Predictive Assays Research Articles

MiR-11903a modulates CLIPB9-mediated pathogen defense and longevity in Aedes aegypti.

Arthropod melanization is a crucial defense mechanism mediated by a complex cascade of CLIP domain serine proteases (CLIPs). In this study, it was confirmed that microRNA-11903a (miR-11903a) targets Aedes-CLIPB9 (AeCLIPB9) by bioinformatics prediction and dual-luciferase reporter assays. Following intrathoracic injection of miR-11903a agomir and antagomir, Real-time quantitative polymerase chain reaction confirmed that AeCLIPB9 is negatively regulated by miR-11903a. Spatiotemporal expression analysis revealed that miR-11903a is most abundant in 4th instar larvae, followed by pupae and adults, and highly expressed in the wings, head, and midgut of female adults. Following pathogen infection, AeCLIPB9 and miR-11903a exhibited opposite expression trends, indicating their potential roles in mosquito innate immunity. To further investigate the relationship between AeCLIPB9 and miR-11903a, double-strand CLIPB9 was synthesized and RNA interference was performed. Seven-d survival assays revealed that both AeCLIPB9 and miR-11903a were crucial immune factors in fighting pathogens. Finally, longevity assays demonstrated that miR-11903a influenced mosquito lifespan.

P-108. Clinical Evaluation of a Sequencing-based Diagnostic for Bacterial and Fungal ID & AST Directly from Patient Blood Samples

Abstract Background Early pathogen ID and targeted treatment are key to reducing bloodstream infection (BSI) morbidity and mortality. Current diagnostics rely on culture which takes 1-2 days for ID and longer for antimicrobial susceptibility testing (AST) results, or molecular assays with limited panels, few resistance markers, and high false positive rates. We report interim results of a first-in-kind comprehensive ID and predictive AST assay directly from patient blood samples. The system can deliver results in ∼8 hrs, uses ultra-high enrichment of microbial DNA directly from blood, whole-genome sequencing, and a predictive AST machine-learning algorithm to identify a broad range of species and pathogen/drug combinations. Methods We enrolled subjects with suspicion of BSI from 3 EDs and 1 ICU/inpatient in 4 Boston area hospitals in 2 IRB approved observational studies. We collected whole blood in SPS vacutainers and 10mL were processed at Day Zero Diagnostics (DZD) and sequenced on an Oxford Nanopore platform. Sequencing data were analyzed by Keynome® algorithms to determine pathogen ID and predict AST profiles. Tables 1 & 2 list current on-panel pathogens and drug models tested. Performance was compared to hospital microbiology lab phenotypic ID/AST results from blood cultures collected within 0-24 hours of the research draw. Results Species-level ID results in 225 subjects (6525 calls) demonstrated 80.0% sensitivity, 99.9% specificity, 64% PPV, and 99.9% agreement with clinical culture. Eight distinct species were identified among the 20 (8.9%) clinical culture positive samples with on-panel organisms. Thirteen samples had sufficient genome coverage for on-panel AST predictions demonstrating 92.3% categoric agreement with phenotypic AST. Conclusion Our data suggests the DZD diagnostic system can provide accurate ID and AST results directly from blood in patients with suspected BSI. To our knowledge these results are the first demonstration of whole genome recovery and comprehensive ID & AST directly from patient blood samples. This assay has the potential to revolutionize speed to diagnosis of BSI, thus facilitating targeted therapy, improved outcomes, and reduced development of antimicrobial resistance. Disclosures Michael R. Filbin, MD, Day Zero Diagnostics: Grant/Research Support|Quidel: Grant/Research Support Peter Hou, MD, Center for Disease Control: Grant/Research Support|Day Zero Diagnostics: Grant/Research Support|iDoc Telehealth Solutions: Ownership Interest Michael Donnino, MD, Day Zero Diagnostics: Grant/Research Support Archana Asundi, MD, Day Zero Diagnostics: Grant/Research Support|Gilead Sciences: Grant/Research Support|GSK/ViiV Healthcare: Grant/Research Support|Theratechnologies: Grant/Research Support Zoe H. Rogers, MPH, Day Zero Diagnostics: employment Emma Briars, PhD, Day Zero Diagnostics: employment Alison Gassett, MPH, Day Zero Diagnostics: employment Alexander Reidel, BS, Day Zero Diagnostics: employment Alexis Campbell, MA, Day Zero Diagnostics: Grant/Research Support Jason Wittenbach, PhD, Day Zero Diagnostics: employment Nicole Billings, PhD, Day Zero Diagnostics: employment

Determining a point of departure for skin sensitization potency and quantitative risk assessment of fragrance ingredients using the GARDskin dose-response assay.

