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BioRxiv : the Preprint Server For... Jan 2024As microtubule-organizing centers, centrosomes direct assembly of the bipolar mitotic spindle required for chromosome segregation and genome stability. Centrosome...
As microtubule-organizing centers, centrosomes direct assembly of the bipolar mitotic spindle required for chromosome segregation and genome stability. Centrosome activity requires the dynamic assembly of pericentriolar material (PCM), the composition and organization of which changes throughout the cell cycle. Recent studies highlight the conserved localization of several mRNAs encoded from centrosome-associated genes enriched at centrosomes, including () mRNA. However, relatively little is known about how RNAs localize to centrosomes and influence centrosome function. Here, we examine mechanisms underlying the subcellular localization of mRNA. We find that mRNA localization is puromycin-sensitive, and the coding sequence is both necessary and sufficient for RNA localization, consistent with a co-translational transport mechanism. We identify regions within the coding sequence that regulate mRNA localization. Finally, we show that protein-protein interactions critical for elaboration of the PCM scaffold permit RNA localization to centrosomes. Taken together, these findings inform the mechanistic basis of mRNA localization and lend insight into the oscillatory enrichment of RNA at centrosomes.
PubMed: 38469150
DOI: 10.1101/2024.01.13.575509 -
Nucleic Acids Research Sep 2023We describe a novel method for in vitro protein display-click display-that does not depend on maintaining RNA integrity during biopanning and yields covalently linked...
We describe a novel method for in vitro protein display-click display-that does not depend on maintaining RNA integrity during biopanning and yields covalently linked protein-cDNA complexes from double-stranded input DNA within 2 h. The display is achieved in a one-pot format encompassing transcription, translation and reverse transcription reactions in series. Stable linkage between proteins and the encoding cDNA is mediated by a modified DNA linker-ML-generated via a click chemistry reaction between a puromycin-containing oligo and a cDNA synthesis primer. Biopanning of a click-displayed mock library coupled with next-generation sequencing analysis revealed >600-fold enrichment of target binders within a single round of panning. A synthetic library of Designed Ankyrin Repeat Proteins (DARPins) with ∼1012 individual members was generated using click display in a 25-μl reaction and six rounds of library panning against a model protein yielded a panel of nanomolar binders. This study establishes click display as a powerful tool for protein binder discovery/engineering and provides a convenient platform for in vitro biopanning selection even in RNase-rich environments such as on whole cells.
Topics: DNA; DNA, Complementary; Peptide Library; Protein Engineering; Proteins; Directed Molecular Evolution
PubMed: 37548398
DOI: 10.1093/nar/gkad643 -
Langenbeck's Archives of Surgery Aug 2023Colorectal liver metastases (CRLM) are the predominant factor limiting survival in patients with colorectal cancer. Multimodal treatment strategies are frequently...
PURPOSE
Colorectal liver metastases (CRLM) are the predominant factor limiting survival in patients with colorectal cancer. Multimodal treatment strategies are frequently necessary to achieve total tumor elimination. This study examines the efficacy of liver resection combined with local ablative therapy in comparison to liver resection only, in the treatment of patients with ≥ 4 CRLM.
METHODS
This retrospective cohort study was conducted at the University Hospital RWTH Aachen, Germany. Patients with ≥ 4 CRLM in preoperative imaging, who underwent curative resection between 2010-2021, were included. Recurrent resections and deaths in the early postoperative phase were excluded. Ablation modalities included radiofrequency or microwave ablation, and irreversible electroporation. Differences in overall- (OS) and recurrence-free-survival (RFS) between patients undergoing combined resection-ablation vs. resection only, were examined.
RESULTS
Of 178 included patients, 46 (27%) underwent combined resection-ablation and 132 (73%) resection only. Apart from increased rates of adjuvant chemotherapy in the first group (44% vs. 25%, p = 0.014), there were no differences in perioperative systemic therapy. Kaplan-Meier and log-rank test analyses showed no statistically significant differences in median OS (36 months for both, p = 0.638) or RFS (9 months for combined resection-ablation vs. 8 months, p = 0.921). Cox regression analysis showed a hazard ratio of 0.891 (p = 0.642) for OS and 0.981 (p = 0.924) for RFS, for patients undergoing resection only.
