (RBS) engineering. [ABSTRACT FROM AUTHOR]

Copyright of China Brewing is the property of China Brewing Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Callus formation induced by auxin accumulation is considered the first step of in vitro plant regeneration. In Arabidopsis, degradation of the Aux/IAA protein, IAA14, in response to auxin signaling, which activates the AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 along with a series of downstream transcription factors, also plays a critical role in this process. However, the specific mechanism by which auxin regulates callus formation remains unclear. By screening mutant library in the solitary root 1 (iaa14/slr) Arabidopsis background we obtained the callus formation related 2 (cfr2) mutant. The cfr2 mutant exhibited a stronger capacity for callus formation, as well as lateral root and adventitious root regeneration from leaf explants than wild type (WT) seedlings, but did not recover gravitropism capability. The auxin signal in cfr2 was significantly enhanced, and the expression of some downstream transcription factors was increased. Map-based cloning, whole genome resequencing, and phenotypic complementation experiments showed that the phenotypes observed in the cfr2 mutant were caused by a point mutation in the IAA14 promoter region. This mutation, which is predicted to disrupt the binding of LBD16, LBD19, and LBD30 to the IAA14 promoter, changed the expression pattern of IAA14 in cfr2. Taken together, our results identified a new mutation in the IAA14 promoter region, which affects the expression pattern of IAA14 and in turn its ability to control plant regeneration. [ABSTRACT FROM AUTHOR]

Background: White lupin (Lupinus albus L.) is a high-protein Old World grain legume with remarkable food and feed production interest. It is sown in autumn or early spring, depending on the local agroclimatic conditions. This study aimed to identify allelic variants associated with vernalization responsiveness, in order to improve our knowledge of legume flowering regulatory pathways and develop molecular selection tools for the desired phenology as required for current breeding and adaptation to the changing climate. Results: Some 120 white lupin accessions originating from a wide range of environments of Europe, Africa, and Asia were phenotyped under field conditions in three environments with different intensities of vernalization, namely, a Mediterranean and a subcontinental climate sites of Italy under autumn sowing, and a suboceanic climate site of France under spring sowing. Two hundred sixty-two individual genotypes extracted from them were phenotyped in a greenhouse under long-day photoperiod without vernalization. Phenology data, and marker data generated by Diversity Arrays Technology sequencing (DArT-seq) and by PCR-based screening targeting published quantitative trait loci (QTLs) from linkage map and newly identified insertion/deletion polymorphisms in the promoter region of the FLOWERING LOCUS T homolog, LalbFTc1 gene (Lalb_Chr14g0364281), were subjected to a genome-wide association study (GWAS). Population structure followed differences in phenology and isolation by distance pattern. The GWAS highlighted numerous loci significantly associated with flowering time, including four LalbFTc1 gene promoter deletions: 2388 bp and 2126 bp deletions at the 5' end, a 264 bp deletion in the middle and a 28 bp deletion at the 3' end of the promoter. Besides LalbFTc1 deletions, this set contained DArT-seq markers that matched previously published major QTLs in chromosomes Lalb_Chr02, Lalb_Chr13 and Lalb_Chr16, and newly discovered QTLs in other chromosomes. Conclusions: This study highlighted novel QTLs for flowering time and validated those already published, thereby providing novel evidence on the convergence of FTc1 gene functional evolution into the vernalization pathway in Old World lupin species. Moreover, this research provided the set of loci specific for extreme phenotypes (the earliest or the latest) awaiting further implementation in marker-assisted selection for spring- or winter sowing. [ABSTRACT FROM AUTHOR]