Potency and quantitative risk assessment are essential for determining safe concentrations for the formulation of potential skin sensitizers into consumer products. Several new approach methodologies (NAMs) for skin sensitization hazard assessment have been developed, validated, and adopted in OECD test guidelines. However, work is ongoing to develop NAMs for predicting skin sensitization potency on a quantitative scale for use as a point of departure (POD) in next-generation risk assessment (NGRA). GARDskin Dose-Response (DR) is an adaptation of the validated GARDskin assay (OECD TG 442E), and the readout of the assay is a quantitative potency prediction similar to the No Expected Sensitization Induction Level (NESIL) value (µg/cm2). The goal of this study was to evaluate the performance of the GARDskin DR assay for potency prediction of fragrance ingredients. One hundred (100) fragrance ingredients from a reference database covering varied structural reactivity domains and potency were tested in GARDskin DR. Materials tested had varied protein-binding reactivity alerts, including Schiff base, Michael addition, SN2, and acylation. Potency categories were predicted with a total accuracy of 37% and an approximate accuracy (exact match or off by 1 category) of 81%. Combining predicted weak and very weak categories increased total accuracy to 53% and approximate accuracy to 98%. The mean prediction error for the NESIL and local lymph node assay (LLNA) EC3 was 3.15- and 3.36-fold, respectively. Based on the results of this study, GARDskin DR is a promising predictor of skin sensitization potency with an applicability domain covering a wide range of fragrance ingredient reaction mechanisms, increasing the confidence in using the assay to conduct NGRA, ultimately reducing the need for animal testing.

Phytochemical, antioxidant, in silico, and in vitro anti-breast cancer effects of Erigeron sumatrensis from Gayo Highlands (Indonesia) against MCF-7 cells

Abstract. Puspa VR, Adista MA, Djufri. 2024. Phytochemical, antioxidant, in silico, and in vitro anti-breast cancer effects of Erigeron sumatrensis from Gayo Highlands (Indonesia) against MCF-7 cells. Biodiversitas 25: 5123-5137. This study investigated the therapeutic potential of Erigeron sumatrensis, a wild herb from the Gayo Highlands, by evaluating its methanol stem extract's phytochemical profile, antioxidant activity, biological activity, molecular docking, ADMET prediction, and MTT assay using MCF-7 cells. Phytochemical analysis revealed the presence of various secondary metabolites, including flavonoids, phenolics, terpenoids, steroids, tannins, and alkaloids. The stem extract exhibited the highest total phenolic content at 959.091 mgQE/g. Antioxidant activity, assessed using the DPPH assay, was strong with an IC50 value of 96 ?g/mL. Gas chromatography-mass spectrometry analysis of the methanol extract of E. sumatrensis stems led to the identification of 24 compounds. Molecular docking studies identified three compounds-Stigmast-7-en-3-ol, (3?,5?,24S)-, Caryophyllene oxide and 1H-Cycloprop[e]azulen-7-ol, decahydro- 1,1,7-trimethyl-4-methylene-, [1ar(1a?,4a?,7?,7a?,7b?)] exhibiting potential anti-proliferative activity against MCF-7 cells. ADMET analysis predicted compliance with all identified compounds, suggesting their suitability as oral drug candidates. The in vitro cytotoxicity assay against MCF-7 cells yielded a moderate cytotoxic classification with an IC50 of 192.90 ?g/mL. These findings highlight the promising potential of E. sumatrensis as a source of bioactive compounds for developing novel anti-breast cancer drugs. However, further research is needed to explore its full therapeutic potential and facilitate its development into pharmaceutical products.

Towards Personalized Radiotherapy in Pelvic Cancer: Patient-Related Risk Factors for Late Radiation Toxicity.