CONCLUSION
For patients with ≥ 4 CRLM, combined resection-ablation is a viable option in terms of OS and RFS. Therefore, combined resection-ablation should be considered for complete tumor clearance, in patients with multifocal disease.
Topics: Humans; Retrospective Studies; Liver Neoplasms; Hepatectomy; Chemotherapy, Adjuvant; Puromycin; Colorectal Neoplasms
PubMed: 37642753
DOI: 10.1007/s00423-023-03082-1 -
American Journal of Physiology. Renal... Jan 2024As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have...
As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes (p16INK4a/p14ARF), (p19INK4d), and (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of , , and In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated β-galactosidase (SA-β-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan black staining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span. Glomerular function is decreased by aging. However, little is known about the molecular mechanisms involved in age-related glomerular changes and which factors could contribute to a worse glomerular aging process. Here, we reported that podocyte injury in young mice and culture podocytes induced senescence, a marker of aging, and accelerates glomerular aging when compared with healthy aging mice.
Topics: Middle Aged; Humans; Mice; Animals; Aged; Podocytes; Glomerulosclerosis, Focal Segmental; Kidney Glomerulus; Kidney Diseases; Aging; Doxorubicin
PubMed: 37855038
DOI: 10.1152/ajprenal.00261.2023 -
BioRxiv : the Preprint Server For... Jul 2023parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. New antimalarial drugs and novel drug...
parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. New antimalarial drugs and novel drug targets are in critical need, along with novel assays to accelerate their identification. Given the essentiality of protein synthesis throughout the complex parasite lifecycle, translation inhibitors are a promising drug class, capable of targeting the disease-causing blood stage of infection, as well as the asymptomatic liver stage, a crucial target for prophylaxis. To identify compounds capable of inhibiting liver stage parasite translation, we developed an assay to visualize and quantify translation in the HepG2 infection model. After labeling infected monolayers with o-propargyl puromycin (OPP), a functionalized analog of puromycin permitting subsequent bioorthogonal addition of a fluorophore to each OPP-terminated nascent polypetide, we use automated confocal feedback microscopy followed by batch image segmentation and feature extraction to visualize and quantify the nascent proteome in individual liver stage parasites and host cells simultaneously. After validation, we demonstrate specific, concentration-dependent liver stage translation inhibition by both parasite-selective and pan-eukaryotic active compounds, and further show that acute pre-treatment and competition modes of the OPP assay can distinguish between direct and indirect translation inhibitors. We identify a Malaria Box compound, MMV019266, as a direct translation inhibitor in liver stages and confirm this potential mode of action in asexual blood stages.
PubMed: 37461595
DOI: 10.1101/2023.07.05.547872 -
Cancer Research May 2024There is an unmet need to improve the efficacy of platinum-based cancer chemotherapy, which is used in primary and metastatic settings in many cancer types. In bladder...
UNLABELLED
There is an unmet need to improve the efficacy of platinum-based cancer chemotherapy, which is used in primary and metastatic settings in many cancer types. In bladder cancer, platinum-based chemotherapy leads to better outcomes in a subset of patients when used in the neoadjuvant setting or in combination with immunotherapy for advanced disease. Despite such promising results, extending the benefits of platinum drugs to a greater number of patients is highly desirable. Using the multiomic assessment of cisplatin-responsive and -resistant human bladder cancer cell lines and whole-genome CRISPR screens, we identified puromycin-sensitive aminopeptidase (NPEPPS) as a driver of cisplatin resistance. NPEPPS depletion sensitized resistant bladder cancer cells to cisplatin in vitro and in vivo. Conversely, overexpression of NPEPPS in sensitive cells increased cisplatin resistance. NPEPPS affected treatment response by regulating intracellular cisplatin concentrations. Patient-derived organoids (PDO) generated from bladder cancer samples before and after cisplatin-based treatment, and from patients who did not receive cisplatin, were evaluated for sensitivity to cisplatin, which was concordant with clinical response. In the PDOs, depletion or pharmacologic inhibition of NPEPPS increased cisplatin sensitivity, while NPEPPS overexpression conferred resistance. Our data present NPEPPS as a druggable driver of cisplatin resistance by regulating intracellular cisplatin concentrations.
