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Background: Increasingly, patient satisfaction scores are being used to assess emergency physicians. We sought to determine whether the patient satisfaction scores collected by our hospital system are lower for patients who are treated in the emergency department (ED) on night shifts as compared to those treated on day shifts.Methods: We performed a cross-sectional analysis of patient satisfaction scores from three EDs in Florida. We obtained satisfaction data from NRC Health (the company that provides our surveys) using a random sample of 1,000 completed surveys from patients treated in 2022; we also performed manual chart review to obtain clinical data. The satisfaction surveys asked patients how likely they would be to recommend the facility (from 0–10). Patients who provided a score of 9 or 10 were considered “promoters.” For our primary analysis, we compared the percentage of promoters for the day shift encounters (7 AM to 7 PM) to the night shift encounters (7 PM to 7 AM). We also performed a multivariable logistic regression analysis using several demographic and clinical variables to further assess the association between night shift arrival and satisfaction scores.Results: Of the 1,000 surveys analyzed, 66.3% of patients arrived during the day shift, and 33.7%arrived during the night shift. Of those who arrived during the day shift, 525 (79.2%) were promoters compared to 228 (67.7%) of those who arrived during the night shift, a difference of 11.5% (95%confidence interval [CI] 5.7–17.4%), P < 0.001. On multivariable analysis, night shift arrival was associated with a lower chance of a patient being a promoter, with adjusted odds ratio 0.60 (95% CI 0.43–0.84), P = 0.003.Conclusion: Patients who presented to the ED during the night shift were less likely to be promoters than patients who arrived during the day shift. Assessments of patient satisfaction data should account for time of visit and other facility-related and operational characteristics.

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.

Precise spatio-temporal expression of genes in organogenesis is regulated by the coordinated interplay of DNA elements such as promoter and enhancers present in the regulatory region of a given locus. POU1F1 transcription factor plays a crucial role in the development of somatotrophs, lactotrophs and thyrotrophs in the anterior pituitary gland, and in maintaining high expression of growth hormone, prolactin and TSH. In mouse, expression of POU1F1 is controlled by a region fenced by two CTCF sites, containing 5 upstream enhancer elements, designated E-A (5 to 3). Elements C, B and A correspond to elements shown previously to play a role in pituitary development and hormonal expression; functional roles for elements E and D have not been reported. We performed comparative sequence analysis of this regulatory region and discovered that three elements, B, C and E, are present in all vertebrate groups except Agnatha. One very long (>2 kb) element (A) is unique to mammals suggesting a specific change in regulation of the gene in this group. Using DNA accessibility assay (ATAC-seq) we showed that conserved elements in anterior pituitary of four non-mammals are open, suggesting functionality as regulatory elements. We showed that, in many non-mammalian vertebrates, an additional upstream exon closely follows element E, leading to alternatively spliced transcripts. Here, element E functions as an alternative promoter, but in mammals this feature is lost, suggesting conversion of alternative promoter to enhancer. Our work shows that regulation of POU1F1 changed markedly during the course of vertebrate evolution, use of a low number of enhancer elements combined with alternative promoters in non-mammalian vertebrates being replaced by use of a unique combination of regulatory units in mammals. Most importantly, our work suggests that evolutionary conversion of alternate promoter to enhancer could be one of the evolutionary mechanisms of enhancer birth.

Plants shed organs such as leaves, petals, or fruits through the process of abscission. Monitoring cues such as age, resource availability, and biotic and abiotic stresses allow plants to abscise organs in a timely manner. How these signals are integrated into the molecular pathways that drive abscission is largely unknown. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene is one of the main drivers of floral organ abscission in Arabidopsis and is known to transcriptionally respond to most abscission-regulating cues. By interrogating the IDA promoter in silico and in vitro, we identified transcription factors that could potentially modulate IDA expression. We probed the importance of ERF- and WRKY-binding sites for IDA expression during floral organ abscission, with WRKYs being of special relevance to mediate IDA up-regulation in response to biotic stress in tissues destined for separation. We further characterized WRKY57 as a positive regulator of IDA and IDA-like gene expression in abscission zones. Our findings highlight the promise of promoter element-targeted approaches to modulate the responsiveness of the IDA signaling pathway to harness controlled abscission timing for improved crop productivity.

An understanding of the enzymatic and scaffolding functions of epigenetic modifiers is important for the development of epigenetic therapies for cancer. The H3K4me2/3 histone demethylase KDM5C has been shown to regulate transcription. The diverse roles of KDM5C are likely determined by its interacting partners, which are still largely unknown. In this study, we screen for KDM5C-binding proteins and show that YY1 interacts with KDM5C. A synergistic antitumor effect is exerted when both KDM5C and YY1 are depleted, and targeting YY1 appears to be a vulnerability in KDM5C-deficient cancer cells. Mechanistically, KDM5C promotes global YY1 chromatin recruitment, especially at promoters. Moreover, an intact KDM5C JmjC domain but not KDM5C histone demethylase activity is required for KDM5C-mediated YY1 chromatin binding. Transcriptional profiling reveals that dual inhibition of KDM5C and YY1 increases transcriptional repression of cell cycle- and apoptosis-related genes. In summary, our work demonstrates a synthetic lethal interaction between YY1 and KDM5C and suggests combination therapies for cancer treatments. Synopsis: This study shows that KDM5C binds to YY1, that it facilitates YY1 chromatin recruitment, and that targeting YY1 increases the vulnerability of KDM5C-low cancer cells. KDM5C promotes global YY1 chromatin recruitment, especially at promoters. An intact JmjC domain is required for KDM5C-mediated YY1 chromatin binding. Dual inhibition of KDM5C and YY1 results in transcriptional repression of cell cycle- and apoptosis-related genes and has a synergistic antitumor effect. This study shows that KDM5C binds to YY1, that it facilitates YY1 chromatin recruitment, and that targeting YY1 increases the vulnerability of KDM5C-low cancer cells. [ABSTRACT FROM AUTHOR]

ABSTRACT Many cellular processes and organismal behaviours are time‐dependent, and asynchrony of these phenomena can facilitate speciation through reinforcement mechanisms. The Mojave and Sonoran desert tortoises (Gopherus agassizii and G. morafkai respectively) reside in adjoining deserts with distinct seasonal rainfall patterns and they exhibit asynchronous winter brumation and reproductive behaviours. We used whole genome sequencing of 21 individuals from the two tortoise species and an outgroup to understand genes potentially underlying these characteristics. Genes within the most diverged 1% of the genome (FST ≥ 0.63) with putatively functional variation showed extensive divergence in regulatory elements, particularly promoter regions. Such genes related to UV nucleotide excision repair, mitonuclear and homeostasis functions. Genes mediating chronobiological (cell cycle, circadian and circannual) processes were also among the most highly diverged regions (e.g., XPA and ZFHX3). Putative promoter variants had significant enrichment of genes related to regulatory machinery (ARC‐Mediator complex), suggesting that transcriptional cascades driven by regulatory divergence may underlie the behavioural differences between these species, leading to asynchrony‐based prezygotic isolation. Further investigation revealed extensive expansion of respiratory and intestinal mucins (MUC5B and MUC5AC) within Gopherus, particularly G. morafkai. This expansion could be a xeric‐adaptation to water retention and/or contribute to differential Mycoplasma agassizii infection rates between the two species, as mucins help clear inhaled dust and bacterial. Overall, results highlight the diverse array of genetic changes underlying divergence, adaptation and reinforcement during speciation. [ABSTRACT FROM AUTHOR]

Juglans mandshurica, a notable woody oil tree species, possesses both fruit and timber value. However, the complete heterodichogamous flowering mechanism in this species remains elusive. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) is a crucial regulator of flower bud development in Arabidopsis thaliana. In this study, we cloned the coding DNA sequence (CDS) of the JmSOC1 gene, revealing a 705 base pair (bp) sequence that encodes a protein of 234 amino acids. The JmSOC1 protein contains a highly conserved MADS-box domain, indicating its role as a transcription factor, and is predominantly localized in the nucleus. The JmSOC1 gene expressed the highest in flower buds. The peak expression level of JmSOC1 during the physiological differentiation phase occurred earlier in male flower buds of protandry (MPD) on April 10th compared to female flower buds of protandry (FPD) on April 14th; similarly, the peak expression in female flower buds of protogyny (FPG) on April 2nd preceded that in male flower buds of protogyny (MPG) on April 14th. This may be the primary reason for the earlier differentiation of the male flowers in protandry individuals and the female flowers in protogyny individuals. Overexpression of JmSOC1 in wild-type A. thaliana resulted in earlier flowering, accompanied by an upregulation of key flowering-related genes such as LEAFY (LFY), APETALA1 (AP1), and FLOWERING LOCUS T (FT). To further explore the function of JmSOC1, a 782 bp promoter sequence of JmSOC1 gene was cloned, which has been verified to have promoter activity by GUS staining. Furthermore, the interaction between the JmSOC1 gene promoter and its upstream regulatory protein JmSVP was verified using a yeast one-hybrid. These results offer valuable insights into the molecular mechanisms underpinning the promotion of early flowering in J. mandshurica and hold promise for laying a theoretical foundation for the flowering regulation network of this species. [ABSTRACT FROM AUTHOR]