Normal tissue reactions vary significantly among patients receiving the same radiation treatment regimen, reflecting the multifactorial etiology of late radiation toxicity. Predicting late radiation toxicity is crucial, as it aids in the initial decision-making process regarding the treatment modalities. For patients undergoing radiotherapy, anticipating late toxicity allows for planning adjustments to optimize individualized care. Various dosimetric parameters have been shown to influence the incidence of late toxicity, and the literature available on this topic is extensive. This narrative review examines patient-related determinants of late toxicity following external beam radiotherapy for pelvic tumors, with a focus on prostate and cervical cancer patients. In Part I, we address various methods for quantifying radiation toxicity, providing context for interpreting toxicity data. Part II examines the current insights into the clinical risk factors for late toxicity. While certain factors-such as previous abdominal surgery, smoking behavior, and severe acute toxicity-have consistently been reported, most of the others show inconsistent associations. In Part III, we explore the influence of genetic factors and discuss promising predictive assays. Single-nucleotide polymorphisms (SNPs) likely elevate the risk in specific combinations. Advances in artificial intelligence now allow for the identification of SNP patterns from large datasets, supporting the development of polygenic risk scores. These innovations hold promise for improving personalized treatment strategies and reducing the burden of late toxicity in cancer survivors.

Galaxy @Sciensano: a comprehensive bioinformatics portal for genomics-based microbial typing, characterization, and outbreak detection

The influx of whole genome sequencing (WGS) data in the public health and clinical diagnostic sectors has created a need for data analysis methods and bioinformatics expertise, which can be a bottleneck for many laboratories. At Sciensano, the Belgian national public health institute, an intuitive and user-friendly bioinformatics tool portal was implemented using Galaxy, an open-source platform for data analysis and workflow creation. The Galaxy @Sciensano instance is available to both internal and external scientists and offers a wide range of tools provided by the community, complemented by over 50 custom tools and pipelines developed in-house. The tool selection is currently focused primarily on the analysis of WGS data generated using Illumina sequencing for microbial pathogen typing, characterization and outbreak detection, but it also addresses specific use cases for other data types. Our Galaxy instance includes several custom-developed 'push-button' pipelines, which are user-friendly and intuitive stand-alone tools that perform complete characterization of bacterial isolates based on WGS data and generate interactive HTML output reports with key findings. These pipelines include quality control, de novo assembly, sequence typing, antimicrobial resistance prediction and several relevant species-specific assays. They are tailored for pathogens with active genomic surveillance programs, and clinical relevance, such as Escherichia coli, Listeria monocytogenes, Salmonella spp. and Mycobacterium tuberculosis. These tools and pipelines utilize internationally recognized databases such as PubMLST, EnteroBase, and the NCBI National Database of Antibiotic Resistant Organisms, which are automatically synchronized on a regular basis to ensure up-to-date results. Many of these pipelines are part of the routine activities of Belgian national reference centers and laboratories, some of which use them under ISO accreditation. This resource is publicly available for noncommercial use at https://galaxy.sciensano.be/ and can help other laboratories establish reliable, traceable and reproducible bioinformatics analyses for pathogens encountered in public health settings.

Divergence in a Eukaryotic Transcription Factor's co-TF Dependence Involves Multiple Intrinsically Disordered Regions Affecting Activation and Autoinhibition.

Combinatorial control by multiple transcription factors (TFs) is a hallmark of eukaryotic gene regulation. Despite its prevalence and crucial roles in enhancing specificity and integrating information, the mechanisms behind why eukaryotic TFs depend on one another, and whether such interdependence evolves, are not well understood. We exploit natural variation in co-TF dependence in the yeast phosphate starvation (PHO) response to address this question. In the model yeast Saccharomyces cerevisiae , the main TF, Pho4, relies on the co-TF Pho2 to regulate ∼28 genes. In a related yeast pathogen, Candida glabrata , its Pho4 exhibits significantly reduced Pho2 dependence and has an expanded target set of ∼70 genes. Biochemical analyses showed C. glabrata Pho4 (CgPho4) binds to the same consensus motif with 3-4-fold higher affinity than ScPho4 does. A machine-learning-based prediction and yeast one-hybrid assay identified two Intrinsically Disordered Regions (IDRs) in CgPho4 that boost the activity of the main activation domain but showed little to no activity on their own. We also found evidence for autoinhibition behind the co-TF dependence in ScPho4. An IDR in ScPho4 next to its DNA binding domain was found to act as a double-edged sword: it both allows for enhanced activity with Pho2, and inhibits Pho4's activity without Pho2. This study provides a detailed molecular picture of how co-TF dependence is mediated and how its evolution, mainly driven by IDR divergence, can lead to significant rewiring of the regulatory network.

