Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Hsp90 Breaks the Deadlock of the Hsp70 Chaperone System.

Protein folding in the cell requires ATP-driven chaperone machines such as the conserved Hsp70 and Hsp90. It is enigmatic how these machines fold proteins. Here, we show that Hsp90 takes a key role in protein folding by breaking an Hsp70-inflicted folding block, empowering protein clients to fold on their own. At physiological concentrations, Hsp70 stalls productive folding by binding hydrophobic, core-forming segments. Hsp90 breaks this deadlock and restarts folding. Remarkably, neither Hsp70 nor Hsp90 alters the folding rate despite ensuring high folding yields. In fact, ATP-dependent chaperoning is restricted to the early folding phase. Thus, the Hsp70-Hsp90 cascade does not fold proteins, but instead prepares them for spontaneous, productive folding. This stop-start mechanism is conserved from bacteria to man, assigning also a general function to bacterial Hsp90, HtpG. We speculate that the decreasing hydrophobicity along the Hsp70-Hsp90 cascade may be crucial for enabling spontaneous folding.

Temperature-dependent intracellular crystallization of firefly luciferase in mammalian cells is suppressed by D-luciferin and stabilizing inhibitors.

Firefly luciferase (Fluc) from Photinus pyralis is one of the most widely used reporter proteins in biomedical research. Despite its widespread use, Fluc's protein phase transition behaviors and phase separation characteristics have not received much attention. Current research uncovers Fluc's intrinsic property to phase separate in mammalian cells upon a simple cell culture temperature change. Specifically, Fluc spontaneously produced needle-shaped crystal-like inclusion bodies upon temperature shift to the hypothermic temperatures ranging from 25 °C to 31 °C. The crystal-like inclusion bodies were not associated with or surrounded by membranous organelles and were likely built from the cytosolic pool of Fluc. Furthermore, the crystal-like inclusion formation was suppressed when cells were cultured in the presence of D-luciferin and its synthetic analog, as well as the benzothiazole family of so-called stabilizing inhibitors. These two classes of compounds inhibited intracellular Fluc crystallization by different modes of action as they had contrasting effects on steady-state luciferase protein accumulation levels. This study suggests that, under substrate insufficient conditions, the excess Fluc phase separates into a crystal-like state that can modulate intracellular soluble enzyme availability and protein turnover rate.

SKAP2 Modular Organization Differently Recognizes SRC Kinases Depending on Their Activation Status and Localization.

Dimerization of SRC kinase adaptor phosphoprotein 2 (SKAP2) induces an increase of binding for most SRC kinases suggesting a fine-tuning with transphosphorylation for kinase activation. This work addresses the molecular basis of SKAP2-mediated SRC kinase regulation through the lens of their interaction capacities. By combining a luciferase complementation assay and extensive site-directed mutagenesis, we demonstrated that SKAP2 interacts with SRC kinases through a modular organization depending both on their phosphorylation-dependent activation and subcellular localization. SKAP2 contains three interacting modules consisting in the dimerization domain, the SRC homology 3 (SH3) domain, and the second interdomain located between the Pleckstrin homology and the SH3 domains. Functionally, the dimerization domain is necessary and sufficient to bind to most activated and myristyl SRC kinases. In contrast, the three modules are necessary to bind SRC kinases at their steady state. The Pleckstrin homology and SH3 domains of SKAP2 as well as tyrosines located in the interdomains modulate these interactions. Analysis of mutants of the SRC kinase family member hematopoietic cell kinase supports this model and shows the role of two residues, Y390 and K7, on its degradation following activation. In this article, we show that a modular architecture of SKAP2 drives its interaction with SRC kinases, with the binding capacity of each module depending on both their localization and phosphorylation state activation. This work opens new perspectives on the molecular mechanisms of SRC kinases activation, which could have significant therapeutic impact.

Pathogenic monoallelic variants in GLIS3 increase type 2 diabetes risk and identify a subgroup of patients sensitive to sulfonylureas.

