Krüppel-like factor 12 decreases progestin sensitivity in endometrial cancer by inhibiting the progesterone receptor signaling pathway.

OBJECTIVE: This study aimed to clarify the mechanism by which Krüppel-like factor 12 (KLF12) affects progesterone sensitivity in endometrial cancer (EC) through the progesterone receptor PGR signaling pathway. METHODS: The relationship of KLF12 with PGR in EC patients was examined by immunohistochemistry, and the expression of KLF12 and PGR in EC cell lines was detected by real-time PCR and western blotting. Cell proliferation assay, plate clone formation, cell apoptosis assay, and cell cycle analysis were conducted to determine the impact of KLF12 intervention on progesterone therapy. CUT&Tag analysis and the dual-luciferase reporter experiment were used to determine the underlying regulatory effect of KLF12 on the PGR DNA sequence. A subcutaneous xenograft nude mouse model was established to validate the in vivo effect of KLF12 on progesterone sensitivity via PGR expression modulation. RESULTS: KLF12 demonstrated decreased progesterone sensitivity and a negative correlation with PGR expression in EC tissues. Progesterone sensitivity was increased by KLF12 deficiency through PGR overexpression, a result that could be significantly reversed by PGR downregulation. PGR was identified as a target gene of KLF12, which could directly bind to the PGR promotor region and inhibit its expression. CONCLUSION: This study is the first to investigate the effect of KLF12 expression on EC cell resistance to progesterone. Our results offer important mechanistic insight into the direct regulation of the PGR promoter region, demonstrating that KLF12 expression strongly suppressed the PGR signaling pathway and, as a result, reduced progesterone sensitivity in EC patients

Development or absence of conjugate fractures in low-permeability sandstones

Natural fractures are ubiquitous in rocks. The Coulomb law of Mohr’s failure theory predicts that the angle between conjugate failure surfaces is a constant. In the Ordos Basin, observing the development of two groups of conjugate fractures in the field, cores and imaging logging is very difficult. In this paper, the directions of paleocurrents in the Upper Triassic Yanchang Formation of the Ordos Basin are determined by measuring the orientations of field bedding. Through the correlation analysis of paleocurrent and natural fracture orientations, when the sediment comes from a single source, a group of fractures with a large angle between conjugate fractures and the paleocurrent direction is found not to develop. When the sediments in the study area have two provenances, both provenance directions affect the development of conjugate fractures. In the southern Ordos Basin, influenced by the direction of paleocurrent flow in the near-north direction, fractures in the near N‒S direction develop. Through rock mechanics experiments in different directions, the planar anisotropy in rock mechanics parameters caused by the direction of paleocurrent flow is found to be the geological factor leading to various degrees of fracture development in different directions within the Ordos Basin

Expression characteristics and transcriptional regulation analysis of VlCKX5 gene in grape