In vitro and In silico Degradation Study Applied to the Carbapenems Meropenem and Imipenem

Background: Imipenem and meropenem are the carbapenem antibiotics most commonly used for the treatment of severe bacterial infections. Their stability behavior, which focuses on degradation products, has been continually reported. Strategies based on in silico prediction have been explored as an additional tool that provides additional information about drug stability. Methods: A comparison between experimental and predictive data, with a focus on stability and degradation products, was performed by examining the literature data and the in silico results. The experimental data were obtained from the literature throughdatabase searches. The in silico degradation prediction was performed through the software Zeneth 9.0, which strictly focused on degradation products. With a focus on stability, degraded samples of meropenem and imipenem were investigated in terms of biological safety, applying an in vitro cytotoxicity assay by MTT reduction and neutral red uptake. Results: From the literature survey, it was found that 11 major degradation products for both carbapenems mainly formed from modifications on the b-lactam ring and dimerization. By using the Zeneth software, a large number of substances were predicted, considering the probability of occurring and the selected stressors. Even in low numbers, the comparative results indicated similarity in 4 degradation products, with high probability. About cytotoxicity, the cell viability by MTT assay was significantly reduced for imipenem samples degraded during 4 hours. For meropenem, applying neutral red uptake assay, a reduction of cell viability was observed in samples stored for 4 hours. Conclusion: Drug stability assay includes different approaches, which can be used favorably from the interaction between all areas of interest, including experimental practice and predictive modelling assay.

Exosomes derived from fibroblasts in DFUs delay wound healing by delivering miR-93-5p to target macrophage ATG16L1.

Diabetes is an extremely costly disease, one-third of which are attributed to the management of diabetic foot disease including chronic, non-healing, diabetic foot ulcers (DFUs). Therefore, much effort is needed to understand the pathogenesis of DFUs and novel therapeutics. We utilized exosome staining to confirm the interaction between fibroblast-derived exosomes and macrophages. Subsequently, we employed public data and qPCR to screen for upregulated miRNAs in fibroblast-derived exosomes in DFUs. The relationship between was validate miR-93-5 and ATG16L1 through data prediction and dual-luciferase reporter assays. A variety of molecular biology experiments were used for subsequent pathway validation. Additionally, we established Atg16l1MKI and Nlrp3MKO mice for further validation. We identified that miR-93-5p derived from fibroblasts played an important role in M1 macrophages polarization. Predicted by database, we found that miR-93-5p can bind to ATG16L1 mRNA, thereby influencing macrophage autophagy mediated by ATG16L1 in the clearance of ROS, thus activating the NLRP3 signaling pathway. In vivo, miR-93-5p antagomir treatment accelerated diabetic wound healing and induced M2 macrophage polarization. Fibroblasts and macrophages show cell crosstalk during the development of DFUs by miR-93-5p, and that antagomir treatment may be a promising and technically advantageous alternative to DFUs therapies.

Mechanism of Action of PARP Inhibitors

The selective vulnerability of BRCA1/2-mutated (BRCA mut) cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors provides one of the best examples of synthetic lethality that has been translated into the clinic. The success of this approach has led to a paradigm shift, with four PARP inhibitors now approved by the US Food and Drug Administration (FDA) for the treatment of ovarian, breast, prostate, and/or pancreatic cancers with BRCA mut. Furthermore, recent preclinical and clinical data suggest that many other types of solid tumors might also benefit from PARP inhibitors, regardless of their BRCA mut status. Despite this progress, resistance to PARP inhibitors is frequently observed in the clinic, which is, at least in part, due to the incomplete understanding of the mechanism of action of PARP inhibitors. In this review, we summarize the diverse processes underlying the signaling mechanisms of the PARP enzymes. We also discuss recent progress in utilizing these mechanistic insights for overcoming PARP inhibitor resistance, developing predictive biomarker assays, designing rational combination therapies, and, finally, developing the next-generation PARP1-targeting agents with a more complete and durable therapeutic response.