Primary open-angle glaucoma (POAG) is the most common form of glaucoma. This condition leads to optic nerve degeneration and eventually to blindness. Tobacco smoking, alcohol consumption, fast-food diets, obesity, heavy weight lifting, high-intensity physical exercises, and many other bad habits are lifestyle-related risk factors for POAG. By contrast, moderate-intensity aerobic exercise and the Mediterranean diet can alleviate POAG. In this work, we for the first time estimated the phylostratigraphic age indices (PAIs) of all 153 POAG-related human genes in the NCBI Gene Database. This allowed us to separate them into two groups: POAG-related genes that appeared before and after the phylum Chordata, that is, ophthalmologically speaking, before and after the camera-type eye evolved. Next, in the POAG-related genes' promoters, we in silico predicted all 3835 candidate SNP markers that significantly change the TATA-binding protein (TBP) affinity for these promoters and, through this molecular mechanism, the expression levels of these genes. Finally, we verified our results against five independent web services—PANTHER, DAVID, STRING, MetaScape, and GeneMANIA—as well as the ClinVar database. It was concluded that POAG is likely to be a symptom of the human self-domestication syndrome, a downside of being civilized. [ABSTRACT FROM AUTHOR]

SAYP and Bap170, subunits of the SWI/SNF remodeling complex, have the ability to support enhancer-dependent transcription when artificially recruited to the promoter on a transgene. We found that the phenomenon critically depends on two subunits of the Mediator kinase module, Med12 and Med13 but does not require the two other subunits of the module (Cdk8 and CycC) or other subunits of the core part of the complex. A cooperation of the above proteins in active transcription was also observed at endogenous loci, but the contribution of the subunits to the activity of a particular gene differed in different loci. The factors SAYP/Bap170 and Med12/Med13 did not form sufficiently stable interactions in the extract, and their cooperation was apparently local at regulatory elements, the presence of SAYP and Bap170 in a locus being necessary for stable recruitment of Med12 and Med13 to the locus. In addition to the above factors, the Nelf-A protein was found to participate in the process. The cooperation of the factors, independent of enzymatic activities of the complexes they are part of, appears to be a novel mechanism that maintains promoter activity and may be used in many loci of the genome. Extended intrinsically disordered regions of the factors were assumed to sustain the mechanism. [ABSTRACT FROM AUTHOR]

Monarda citriodora Cerv. ex Lag. is a rich source of industrially important compounds like γ-terpinene, carvacrol, thymol and thymoquinone. Understanding the regulation of γ-terpinene biosynthesis, a precursor for other monoterpenes, could facilitate upscaling of these metabolites in M. citriodora. Therefore, the present study aimed to unravel and characterize the terpene synthase (TPS) involved in γ-terpinene biosynthesis. Homology searches revealed 33 TPS members in the transcriptome assembly of M. citriodora. Based on the correlation of expression patterns and phytochemical profile, McTPS22 emerged as the putative TPS for γ-terpinene biosynthesis. Molecular docking suggested geranyl diphosphate (GPP) as a potential substrate for McTPS22. Heterologous expression in Escherichia coli and Nicotiana benthamiana confirmed the role of McTPS22 in γ-terpinene biosynthesis. Both in-silico prediction and confocal microscopy indicated plastidial localization of the McTPS22. Gene co-expression network analysis revealed 507 genes interacting with McTPS22, including 80 transcription factors (TFs). Of these, 46 TFs had binding sites in the McTPS22 promoter, and 36 showed significant correlations with γ-terpinene accumulation, suggesting they may be potential regulators. Promoter analysis indicated regulation by phytohormones and abiotic factors, confirmed by phytohormone elicitation and QRT-PCR. The histochemical GUS staining suggested that McTPS22 is primarily active in the glandular trichomes of M. citriodora. The present work provides insights into the molecular regulation of biosynthesis of γ-terpinene in M. citriodora. [ABSTRACT FROM AUTHOR]

Potassium hydroxide and potassium carbonate, being cost‐effective and environmentally friendly CO2 capture solvents, are promising candidates for carbon capture applications. Their slow absorption kinetics, however, necessitate strategies to enhance their rates, thereby reducing the capital costs of absorption equipment and saving energy for regenerating large volumes of solvent. Glycine, a potential additive, is explored for this purpose. While glycine‐based solvents are more stable than MEA, their amino functional group renders them susceptible to oxidative degradation. This study investigates the degradation of these solvents and the influence of potassium hydroxide and potassium carbonate on their stability. The experiment was performed under 100% O2 at 90 °C and 3 bar for about 3 weeks. It was observed that glycinate degraded by 53% for the glycinate‐only solution. The results also show that the addition of potassium hydroxide and potassium carbonate to a glycinate‐only solution had a mixed effect on the degradation of glycinate. Potassium hydroxide increased degradation by 5% compared to the glycinate‐only solution, while potassium carbonate decreased degradation by 4%. This order is supported by the degradation rate constants. Meanwhile, under N2, no significant change was observed in glycine concentration. Glycine's susceptibility to oxidative degradation is likely attributed to its less compact and rigid structure, resulting in weaker bonding and increased vulnerability to external factors. This instability leads to the formation of formate, carbonate, acetate, and oxalate as the primary degradation products across all studied solutions. A proposed mechanism for glycinate oxidative degradation sheds light on this process. These findings are crucial for informed decision making regarding performance trade‐offs in point source carbon capture and direct air capture, where oxygen is a prevalent gas component and potassium‐based solutions are commonly employed as absorbents. © 2024 Society of Chemical Industry and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Bread wheat is an important crop for the human diet, but the increasing soil salinization is reducing the yield. The Ca2+ signaling events at the early stages of the osmotic phase of salt stress are crucial for the acclimation response of the plants through the performance of calcium-sensing proteins, which activate or repress transcription factors (TFs) that affect the expression of downstream genes. Physiological, genetic mapping, and transcriptomics studies performed with the contrasting genotypes Syn86 (synthetic, salt-susceptible) and Zentos (elite cultivar, salt-tolerant) were integrated to gain a comprehensive understanding of the salt stress response. The MACE (Massive Analysis of cDNA 3ʹ-Ends) based transcriptome analysis until 4 h after stress exposure revealed among the salt-responsive genes, the over-representation of genes coding for calcium-binding proteins. The functional and structural diversity within this category was studied and linked with the expression levels during the osmotic phase in the contrasting genotypes. The non-EF-hand category from calcium-binding proteins was found to be enriched for the susceptibility response. On the other side, the tolerant genotype was characterized by a faster and higher up-regulation of genes coding for proteins with EF-hand domain, such as RBOHD orthologs, and TF members. This study suggests that the interplay of calcium-binding proteins, WRKY, and AP2/ERF TF families in signaling pathways at the start of the osmotic phase can affect the expression of downstream genes. The identification of SNPs in promoter sequences and 3ʹ -UTR regions provides insights into the molecular mechanisms controlling the differential expression of these genes through differential transcription factor binding affinity or altered mRNA stability.Key message: The fine-tuned expression of calcium-binding genes and transcription factors during the osmotic phase underlies the susceptibility and tolerance to salt stress responses of contrasting bread wheat genotypes. [ABSTRACT FROM AUTHOR]

The SWR1 chromatin remodeling complex is recruited to +1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the +1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes. [Display omitted] • Cryo-EM reveals a unique open conformation of the Swr1 ATPase adopted upon DNA binding • Swc2 subunit structurally organizes core SWR1 and orients it on the nucleosome • Swc3 subunit is essential for SWR1-promoter-specific recruitment and activity in cells • Flexible extended structure of N-SWR1 subcomplex allows readout of acetyl-histone marks This study elucidates the structural and molecular mechanisms by which the SWR1 chromatin remodeler is specifically recruited to accessible promoter DNA of active or poised genes and subsequently captures the +1 nucleosome for targeted histone H2A.Z exchange. [ABSTRACT FROM AUTHOR]