[Objective] Grapes (Vitis vinifera L.) are an economically important fruit crop in the world, and severe berry drop can affect grape yield and quality. The synthetic cytokinin analog N- (2-chloro-4-pyridyl) -N'-phenylurea (CPPU) is known to enhance berry set in grapes. Cytokinin oxidase/dehydrogenase (CKX) enzymes, which are responsible for the irreversible degradation of cytokinin, are pivotal in modulating plant growth and development. In the present investigation, the cytokinin oxidase/dehydrogenase 5 (VlCKX5) gene and its promoter were cloned, and bioinformatic analysis, expression specificity and transcriptional regulation were performed to illustrate its role in grape berry setting. [Methods] In this study, we conducted experiments using Kyoho grapes (V. vinifera L. × V. labrusca L.) as the experimental material. The young berries were treated with 10 mg·L-1 of cytokinin-like growth regulator CPPU 5 days after anthesis. The treated berries were sampled at 1, 2, 4 and 8 days after treatment. Furthermore, at 13th day after anthesis, we systematically harvested roots, stems, leaves, inflorescences, tendrils and young berries from grapevines for subsequent tissue-specific analysis. The VlCKX5 gene region was amplified via polymerase chain reaction (PCR). Bioinformatic analysis of the VlCKX5 protein sequence, including various physicochemical properties, was performed using the Expasy web tool. The identification of conserved domains within VlCKX5 was conducted through the InterPro database. Furthermore, the phylogenetic relationship among VlCKX5 and its homologs was examined using MEGA software. Protein domain architecture of VlCKX5 and its orthologous proteins was examined utilizing the GeneDoc 2.7. Expression levels of VlCKX5 in grapevine tissues, including roots, canes, leaves, inflorescences, tendrils and young berries under natural growth conditions, as well as in young berries following treatment with the CPPU, were quantified using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The activity of the VlCKX5 promoter was evaluated through histochemical staining with β-glucuronidase (GUS). To predict the transcriptional regulatory interactions involving VlCKX5, we utilized the PlantTFDB, CIS-BP and JASPAR databases to identify potential key transcription factors that may modulate its expression. The coding sequence (CDS) of VlAGL6, with the termination codon excised, was cloned into the 101LYFP vector. The construct was then transformed into Agrobacterium Competent Cells (GV3101), which were subsequently mixed with a selection marker and used to infiltrate Nicotiana benthamiana plants. At 72 hours post-transformation, the subcellular localization of fluorescence within N. benthamiana leaf cells was analyzed using laser scanning confocal microscopy. RT-qPCR was used to analyze the expression pattern of VlAGL6a in grape tissues after CPPU treatment. A fragment of the VlCKX5 promoter containing the VlAGL6a binding site, designated as P, was cloned into the pAbAi vector, generating the recombinant bait plasmid pAbAi-proVlCKX5/P. This plasmid was then transfected into the Y1HGold yeast strain. Subsequently, the VlAGL6a gene was cloned into the pGADT7 vector to create the recombinant prey plasmid pGADT7-VlAGL6a, which was transfected into a yeast strain positive for the bait genome to perform yeast one-hybrid (Y1H) interaction detection. A 1566 base pair segment of the VlCKX5 promoter, located in upstream of the ATG start codon, was cloned into the pGreenII0800-LUC vector to create a reporter construct. The VlAGL6a CDS was subcloned into the pSAK277 vector to produce an effector construct. Agrobacterium tumefaciens strains carrying these constructs were co-infiltrated into N. benthamiana leaves. The luciferase activity in the infiltrated samples was measured 48 hours post-infiltration using a dual-luciferase reporter assay system. [Results] VlCKX5 was 1641 bp in length and encoded 546 amino acids. The molecular weight of VlCKX5 was 61.516 62 ku, the isoelectric point was 8.36, the instability index was 36.64, the fat coefficient was 94.27, and the protein structure was stable. VlCKX5 had the closest homology to CKX amino acids in Chinese kiwifruit, and had a FAD domain and cytokinin binding site (CK-binding), belonging to a typical CKX family. VlCKX5 was highly expressed in roots and leaves, followed by inflorescences, and the expression of VlCKX5 was significantly reduced at 1, 2, 4 and 8 d after CPPU treatment. Prediction of the cis-acting elements of the VlCKX5 promoter revealed elements containing hormones responsive to IBA, SA and ABA. GUS chemical tissue staining test results showed that VlCKX5 activated its promoter activity in response to the treatment of CPPU, SA, IBA and ABA. Transcriptional regulation analysis showed that BPC, DOF, MADS and FLC family transcription factors may be involved in the transcriptional regulation of VlCKX5, and VlAGL6a was a key candidate transcription factor for VlCKX5. Subcellular localization assay verified that VlAGL6a was localized in the nucleus. The results of fluorescence quantification showed that VlAGL6a was the highest in inflorescences, followed by berries and tendrils, and the lowest in roots, stems and leaves. The RT-qPCR results after CPPU treatment showed that the expression levels of VlAGL6a were significantly reduced on the 1st, 2nd, 4th and 8th days, which was consistent with the expression pattern of VlCKX5. Y1H and double luciferase assay showed that VlAGL6a could interact with VlCKX5 and promote its expression. [Conclusion] The VlCKX5 gene of grape responded to the signal of CPPU, and the transcription factor VlAGL6a was specifically bound to the promoter of VlCKX5 gene and promoted the transcription of VlCKX5, which affected grape berry setting by regulating the level of cytokinin, which provides a theoretical basis for further analysis of the mechanism of grape berry set