Network pharmacology and transcriptomics reveal androgen receptor as a potential protein target for 6PPD-quinone

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone (6PPD-quinone, or 6PPD-Q) has received increasing attention as an emerging hotspot contaminant. The occurrence of 6PPD-Q in dust and fine atmospheric particles indicates substantial human exposure to this toxicant but the hazards of 6PPD-Q to human health is unknown. We used in silico approaches to identify potential human protein targets of 6PPD-Q and conducted preliminary validation through an in vitro cell proliferation assay and an in vivo transcriptomic analysis of prostate tissues from 6PPD-Q-treated mice. Receptor-based reverse screening and network pharmacology identified four hub targets of 6PPD-Q that were closely related to prostate carcinogenesis. Among these four targets, 6PPD-Q exhibited a strong binding tendency to androgen receptor (AR) with a binding free energy of −23.04 kcal/mol. A support vector machine (SVM) model for predicting chemicals with AR agonism or AR-inactivity was established with good prediction performance (mean prediction accuracy: 0.92). SVM prediction and AR-mediated cell-based assays, with a known AR agonist and a proposed AR inactive agent as positive and negative controls, confirmed that 6PPD-Q displayed AR agonism. Upregulation of Ar mRNA expression (FC = 1.29, p = 0.0404) and its related prostate cancer pathway was observed in the prostate of mice exposed to environmentally realistic concentrations of 6PPD-Q, suggesting a potential role in promoting prostate carcinogenesis. These findings provide evidence that 6PPD-Q agonized AR to exert downstream gene transactivation and imply its prostate cancer risks to humans.

Cryo-EM structure of PML RBCC dimer reveals CC-mediated octopus-like nuclear body assembly mechanism

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are essential in regulating tumor suppression, antiviral response, inflammation, metabolism, aging, and other important life processes. The re-assembly of PML NBs might lead to an ~100% cure of acute promyelocytic leukemia. However, until now, the molecular mechanism underpinning PML NB biogenesis remains elusive due to the lack of structural information. In this study, we present the cryo-electron microscopy (cryo-EM) structure of the PML dimer at an overall resolution of 5.3 Å, encompassing the RING, B-box1/2 and part of the coiled-coil (RBCC) domains. The integrated approach, combining crosslinking and mass spectrometry (XL-MS) and functional analyses, enabled us to observe a unique folding event within the RBCC domains. The RING and B-box1/2 domains fold around the α3 helix, and the α6 helix serves as a pivotal interface for PML dimerization. More importantly, further characterizations of the cryo-EM structure in conjugation with AlphaFold2 prediction, XL-MS, and NB formation assays, help unveil an unprecedented octopus-like mechanism in NB assembly, wherein each CC helix of a PML dimer (PML dimer A) interacts with a CC helix from a neighboring PML dimer (PML dimer B) in an anti-parallel configuration, ultimately leading to the formation of a 2 µm membrane-less subcellular organelle.

Inhibition of CRLF1 expression by miR-8485 alleviates IL-1β-induced chondrocyte inflammation, apoptosis, and extracellular matrix degradation

The aim of this study was to investigate the impact of differentially expressed miR-8485 on chondrocyte inflammation in osteoarthritis (OA) and its underlying pathological mechanisms. MiR-8485, which was downregulated in OA, was identified by microarray analysis, and was also found to be decreased in IL-1β-induced C28/I2 cells. miR-8485 down-regulation or IL-1β treated of C28/I2 cells induces a decrease in cellular activity, an increase in apoptosis, an elevation in Cleaved caspase-3, MMP13, and ADAMTS5 protein levels, a decrease in Collagen II and Aggrecan levels, and an increase in the levels of pro-inflammatory factors TNF-α and IL-6. CRLF1 was identified to be a downstream target gene of miR-8485 using bioinformatics prediction and dual luciferase reporter gene assays. CRLF1 was shown to be increased in IL-1β-treated C28/I2 cells, and CRLF1 overexpression partially abrogated the suppressive effect of upregulated miR-8485 on chondrocyte inflammation. In addition, miR-8485 was able to inhibit MAPK/ERK and PI3K/AKT signaling activation by inhibiting CRLF1. In conclusion, miR-8485 was able to inhibit CRLF1 expression and thus inhibit IL-1β-triggered inflammation in chondrocytes, potentially through the inhibition of MAPK/ERK and PI3K/AKT signaling pathways.

A coronaviral pore-replicase complex links RNA synthesis and export from double-membrane vesicles.

Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the nonstructural viral membrane protein nsp4 is the key pore organizer, spanning the double membrane and forming most of the pore lining. Nsp4 interacts with nsp3 on the cytoplasmic side and with the viral replicase inside the DMV. Newly synthesized mRNAs exit the DMV into the cytoplasm, passing through a narrow ring of conserved nsp4 residues. Steric constraints imposed by the ring predict that modified nucleobases block mRNA transit, resulting in broad-spectrum anticoronaviral activity.