AIMS/HYPOTHESIS: GLIS3 encodes a transcription factor involved in pancreatic beta cell development and function. Rare pathogenic, bi-allelic mutations in GLIS3 cause syndromic neonatal diabetes whereas frequent SNPs at this locus associate with common type 2 diabetes risk. Because rare, functional variants located in other susceptibility genes for type 2 diabetes have already been shown to strongly increase individual risk for common type 2 diabetes, we aimed to investigate the contribution of rare pathogenic GLIS3 variants to type 2 diabetes. METHODS: GLIS3 was sequenced in 5471 individuals from the Rare Variants Involved in Diabetes and Obesity (RaDiO) study. Variant pathogenicity was assessed following the criteria established by the American College of Medical Genetics and Genomics (ACMG). To address the pathogenic strong criterion number 3 (PS3), we conducted functional investigations of these variants using luciferase assays, focusing on capacity of GLIS family zinc finger 3 (GLIS3) to bind to and activate the INS promoter. The association between rare pathogenic or likely pathogenic (P/LP) variants and type 2 diabetes risk (and other metabolic traits) was then evaluated. A meta-analysis combining association results from RaDiO, the 52K study (43,125 individuals) and the TOPMed study (44,083 individuals) was finally performed. RESULTS: Through targeted resequencing of GLIS3, we identified 105 rare variants that were carried by 395 participants from RaDiO. Among them, 49 variants decreased the activation of the INS promoter. Following ACMG criteria, 18 rare variants were classified as P/LP, showing an enrichment in the last two exons compared with the remaining exons (p<5×10-6; OR>3.5). The burden of these P/LP variants was strongly higher in individuals with type 2 diabetes (p=3.0×10-3; OR 3.9 [95% CI 1.4, 12]), whereas adiposity, age at type 2 diabetes diagnosis and cholesterol levels were similar between variant carriers and non-carriers with type 2 diabetes. Interestingly, all carriers with type 2 diabetes were sensitive to oral sulfonylureas. A total of 7 P/LP variants were identified in both 52K and TOPMed studies. The meta-analysis of association studies obtained from RaDiO, 52K and TOPMed showed an enrichment of P/LP GLIS3 variants in individuals with type 2 diabetes (p=5.6×10-5; OR 2.1 [95% CI 1.4, 2.9]). CONCLUSIONS/INTERPRETATION: Rare P/LP GLIS3 variants do contribute to type 2 diabetes risk. The variants located in the distal part of the protein could have a direct effect on its functional activity by impacting its transactivation domain, by homology with the mouse GLIS3 protein. Furthermore, rare P/LP GLIS3 variants seem to have a direct clinical effect on beta cell function, which could be improved by increasing insulin secretion via the use of sulfonylureas.

High sensitivity and low-cost flavin luciferase (FLUXVc)-based reporter gene for mammalian cell expression.

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from V. campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the nonengineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUXVc/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We also demonstrated that FLUXVc can be used for testing inhibitors of the NF-κB signaling pathway. Collectively, our results provide an optimized method for using the more economical flavin-dependent luciferase in mammalian cells.

The ubiquitination status of the glucagon receptor determines signal bias.

The pancreatic hormone glucagon activates the glucagon receptor (GCGR), a class B seven-transmembrane G protein-coupled receptor that couples to the stimulatory heterotrimeric G protein and provokes PKA-dependent signaling cascades vital to hepatic glucose metabolism and islet insulin secretion. Glucagon-stimulation also initiates recruitment of the endocytic adaptors, ßarrestin1 and ßarrestin2, which regulate desensitization and internalization of the GCGR. Unlike many other G protein-coupled receptors, the GCGR expressed at the plasma membrane is constitutively ubiquitinated and upon agonist-activation, internalized GCGRs are deubiquitinated at early endosomes and recycled via Rab4-containing vesicles. Herein we report a novel link between the ubiquitination status and signal transduction mechanism of the GCGR. In the deubiquitinated state, coupling of the GCGR to Gs is diminished, while binding to ßarrestin is enhanced with signaling biased to a ßarrestin1-dependent p38 mitogen activated protein kinase (MAPK) pathway. This ubiquitin-dependent signaling bias arises through the modification of lysine333 (K333) on the cytoplasmic face of transmembrane helix V. Compared with the GCGR-WT, the mutant GCGR-K333R has impaired ubiquitination, diminished G protein coupling, and PKA signaling but unimpaired potentiation of glucose-stimulated-insulin secretion in response to agonist-stimulation, which involves p38 MAPK signaling. Both WT and GCGR-K333R promote the formation of glucagon-induced ßarrestin1-dependent p38 signaling scaffold that requires canonical upstream MAPK-Kinase3, but is independent of Gs, Gi, and ßarrestin2. Thus, ubiquitination/deubiquitination at K333 in the GCGR defines the activation of distinct transducers with the potential to influence various facets of glucagon signaling in health and disease.

