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Blood Advances Mar 2024Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth...
Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.
Topics: Mice; Humans; Animals; Platelet Aggregation; Thrombin; Thrombosis; Thromboxanes; Puromycin
PubMed: 38163324
DOI: 10.1182/bloodadvances.2023011734 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Dec 2023With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its...
With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 () is believed to have anti-brucella potential. Exploring the impack of on goat SSCs not only helps investigate the ability of to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a overexpression vector was constructed by homologous recombination. The overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (, , ), and , a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of in goat spermatogonia and provided a cell platform for further study into the biological function of . These findings also establish a foundation for breeding overexpressing goats.
Topics: Animals; Male; Goats; Stem Cells; Spermatogonia; Cell Proliferation; Flow Cytometry; Testis
PubMed: 38147990
DOI: 10.13345/j.cjb.230570 -
Zhongguo Zhong Yao Za Zhi = Zhongguo... Nov 2023This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted...
This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.
Topics: Humans; Child; Rats; Animals; Puromycin Aminonucleoside; Metabolomics; Biomarkers; Chromatography, High Pressure Liquid; Acetophenones; Glomerulonephritis; Phenylalanine; Amino Acids
PubMed: 38114186
DOI: 10.19540/j.cnki.cjcmm.20230707.406 -
BioRxiv : the Preprint Server For... Dec 2023Protein synthesis is a core cellular process, necessary throughout the complex lifecycle of parasites, thus specific translation inhibitors would be a valuable class of...
Protein synthesis is a core cellular process, necessary throughout the complex lifecycle of parasites, thus specific translation inhibitors would be a valuable class of antimalarial drugs, capable of both treating symptomatic infections in the blood and providing chemoprotection by targeting the initial parasite population in the liver, preventing both human disease and parasite transmission back to the mosquito host. As increasing numbers of antiplasmodial compounds are identified that converge mechanistically at inhibition of cytoplasmic translation, regardless of molecular target or mechanism, it would be useful to gain deeper understanding of how their effectiveness as liver stage translation inhibitors relates to their chemoprotective potential. Here, we probed that relationship using the -HepG2 liver stage infection model. Using o-propargyl puromycin-based labeling of the nascent proteome in infected HepG2 monolayers coupled with automated confocal feedback microscopy to generate unbiased, single parasite image sets of liver stage translation, we determined translation inhibition EC for five compounds, encompassing parasite-specific aminoacyl tRNA synthetase inhibitors, compounds targeting the ribosome in both host and parasite, as well as DDD107498, which targets eEF2, and is a leading antimalarial candidate compound being clinically developed as cabamiquine. Compounds were then tested at equivalent effective concentrations to compare the parasite response to, and recovery from, a brief period of translation inhibition in early schizogony, with parasites followed up to 120 hours post-infection to assess liver stage antiplasmodial effects of the treatment. Our data conclusively show that translation inhibition efficacy does not determine a translation inhibitor's antiplasmodial efficacy. DDD107498 was the least effective translation inhibitor, yet exerted the strongest antimalarial effects at both 5x- and 10x EC concentrations. We show compound-specific heterogeneity in single parasite and population responses to translation inhibitor treatment, with no single metric strongly correlated to release of hepatic merozoites for all compound, demonstrate that DDD107498 is capable of exerting antiplasmodial effects on translationally arrested liver stage parasites, and uncover unexpected growth dynamics during the liver stage. Our results demonstrate that translation inhibition efficacy cannot function as a proxy for antiplasmodial effectiveness, and highlight the importance of exploring the ultimate, as well as proximate, mechanisms of action of these compounds on liver stage parasites.
PubMed: 38106175
DOI: 10.1101/2023.12.07.570699 -
Biochemistry. Biokhimiia Nov 2023In response to stress stimuli, eukaryotic cells typically suppress protein synthesis. This leads to the release of mRNAs from polysomes, their condensation with...
In response to stress stimuli, eukaryotic cells typically suppress protein synthesis. This leads to the release of mRNAs from polysomes, their condensation with RNA-binding proteins, and the formation of non-membrane-bound cytoplasmic compartments called stress granules (SGs). SGs contain 40S but generally lack 60S ribosomal subunits. It is known that cycloheximide, emetine, and anisomycin, the ribosome inhibitors that block the progression of 80S ribosomes along mRNA and stabilize polysomes, prevent SG assembly. Conversely, puromycin, which induces premature termination, releases mRNA from polysomes and stimulates the formation of SGs. The same effect is caused by some translation initiation inhibitors, which lead to polysome disassembly and the accumulation of mRNAs in the form of stalled 48S preinitiation complexes. Based on these and other data, it is believed that the trigger for SG formation is the presence of mRNA with extended ribosome-free segments, which tend to form condensates in the cell. In this study, we evaluated the ability of various small-molecule translation inhibitors to block or stimulate the assembly of SGs under conditions of severe oxidative stress induced by sodium arsenite. Contrary to expectations, we found that ribosome-targeting elongation inhibitors of a specific type, which arrest solitary 80S ribosomes at the beginning of the mRNA coding regions but do not interfere with all subsequent ribosomes in completing translation and leaving the transcripts (such as harringtonine, lactimidomycin, or T-2 toxin), completely prevent the formation of arsenite-induced SGs. These observations suggest that the presence of even a single 80S ribosome on mRNA is sufficient to prevent its recruitment into SGs, and the presence of extended ribosome-free regions of mRNA is not sufficient for SG formation. We propose that mRNA entry into SGs may be mediated by specific contacts between RNA-binding proteins and those regions on 40S subunits that remain inaccessible when ribosomes are associated.