Background: The laccase Lcc9 from Coprinopsis cinerea has optimal catalytic activity at moderate to alkaline pH conditions, making it invaluable for industrial applications. However, C. cinerea naturally secretes Lcc9 at low expression levels, which limits the industrial application of Lcc9 on a large scale. Recombinant production of Lcc9 using Aspergillus niger would be an effective way to achieve its high production. Results: This study achieved the secretory production of Lcc9 in A. niger and established an efficient transformation procedure for A. niger by optimizing its protoplast preparation system. The transformation efficiency of A. niger was increased 3.8-fold under the optimal system (cell wall digestion enzyme solution: 2% cellulase, 1% snailase, 1% lyticase, and 0.5% lysozyme; incubation time: 3 h; incubation temperature: 37 ℃; culture time: 48 h). The extracellular yield of Lcc9 was enhanced by optimizing gene expression cassette and bioprocess. First, the strain AnGgcL (containing PgpdA) mediated by the SPCAT, a signal peptide of the extracellular high abundance protein catalase, had an extracellular laccase activity of 10 U/L after shake flask fermentation. Then, by optimizing promoter and signal peptide combinations that regulate lcc9 expression, the strain AnGcgL mediated by PcitA-SPGlaA had an extracellular laccase activity of 20 U/L. Subsequently, the strain AnRcgL1 (containing PcitA-SPGlaA) obtained by random integration had an extracellular laccase activity of 86 U/L. Sequencing revealed that the lcc9 expression cassette was integrated into the citrate synthase gene locus in the AnRcgL1 genome in a 9-copy form. By optimizing the microparticle, osmolyte, and Cu2+ in the fermentation medium, the AnRcgL1 extracellular laccase activity was further increased to 1566.7 U/L, which was 156.7-fold higher than that of AnGgcL. Furthermore, its extracellular laccase activity was increased to 1961 U/L in a 1-L fermenter. Conclusions: To our knowledge, this study is the first to report the recombinant extracellular production of the C. cinerea laccase Lcc9 in A. niger and to use SPCAT in the A. niger expression system. The results of this study will help accelerate the industrial application of Lcc9. Moreover, the strategy used in this work provides methodological guidance for increasing other exogenous protein yields in A. niger. [ABSTRACT FROM AUTHOR]

Gene therapy is a promising method for treating inherited diseases by directly delivering the correct genetic material into patient cells. However, the limited packaging capacity of vectors poses a challenge. Minimizing promoter size is a viable strategy among various approaches to address this issue. This study aims to optimize the bovine myostatin (MSTN) promoter, enhancing its utility in gene therapy applications. We identified the primary driver of activity as the proximal regulatory region. Isolated from the native promoter and termed M243, this 243-bp sequence was assessed for its potential as a ubiquitous promoter. In a variety of cell types, including bovine embryos and embryonic stem cells, the M243 promoter showed consistent expression, highlighting its suitability for applications requiring compact promoters. The isolation of a highly conserved, compact 243-bp sequence serving as a promoter suggests a solution for overcoming the size constraints of AAV vectors, suggesting potential contributions to the field of gene therapy. [ABSTRACT FROM AUTHOR]

Pathogenesis-related (PR) proteins, found in plants, play a crucial role in responding to both biotic and abiotic stresses and are categorized into 17 distinct families based on their properties and functions. We have conducted a phylogenetic analysis of OsPR1 genes (rice PR1 genes) in conjunction with 58 putative PR1 genes identified in Brachypodium distachyon , Hordeum vulgare , Brassica rapa , and Zea mays through BLASTP predictions. We extensively investigated the responses of the remaining 11 rice PR1 genes, using OsPR1a as a reference, under various stress conditions, including phytohormone treatments (salicylic acid and brassinosteroid [BR]), wounding, and heat stress (HS). In rice, of the 32 predicted OsPR1 genes, 12 have been well-characterized for their roles in disease resistance, while the functions of the remaining genes have not been studied extensively. In our study, we selected an additional 11 OsPR1 genes for further analysis and constructed a phylogenetic tree based on the presence of a 10-amino-acid-long conserved motif within these proteins. The phylogenetic analysis revealed that both OsPR1a from earlier studies and OsPR1-74 from our current study belong to the same clade. These genes consistently exhibit upregulation in response to diverse stress treatments such as biotic stress and abiotic stresses such as heat, drought, and salinity, indicating their potential roles in enhancing stress tolerance in rice. Significantly, this study delves into the previously unexplored role of OsPR1 genes in responding to Brassinosteroid (BR) and heat stress (HS) treatments, confirming their involvement in stress responses through qRT-PCR analysis. We found that seven genes were upregulated by EBR treatment. During heat stress (HS), six and seven genes were upregulated at 1hand 4h HS, respectively. The remaining genes OsPR1-22 and OsPR1-75 were upregulated at 1h but downregulated at 4h HS and under EBR treatment. In contrast, OsPR1-76 was upregulated at both 1h and 4h HS, but downregulated under EBR treatment. Promoters of PR1 genes in rice and other crops are rich in transcription factor binding sites (TFBSs) and feature a conserved Cysteine-rich secretory protein (SCP or CAP) motif. This study advances our understanding of PR1 gene regulation and its potential to enhance stress tolerance in rice. [ABSTRACT FROM AUTHOR]

Oxidative transformation of phenols is an efficient route towards the production of moieties for naturally occurring quinones with a wide range of biological activities. Silica supported Ru−Bi catalysts were prepared by microwave‐assisted loading (MW) and deposition (DP) methods. The as‐prepared catalysts were characterized by XRD (X‐ray diffraction), N2 physisoption, SEM (Scanning electron microscopy), TEM (Transmission electron microscopy) and XPS (X‐ray photoelectron spectrometry) techniques. Catalysts with low Bi loading, 5 %Ru‐1 %Bi, have narrow particle size distributions that correspond to an average particle size of 2.21–2.59 nm. 5 %Ru‐5 %Bi catalysts consist of Ru−Bi nanochains. The outcome of the catalytic oxidation of 2,3,5‐trimethylhydroquinone (TMHQ, 1) to 2,3,5‐trimethyl‐1,4‐benzoquinone (TMBQ, 2) depends on the catalyst preparation method, promoter loading, solvent and temperature used. MW‐5Ru1Bi is the most active catalyst with conversions that range between 99–100 % at both room temperature (r.t) and under refluxing MeNO2 and MeOH. The highest yields of TMBQ (100 %) were obtained over MW‐5 %Ru‐1 %Bi and DP‐5 %Ru‐5 %Bi catalysts. [ABSTRACT FROM AUTHOR]

Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin (A2M) is increased in DKD and is a critical regulator of the fibrotic response in glomerular mesangial cells (MC), an initial site of injury during DKD development. How A2M is regulated by high glucose (HG) has not yet been elucidated and is the focus of this investigation. Using serial deletions of the full A2M promoter, we identified the −405 bp region as HG-responsive in MC. Site-directed mutagenesis, siRNA, and ChIP studies showed that the transcription factor, nuclear factor of activated T cells 5 (NFAT5), regulated A2M promoter activity and protein expression in response to HG. Forkhead box P1 (FOXP1) served as a cooperative binding partner for NFAT5, required for A2M upregulation. Lastly, we showed that Smad3, known for its role in kidney fibrosis, regulated A2M promoter activity and protein production independently of HG. The importance of NFAT5, FOXP1, and Smad3 in A2M regulation was confirmed in ex vivo studies using isolated glomeruli. In conclusion, Smad3 is required for basal and HG-induced A2M expression, while NFAT5 and FOXP1 cooperatively regulate increased A2M transcription in response to HG. Inhibition of NFAT5/FOXP1 will be further evaluated as a potential therapeutic strategy to inhibit A2M production and attenuate profibrotic signaling in DKD. [ABSTRACT FROM AUTHOR]

Catheter-associated urinary tract infections (CAUTIs) are a significant burden on healthcare systems, accounting for up to 40% of hospital-acquired infections globally. A prevalent CAUTI pathogen, Proteus mirabilis, is an understudied Gram-negative bacterium. One sequela of P. mirabilis CAUTI is the production of urinary stones, which complicates treatment and clearing of the infection. Stone formation is induced by the activity of urease, a nickel-metalloenzyme that is regulated by UreR in a urea-dependent manner. As urea is abundant in the urinary tract, urease genes are highly expressed during experimental UTI. We sought to leverage the urease promoter to create an expression system that would enable urea-inducible expression of genes during in vitro experiments as well as during experimental UTI. During preliminary studies, we observed unexpectedly high levels of basal expression of the urease promoter. This was somewhat dependent on the presence of regulator UreR. To further develop this expression system, we generated a series of reporter constructs to assess the impact of specific promoter elements on promoter activity in the presence and absence of urea. Elements of interest included known regulatory binding sites, alternative translational start sites, and single-nucleotide polymorphisms identified through comparative genomics. This work describes a suite of urea-inducible promoters, constructed during this study, that exhibit a variety of expression dynamics, providing a customizable platform for gene expression. IMPORTANCE Urea is an inexpensive molecule that can easily be supplied during in vitro experiments. A urea-inducible promoter would also be activated by environments where urea naturally occurs, such as in the urinary tract. Thus, the development of a urea-inducible system for selective gene expression is of great interest to the field of uropathogenesis as it would enable selective gene induction during experimental urinary tract infection. This expression system would also have important applications for recombinant protein production in biotech and manufacturing. [ABSTRACT FROM AUTHOR]