Development of dual-visible reporter assays to determine the DNA-protein interaction.

The application of DNA-protein interaction reporter assays for relational or ratiometric measurements within an experimental system is popular in biological research. However, the existing reporter-based interaction assays always require special equipment, expensive chemicals, and a complicated operation. Here, we developed a DNA-protein interaction technology integrating two visible reporters, RUBY and UV-visible GFP (eYGFPuv), which allows the expression of the cassette reporter contained cis-acting DNA element (DE) fused upstream of TATA box and RUBY, and a constitutive promoter regulating eYGFPuv in the same construct. The interaction of transcription factor (TF) and the DE can be detected by co-expressed the cassette reporter and TF in tobacco leaves where the cassette reporter alone serves as a control. We also revealed that eight function-unknown bamboo AP2/ERFs interacted with the DE of ANT-AP2R1R2 (ABE), DRE (DBE), GCC-box (EBE), and RAV1 binding element (RBE), respectively, which are consistent with the results by dual-luciferase reporter assays. Thus, the dual-visible reporters offer a convenient, visible, and cost-saving alternative to other existing techniques for DNA-protein interaction in plants.

Insights into Phakopsora pachyrhizi Effector-Effector Interactions.

The multifaceted role of pathogen-encoded effectors in plant-pathogen interactions is complex and not fully understood. Effectors operate within intricate host environments, interacting with host proteins and other effectors to modulate virulence. The complex interplay between effectors raises the concept of metaeffectors, wherein some effectors regulate the activity of others. While previous research has demonstrated the importance of effector repertoires in pathogen virulence, only a limited number of studies have investigated the interactions between these effectors. This study explores the interactions among Phakopsora pachyrhizi effector candidates (PpECs). P. pachyrhizi haustorial transcriptome analysis identified a collection of predicted PpECs. Among these, PpEC23 was found to interact with PpEC48, prompting further exploration into their potential interaction with other effectors. Here, we utilized a yeast two-hybrid screen to explore protein-protein interactions between PpECs. A split-luciferase complementation assay also demonstrated that these interactions could occur within soybean cells. Interestingly, PpEC48 displayed the ability to interact with several small cysteine-rich proteins (SCRPs), suggesting its affinity for this specific class of effectors. We show that these interactions involve a histidine-rich domain within PpEC48, emphasizing the significance of structural motifs in mediating effector interactions. The unique nature of PpEC48, showing no sequence matches in other organisms, suggests its relatively recent evolution and potential orphan gene status. Our work reveals insights into the intricate network of interactions among P. pachyrhizi effector-effector interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Development of a split-luciferase assay to establish optimal protein secretion conditions for protein production by Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by B. subtilis, a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner. In this study, we introduce a split-luciferase-based method for measuring protein secretion by B. subtilis. The NanoBiT system was used to monitor secretion of four different proteins: xylanase A, amylase M, protein glutaminase A, and GFP nanobody. Our findings underscore the split-luciferase system as a quick, sensitive, and user-friendly method.

Subcellular imaging of MGAT1/MGAT2 homo- and heteromers in living cells using bioluminescence microscopy.