High Mobility Group A2 (HMGA2) oncofetal proteins are a distinct category of Transcription Factors (TFs) known as "architectural factors" due to their lack of direct transcriptional activity. Instead, they modulate the three-dimensional structure of chromatin by binding to AT-rich regions in the minor grooves of DNA through their AT-hooks. This binding allows HMGA2 to interact with other proteins and different regions of DNA, thereby regulating the expression of numerous genes involved in carcinogenesis. Consequently, multiple mechanisms exist to finely control HMGA2 protein expression at various transcriptional levels, ensuring precise concentration adjustments to maintain cellular homeostasis. During embryonic development, HMGA2 protein is highly expressed but becomes absent in adult tissues. However, recent studies have revealed its re-elevation in various cancer types. Extensive research has demonstrated the involvement of HMGA2 protein in carcinogenesis at multiple levels. It intervenes in crucial processes such as cell cycle regulation, apoptosis, angiogenesis, epithelial-to-mesenchymal transition, cancer cell stemness, and DNA damage repair mechanisms, ultimately promoting cancer cell survival. This comprehensive review provides insights into the HMGA2 protein, spanning from the genetic regulation to functional protein behavior. It highlights the significant mechanisms governing HMGA2 gene expression and elucidates the molecular roles of HMGA2 in the carcinogenesis process. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The AT-hook motif nuclear-localized (AHL) family is pivotal for the abiotic stress response in plants. However, the function of the cassava AHL genes has not been elucidated. Promoters, as important regulatory elements of gene expression, play a crucial role in stress resistance. In this study, the promoter of the cassava MeAHL31 gene was cloned. The MeAHL31 protein was localized to the cytoplasm and the nucleus. qRT-PCR analysis revealed that the MeAHL31 gene was expressed in almost all tissues tested, and the expression in tuber roots was 321.3 times higher than that in petioles. Promoter analysis showed that the MeAHL31 promoter contains drought, methyl jasmonate (MeJA), abscisic acid (ABA), and gibberellin (GA) cis-acting elements. Expression analysis indicated that the MeAHL31 gene is dramatically affected by treatments with salt, drought, MeJA, ABA, and GA3. Histochemical staining in the proMeAHL31-GUS transgenic Arabidopsis corroborated that the GUS staining was found in most tissues and organs, excluding seeds. Beta-glucuronidase (GUS) activity assays showed that the activities in the proMeAHL31-GUS transgenic Arabidopsis were enhanced by different concentrations of NaCl, mannitol (for simulating drought), and MeJA treatments. The integrated findings suggest that the MeAHL31 promoter responds to the abiotic stresses of salt and drought, and its activity is regulated by the MeJA hormone signal. [ABSTRACT FROM AUTHOR]

Recombinant collagen is a biopolymer with broad potential applications in various fields, such as biomedicine and tissue engineering. The triple-helix structure of collagen imparts unique biological functions and biocompatibilit but also increases the complexity of its expression in microbial systems. In this study, bacterial collagen V-B was used as a model protein to promote the expression of recombinant collagen in Corynebacterium glutamicum through optimization of expression elements. Firstly, the most efficient promoter tac-R0 was obtained through promoter screening and fermentation duration optimization. Subsequently, the RBS calculator was used to design a mutation library for ribosome binding sites (RBS) and aligned spacing (AS). The RBS and AS with the highest strength were identified, which increased the yield of V-B to 514 mg/L. Furthermore, through the tandem combination of multi-gene expression cassettes, the final yield of V-B increased by 8.4-fold compared to the initial level, reaching 697 mg/L. This study lays a foundation for the industrial production of recombinant collagen. [ABSTRACT FROM AUTHOR]

