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BioRxiv : the Preprint Server For... Apr 2024The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid...
The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin () as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.
PubMed: 38617237
DOI: 10.1101/2024.04.02.587756 -
Cells Apr 2024GDF15, also known as MIC1, is a member of the TGF-beta superfamily. Previous studies reported elevated serum levels of GDF15 in patients with kidney disorder, and its...
GDF15, also known as MIC1, is a member of the TGF-beta superfamily. Previous studies reported elevated serum levels of GDF15 in patients with kidney disorder, and its association with kidney disease progression, while other studies identified GDF15 to have protective effects. To investigate the potential protective role of GDF15 on podocytes, we first performed in vitro studies using a -deficient podocyte cell line. The lack of GDF15 intensified puromycin aminonucleoside (PAN)-triggered endoplasmic reticulum stress and induced cell death in cultivated podocytes. This was evidenced by elevated expressions of and ER-associated chaperones, alongside AnnexinV/PI staining and LDH release. Additionally, we subjected mice to nephrotoxic PAN treatment. Our observations revealed a noteworthy increase in both GDF15 expression and secretion subsequent to PAN administration. knockout mice displayed a moderate loss of WT1+ cells (podocytes) in the glomeruli compared to wild-type controls. However, this finding could not be substantiated through digital evaluation. The parameters of kidney function, including serum BUN, creatinine, and albumin-creatinine ratio (ACR), were increased in knockout mice as compared to wild-type mice upon PAN treatment. This was associated with an increase in the number of glomerular macrophages, neutrophils, inflammatory cytokines, and chemokines in -deficient mice. In summary, our findings unveil a novel renoprotective effect of GDF15 during kidney injury and inflammation by promoting podocyte survival and regulating endoplasmic reticulum stress in podocytes, and, subsequently, the infiltration of inflammatory cells via paracrine effects on surrounding glomerular cells.
Topics: Humans; Mice; Animals; Podocytes; Puromycin Aminonucleoside; Growth Differentiation Factor 15; Creatinine; Kidney Diseases; Inflammation; Mice, Knockout
PubMed: 38607075
DOI: 10.3390/cells13070637 -
Scientific Reports Apr 2024The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside)...
The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside) yielded very stable preparations of modified biomaterials. The main emphasis was placed on analyses of their antimicrobial activity and potential immunomodulatory and non-cytotoxic properties towards the CCD841 CoTr model cell line. Cecropin A did not significantly affect the viability or proliferation of the CCD 841 CoTr cells, regardless of its soluble or immobilized form. In contrast, puromycin did not induce a significant decrease in the cell viability or proliferation in the immobilized form but significantly decreased cell viability and proliferation when administered in the soluble form. The covalent immobilization of these two molecules on the surface of biomaterials resulted in stable preparations that were able to inhibit the multiplication of Staphylococcus aureus and S. epidermidis strains. It was also found that the preparations induced the production of cytokines involved in antibacterial protection mechanisms and stimulated the immune response. The key regulator of this activity may be related to TLR4, a receptor recognizing bacterial LPS. In the present study, these factors were produced not only in the conditions of LPS stimulation but also in the absence of LPS, which indicates that cecropin A- and puromycin-modified biomaterials may upregulate pathways leading to humoral antibacterial immune response.
Topics: Biocompatible Materials; Lipopolysaccharides; Anti-Infective Agents; Anti-Bacterial Agents; Polymers; Staphylococcus epidermidis; Puromycin
PubMed: 38580807
DOI: 10.1038/s41598-024-58730-3 -
The Journal of General Virology Apr 2024The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing...
The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.
Topics: Viral Proteins; Ribonucleases; DNA Helicases; Poly-ADP-Ribose Binding Proteins; RNA Helicases; RNA Recognition Motif Proteins; Herpesvirus 1, Human; Endoribonucleases; RNA Stability; Virion; RNA, Messenger
PubMed: 38572740
DOI: 10.1099/jgv.0.001976 -
Environmental Science & Technology Apr 2024Methane emissions present a significant environmental challenge in both natural and engineered aquatic environments. Denitrifying anaerobic methane oxidation (N-DAMO)...
