Human induced pluripotent stem cell (hiPSC) technology has revolutionized studies on human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human ...diseases. Here, we use PiggyBac‐mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl‐tRNA synthetase (PylRS), pyrrolysyl‐tRNA (PylT) and the target protein of interest enables site‐specific incorporation of a non‐canonical amino acid (ncAA) in response to an amber stop codon. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA‐bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site‐directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC‐derived cell types and complex tissue models.
Brain organoids derived from human induced pluripotent stem cells (hiPSC) recapitulate key features of the developing brain and can model complex neurological disorders. Integrating the PylRS/PylT pair into the genome of hiPSCs allows efficient site‐directed incorporation of non‐canonical amino acids (ncAAs) and allows derivatization of neurons and brain organoids with an expanded genetic code. The method will make existing ncAA‐based strategies to probe and manipulate proteins in live cells accessible in hiPSC‐derived complex human disease models.
Introduction: Coumarins belong to the benzopyrones family. They are naturally plant-derived and synthetically taken polyphenolic substances, presenting a wide variety of biological activities and ...behaviours, supporting their use as therapeutic agents for multiple diseases. Their structural characteristics correlated to physicochemical properties seem to define the extent of the biological activity.Areas covered: Recent patent publications (2012 - 2014), describing coumarins and their derivatives are analyzed. Synthesis, hybridization techniques and biological evaluation in vitro/in vivo, for example, antimitotic, antiviral, anticancer, cytotoxic, anti-acne and antioxidant coumarin macromolecule polymer agents are included. Furthermore, a wide range of pharmaceutical applications and pharmaceutical compositions are also summarized.Expert opinion: Several natural and synthetic coumarins, hybrids and derivatives appear to have promising anticancer-antitumor activities. Their clinical evaluation will be critical to assess therapeutic utility. The compounds for which the mechanism of action is well defined can serve as lead compounds for the design of new more potent molecules.
Introduction:
Chalcones are a group of plant-derived polyphenolic compounds belonging to the flavonoids family that possess a wide variety of cytoprotective and modulatory functions, which may have ...therapeutic potential for multiple diseases. Their physicochemical properties seem to define the extent of their biological activity.
Areas covered:
A comprehensive synopsis of recent patent literature (2005 - 2011) describing chalcones and their derivatives on selected activities (e.g., anti-inflammatory, antimitotic, cytotoxic, antioxidant, anti-infection) is provided in this paper. Synthesis, combinatorial techniques, biological evaluation in vitro/in vivo, and new biological assays are discussed. In addition to selected biological data, a wide range of pharmaceutical applications and pharmaceutical compositions are also summarized.
Expert opinion:
Several natural and synthetic chalcones and their derivatives appear as promising anti-inflammatory and anticancer activities. Their clinical evaluation will be critical to assess their therapeutic utility. Those for which the mechanism of action is well defined can serve as lead compounds for the design of new, more promising molecules.
RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains ...elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.
In this study a series of curcumin analogues were evaluated for their ability to inhibit the activation of NF-κΒ, a transcription factor at the crossroads of cancer-inflammation. Our novel curcumin ...analogue BAT3 was identified to be the most potent NF-κB inhibitor and EMSA assays clearly showed inhibition of NF-κB/DNA-binding in the presence of BAT3, in agreement with reporter gene results. Immunofluorescence experiments demonstrated that BAT3 did not seem to prevent nuclear p65 translocation, so our novel analogue may interfere with NF-κB/DNA-binding or transactivation, independently of IKK2 regulation and NF-κB-translocation. Gene expression studies on endogenous NF-κB target genes revealed that BAT3 significantly inhibited TNF-dependent transcription of IL6, MCP1 and A20 genes, whereas an NF-κB independent target gene heme oxygenase-1 remained unaffected. In conclusion, we demonstrate that BAT3 seems to inhibit different cancer-related inflammatory targets in the NF-κB signaling pathway through a different mechanism in comparison to similar analogues, previously reported.
The tumor microenvironment (TME) is composed of highly heterogeneous structures and cell types that dynamically influence and communicate with each other. The constant interaction between a tumor and ...its microenvironment plays a critical role in how the cancer develops, progresses, and responds to therapies. Traditionally, Hematoxylin and Eosin (H&E) and immunohistochemistry staining have been used to annotate and characterize tissues and associated pathologies. Recent single analyte approaches spatially interrogate targeted or transcriptome‐wide expression of RNA in tissue sections, while others capture phenotypes using a limited number of protein markers. However, for a more comprehensive understanding of the unique characteristics of cell types, cell states, and cell‐cell interactions within the TME, analysis of multiple analytes is necessary.