The Chinese gentian, Gentiana sino-ornata produces brilliant blue flowers. To investigate the biological function and transcriptional regulation mechanism of the anthocyanin 5-O-acyltransferase gene (Gs5AT) in the corolla, it is beneficial to analyse the mechanism of blue flower colour presentation. In this investigation, we obtained the CDS and promoter sequences of the gene Gs5AT. Yeast one-hybrid experiments were used to identify the transcription factor GsbHLH7 that activates the gene Gs5AT. According to quantitive reverse transcription polymerase chain reaction analysis, the expression of the gene Gs5AT was significantly and positively correlated with the gene GsbHLH7. The colour phenotype of the flowers was significantly altered by the virus-induced gene silencing transduction of Gs5AT and GsbHLH7 , with GsbHLH7 silencing producing more pronounced changes in the corolla colour than Gs5AT. The expression of GsF3′5′H , GsDFR , GsANS , Gs3GT , and Gs5GT all fell to varying degrees after GsbHLH7 silencing, indicating that GsbHLH7 may regulate transcription of these genes as well as Gs5AT. The results of this study indicate that Gs5AT was positively regulated by the GsbHLH7 , and thus affects the colour presentation of the blue corolla. The Chinese gentian, Gentiana sino-ornata , is a commercially important flower that produces brilliant blue blossoms. We identified the responsible gene, Gs5AT , and discovered that another gene, GsbHLH7 , can regulate the expression of the first gene. This study provides a further basis for molecular breeding of blue flowers. [ABSTRACT FROM AUTHOR]

The precise spatiotemporal expression of the hematopoietic ETS transcription factor PU.1, a key determinant of hematopoietic cell fates, is tightly regulated at the chromatin level. However, how chromatin signatures are linked to this dynamic expression pattern across different blood cell lineages remains uncharacterized. Here, we performed an in-depth analysis of the relationships between gene expression, chromatin structure, 3D architecture, and trans-acting factors at PU.1 cis-regulatory elements (PCREs). By identifying phylogenetically conserved DNA elements within chromatin-accessible regions in primary human blood lineages, we discovered multiple novel candidate PCREs within the upstream region of the human PU.1 locus. A subset of these elements localizes within an 8-kb-wide cluster exhibiting enhancer features, including open chromatin, demethylated DNA, enriched enhancer histone marks, present enhancer RNAs, and PU.1 occupation, presumably mediating PU.1 autoregulation. Importantly, we revealed the presence of a common 35-kb-wide CTCF-flanked insulated neighborhood that contains the PCRE cluster (PCREC), forming a chromatin territory for lineage-specific and PCRE-mediated chromatin interactions. These include functional PCRE-promoter interactions in myeloid and B cells that are absent in erythroid and T cells. By correlating chromatin structure and 3D architecture with PU.1 expression in various lineages, we were able to attribute enhancer versus silencer functions to individual elements. Our findings provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in the chromatin regulation of PU.1 expression. This study lays crucial groundwork for additional experimental studies that validate and dissect the role of PCREs in epigenetic regulation of normal and malignant hematopoiesis. Main points: An unbiased, multiomics comparison between multiple blood cell types identified novel PU.1 cis regulatory elements with specific chromatin signatures. A 35-kb insulated neighborhood forms a territory for lineage-specific interactions involving an 8-kb PU.1 cis regulatory element cluster in 3D chromatin space. [ABSTRACT FROM AUTHOR]

Background: Morganella morganii is an emerging nosocomial opportunistic pathogen with increasing multidrug resistance. Antibiotic resistance, driven primarily by the horizontal transfer of resistance genes, has become a global health crisis. Integrons, mobile genetic elements, are now understood to facilitate the transfer of these genes, contributing to the rapid proliferation of resistant strains. Understanding the regulatory role of integrons in drug resistance gene expression is crucial for developing novel strategies to combat this pressing public health issue. Objective: To investigate the distribution of promoter types in the variable regions of class 1 integrons isolated from clinical isolates of M. morganii and their regulatory role in the expression of downstream drug resistance gene cassettes. Methods: Ninety seven clinical isolates of M. morganii were screened for the presence of class 1 integrons (intI1) using polymerase chain reaction (PCR). Gene cassettes within the variable regions of positive isolates were characterized, and the gene cassette promoter Pc variants and downstream auxiliary promoter P2 were identified. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was employed for homology analysis. Recombinant plasmids containing different variable region promoters and gene cassettes were constructed to evaluate drug resistance genes and integrase (intI1) expression levels using reverse transcription-quantitative PCR (RT-qPCR) and antimicrobial susceptibility testing. Results: Of the clinical isolates, 28.9% (n = 28/97) were positive for class 1 integrons. 24.7% (n = 24/97) of these isolates carried gene cassettes encoding resistance to aminoglycosides and trimethoprim. Three Pc promoter types (PcH1, PcS, and PcW) were identified, while all P2 promoters were inactive with a 14-base pair spacing between the -35 and -10 regions. ERIC-PCR analysis classified the integron-positive strains into 6 genotypes, with high consistency in promoter types and gene cassettes within each genotype. RT-qPCR and antimicrobial susceptibility testing demonstrated that strong promoters significantly enhanced the expression of downstream drug resistance gene cassettes compared to weak promoters. Additionally, RT-qPCR revealed a negative correlation between intI1 expression and Pc promoter strength. Conclusion: Class 1 integrons are prevalent in M. morganii. The promoter types within these integrons are diverse, and promoter strength is closely linked to downstream gene cassette expression. Integron-positive strains exhibit high homology, suggesting horizontal gene transfer and dissemination in clinical settings. [ABSTRACT FROM AUTHOR]

During marine oil–gas extraction, significant amounts of carbon dioxide (CO2) gas are often produced. Effectively separating these associated CO2 gases during extraction has become a critical technical challenge. Therefore, this paper aims to enhance the efficiency of CO2 hydrate-based capture technology and conduct relevant research. The goal is to increase the driving force for hydrate formation by combining the traditional thermodynamic additive TBAB with pressure modulation and to improve the hydrate formation rate through the use of multiple kinetic promoters. This paper presents the initial investigation into the effect of the thermodynamic accelerator tetrabutylammonium bromide (TBAB) on the characteristics of CO2 hydrate formation. The promotion effects of TBAB solutions with varying mass concentrations (3%, 5%, 7.5%, 10%) and reaction pressures (3 MPa, 4 MPa) were subjected to a systematic analysis, and the optimal conditions were identified as 4 MPa and a 5 wt% TBAB concentration. Subsequently, the impact of combining TBAB with kinetic promoters (SDS, nano Al2O3, L-methionine, L-leucine) on CO2 hydrate generation characteristics was further investigated. In this paper, the effect of a single promoter on the generation characteristics of CO2 hydrate was investigated, and the efficient carbon trapping ability of the complex promoter was verified, which provides theoretical support for the application of CO2 trapping technology using the hydrate method. [ABSTRACT FROM AUTHOR]

Aberrant DNA methylation plays a crucial role in breast cancer progression by regulating gene expression. However, the regulatory pattern of DNA methylation in long noncoding RNAs (lncRNAs) for breast cancer remains unclear. In this study, we integrated gene expression, DNA methylation, and clinical data from breast cancer patients included in The Cancer Genome Atlas (TCGA) database. We examined DNA methylation distribution across various lncRNA categories, revealing distinct methylation characteristics. Through genome-wide correlation analysis, we identified the CpG sites located in lncRNAs and the distally associated CpG sites of lncRNAs. Functional genome enrichment analysis, conducted through the integration of ENCODE ChIP-seq data, revealed that differentially methylated CpG sites (DMCs) in lncRNAs were mostly located in promoter regions, while distally associated DMCs primarily acted on enhancer regions. By integrating Hi-C data, we found that DMCs in enhancer and promoter regions were closely associated with the changes in three-dimensional chromatin structures by affecting the formation of enhancer–promoter loops. Furthermore, through Cox regression analysis and three machine learning models, we identified 11 key methylation-driven lncRNAs (DIO3OS, ELOVL2-AS1, MIAT, LINC00536, C9orf163, AC105398.1, LINC02178, MILIP, HID1-AS1, KCNH1-IT1, and TMEM220-AS1) that were associated with the survival of breast cancer patients and constructed a prognostic risk scoring model, which demonstrated strong prognostic performance. These findings enhance our understanding of DNA methylation's role in lncRNA regulation in breast cancer and provide potential biomarkers for diagnosis. [ABSTRACT FROM AUTHOR]