Protein-protein interactions (PPIs) play fundamental roles in many biological processes including the functioning of glycosylation machineries present in the endoplasmic reticulum (ER) and Golgi apparatus of mammalian cells. For the last couple of years, we have been successfully employing the most advanced version of the split luciferase complementation assay, termed NanoBiT, to demonstrate PPIs between solute carrier 35 (SLC35) family members with nucleotide sugar transporting activity and functionally related glycosyltransferases. NanoBiT has several unmatched advantages as compared with other strategies for studying PPIs. Firstly, the tendency of the free luciferase fragments to spontaneously associate is strongly reduced. As a consequence, the fragments of the reconstituted luciferase may dissociate upon the disruption of the PPI of interest. Secondly, the recombinant fusion proteins are expressed at low (near-endogenous) levels. Both of these features significantly minimize the possibility of obtaining false positive results. In this study we pushed the boundaries of this already powerful technique even further by coupling it with bioluminescence imaging of PPIs. Specifically, we visualized homo- and heterologous complexes formed by MGAT1 and MGAT2 glycosylation enzymes tagged with NanoBiT fragments and demonstrated ER-to-Golgi transitions between enzyme homo- and heteromers.

Restoring microRNA-34a overcomes acquired drug resistance and disease progression in human breast cancer cell lines via suppressing the ABCC1 gene.

PURPOSE: Breast cancer is one of the leading types of cancer diagnosed in women. Despite the improvements in chemotherapeutic cure strategies, drug resistance is still an obstacle leading to disease aggressiveness. The small non-coding RNA molecules, miRNAs, have been implicated recently to be involved as regulators of gene expression through the silencing of mRNA targets that contributed to several cellular processes related to cancer metastasis. Hence, the present study aimed to investigate the beneficial role and mechanism of miRNA-34a-based gene therapy as a novel approach for conquering drug resistance mediated by ATP-binding cassette (ABC) transporters in breast cancer cells, besides exploring the associated invasive behaviors. MATERIAL AND METHODS: Bioinformatics tools were used to predict miRNA ABC transporter targets by tracking the ABC transporter pathway. After the establishment of drug-resistant breast cancer MCF-7 and MDA-MB-231 sublines, cells were transfected with the mimic or inhibitor of miRNA-34a-5p. The quantitative expression of genes involved in drug resistance was performed by QRT-PCR, and the exact ABC transporter target specification interaction was confirmed by dual-luciferase reporter assay. Furthermore, flow cytometric analysis was utilized to determine the ability of miRNA-34a-treated cells against doxorubicin uptake and accumulation in cell cycle phases. The spreading capability was examined by colony formation, migration, and wound healing assays. The apoptotic activity was estimated as well. RESULTS: Our findings firstly discovered the mechanism of miRNA-34a-5p restoration as an anti-drug-resistant molecule that highly significantly attenuates the expression of ABCC1 via the direct targeting of its 3'- untranslated regions in resistant breast cancer cell lines, with a significant increase of doxorubicin influx by MDA-MB-231/Dox-resistant cells. Additionally, the current data validated a significant reduction of metastatic potentials upon miRNA-34a-5p upregulation in both types of breast cancer-resistant cells. CONCLUSION: The ectopic expression of miRNA-34a ameliorates the acquired drug resistance and the migration properties that may eventually lead to improved clinical strategies and outcomes for breast cancer patients. Additionally, miRNA-34a could be monitored as a diagnostic/prognostic biomarker for resistant conditions.

Multiplexed effector screening for recognition by endogenous resistance genes using positive defense reporters in wheat protoplasts.

Plant resistance (R) and pathogen avirulence (Avr) gene interactions play a vital role in pathogen resistance. Efficient molecular screening tools for crops lack far behind their model organism counterparts, yet they are essential to rapidly identify agriculturally important molecular interactions that trigger host resistance. Here, we have developed a novel wheat protoplast assay that enables efficient screening of Avr/R interactions at scale. Our assay allows access to the extensive gene pool of phenotypically described R genes because it does not require the overexpression of cloned R genes. It is suitable for multiplexed Avr screening, with interactions tested in pools of up to 50 Avr candidates. We identified Avr/R-induced defense genes to create a promoter-luciferase reporter. Then, we combined this with a dual-color ratiometric reporter system that normalizes read-outs accounting for experimental variability and Avr/R-induced cell death. Moreover, we introduced a self-replicative plasmid reducing the amount of plasmid used in the assay. Our assay increases the throughput of Avr candidate screening, accelerating the study of cellular defense signaling and resistance gene identification in wheat. We anticipate that our assay will significantly accelerate Avr identification for many wheat pathogens, leading to improved genome-guided pathogen surveillance and breeding of disease-resistant crops.