Here we demonstrate a novel, streamlined multiomic spatial assay that integrates histological staining and imaging with simultaneous transcriptome‐wide gene expression and highly multiplexed protein expression profiling from the same formalin‐fixed paraffin embedded (FFPE) tissue section. In short, tissue sections from archived FFPE samples were placed on slides containing arrayed capture oligos with unique positional barcodes. The H&E stained tissues were then imaged, followed by incubation with transcriptome‐wide probes and a high‐plex DNA‐barcoded antibody panel containing intra‐ and extracellular markers. Transcriptome probes and antibody‐barcodes were then spatially captured on the slide and converted into sequencing‐ready libraries. Our data analysis and interactive visualization software enable interrogation of all data layers (H&E morphology, RNA, protein) from the same tissue section.
We apply this method to simultaneously measure gene and protein expression within the TME of human breast cancer and melanoma FFPE samples using whole transcriptome probes and an immune‐oncology antibody panel. The data enables comparison and correlation of multiple analytes and their patterns within the same sample section. In addition, this simultaneous detection enables marker‐guided regional selection and differential gene expression analysis on the defined areas. Taken together, our data demonstrates that a spatially resolved, multiomic approach provides a more comprehensive understanding of cellular behavior in and around tumors, yielding new insights into disease progression, predictive biomarkers, drug response and resistance, and therapeutic development.
Eukaryotic initiation factor 4A-III (eIF4A3), a core helicase component of the exon junction complex, is essential for splicing, mRNA trafficking, and nonsense-mediated decay processes emerging as ...targets in cancer therapy. Here, we unravel eIF4A3's tumor-promoting function by demonstrating its role in ribosome biogenesis (RiBi) and p53 (de)regulation. Mechanistically, eIF4A3 resides in nucleoli within the small subunit processome and regulates rRNA processing via R-loop clearance.
depletion induces cell cycle arrest through impaired RiBi checkpoint-mediated p53 induction and reprogrammed translation of cell cycle regulators. Multilevel omics analysis following
depletion pinpoints pathways of cell death regulation and translation of alternative mouse double minute homolog 2 (
) transcript isoforms that control p53. EIF4A3 expression and subnuclear localization among clinical cancer specimens correlate with the RiBi status rendering eIF4A3 an exploitable vulnerability in high-RiBi tumors. We propose a concept of eIF4A3's unexpected role in RiBi, with implications for cancer pathogenesis and treatment.
Abstract
9-Substituted (pyrazol-5-yl)methyl- or (2-pyrazolin-5-yl)methyl-9H-purines were synthesized from 9-allyl-6-chloro-9H-purine through the 1,3-dipolar cycloaddition reaction with nitrile ...imines, prepared in situ from the corresponding hydrazone and NBS/Et3N under MW or from hydrazinoylchloride and Et3N under reflux. The coupling of new 6-chloropurines with amines in H2O under microwaves resulted quantitatively to modified pyrazol-5-yl- or 2-pyrazolin-5-yl adenine homo-N-nucleosides. The new compounds were tested in vitro for their ability to: (i) interact with 1,1-diphenyl-2-picryl-hydrazyl (DPPH), (ii) inhibit lipid peroxidation, (iii) inhibit the activity of soybean lipoxygenase, (iv) inhibit in vitro thrombin and for (v) their antiproliferative and cytotoxic activity. Pyrazolines were found to be more potent in vitro. Compound 7a exhibited satisfactory combined antioxidant and anti-lipid peroxidation activity, inhibition of lipoxygenase (89%) and thrombin inhibitory ability, whereas compound 7b exhibited high lipoxygenase inhibitory activity in combination to significant anti-thrombin activity. No compound exhibited a significant cytotoxic activity, while all showed moderate antiproliferative activity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Brain organoids derived from human induced pluripotent stem cells (hiPSC) recapitulate key features of the developing brain and can model complex neurological disorders. Integrating the PylRS/PylT ...pair into the genome of hiPSCs enables efficient site‐directed incorporation of non‐canonical amino acids (ncAAs) and allows derivatization of neurons and brain organoids with an expanded genetic code. The cover feature demonstrates how PylT recodes an amber stop codon within a GFP reporter to direct site‐specific incorporation of an ncAA, leading to GFP fluorescence throughout the organoid, as visible by light‐sheet microscopy. The method will help to implement existing ncAA‐based strategies for probing and manipulating proteins in hiPSC‐derived complex human disease models. More information can be found in the Full Paper by L. van Husen et al.
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