SIGNIFICANCE
Targeting NPEPPS, which induces cisplatin resistance by controlling intracellular drug concentrations, is a potential strategy to improve patient responses to platinum-based therapies and lower treatment-associated toxicities.
Topics: Humans; Drug Resistance, Neoplasm; Cisplatin; Urinary Bladder Neoplasms; Animals; Mice; Cell Line, Tumor; Aminopeptidases; Xenograft Model Antitumor Assays; Antineoplastic Agents; Organoids
PubMed: 38535994
DOI: 10.1158/0008-5472.CAN-23-1976 -
STAR Protocols Dec 2023Protein synthesis, or mRNA translation, is the biological process through which genetic information stored in messenger RNAs is encoded into proteins. Here, we present...
Protein synthesis, or mRNA translation, is the biological process through which genetic information stored in messenger RNAs is encoded into proteins. Here, we present an optimized protocol for assessing the translation rate in mouse adult microglia and cultured bone-marrow-derived macrophages. We describe steps for isolating cells, treating them with a puromycin-analog probe, and fluorescently labeling the puromycylated-polypeptide chains. We then detail their quantification by flow cytometry or with a fluorescent plate reader. For complete details on the use and execution of this protocol, please refer to Keane et al. (2021)..
Topics: Animals; Mice; Microglia; Bone Marrow; Macrophages; Coloring Agents; Protein Biosynthesis
PubMed: 37713309
DOI: 10.1016/j.xpro.2023.102559 -
Cancer Cell International Mar 2024Resistance to targeted therapies represents a significant hurdle to successfully treating hepatocellular carcinoma (HCC). While epigenetic abnormalities are critical...
BACKGROUND
Resistance to targeted therapies represents a significant hurdle to successfully treating hepatocellular carcinoma (HCC). While epigenetic abnormalities are critical determinants of HCC relapse and therapeutic resistance, the underlying mechanisms are poorly understood. We aimed to address whether and how dysregulated epigenetic regulators have regulatory and functional communications in establishing and maintaining drug resistance.
METHODS
HCC-resistant cells were characterized by CCK-8, IncuCyte Live-Cell analysis, flow cytometry and wound-healing assays. Target expression was assessed by qPCR and Western blotting. Global and promoter DNA methylation was measured by dotblotting, methylated-DNA immunoprecipitation and enzymatic digestion. Protein interaction and promoter binding of DNMT3a-TET2 were investigated by co-immunoprecipitation, ChIP-qPCR. The regulatory and functional roles of DNMT3a and TET2 were studied by lentivirus infection and puromycin selection. The association of DNMT and TET expression with drug response and survival of HCC patients was assessed by public datasets, spearman correlation coefficients and online tools.
RESULTS
We identified the coordination of DNMT3a and TET2 as an actionable mechanism of drug resistance in HCC. The faster growth and migration of resistant HCC cells were attributed to DNMT3a and TET2 upregulation followed by increased 5mC and 5hmC production. HCC patients with higher DNMT3a and TET2 had a shorter survival time with a less favorable response to sorafenib therapy than those with lower expression. Cancer stem cell-like cells (CSCs) displayed DNMT3a and TET2 overexpression, which were insensitive to sorafenib. Either genetic or pharmacological suppression of DNMT3a or/and TET2 impaired resistant cell growth and oncosphere formation, and restored sorafenib sensitivity. Mechanistically, DNMT3a did not establish a regulatory circuit with TET2, but formed a complex with TET2 and HDAC2. This complex bound the promoters of oncogenes (i.e., CDK1, CCNA2, RASEF), and upregulated them without involving promoter DNA methylation. In contrast, DNMT3a-TET2 crosstalk silences tumor suppressors (i.e., P15, SOCS2) through a corepressor complex with HDAC2 along with increased promoter DNA methylation.