Genome-Wide Identification, Characterization and Expression Patterns of the DBB Transcription Factor Family Genes in Wheat.

Double B-box (DBB) proteins are plant-specific transcription factors (TFs) that play crucial roles in plant growth and stress responses. This study investigated the classification, structure, conserved motifs, chromosomal locations, cis-elements, duplication events, expression levels, and protein interaction network of the DBB TF family genes in common wheat (Triticum aestivum L.). In all, twenty-seven wheat DBB genes (TaDBBs) with two conserved B-box domains were identified and classified into six subgroups based on sequence features. A collinearity analysis of the DBB family genes among wheat, Arabidopsis, and rice revealed some duplicated gene pairs and highly conserved genes in wheat. An expression pattern analysis indicated that wheat TaDBBs were involved in plant growth, responses to drought stress, light/dark, and abscisic acid treatment. A large number of cis-acting regulatory elements related to light response are enriched in the predicted promoter regions of 27 TaDBBs. Furthermore, some of TaDBBs can interact with COP1 or HY5 based on the STRING database prediction and yeast two-hybrid (Y2H) assay, indicating the potential key roles of TaDBBs in the light signaling pathway. Conclusively, our study revealed the potential functions and regulatory mechanisms of TaDBBs in plant growth and development under drought stress, light, and abscisic acid.

A multi-model approach integrating whole-slide imaging and clinicopathologic features to predict breast cancer recurrence risk.

Breast cancer is the most common malignancy affecting women worldwide and is notable for its morphologic and biologic diversity, with varying risks of recurrence following treatment. The Oncotype DX Breast Recurrence Score test is an important predictive and prognostic genomic assay for estrogen receptor positive/HER2 negative breast cancer that guides therapeutic strategies; however, such tests can be expensive, delay care, and are not widely available. The aim of this study was to develop a multi-model approach integrating the analysis of whole-slide images and clinicopathologic data to predict their associated breast cancer recurrence risks and categorize these patients into two risk groups according to the predicted score: low-risk and high-risk. The proposed novel methodology uses convolutional neural networks for feature extraction and vision transformers for contextual aggregation, complemented by a logistic regression model that analyzes clinicopathologic data for classification into two risk categories. This method was trained and tested on 956 hematoxylin and eosin-stained whole-slide images of 950 ER+/HER2- breast cancer patients with corresponding clinicopathological features that had prior Oncotype DX testing. The model's performance was evaluated using an internal test set of 192 patients from Dartmouth Health and an external test set of 405 patients from the University of Chicago. The multi-model approach achieved an AUC of 0.91 (95% CI: 0.87-0.95) on the internal set and an AUC of 0.84 (95% CI: 0.78-0.89) on the external cohort for predicting low- and high-breast cancer recurrence risk categories based on the Oncotype DX recurrence score. With further validation, the proposed methodology could provide an alternative to assist clinicians in personalizing treatment for breast cancer patients and potentially improving their outcomes.

Flavin-Dependent Monooxygenase Kmy13 Mediates Formation of the Carbocyclic ansa System during Kendomycin B Biosynthesis.

Kendomycin B is distinguished from other ansamycins by its unique, fully carbogenic ansa scaffold. We show here that FAD-dependent monooxygenase Kmy13 is solely responsible for installing the rare ansa structural framework; in vivo gene disruption/complementation experiments and in vitro enzymatic assays are described in detail. Moreover, the compound with a β-keto ester, kendolactone A (2), was confirmed as the natural substrate of Kmy13 and a bona fide biosynthetic intermediate en route to kendomycin B. Further structural prediction and biochemical assays have provided significant insights into the catalytic mechanism of Kmy13.