Background: Malignant cells exhibit reduced period circadian regulator 3 (PER3) expression. However, the underlying mechanisms of variations in PER3 expression in cancers and the specific function of PER3 in tumor progression remain poorly understood. Results: We explored multiple public databases, conducted bioinformatics analyses, and performed in vitro and in vivo experiments for validation. We found PER3 expression was decreased in most types of cancers, and PER3 downregulation was associated with a poor prognosis in 8 types of cancer. PER3 promoter methylation levels were increased in 11 types of cancer. Promoter hypermethylation (CpG islands [CGIs] cg12258811 and cg14204433) correlated with decreased PER3 expression in six cancers (breast invasive carcinoma, colon adenocarcinoma, head and neck squamous cell carcinoma, kidney renal papillary cell carcinoma [KIRP], lung adenocarcinoma [LUAD], and uterine corpus endometrial carcinoma). CGI cg12258811 hypermethylation was associated with reduced survival time and advanced cancer stages. Moreover, the bisulfite pyrosequencing assay confirmed CGI cg12258811 hypermethylation and its negative correlation with PER3 expression. In vitro and in vivo experiments demonstrated that PER3 inhibited KIRP and LUAD progression. Decitabine enhanced PER3 expression and inhibited KIRP cell functions by reducing promoter (cg12258811) methylation level. Conclusions: Our findings advanced the mechanistic understanding of variations in PER3 expression in cancers and confirmed the tumor-associated function of PER3 hypermethylation and downregulation. [ABSTRACT FROM AUTHOR]

Translation elongation factor P, expressed by the efp gene, is a conserved protein closely related to bacterial virulence and environmental stress regulation responses, however, little is known about the efp gene expression regulations. Here, the strain of Staphylococcus aureus subsp. aureus NCTC 8325 was taken as the research object and cultured under different conditions, including different culture temperatures, pH, and antibiotics, to study the expression of the efp gene in S. aureus by qRT-PCR, the results showed that the expression of the efp gene is upregulated under high temperature (40 °C), acidic (pH 5.4) or alkaline (pH 9.4) culture conditions, but upregulated early and downregulated later under the conditions of 0.5 MIC antibiotics (chloramphenicol at the final concentration of 2 μg/mL and vancomycin at the final concentration of 0.25 μg/mL), indicating that the efp promoter in S. aureus is inducible. The efp promoter sequence and structure in S. aureus were predicted by bioinformatics methods, and the predicted promoter was validated by constructing a promoter-probe vector and a series of promoter mutants, the results showed that the efp promoter sequence in S. aureus, named Pro, located in 1,548,179–1,548,250 of the S. aureus genome (NC_007795.1), and the sequence of − 10 element is CCTTATAGT, − 35 element is TTTACT. The results above could lay a foundation for screening transcription factors involved in the expression of the efp gene and then exploring the transcriptional regulation mechanism of EF-P in S. aureus. [ABSTRACT FROM AUTHOR]

The occurrence of extreme weather patterns induced by climate change has resulted in abiotic stress problems impacting the growth and productivity of plants. Rice (Oryza sativa), a staple food source for most Asians, is similarly affected by these challenges. Previous studies have identified the Oryza sativa Stress Associated Protein (OsSAP) genes to play a significant role in responding to abiotic stress. Among the 18 Stress Associated Protein members, OsSAP4 was highly expressed during drought and salinity conditions. Therefore, further experiments have been conducted, focusing specifically on the promoter region, to comprehend its regulation in response to abiotic stresses. Various types of cis-elements binding sites have been identified within the OsSAP4 promoter, encompassing MYB, CAMTA, CPP, C3H, HDZIP, bZIP, WRKY, and ERF. However, promoter analysis revealed that the distribution of the Cis-Regulatory elements bound by the Ethylene Response Factor (ERF) was the most prominent in the OsSAP4 promoter. Consequently, an analysis of promoter regulation was conducted using GUS reporter in Arabidopsis thaliana (A. thaliana) on two different sizes of OsSAP4 promoter sequences, each containing different quantities of ERF transcription factor binding sites. A noticeable difference in GUS staining activity was observed between pOsSAP4(1524 pb)::GUS and pOsSAP4(460 pb)::GUS, where pOsSAP4(1524 pb)::GUS exhibited higher GUS staining activity than pOsSAP4(460 pb)::GUS. The differences in GUS staining analysis are evident at the vegetative stage (leaf), silique, and inflorescence stages. This implies the participation of various other cis-element binding sites that influence the expression pattern of the OsSAP4 promoter during abiotic stress. [ABSTRACT FROM AUTHOR]

【Objective】This work is aimed at establishing a library of endogenous expression elements of Streptococcus epizooticus, and investigating the application of it in increasing hyaluronic acid yields.【Method】The 32 kinds of high, medium and low strength promoter and RBS combination (expression element, PR) were preliminarily screened, through transcriptomics analysis of S. zooepidemicus in the exponential or stationary stage of fermentation. Then, the strength of the 32 PRs was verified via detecting the transcriptional level of gfp gene and fluorescence intensity of GFP. Eventually, the key hyaluronic acid synthesis gene hasA, hasB, hasC, hasD, and hasE was overexpressed respectively with the selected strong expression element PR31, and the impact of the strong PR on enhancing hyaluronic acid production was assessed through a 2 L fermentation.【Result】The relative transcription of the aforementioned genes were elevated to 8.17, 7.32, 3.72, 39.48 and 9-fold. Among them, the overexpression of hasA and hasD increased the hyaluronic acid yield by 43% and 31%, respectively, compared to the wild type, reaching 5.654 g/L and 5.283 g/L. 【Conclusion】A library of endogenous expression elements of S. zooepidemicus had been constructed successfully, which can be employed for metabolic engineering, such as reinforcing the hyaluronic acid synthesis pathway or weaking competitive pathways. [ABSTRACT FROM AUTHOR]

Various reporter genes have been developed to study gene expression pattern and gene regulation. The RUBY reporter gene was recently developed and widely used, because of its visible and noninvasive advantages. However, quantitative analysis of RUBY gene expression levels was lacking. In this study, we introduce a novel betalain quantification method in combination with the tobacco transient expression system. The betalain produced in tobacco leaves was extracted and purified, and its concentration was quantitatively measured. We successfully applied this approach in studying the transcriptional regulation of ARC5 gene by transcription factors CPD25 and CPD45. Furthermore, with this method, we showed that the gene expression of RCA and Rbcs1A gene were regulated by light, transcription factors HY5 and PIFs through G‐box and I‐box elements. The development of this betalain quantification approach with the tobacco transient expression system offers a cost‐effective and intuitive strategy for studying the regulatory mechanism of gene expression. Summary statement: A quantitative method was developed to determine the betalain content produced by the RUBY reporter, which is convenient, accurate, and costless. This method is well‐suited for studying the regulation of environmental signals, such as light, on the expression of genes related to chloroplast function. [ABSTRACT FROM AUTHOR]

Background: The Jun proto-oncogene (JUN), also referred to as C-JUN, is an integral component of the JNK signaling pathway, which is crucial for the formation and differentiation of spermatogonial stem cells (SSCs). Investigations into the transcriptional regulation of chicken JUN can offer a molecular foundation for elucidating its mechanistic role in SSCs. Methods: In this study, we successfully cloned a 2000 bp upstream sequence of the JUN transcription start site and constructed a series of pGL3 recombinant vectors containing JUN promoters of varying lengths. Results: We verified the promoter activity of the 2000 bp upstream sequence by assessing the fluorescence intensity of DF-1 and identified the promoter activities of different regions via dual-luciferase assays. The transcription of JUN and its promoter region spanning −700 to 0 bp was modulated by an activator of the JNK signaling pathway. Bioinformatics analysis revealed that this −700 to 0 bp region was highly conserved among avian species and predicted the presence of binding sites for Wilms tumor 1 (WT1) and CCAAT/enhancer binding protein alpha (CEBPA). The JNK signaling pathway activator was found to upregulate the expression of these transcription factors in DF-1 cells. Through the deletion of binding sites and the overexpression of WT1 and CEBPA, we demonstrated that WT1 inhibited the transcription of JUN, while CEBPA promoted it. Conclusions: In conclusion, the −700 to 0 bp region is the key region of the JUN promoter, with WT1 inhibiting JUN transcription. The results of the study not only provide ideas for exploring the regulatory mechanism of JUN in chicken SSCs, but also lay an important foundation for the study of avian SSCs. [ABSTRACT FROM AUTHOR]