Topics: RNA, Messenger; Protein Biosynthesis; Stress Granules; Cytoplasmic Granules; Ribosomes; Protein Synthesis Inhibitors; RNA-Binding Proteins
PubMed: 38105199
DOI: 10.1134/S000629792311010X -
Inflammation Apr 2024To explore the role of Notch1 pathway in the pathogenesis of podocyte injury, and to provide novel strategy for podocyte repair in lupus nephritis (LN). Bioinformatics...
To explore the role of Notch1 pathway in the pathogenesis of podocyte injury, and to provide novel strategy for podocyte repair in lupus nephritis (LN). Bioinformatics analysis and immunofluorescence assay were applied to determine the expression and localization of Notch1 intracellular domain1 (NICD1) in kidneys of LN patients and MRL/lpr mice. The stable podocyte injury model in vitro was established by puromycin aminonucleoside (PAN) treatment. Expression of inflammasome activation related gene was detected by qPCR. The podocytes with PAN treatment were cultured with or without N-S-phenyl-glycine-t-butylester (DAPT), an inhibitor of Notch1 pathway. NICD1, Wilm'stumor1 (WT1), nucleotide-binding oligomerization domain-like receptors 3 (NLRP3), and absent in melanoma-like receptors 2 (AIM2) were detected by western blot. In vivo, MRL/lpr mice were administrated with DAPT or vehicle. The LN symptoms were assessed. The podocyte injury was evaluated, and the NLRP3 in podocytes of mice was detected. Notch1 pathway was overactivated in glomeruli of LN patients. NICD1 was colocalized with podocytes of LN patients and MRL/lpr mice. The inflammasome-related genes were significantly increased in podocytes with PAN treatment. NICD1 and NLRP3 were significantly decreased, while WT1 was significantly increased in injured podocytes treated with DAPT in vitro. In vivo, lupus-like symptoms were alleviated in DAPT treatment group. Notch1 pathway was inhibited in kidneys of mice treated with DAPT. The renal inflammation was reduced and the podocyte injury was mitigated in DAPT treatment group. The NLRP3 was decreased in podocytes of mice treated with DAPT. Notch1 pathway was overactivated in podocytes of LN patients and MRL/lpr mice. Blockade of Notch1 pathway reduced renal inflammation and alleviated podocyte injury via inhibition of NLRP3 inflammasome activation in LN.
Topics: Podocytes; Animals; Lupus Nephritis; NLR Family, Pyrin Domain-Containing 3 Protein; Receptor, Notch1; Mice; Inflammasomes; Signal Transduction; Humans; Mice, Inbred MRL lpr; Female; Dipeptides; Adult
PubMed: 38085465
DOI: 10.1007/s10753-023-01935-x -
Liver Cancer Dec 2023Complete resection is the only possible treatment for cholangiocarcinoma in the extrahepatic biliary tree (eCCA), although current imaging modalities are limited in...
INTRODUCTION
Complete resection is the only possible treatment for cholangiocarcinoma in the extrahepatic biliary tree (eCCA), although current imaging modalities are limited in their ability to accurately diagnose longitudinal spread. We aimed to develop fluorescence imaging techniques for real-time identification of eCCA using an enzyme-activatable probe, which emits fluorescence immediately after activation by a cancer-specific enzyme.
METHODS
Using lysates and small tissue fragments collected from surgically resected specimens, we selected the most specific probe for eCCA from among 800 enzyme-activatable probes. The selected probe was directly sprayed onto resected specimens and fluorescence images were acquired; these images were evaluated for diagnostic accuracy. We also comprehensively searched for enzymes that could activate the probe, then compared their expression levels in cancer and non-cancer tissues.
RESULTS
Analyses of 19 samples (four cancer lysates, seven non-cancer lysates, and eight bile samples) and 54 tissue fragments (13 cancer tissues and 41 non-cancer tissues) revealed that PM-2MeSiR was the most specific fluorophore for eCCA. Fluorescence images of 7 patients were obtained; these images enabled rapid identification of cancerous regions, which closely matched histopathology findings in 4 patients. Puromycin-sensitive aminopeptidase was identified as the enzyme that might activate the probe, and its expression was upregulated in eCCA.
CONCLUSION
Fluorescence imaging with PM-2MeSiR, which may be activated by puromycin-sensitive aminopeptidase, yielded generally high accuracy. This technique may be useful for real-time identification of the spread of eCCA during surgery and endoscopic examinations.