To date, clinical genetic testing for Mendelian disease variants has focused heavily on exonic coding and intronic gene regions. This multi-step study was undertaken to provide an evidence base for selecting and applying computational approaches for use in clinical classification of 5′ cis -regulatory region variants. Curated datasets of clinically reported disease-causing 5′ cis -regulatory region variants and variants from matched genomic regions in population controls were used to calibrate six bioinformatic tools as predictors of variant pathogenicity. Likelihood ratio estimates were aligned to code weights following ClinGen recommendations for application of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification scheme. Considering code assignment across all reference dataset variants, performance was best for CADD (81.2%) and REMM (81.5%). Optimized thresholds provided moderate evidence toward pathogenicity (CADD, REMM) and moderate (CADD) or supporting (REMM) evidence against pathogenicity. Both sensitivity and specificity of prediction were improved when further categorizing variants based on location in an EPDnew-defined promoter region. Combining predictions (CADD, REMM, and location in a promoter region) increased specificity at the expense of sensitivity. Importantly, the optimal CADD thresholds for assigning ACMG/AMP codes PP3 (≥10) and BP4 (≤8) were vastly different from recommendations for protein-coding variants (PP3 ≥25.3; BP4 ≤22.7); CADD <22.7 would incorrectly assign BP4 for >90% of reported disease-causing cis -regulatory region variants. Our results demonstrate the need to consider a tiered approach and tailored score thresholds to optimize bioinformatic impact prediction for clinical classification of 5′ cis -regulatory region variants. [Display omitted] Based on results of region-specific bioinformatic tool calibration, we report the conditions under which several impact prediction scores can be applied as computational evidence for variants in 5′ cis -regulatory regions. This work provides evidence-based methods for informing the classification of 5′ cis -regulatory region variants. [ABSTRACT FROM AUTHOR]

Background/aim: Aging, a multifaceted biological process, leads to diminished physical performance, especially in older adults with diabetes, where a mismatch between biological and chronological age is noticeable. Numerous studies have demonstrated that diabetes accelerates aging at the cellular and organ levels. Notable aging markers are telomerase reverse transcriptase (TERT), related to telomere length, and type 1 chain collagen (COL1A1), a key component of skin collagen. Additionally, age-related methylation increases, as revealed through methylation analysis, augmenting aspects of aging. However, the detailed interplay between aging and diabetes, particularly regarding methylation, remains underexplored and warrants further study to elucidate the biological links between the two. Materials and methods: In this study, we elucidate the modulatory influence of diabetes on the aging process, focusing specifically on the modifications in TERT in the kidney and COL1A1 in the skin using mice of Swiss Webster strain as the diabetes model. Specimens were categorized into three distinct chronological cohorts: chronologically young (16 weeks; n = 5), chronologically old (40 weeks; n = 5), and a periodically assessed group (16 weeks; n = 30), from which five mice were systematically sacrificed on a weekly basis. Results: Our findings reveal a marked impact of diabetes on the methylation statuses of TERT and COL1A1, characterized by an elevation in methylation levels within the periodic group (1st-6th week) and a simultaneous, progressive attenuation in the expression of TERT and COL1A1 genes. Conclusion: The observed alterations in the methylation levels of TERT and COL1A1 propound the hypothesis that diabetes potentially expedites the aging process, concomitantly impinging on the production of TERT and COL1A, ostensibly through the mechanism of promoter gene hypermethylation. [ABSTRACT FROM AUTHOR]

Genetic toolkit is now extended to disease vectors. For genetic control methods such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-based editing and gene drives, promoters with robust and tissue-specific expression are needed. The availability of genomes and machine-learning technologies provides an opportunity to identify promoters in non-model vectors. Vector-borne diseases (VBDs) impose devastating effects on human health and a heavy financial burden. Malaria, Lyme disease, and dengue fever are just a few examples of VBDs that cause severe illnesses. The current strategies to control VBDs consist mainly of environmental modification and chemical use, and to a small extent, genetic approaches. The genetic approaches, including transgenesis/genome modification and gene-drive technologies, provide the basis for developing new tools for VBD prevention by suppressing vector populations or reducing their capacity to transmit pathogens. The regulatory elements such as promoters are required for a robust sex-, tissue-, and stage-specific transgene expression. As discussed in this review, information on the regulatory elements is available for mosquito vectors but is scant for other vectors. [ABSTRACT FROM AUTHOR]