Methane emissions present a significant environmental challenge in both natural and engineered aquatic environments. Denitrifying anaerobic methane oxidation (N-DAMO) has the potential for application in wastewater treatment plants. However, our understanding of the N-DAMO process is primarily based on studies conducted on environmental samples or enrichment cultures using metagenomic approaches. To gain deeper insights into N-DAMO, we used antimicrobial compounds to study the function and physiology of ' Methanoperedens nitroreducens' and ' Methylomirabilis oxyfera' in N-DAMO enrichment cultures. We explored the effects of inhibitors and antibiotics and investigated the potential application of N-DAMO in wastewater contaminated with ammonium and heavy metals. Our results showed that ' M. nitroreducens' was susceptible to puromycin and 2-bromoethanesulfonate, while the novel methanogen inhibitor 3-nitrooxypropanol had no effect on N-DAMO. Furthermore, ' M. oxyfera' was shown to be susceptible to the particulate methane monooxygenase inhibitor 1,7-octadiyne and a bacteria-suppressing antibiotic cocktail. The N-DAMO activity was not affected by ammonium concentrations below 10 mM. Finally, the N-DAMO community appeared to be remarkably resistant to lead (Pb) but susceptible to nickel (Ni) and cadmium (Cd). This study provides insights into microbial functions in N-DAMO communities, facilitating further investigation of their application in methanogenic, nitrogen-polluted water systems.
Topics: Nitrates; Wastewater; Anaerobiosis; Methane; Bacteria; Ammonium Compounds; Oxidation-Reduction; Anti-Infective Agents; Nitrites; Bioreactors; Denitrification
PubMed: 38551595
DOI: 10.1021/acs.est.3c07197 -
Micromachines Feb 2024We developed a 3D glomeruli tissue chip for glomerulonephritis (GN) testing, featuring a gravity-driven glomerular filtration barrier (GFB) with human podocytes and...
We developed a 3D glomeruli tissue chip for glomerulonephritis (GN) testing, featuring a gravity-driven glomerular filtration barrier (GFB) with human podocytes and endothelial cells with a bidirectional flow in the bottom channel. Using puromycin-induced GN, we observed decreased cell viability, increased albumin permeability, and reduced WT1 and nephrin compared to the normal GFB. Tacrolimus restored cell viability, reduced albumin permeability, and increased WT1 expression. Using serum from five membranous nephropathy (MN) patients, we created MN models using a GFB-mimicking chip. A notable decline in cell viability was observed in the serum-induced MN1 and MN2 models. However, tacrolimus restored it. Albumin permeability was reduced in the MN1, MN2, and MN5 models by tacrolimus treatment. MN1 displayed the best clinical response to tacrolimus, exhibiting increased expression of WT1 in chip-based evaluations after tacrolimus treatment. We successfully evaluated the efficacy of tacrolimus using puromycin-induced and serum-induced GN models on a chip that mimicked the structure and function of the GFB. The GFB-mimicking chip holds promise as a personalized platform for assessing drug efficacy using patient serum samples.
PubMed: 38542564
DOI: 10.3390/mi15030317 -
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 -
Antibodies (Basel, Switzerland) Mar 2024There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody-drug conjugates for research and therapeutic use....
There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody-drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows the rapid generation of stable Expi293 cell pools by episomal retention of transfected DNA. By linking protein expression to puromycin resistance through an attenuated internal ribosome entry site, we achieve stable cell pools producing proteins of interest. In addition, split intein-split puromycin-mediated selection of two separate protein expression cassettes allows the stable production of bispecific antibody-like molecules or antibodies with distinct C-terminal heavy chain modifications, such as an antigen on one chain and a sortase tag on the other chain. We also use this novel expression system to generate stable Expi293 cell pools that secrete sortase A Δ59 variant Srt4M. Using these reagents, we prepared a site-specific drug-to-antibody ratio of 1 antibody-siRNA conjugate. We anticipate the simple, robust, and rapid stable protein expression systems described here being useful for a wide variety of applications.
PubMed: 38534208
DOI: 10.3390/antib13010018 -
Journal of Biomedical Science Mar 2024The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity...
BACKGROUND
The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency.
METHOD
Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies.
RESULT
We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration.
CONCLUSION
Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.
Topics: Humans; CRISPR-Cas Systems; Gene Editing; DNA Breaks, Double-Stranded; Transgenes; DNA
PubMed: 38532479
DOI: 10.1186/s12929-024-01020-x -
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