G6PC2 encodes a glucose-6-phosphatase catalytic subunit that opposes the action of glucokinase in pancreatic islets, thereby modulating the sensitivity of insulin and glucagon secretion to glucose. In mice, G6pc2 is expressed at ~20-fold higher levels in ß-cells than in a-cells, whereas in humans G6PC2 is expressed at only ~5-fold higher levels in ß-cells. We therefore hypothesize that G6PC2 likely influences glucagon secretion to a greater degree in humans. With a view to generating a humanized mouse that recapitulates augmented G6PC2 expression levels in a-cells, we sought to identify the genomic regions that confer differential mouse G6pc2 expression in a-cells versus ß-cells as well as the evolutionary changes that have altered this ratio in humans. Studies in islet-derived cell lines suggest that the elevated G6pc2 expression in mouse ß-cells versus a-cells is mainly due to a difference in the relative activity of the proximal G6pc2 promoter in these cell types. Similarly, the smaller difference in G6PC2 expression between a-cells and ß-cells in humans is potentially explained by a change in relative proximal G6PC2 promoter activity. However, we show that both glucocorticoid levels and multiple differences in the relative activity of eight transcriptional enhancers between mice and humans likely contribute to differential G6PC2 expression. Finally, we show that a mouse-specific non-coding RNA, Gm13613, whose expression is controlled by G6pc2 enhancer I, does not regulate G6pc2 expression, indicating that altered expression of Gm13613 in a humanized mouse that contains both the human promoter and enhancers should not affect G6PC2 function. [ABSTRACT FROM AUTHOR]

Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 × 10− 24 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D' = 1.0; R2 > 0.90) revealed marker rs2303751 (p value 8.12 × 10− 20) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding site for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2−/− knockout (KO) zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2−/− ko zebrafish Tg(wt1ß:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation. [ABSTRACT FROM AUTHOR]

Here, the Zn/titanosilicate-1 (TS-1) catalysts with different loadings and promotors were prepared to investigate catalytic performance in the dehydrogenation of propane. Derived from the alteration of the strength and distribution of acid sites due to the variation of the ZnO loadings, the optimal catalytic activity and selectivity of 2Zn/TS-1 catalyst (2 wt% ZnO) can be well reasonably attributed to the appropriate number of acid sites and the acidic strength. The addition of promoters such as 0.5 wt% MgO and 2.0 wt% ZrO2 may improve the stability of Zn/TS-1 catalyst due to the strong interaction between Zn2+ and promoter ions, which is believed to partially prevent the reduction of Zn2+ to metallic Zn. 2Zn/TS-1 catalyst exhibited good recycle stability. The loss of active Zn species on the used catalysts can be suppressed by regeneration treatment in air, in which the interaction between the Zn2+ and TS-1 support could become stronger. Well dispersed titanosilicate-1 (TS-1) supported ZnO catalysts were prepared, among which 2Zn/TS-1 catalyst exhibited good catalytic performance and recycle stability. [ABSTRACT FROM AUTHOR]

SUMMARY: Deep learning offers new approaches to investigate the mechanisms underlying complex biological phenomena, such as subgenome dominance. Subgenome dominance refers to the dominant expression and/or biased fractionation of genes in one subgenome of allopolyploids, which has shaped the evolution of a large group of plants. However, the underlying cause of subgenome dominance remains elusive. Here, we adopt deep learning to construct two convolutional neural network (CNN) models, binary expression model (BEM) and homoeolog contrast model (HCM), to investigate the mechanism underlying subgenome dominance using DNA sequence and methylation sites. We apply these CNN models to analyze three representative polyploidization systems, Brassica, Gossypium, and Cucurbitaceae, each with available ancient and neo/synthetic polyploidized genomes. The BEM shows that DNA sequence of the promoter region can accurately predict whether a gene is expressed or not. More importantly, the HCM shows that the DNA sequence of the promoter region predicts dominant expression status between homoeologous gene pairs retained from ancient polyploidizations, thus predicting subgenome dominance associated with these events. However, HCM fails to predict gene expression dominance between new homoeologous gene pairs arising from the neo/synthetic polyploidizations. These results are consistent across the three plant polyploidization systems, indicating broad applicability of our models. Furthermore, the two models based on methylation sites produce similar results. These results show that subgenome dominance is associated with long‐term sequence differentiation between the promoters of homoeologs, suggesting that subgenome expression dominance precedes and is the driving force or even the determining factor for sequence divergence between subgenomes following polyploidization. Significance Statement: Our study demonstrates that deep learning models can predict subgenome dominance in ancient polyploids but not in newly synthesized polyploids, highlighting the importance of long‐term sequence divergence. These findings provide valuable insights into the evolutionary mechanisms of polyploidy and offer a novel approach for studying complex genomic interactions. [ABSTRACT FROM AUTHOR]

Key message: Promoters of moso bamboo silicon transporter genes PeLsi1-1 and PeLsi1-2 contain elements in response to hormone, silicon, and abiotic stresses, and can drive the expression of PeLsi1-1 and PeLsi1-2 in transgene Arabidopsis. Low silicon 1 (Lsi1) transporters from different species have been shown to play an important role in influxing silicon from soil. In previous study, we cloned PeLsi1-1 and PeLsi1-2 from Phyllostachys edulis and verified that PeLsi1-1 and PeLsi1-2 have silicon uptake ability. Furthermore, in this study, the promoters of PeLsi1-1(1910 bp) and PeLsi1-2(1922 bp) were cloned. Deletion analysis identified the key regions of the PeLsi1-1 and PeLsi1-2 promoters in response to hormone, silicon, and abiotic stresses. RT-qPCR analysis indicated that PeLsi1-1 and PeLsi1-2 were regulated by hormones, salt stress and osmotic stress. In addition, we found that the driving activity of the PeLsi1-1 and PeLsi1-2 promoters was regulated by 2 mM K2SiO3 and PeLsi1-1-P3 ~ P4 and PeLsi1-2-P4 ~ 5 were the regions regulated by silicon. Overexpression of PeLsi1-1 or PeLsi1-2 driven by 35S promoter in Arabidopsis resulted in a threefold increase of Si accumulation, whereas transgenic plants showed deleterious symptoms and dwarf seedlings and shorter roots under 2 mM Si treatment. When the 35S promoter was replaced by PeLsi1-1 or PeLsi1-2 promoter, a similar Si absorption was achieved and the transgene plants grew normally. This study, therefore, demonstrates that the promoters of PeLsi1-1 and PeLsi1-2 are indeed effective in driving the expression of moso bamboo Lsi1 genes and leading to silicon uptake. [ABSTRACT FROM AUTHOR]

The cyclic lipopeptide bacillomycin D exhibits potent antifungal activity against a wide range of fungi, particularly filamentous fungi, thereby exhibiting significant potential for application in food preservation. However, the limited yield obtained from wild strains hinders the practical application of bacillomycin D. In this study, we used different strategies to modify the original strain Bacillus amyloliquefaciens fmbJ, including promoter replacement, competitive synthetic gene knockout, and overexpression of the lipopeptide transporter gene. Subsequently, we analyzed changes in the production and expression of bacillomycin D genes in the engineered strain. The results demonstrated that P bacA exhibits the highest promoter activity, leading to a significant increase (2.05-fold) in bacillomycin D production compared with control group. Additionally, deletion of the epsA- O gene cluster also significantly enhances the synthesis of bacillomycin D. However, when amyloid gene cluster t asA-sipW-yqxM was deleted,bacillomycin D accumulation time was shortened, resulting in no significant increase in bacillomycin D production. In addition, lipopeptide transporters SwrC, KrsE and YcxA did not promote bacillomycin D production. Finally, by replacing the promoter P bacA , we successfully disrupted the epsA -O gene cluster, and bacillomycin D production increased by 2.55 times compared to the initial bacillomycin D strain. In conclusion, this study indicated that genetic engineering of regulatory genes was an effective strategy to improve the yields of bacillomycin D and provided promising strains for industrial production of bacillomycin D. [Display omitted] • The Bacillomycin D, as an antimicrobial lipopeptide. • Effect of promoter replacement on bacillomycin D synthesis. • Effect of competing pathway gene knockout on bacillomycin D synthesis. • Effect of lipopeptide transporter overexpression on bacillomycin D synthesis. • Production of bacillomycin D in superimposed engineered bacteria. [ABSTRACT FROM AUTHOR]