Dry reforming of methane (DRM) has garnered significant attention because it is featured by the conversion of the two most representative greenhouse gases CH4 and CO2 into syngas composed of H2 and CO as target products. In our previous studies, we demonstrated the high catalytic activity and stability in DRM for the nickel silicate BEA-type zeolite (Ni-BEA) catalyst synthesized by interzeolite transformation. Here, the molybdenum modified Mo/Ni-BEA was prepared by deposition precipitation method on Ni-BEA, and its DRM activity was evaluated. Compared to the bare Ni-BEA, the modified Mo/Ni-BEA showed the more enhanced activity and stability with low carbon deposition, e.g., 9% lower coke content after 12 h on stream under harsh DRM reaction conditions, because of the promoter effect of molybdenum. Furthermore, Mo/Ni-BEA exhibited the stronger stability, i.e., both the deactivation rates for CH4 and CO2 were less than half compared to those of Ni-BEA, in a long-term DRM test for 55 h. To investigate the role of molybdenum as a promoter, the catalysts before and after reactions were analyzed through various characterization methods. [ABSTRACT FROM AUTHOR]

The present study was designed to functionally characterize the promoters associated with AtMYB42, AtMYB85, and BjuMYB85. These genes are well known to be involved in lignin synthesis via phenylpropanoids, which are crucial for secondary cell wall development. We previously reported the complete absence of homologs of MYB42 from Brassica species. Inspite of their known role in secondary cell wall development, detailed knowledge about cis-element and transcriptional regulation of AtMYB42, AtMYB85 and BjMYB85 (BjuA013029) is lacking. It is therefore crucial investigating the transcriptional regulation of AtMYB42, AtMYB85, and BjMYB85 (BjuA013029), analyze functional and regulatory conservation and divergence and address whether BjMYB85 potentially compensates for the absence of MYB42 homologs in Brassica. In silico analysis revealed differences in the promoter sequences but shared transcription factor-binding sites and motifs, suggesting a common cis-regulatory pathway. Functional characterization using transcriptional fusion constructs revealed tissue-specific expression patterns not only in the stem, as has been reported earlier, but also in anther walls and siliques where lignin deposition plays an important role in dehiscence. Hormone and stress responsiveness of these promoters were assessed in seedlings. The AtMYB42 promoter displayed greater responsiveness to ethylene, cytokinin, and salicylic acid compared to AtMYB85 and BjuA013029MYB85. Expression was observed in various tissues, including seedlings, anthers, and silique and leaf midribs. This study provides novel insights into the expression patterns of these promoters, shedding light on their roles in non-stem tissues and contributing to our understanding of secondary cell wall formation. [ABSTRACT FROM AUTHOR]

The main components of sandalwood heartwood essential oil are terpenoids, approximately 80% of which are α-santalol and β-santalol. In the synthesis of the main secondary metabolites of sandalwood heartwood, the key gene, santalene synthase (SaSSY), can produce α-santalene and β-santalene by catalyzed (E, E)-FPP. Furthermore, santalene is catalyzed by the cytochrome monooxygenase SaCYP736A167 to form sandalwood essential oil, which then produces a fragrance. However, the upstream regulatory mechanism of the key gene santalene synthase remains unclear. In this study, SaSSY (Sal3G10690) promoter transcription factors and SaSSY cis-elements were screened. The results showed that the titer of the sandalwood cDNA library was 1.75 × 107 CFU/mL, 80% of the inserted fragments identified by PCR were over 750 bp in length, and the positivity rate of the library was greater than 90%. The promoter region of the SaSSY gene was shown to have the structural basis for potential regulatory factor binding. After sequencing and bioinformatics analysis, we successfully obtained 51 positive clones and identified four potential SaSSY transcriptional regulators. Sal6G03620 was annotated as the transcription factor MYB36-like, and Sal8G07920 was annotated as the small heat shock protein HSP20 in sandalwood. Sal1G00910 was annotated as a hypothetical protein of sandalwood. Sal4G10880 was annotated as a homeobox-leucine zipper protein (ATHB-15) in sandalwood. In this study, a cDNA library of sandalwood was successfully constructed using a yeast one-hybrid technique, and the transcription factors that might interact with SaSSY gene promoters were screened. This study provides a foundation for exploring the molecular regulatory mechanism involved in the formation of sandalwood heartwood. [ABSTRACT FROM AUTHOR]