A promoter polymorphism defines distinct roles in anther development for Col-0 and Ler-0 alleles of Arabidopsis ACYL-COA BINDING PROTEIN3.

Acyl-CoA-Binding Proteins (ACBPs) bind acyl-CoA esters and function in lipid metabolism. Although acbp3-1, the ACBP3 mutant in Arabidopsis thaliana ecotype Col-0, displays normal floral development, the acbp3-2 mutant from ecotype Ler-0 characterized herein exhibits defective adaxial anther lobes and improper sporocyte formation. To understand these differences and identify the role of ERECTA in ACBP3 function, the acbp3 mutants and acbp3-erecta (er) lines were analyzed by microscopy for anther morphology and high-performance liquid chromatography for lipid composition. Defects in Landsberg anther development were related to the ERECTA-mediated pathway because the progenies of acbp3-2 × La-0 and acbp3-1 × er-1 in Col-0 showed normal anthers, contrasting to that of acbp3-2 in Ler-0. Polymorphism in the regulatory region of ACBP3 enabled its function in anther development in Ler-0 but not Col-0 which harbored an AT-repeat insertion. ACBP3 expression and anther development in acbp3-2 were restored using ACBP3pro (Ler)::ACBP3 not ACBP3pro (Col)::ACBP3. SPOROCYTELESS (SPL), a sporocyte formation regulator activated ACBP3 transcription in Ler-0 but not Col-0. For anther development, the ERECTA-related role of ACBP3 is required in Ler-0, but not Col-0. The disrupted promoter regulatory region for SPL binding in Col-0 eliminates the role of ACBP3 in anther development.

Functional characterization of variants found in Japanese patients with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant form of vascular dysplasia. Genetic diagnosis is made by identifying loss-of-function variants in genes, such as ENG and ACVRL1. However, the causal mechanisms of various variants of unknown significance remains unclear. In this study, we analyzed 12 Japanese patients from 11 families who were clinically diagnosed with HHT. Sequencing analysis identified 11 distinct variants in ACVRL1 and ENG. Three of the 11 were truncating variants, leading to a definitive diagnosis, whereas the remaining eight were splice-site and missense variants that required functional analyses. In silico splicing analyses demonstrated that three variants, c.526-3C > G and c.598C > G in ACVRL1, and c.690-1G > A in ENG, caused aberrant splicing, as confirmed by a minigene assay. The five remaining missense variants were p.Arg67Gln, p.Ile256Asn, p.Leu285Pro, and p.Pro424Leu in ACVRL and p.Pro165His in ENG. Nanoluciferase-based bioluminescence analyses demonstrated that these ACVRL1 variants impaired cell membrane trafficking, resulting in the loss of bone morphogenetic protein 9 (BMP9) signal transduction. In contrast, the ENG mutation impaired BMP9 signaling despite normal cell membrane expression. The updated functional analysis methods performed in this study will facilitate effective genetic testing and appropriate medical care for patients with HHT.

A reproducible and affordable method of conducting luciferase assay.

Commercially available glow luciferase assay kits are widely popular and convenient to use. However, concerning high-throughput screening, commercial kits are limited by huge running costs. As an alternative to commercial luciferase assay kits, this study presents a cost-effective and efficient methodology of performing a simple and rapid laboratory flash luciferase assay. The proposed luciferase assay method has a versatile use ranging from screening lysates in a microplate reader for quantitative assay as well as screening live cells qualitatively or quantitatively under an imaging system.

Development of a reporter gene-based assay for the bioactivity determination of rhLH pharmaceutical products.

To establish an in vitro biological activity detection method for luteinizing hormone (LH), the hLHCGR-CREB-HEK293 cell line was constructed to stably express human luteinizing hormone/chorionic gonadotropin receptor (hLHCGR). After optimization, the rhLH starting working concentration was 800 mIU/mL with 4-fold serial dilutions, 10 concentrations and an incubation time of 5 h. The method was confirmed to be highly specific, with good accuracy, precision and linearity, meeting the needs of process research and release testing, and can be used as a routine detection method for LH biological activity. With the increasing demand for research and development of rhLH biologically similar drugs, establishing a stable and simple activity assay method to evaluate the biological activity of rhLH can provide technical support for quality control of rhLH products and powerful tools for comparability research of similar products.