An increase in GDH activity has been established in the first six hours after plants are exposed to hypoxic conditions, which ensures the formation of an adaptive response of cellular metabolism to the lack of oxygen in the cell. A difference in the transcriptional activity of homologous GDH-1 genes in wheat leaves under stress conditions was shown. An increase in the mRNA content of the GDH-1(5A) gene in wheat leaves after plants are exposed to hypoxic conditions is observed in the first hours of the experiment, which correlates with a change in the catalytic activity of glutamate dehydrogenase. Regulation of this gene is carried out by the transcription factor HIF, the specific landing site of which is found in the transcription initiation site of the promoter of this gene. [ABSTRACT FROM AUTHOR]

A promoter is a crucial component in driving the expression of a transgene of interest for biotechnological applications in crop improvement and thus characterization of varied regulatory regions is essential. Here, we identified the promoter of COR2-like (codeinone reductase-like) from banana and characterized its tissue specific and stress inducible nature. MusaCOR2-like of banana is closely related to COR2 and CHR (chalcone reductase) sequences from different plant species and contains signature sequences including a catalytic tetrad typical of proteins with aldo–keto reductase activity. Transcript level of MusaCOR2-like was strongly induced in response to drought, salinity and exposure of signaling molecules such as abscisic acid, methyl-jasmonate and salicylic acid. Induction of MusaCOR2-like under stress strongly correlated with the presence of multiple cis-elements associated with stress responses in the PMusaCOR2-like sequence isolated from Musa cultivar Rasthali. Transgenic tobacco lines harbouring PMusaCOR2-like-GUS displayed visible GUS expression in vascular tissue of leaves and stem while its expression was undetectable in roots under control conditions. Exposure to drought, salinity and cold strongly induced GUS expression from PMusaCOR2-like-GUS in transgenic tobacco shoots in a window period of 3H to 12H. Applications of salicylic acid, methyl-jasmonate, abscisic acid and ethephon also activate GUS in transgenic shoots at different period, with salicylic acid and abscisic acid being the stronger stimulants of PMusaCOR2-like. Using PMusaCOR2-like-GUS fusion and expression profiling, the current study sheds insights into a complex regulation of COR2-like, one of the least studied genes of secondary metabolite pathway in plants. [ABSTRACT FROM AUTHOR]

The primary constituents of the essential oil derived from Santalum album L. are (Z)-α-santalol, (Z)-β-santalol, (Z)-α-exo-bergamotol, and (Z)-epi-β- santalol. SaCYP736A167 plays a pivotal role in the biosynthesis of these sesquiterpene alcohols within S. album, but the mechanisms governing the expression of the SaCYP736A167 gene is far from being deciphered. In this research, a promoter sequence of the SaCYP736A167 gene, spanning 2808 base pairs, was isolated from S. album. A bioinformatics analysis of the 2384-bp SaCYP736A167 promoter (PSaCYP736A167) showed that abundant stress-inducible cis-acting elements were distributed in different regions of PSaCYP736A167. The histochemical β-glucuronidase (GUS) staining of T1 transgenic Nicotiana tabacum plants harboring PSaCYP736A167 demonstrated that the predominant GUS activity was exhibited in the parenchyma cells of the stem cortex and phloem, suggesting that PSaCYP736A167 is a tissue-specific expression promoter. GUS fluorometric assays of transiently transgenic N. benthamiana leaves revealed that seven distinct segments of PSaCYP736A167 exhibited notably varied levels of GUS activity. A 936-base pair sequence upstream of the transcription initiation codon ATG constitutes the core promoter section of PSaCYP736A167. Our findings shed light on the regulatory mechanisms controlling the transcription of the SaCYP736A167 gene, potentially serving as a novel tissue-specific promoter for applications in transgenic plant biotechnology. [ABSTRACT FROM AUTHOR]

Abstract Background The laccase Lcc9 from Coprinopsis cinerea has optimal catalytic activity at moderate to alkaline pH conditions, making it invaluable for industrial applications. However, C. cinerea naturally secretes Lcc9 at low expression levels, which limits the industrial application of Lcc9 on a large scale. Recombinant production of Lcc9 using Aspergillus niger would be an effective way to achieve its high production. Results This study achieved the secretory production of Lcc9 in A. niger and established an efficient transformation procedure for A. niger by optimizing its protoplast preparation system. The transformation efficiency of A. niger was increased 3.8-fold under the optimal system (cell wall digestion enzyme solution: 2% cellulase, 1% snailase, 1% lyticase, and 0.5% lysozyme; incubation time: 3 h; incubation temperature: 37 ℃; culture time: 48 h). The extracellular yield of Lcc9 was enhanced by optimizing gene expression cassette and bioprocess. First, the strain AnGgcL (containing P gpdA ) mediated by the SPCAT, a signal peptide of the extracellular high abundance protein catalase, had an extracellular laccase activity of 10 U/L after shake flask fermentation. Then, by optimizing promoter and signal peptide combinations that regulate lcc9 expression, the strain AnGcgL mediated by P citA -SPGlaA had an extracellular laccase activity of 20 U/L. Subsequently, the strain AnRcgL1 (containing P citA -SPGlaA) obtained by random integration had an extracellular laccase activity of 86 U/L. Sequencing revealed that the lcc9 expression cassette was integrated into the citrate synthase gene locus in the AnRcgL1 genome in a 9-copy form. By optimizing the microparticle, osmolyte, and Cu2+ in the fermentation medium, the AnRcgL1 extracellular laccase activity was further increased to 1566.7 U/L, which was 156.7-fold higher than that of AnGgcL. Furthermore, its extracellular laccase activity was increased to 1961 U/L in a 1-L fermenter. Conclusions To our knowledge, this study is the first to report the recombinant extracellular production of the C. cinerea laccase Lcc9 in A. niger and to use SPCAT in the A. niger expression system. The results of this study will help accelerate the industrial application of Lcc9. Moreover, the strategy used in this work provides methodological guidance for increasing other exogenous protein yields in A. niger.

Abstract Gene therapy is a promising method for treating inherited diseases by directly delivering the correct genetic material into patient cells. However, the limited packaging capacity of vectors poses a challenge. Minimizing promoter size is a viable strategy among various approaches to address this issue. This study aims to optimize the bovine myostatin (MSTN) promoter, enhancing its utility in gene therapy applications. We identified the primary driver of activity as the proximal regulatory region. Isolated from the native promoter and termed M243, this 243-bp sequence was assessed for its potential as a ubiquitous promoter. In a variety of cell types, including bovine embryos and embryonic stem cells, the M243 promoter showed consistent expression, highlighting its suitability for applications requiring compact promoters. The isolation of a highly conserved, compact 243-bp sequence serving as a promoter suggests a solution for overcoming the size constraints of AAV vectors, suggesting potential contributions to the field of gene therapy.

Background Radioiodine (RI) ablation following thyroid-stimulating hormone suppression is an effective treatment for papillary thyroid cancer (PTC), typically leading to favorable outcomes. However, RI-refractory tumors exhibit aggressive behavior and poor prognoses. Recent studies highlight the role of genetic abnormalities in PTC signaling pathways, including the activation of telomerase reverse transcriptase (TERT), and the correlation of mutations with adverse outcomes. Methods This study analyzed mutations in BRAF V600E and the TERT-promoter genes, comparing clinicopathological features between RI-refractory and RI-responsive PTCs. Among 82 RI-refractory patients, formalin-fixed, paraffin-embedded tissues from initial surgeries were available for 26. Another 89 without distant metastasis over 5 years formed a matched RI-responsive control group. Results Histopathologically, RI-refractory PTCs showed increased frequencies of small tumor clusters without fibrovascular cores, hobnail features, and a high height-to-width ratio of tumor cells. These tumors were more likely to exhibit necrosis, mitosis, lymph node metastasis, extrathyroidal extension, and involvement of resection margins. TERT-promoter mutations were statistically significantly associated with these aggressive clinicopathologic features. Immunohistochemically, decreased expression of sodium iodide symporter and thyroglobulin stimulating hormone receptor proteins was common in RI-refractory PTCs, along with lower levels of oncogenic proteins such as vascular endothelial cell growth factor, vascular endothelial cell growth factor receptor 2, and nuclear factor kappa-light-chain-enhancer of activated B cells. Total loss of PTEN expression was occasionally observed. In contrast, all cases tested positive for cytoplasmic β-catenin. Conclusions RI-refractory PTCs are linked to TERT mutations and exhibit specific aggressive histopathologic features, particularly in tumor centers.