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Enhancement of aminoacylase activity by sodium citrate

Kidney and other tissues of animals and humans have a high concentration of citrate which is an important intermediate substance in the citrate cycle. Citrate may play an important physiological role in metabolism. In this paper, we studied the interaction of the sodium salt of citrate with aminoacylase which is an important enzyme in metabolism and found sodium citrate can enhance the activity of aminoacylase. The maximum enzyme activity induced by sodium citrate increased approximately 3 folds over the enzyme activity without sodium citrate. The initial reaction rates for different concentrations of sodium citrate were obtained, showing that sodium citrate is a non-competitive activator. The result of the ANS binding fluorescence measurements for aminoacylase indicated that increasing sodium citrate concentrations markedly increased the ANS binding fluorescence with a blue shift of the emission spectra peak. This suggests the formation of more hydrophobic regions. Aggregates formed quickly when aminoacylase was incubated with sodium citrate (0.3 mol/L) and guanidinium chloride (0-3.5 mol/L). Aminoacylase lost enzyme activity in the guanidinium chloride more quickly in the presence of sodium citrate than in the absence of sodium citrate. The intrinsic fluorescence emission intensity decreased more quickly and the red shift of the emission spectra peak was larger than that without sodium citrat

Does digitalization mitigate regional inequalities? Evidence from China

Regional inequality significantly influences sustainable development and human well-being. In China, there exists pronounced regional disparities in economic and digital advancements; however, scant research delves into the interplay between them. By analyzing the economic development and digitalization gaps at regional and city levels in China, extending the original Cobb-Douglas production function, this study aims to evaluate the impact of digitalization on China’s regional inequality using seemingly unrelated regression. The results indicate a greater emphasis on digital inequality compared to economic disparity, with variable coefficients of 0.59 for GDP per capita and 0.92 for the digitalization index over the past four years. However, GDP per capita demonstrates higher spatial concentration than digitalization. Notably, both disparities have shown a gradual reduction in recent years. The southeastern region of the Hu Huanyong Line exhibits superior levels and rates of economic and digital advancement in contrast to the northwestern region. While digitalization propels economic growth, it yields a nuanced impact on achieving balanced regional development, encompassing both positive and negative facets. Our study highlights that the marginal utility of advancing digitalization is more pronounced in less developed regions, but only if the government invests in the digital infrastructure and education in these areas. This study’s methodology can be utilized for subsequent research, and our findings hold the potential to the government’s regional investment and policy-making

Enhancement of aminoacylase activity by sodium citrate

Kidney and other tissues of animals and humans have a high concentration of citrate which is an important intermediate substance in the citrate cycle. Citrate may play an important physiological role in metabolism. In this paper, we studied the interaction of the sodium salt of citrate with aminoacylase which is an important enzyme in metabolism and found sodium citrate can enhance the activity of aminoacylase. The maximum enzyme activity induced by sodium citrate increased approximately 3 folds over the enzyme activity without sodium citrate. The initial reaction rates for different concentrations of sodium citrate were obtained, showing that sodium citrate is a non-competitive activator. The result of the ANS binding fluorescence measurements for aminoacylase indicated that increasing sodium citrate concentrations markedly increased the ANS binding fluorescence with a blue shift of the emission spectra peak. This suggests the formation of more hydrophobic regions. Aggregates formed quickly when aminoacylase was incubated with sodium citrate (0.3 mol/L) and guanidinium chloride (0-3.5 mol/L). Aminoacylase lost enzyme activity in the guanidinium chloride more quickly in the presence of sodium citrate than in the absence of sodium citrate. The intrinsic fluorescence emission intensity decreased more quickly and the red shift of the emission spectra peak was larger than that without sodium citrat