PubMed: 38058421
DOI: 10.1159/000530645 -
Journal of Experimental & Clinical... Dec 2023Colorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism. Ferroptosis, a programmed cell death dependent on iron,...
BACKGROUND
Colorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism. Ferroptosis, a programmed cell death dependent on iron, has emerged as a promising strategy for CRC treatment. Although small nucleolar RNAs are extensively involved in carcinogenesis, it is unclear if they regulate ferroptosis during CRC pathogenesis.
METHODS
The dysregulated snoRNAs were identified using published sequencing data of CRC tissues. The expression of the candidate snoRNAs, host gene and target gene were assessed by real-time quantitative PCR (RT-qPCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and western blots. The biological function of critical molecules was investigated using in vitro and in vivo strategies including Cell Counting Kit-8 (CCK8), colony formation assay, flow cytometry, Fe/Fe, GSH/GSSG and the xenograft mice models. The ribosomal activities were determined by polysome profiling and O-propargyl-puromycin (OP-Puro) assay. The proteomics was conducted to clarify the downstream targets and the underlying mechanisms were validated by IHC, Pearson correlation analysis, protein stability and rescue assays. The clinical significance of the snoRNA was explored using the Cox proportional hazard model, receiver operating characteristic (ROC) and survival analysis.
RESULTS
Here, we investigated the SNORA56, which was elevated in CRC tissues and plasma, and correlated with CRC prognosis. SNORA56 deficiency in CRC impaired proliferation and triggered ferroptosis, resulting in reduced tumorigenesis. Mechanistically, SNORA56 mediated the pseudouridylation of 28 S rRNA at the U1664 site and promoted the translation of the catalytic subunit of glutamate cysteine ligase (GCLC), an indispensable rate-limiting enzyme in the biosynthesis of glutathione, which can inhibit ferroptosis by suppressing lipid peroxidation.
CONCLUSIONS
Therefore, the SNORA56/28S rRNA/GCLC axis stimulates CRC progression by inhibiting the accumulation of cellular peroxides, and it may provide biomarker and therapeutic applications in CRC.
Topics: Animals; Humans; Mice; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Ferroptosis; Glutamate-Cysteine Ligase; In Situ Hybridization, Fluorescence; RNA, Ribosomal; RNA, Small Nuclear
PubMed: 38049865
DOI: 10.1186/s13046-023-02906-8 -
Nephrology, Dialysis, Transplantation :... Jun 2024Glucocorticoids are the treatment of choice for proteinuric patients with minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS)....
BACKGROUND
Glucocorticoids are the treatment of choice for proteinuric patients with minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS). Immunosuppressive as well as direct effects on podocytes are believed to mediate their actions. In this study, we analyzed the anti-proteinuric effects of inhibition of the glucocorticoid receptor (GR) in glomerular epithelial cells, including podocytes.
METHODS
We employed genetic and pharmacological approaches to inhibit the GR. Genetically, we used Pax8-Cre/GRfl/fl mice to specifically inactivate the GR in kidney epithelial cells. Pharmacologically, we utilized a glucocorticoid antagonist called mifepristone.
RESULTS
Genetic inactivation of GR, specifically in kidney epithelial cells, using Pax8-Cre/GRfl/fl mice, ameliorated proteinuria following protein overload. We further tested the effects of pharmacological GR inhibition in three models and species: the puromycin aminonucleoside-induced nephrosis model in rats, the protein overload model in mice and the inducible transgenic NTR/MTZ zebrafish larvae with specific and reversible podocyte injury. In all three models, both pharmacological GR activation and inhibition consistently and significantly ameliorated proteinuria. Additionally, we translated our findings to humans, where three nephrotic adult patients with MCD or primary FSGS with contraindications or insufficient responses to corticosteroids were treated with mifepristone. This treatment resulted in a clinically relevant reduction of proteinuria.
CONCLUSIONS
Thus, across multiple species and proteinuria models, both genetic and pharmacological GR inhibition was at least as effective as pronounced GR activation. While the mechanism remains perplexing, GR inhibition may be a novel and targeted therapeutic approach to treat glomerular proteinuria potentially bypassing adverse actions of steroids.
Topics: Animals; Receptors, Glucocorticoid; Mice; Proteinuria; Humans; Rats; Podocytes; Zebrafish; Male; Mifepristone; Disease Models, Animal; Glomerulosclerosis, Focal Segmental; Female; Kidney Diseases; Puromycin Aminonucleoside; Hormone Antagonists; Nephrosis, Lipoid; Mice, Inbred C57BL; Mice, Transgenic
PubMed: 38037533
DOI: 10.1093/ndt/gfad254 -
Gene Feb 2024Hemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will...
BACKGROUND
Hemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease.
METHODS
Initially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression.
RESULTS
We identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro.
CONCLUSIONS
Knock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases.
Topics: Animals; Mice; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Gene Editing; Hemophilia A; RNA, Guide, CRISPR-Cas Systems; Factor VIII
PubMed: 38036077
DOI: 10.1016/j.gene.2023.148038