Abstract The precise spatiotemporal expression of the hematopoietic ETS transcription factor PU.1, a key determinant of hematopoietic cell fates, is tightly regulated at the chromatin level. However, how chromatin signatures are linked to this dynamic expression pattern across different blood cell lineages remains uncharacterized. Here, we performed an in-depth analysis of the relationships between gene expression, chromatin structure, 3D architecture, and trans-acting factors at PU.1 cis-regulatory elements (PCREs). By identifying phylogenetically conserved DNA elements within chromatin-accessible regions in primary human blood lineages, we discovered multiple novel candidate PCREs within the upstream region of the human PU.1 locus. A subset of these elements localizes within an 8-kb-wide cluster exhibiting enhancer features, including open chromatin, demethylated DNA, enriched enhancer histone marks, present enhancer RNAs, and PU.1 occupation, presumably mediating PU.1 autoregulation. Importantly, we revealed the presence of a common 35-kb-wide CTCF-flanked insulated neighborhood that contains the PCRE cluster (PCREC), forming a chromatin territory for lineage-specific and PCRE-mediated chromatin interactions. These include functional PCRE-promoter interactions in myeloid and B cells that are absent in erythroid and T cells. By correlating chromatin structure and 3D architecture with PU.1 expression in various lineages, we were able to attribute enhancer versus silencer functions to individual elements. Our findings provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in the chromatin regulation of PU.1 expression. This study lays crucial groundwork for additional experimental studies that validate and dissect the role of PCREs in epigenetic regulation of normal and malignant hematopoiesis.

CO2 reformation of methane (CRM) and CO2 methanation are two interconnected processes with significant implications for greenhouse gas reduction and sustainable energy production for industrial purposes. While Ni-based catalysis suffers from poor stability due to coke formation or sintering, we report a super stable remedy. The active sites of mesoporous MgO were loaded using wet impregnation. The incorporation of Ni and promoters altered the physical features of the catalysts. Sm–Ni/MgO showed the smallest crystallite size, specific surface area, and pore volume. The Sm–Ni/MgO catalyst was selected as the most suitable candidate for CRM, with 82 ​% CH4 and H2/CO ratio of approximately 100 ​% and also for CO2 methanation with the conversion of carbon dioxide (82 ​%) and the selectivity toward methane reaches 100 ​% at temperatures above 300 ᵒC. Furthermore, the Sm–Ni/MgO catalyst was stable for 900 ​min of continuous reaction, without significant carbon deposition. This stability was largely due to the high oxygen mobility on the catalyst surface in the presence of Sm. Overall, we demonstrated the efficacy of using promoted Ni catalysts supported by mesoporous magnesia for the improved reformation of greenhouse gases.

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 socio-demographic 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 Background Malignant cells exhibit reduced period circadian regulator 3 (PER3) expression. However, the underlying mechanisms of variations in PER3 expression in cancers and the specific function of PER3 in tumor progression remain poorly understood. Results We explored multiple public databases, conducted bioinformatics analyses, and performed in vitro and in vivo experiments for validation. We found PER3 expression was decreased in most types of cancers, and PER3 downregulation was associated with a poor prognosis in 8 types of cancer. PER3 promoter methylation levels were increased in 11 types of cancer. Promoter hypermethylation (CpG islands [CGIs] cg12258811 and cg14204433) correlated with decreased PER3 expression in six cancers (breast invasive carcinoma, colon adenocarcinoma, head and neck squamous cell carcinoma, kidney renal papillary cell carcinoma [KIRP], lung adenocarcinoma [LUAD], and uterine corpus endometrial carcinoma). CGI cg12258811 hypermethylation was associated with reduced survival time and advanced cancer stages. Moreover, the bisulfite pyrosequencing assay confirmed CGI cg12258811 hypermethylation and its negative correlation with PER3 expression. In vitro and in vivo experiments demonstrated that PER3 inhibited KIRP and LUAD progression. Decitabine enhanced PER3 expression and inhibited KIRP cell functions by reducing promoter (cg12258811) methylation level. Conclusions Our findings advanced the mechanistic understanding of variations in PER3 expression in cancers and confirmed the tumor-associated function of PER3 hypermethylation and downregulation.

Abstract Background Cervical cancer is the fourth leading cause of cancer-related death among women worldwide, and effective therapeutic strategies for its treatment are limited. Recent studies have indicated that ferroptosis, a form of regulated cell death, is a promising therapeutic strategy. KLF14 has been shown to regulate both cell proliferation and apoptosis in cervical cancer. However, its role in modulating lipid peroxidation and ferroptosis remains largely unexplored and enigmatic. Methods SiHa and HeLa cells were transduced with lentiviral vectors to overexpress KLF14. Protein levels were analyzed via western blotting and immunohistochemistry (IHC). LDH assays, calcein-AM/propidium iodide (PI) staining, and generation of cell growth curves using a real-time cell analysis (RTCA) system were used to detect cell damage and proliferation. Cellular ROS, lipid ROS, transmission electron microscopy (TEM), and Fe2+ assays and a xenograft mouse model were used to measure the level of ferroptosis. Proteomics combined with bioinformatics methods was used to screen target genes regulated by KLF14, and CUT&Tag and dual-luciferase assays confirmed the repression of GPX4 by KLF14 via direct binding to its promoter. Results KLF14 is abnormally expressed in various tumors and downregulated in cervical cancer. Overexpression of KLF14 induced ferroptosis and inhibited cell proliferation in vitro as well as xenograft tumorigenicity in vivo. Mechanistic studies revealed that KLF14 binds to the promoter of GPX4, suppressing its transcriptional activity and thereby decreasing its expression, which contributes to the induction of ferroptosis. Truncation and point mutation analyses of the GPX4 promoter revealed multiple binding sites for KLF14 within the − 1000 bp to + 35 bp region, which are responsible for its inhibitory effect on GPX4 transcription. Additionally, deletion of the zinc finger motif in KLF14 abolished its inhibitory effect on GPX4 promoter activity and cell proliferation. Conclusion Our data revealed a previously unidentified function of KLF14 in promoting ferroptosis, which results in the suppression of cell proliferation. Mechanistically, we revealed a novel regulatory mechanism by which KLF14 targets GPX4. These findings suggest a novel strategy to induce ferroptosis through the targeting of KLF14 in human cervical cancer cells.

Abstract Background The UDP-glucuronosyltransferase 91D2 (SrUGT91D2) gene is a crucial element in the biosynthetic pathway of steviol glycosides (SGs) and is responsible for creating 1,2-β-D glucosidic bonds at the C19 and C13 positions. This process plays a vital role in the synthesis of rebaudioside M (RM) and rebaudioside D (RD). The promoter, which regulates gene expression, requires functional analysis to understand gene expression regulation. However, investigations into the function of the promoter of SrUGT91D2 (pSrUGT91D2) have not been reported. Results The pSrUGT91D2 was isolated from six S. rebaudiana lines, and subsequent multiple sequence comparisons revealed the presence of a 26 bp inDel fragment (pSrUGT91D2-B1188 type) in lines GP, GX, 110, 1114, and B1188 but not in the pSrUGT91D2 of line 023 (pSrUGT91D2-023 type). Bioinformatics analysis revealed a prevalence of significant cis-regulatory elements (CREs) within the promoter sequences, including those responsive to abscisic acid, light, anaerobic conditions, auxin, drought, low temperature, and MeJA. To verify the activity of pSrUGT91D2, the full-length promoter and a series of 5’ deletion fragments (P1–P7) and a 3’ deletion fragment (P8) from various lines were fused with the reporter β-glucuronidase (GUS) gene to construct the plant expression vector, pCAMBIA1300-pro∷GUS. The transcriptional activity of these genes was examined in tobacco leaves through transient transformation. GUS tissue staining analysis and enzyme activity assays demonstrated that both the full-length promoter and truncated pSrUGT91D2 were capable of initiating GUS expression in tobacco leaves. Interestingly, P8-pSrUGT91D2-B1188 (containing the inDel segment, 301 bp) exhibited enhanced activity in driving GUS gene expression. Transient expression studies of P8-pSrUGT91D2-B1188 and P8-pSrUGT91D2-023 in response to exogenous hormones (abscisic acid and indole-3-acetic acid) and light indicated the necessity of the inDel region for P8 to exhibit transcriptional activity, as it displayed strong responsiveness to abscisic acid (ABA), indole-3-acetic acid (IAA), and light induction. Conclusions These findings contribute to a deeper understanding of the regulatory mechanism of the upstream region of the SrUGT91D2 gene and provide a theoretical basis for future studies on the interaction between CREs of pSrUGT91D2 and related transcription factors.

Abstract Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 × 10− 24 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D’ = 1.0; R2 > 0.90) revealed marker rs2303751 (p value 8.12 × 10− 20) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding site for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2 −/− knockout (KO) zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2 −/− ko zebrafish Tg(wt1ß:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.