LncRNA TUG1 regulates mir-34a-5p / SIRT6 to participate in benzene-induced hematotoxicity through PI3K / AKT /mTOR signaling pathway

LncRNA TUG1 plays pivotal roles in various diseases. However, its exact roles in benzene - induced hematotoxicity remain unclear. Herein, we aimed to investigate the role and mechanism of TUG1 in hematoxic injuries caused by benzene. In the current study, TUG1 was found dramatically decreased in WBCs of benzene exposure workers and negatively correlated with benzene exposure duration and urine SPMA. In vitro assays demonstrated that TUG1 overexpression attenuated 1,4-BQ-caused suppression of cell viability and proliferation, and promotion of ROS generation and apoptosis via PI3K/AKT/mTOR pathway. Bioinformatic prediction and molecular assay validated miR-34a-5p was negatively regulated by TUG1. The miR-34a-5p was upregulated in 1,4-BQ treated cells and downregulated in TUG1 overexpression cells. Moreover, miR-34a-5p upregulation partially reversed the protective effects of TUG1 overexpression on 1,4-BQ - caused cytotoxicity. Furthermore, SIRT6 was a downstream target gene of miR-34a-5p, whose expression was reduced in miR-34a-5p upregulation cells and elevated in TUG1 overexpression cells. Upregulated SIRT6 could counteract accelerated cytotoxicity mediated by miR-34a-5p upregulation after 1,4-BQ treatment. Taken together, our study revealed that the critical role of the TUG1/miR-34a-5p/SIRT6 axis in benzene-caused hematotoxicity, and provided scientific basis for further understanding the epigenetic regulatory mechanisms underlying benzene hematotoxicity.

Rutaecarpine alleviates inflammation and fibrosis by targeting CK2α in diabetic nephropathy

Diabetic nephropathy (DN) is one of the serious microvascular complications of diabetes mellitus. During the progression of DN, the proliferation of glomerular mesangial cells (GMCs) leads to the deposition of excessive extracellular matrix (ECM) in the mesangial region, eventually resulting in glomerulosclerosis. Rutaecarpine (Rut), an alkaloid found in the traditional Chinese medicinal herb Fructus Evodiae (Euodia rutaecarpa (Juss.) Benth.), has many biological activities. However, its mechanism of action in DN remains unknown. This study used db/db mice and high glucose (HG)-treated mouse mesangial cells (SV40 MES-13) to evaluate the protective effects of Rut and underlying mechanisms on GMCs in DN. We found that Rut alleviated urinary albumin and renal function and significantly relieved renal pathological damage. In addition, Rut decreased the ECM production, and renal inflammation and suppressed the activation of TGF-β1/Smad3 and NF-κB signaling pathways in vitro and in vivo. Protein kinase CK2α (CK2α) was identified as the target of Rut by target prediction, molecular docking, and cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR). Furthermore, Rut could not continue to play a protective role in HG-treated SV40 cells after silencing CK2α. In summary, this study is the first to find that Rut can suppress ECM production and inflammation in HG-treated SV40 cells by inhibiting the activation of TGF-β1/Smad3 and NF-κB signaling pathways and targeting CK2α. Thus, Rut can potentially become a novel treatment option for DN.

MiR-2110 Affects the Biological Behaviors of Lung Adenocarcinoma by Regulating CDT1

To investigate the effect of miR-2110 on the biological behaviors, such as cell proliferation, apoptosis, and metastasis, of lung adenocarcinoma (LUAD) cells by means of cell and animal experiments. Bioinformatics websites, including ENCORI, TargetScan, miRTarBase, and Tarbase, were used to analyze the changes in the expression of miR-2110 in LUAD samples and to predict miR-2110 target. LUAD tissue samples and cells were collected and the changes in the expression of miR-2110 were verified through PCR technology. CCK-8 assay, clonogenic assay, Transwell assay, and flow cytometry were conducted to analyze alterations in the functions of LUAD cells. In addition, 10 BALB/c female nude mice aged 6 to 8 weeks were randomly divided into 2 groups, and the effect of miR-2110 on LUAD was investigated by in vivo experiments. miR-2110 was significantly decreased in LUAD tissues and cells compared with the normal lung tissues. miR-2110 overexpression inhibited the proliferation and metastasis of LUAD cells and promoted the apoptosis of tumor cells (P<0.05). Bioinformatics prediction and dual luciferase reporter gene assay results confirmed that miR-2110 could target and bind to CDT1. In addition, overexpression of CDT1 gene reversed the proliferation, metastasis, and apoptosis of miR-2110 compared with the miR-2110 overexpression group (P<0.05). Nude mice in vivo experiments showed that miR-2110 overexpression significantly decreased the expression of Ki67, a tumor proliferation index, and vimentin and MMP9, two metastasis indices, compared with the control group. miR-2110 can inhibit proliferation and metastasis of LUAD by targeting CDT1, providing a new rationale for the treatment of LUAD.