CONCLUSIONS
We demonstrate that DNMT3a and TET2 act coordinately to regulate HCC cell fate in DNA methylation-dependent and -independent manners, representing strong predictors for drug resistance and poor prognosis, and thus are promising therapeutic targets for refractory HCC.
PubMed: 38528605
DOI: 10.1186/s12935-024-03288-3 -
Environmental Toxicology and... Nov 2023Our study aimed to verify the hypothesis concerning low-frequency magnetic fields (LF-MFs)-related changes in cell viability through the biomechanism(s) based on...
Our study aimed to verify the hypothesis concerning low-frequency magnetic fields (LF-MFs)-related changes in cell viability through the biomechanism(s) based on calcineurin (CaN)-mediated signaling pathways triggered via ROS-like molecules. For experiments, Mono Mac 6 and U937 leukocytic cell lines were chosen and exposed to various LF-MFs and/or puromycin (PMC). The protein expression level of key regulatory proteins of calcium metabolism was examined by Western Blot analysis. In turn, the reactive oxygen species (ROS) and cell viability parameters were evaluated by cytochrome C reduction assay and flow cytometry, respectively. The simultaneous action of applied MF and PMC influenced cell viability in a MF-dependent manner. The changes in cell viability were correlated with protein expression and ROS levels. It was verified experimentally that applied stress stimuli influence cell susceptibility to undergo cell death. Moreover, the evoked bioeffects might be recognized as specific to both types of leukocyte populations.
Topics: Electromagnetic Fields; Reactive Oxygen Species; Calcium; Cell Line; Puromycin; Leukocytes
PubMed: 37984675
DOI: 10.1016/j.etap.2023.104320 -
Journal of Pediatric Surgery Jul 2023To induce the expression of Amphinase, an antitumor ribonuclease from Rana pipiens oocytes, in neuroblastoma cell lines and build a foundation for mechanism study.
PURPOSE
To induce the expression of Amphinase, an antitumor ribonuclease from Rana pipiens oocytes, in neuroblastoma cell lines and build a foundation for mechanism study.
METHODS
A loxP-cassette vector was constructed comprising a sequence of loxP -Puro-3∗polyA-loxP, followed by amphinase cDNA. The vector was transfected into neuroblastoma cell lines, SK-N-BE(2)-C, by Lipofectamine LTX. The transfected cells were selected by puromycin for two weeks. Polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) were conducted to verify that the loxP-cassette vector was stably transfected. The expression of amphinase was activated by the addition of Cre recombinase delivered by a lentiviral vector and identified by qPCR and Western blotting (WB). CCK8 assay and colony formation assay were conducted to check the effect of amphinase on cell proliferation. RNA sequencing (RNA-seq) was conducted to explore the targeted pathway of Cre/loxP-mediated amphinase and recombinant amphinase.
RESULTS
Stably transfected cell clones were achieved through puromycin selection. After Cre recombinase was delivered to the cells, the loxP-flanked fragment was deleted and the expression of amphinase was induced, which were tested by PCR and qPCR. It was shown that cell proliferation was significantly inhibited by the amphinase mediated by the Cre/loxP system. KEGG enrichment and GSEA analysis indicated that amphinase had an impact on the ER function of neuroblastoma cells, which was identical to the effect of the recombinant amphinase.
CONCLUSION
We successfully induce the expression of amphinase in neuroblastoma cell lines via Cre/loxP system. The Cre/loxP-mediated amphinase had a similar antitumor mechanism to the recombinant amphinase, providing a powerful tool for the mechanism study of amphinase.
Topics: Humans; Genetic Vectors; Integrases; Cell Line; Neuroblastoma
PubMed: 36973104
DOI: 10.1016/j.jpedsurg.2023.02.037