DNA-Dependent Protein Kinase Mediates YB-1 (Y-Box Binding Protein)-Induced Double Strand Break Repair.

BACKGROUND: DNA-PK (DNA-dependent protein kinase) is a stress-activated serine/threonine kinase that plays a central role in vascular smooth muscle cell proliferation and vascular proliferative disease processes such as neointimal formation. In this study, we link the activation of DNA-PK to the function of the transcription factor YB-1 (Y-box binding protein). METHODS: To identify YB-1 phosphorylation by DNA-PK, we generated different YB-1-expressing vectors. YB-1 nuclear translocation was investigated using immunoblotting and immunofluorescence staining. For YB-1 activity, luciferase assays were performed. RESULTS: We show by mutational analysis and kinase assay that the transcriptional regulator YB-1 is a substrate of DNA-PK. Blockade of DNA-PK by specific inhibitors revealed its critical involvement in YB-1phosphorylation as demonstrated by inhibition of an overexpressed YB-1 reporter construct. Using DNA-PK-deficient cells, we demonstrate that the shuttling of YB-1 from the cytoplasm to the nucleus is dependent on DNA-PK and that the N-terminal domain of YB-1 is phosphorylated at threonine 89. Point mutation of YB-1 at this residue abrogated the translocation of YB-1 into the nucleus. The phosphorylation of YB-1 by DNA-PK increased cellular DNA repair after exposure to ionizing radiation. Atherosclerotic tissue specimens were analyzed by immunohistochemistry. The DNA-PK subunits and YB-1 phosphorylated at T89 were found colocalized suggesting their in vivo interaction. In mice, the local application of the specific DNA-PK inhibitor NU7026 via thermosensitive Pluronic F-127 gel around dilated arteries significantly reduced the phosphorylation of YB-1. CONCLUSIONS: DNA-PK directly phosphorylates YB-1 and, this way, modulates YB-1 function. This interaction could be demonstrated in vivo, and colocalization in human atherosclerotic plaques suggests clinical relevance of our finding. Phosphorylation of YB-1 by DNA-PK may represent a novel mechanism governing atherosclerotic plaque progression.

Characterization of genital chlamydia trachomatis infection among women attending infertility and gynecology clinics in Hunan, China.

BACKGROUND: Genital infection with Chlamydia trachomatis (C. trachomatis) is a major public health issue worldwide. It can lead to cervicitis, urethritis, and infertility. This study was conducted to determine the characteristics of genital C. trachomatis infection among women attending to the infertility and gynecology clinics. METHODS: Endocervical swabs were collected from 8,221 women for C. trachomatis nucleotide screening and genotyping, while serum samples were collected for C. trachomatis pgp3 antibody determination using luciferase immunosorbent assays. RESULTS: High C. trachomatis DNA prevalence (3.76%) and seroprevalence (47.46%) rates were found, with genotype E (27.5%) being the most prevalent. C. trachomatis omp1 sense mutation was associated with cervical intraepithelial neoplasia (CIN) (odds ratio [OR] = 6.033, 95% confidence interval [CI] = 1.219-39.185, p = 0.045). No significant differences in C. trachomatis seroprevalence rates were observed between women with detectable C. trachomatis DNA in the infertility and routine physical examination groups (86.67% vs. 95%, p > 0.05); however, among women with negative C. trachomatis DNA, the former group had a markedly higher seroprevalence than the latter group (56.74% vs. 20.17%, p < 0.001). C. trachomatis DNA, but not pgp3 antibody, was significantly associated with CIN (OR = 4.087, 95% CI = 2.284-7.315, p < 0.001). CONCLUSION: Our results revealed a high prevalence, particularly seroprevalence, of C. trachomatis among women with infertility. Furthermore, we found an association between C. trachomatis omp1 sense mutations and CIN. Therefore, C. trachomatis serves as a risk factor for CIN.

Sensing chemical-induced DNA damage using CRISPR/Cas9-mediated gene-deletion yeast-reporter strains.

Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid-based reporter assays in budding yeast using a RNR3 promoter-driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid-based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid-based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. KEY POINTS: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast.