Mechanistic analysis and experimental verification of bicarbonate-controlled enteric coat dissolution: Potential in vivo implications

: Chagas disease, Sleeping sickness and Leishmaniasis, caused by trypanosomatids Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively, are considered neglected tropical diseases, and they especially affect impoverished populations in the developing world. The available chemotherapies are very limited and a search for alternatives is still necessary. In folk medicine, natural naphthoquinones have been employed for the treatment of a great variety of illnesses, including parasitic infections. This review is focused on the anti-trypanosomatid activity and mechanistic analysis of naphthoquinones and derivatives. Among all the series of derivatives tested in vitro, naphthoquinone-derived 1,2,3-triazoles were very active on T. cruzi infective forms in blood bank conditions, as well as in amastigotes of Leishmania spp. naphthoquinones containing a CF3 on a phenyl amine ring inhibited T. brucei proliferation in the nanomolar range, and naphthopterocarpanquinones stood out for their activity on a range of Leishmania species. Some of these compounds showed a promising selectivity index (SI) (30 to 1900), supporting further analysis in animal models. Indeed, high toxicity to the host and inactivation by blood components are crucial obstacles to be overcome to use naphthoquinones and/or their derivatives for chemotherapy. Multidisciplinary initiatives embracing medicinal chemistry, bioinformatics, biochemistry, and molecular and cellular biology need to be encouraged to allow the optimization of these compounds. Large scale automated tests are pivotal for the efficiency of the screening step, and subsequent evaluation of both the mechanism of action in vitro and pharmacokinetics in vivo are essential for the development of a novel, specific and safe derivative, minimizing adverse effects.

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PPM1G promotes the progression of hepatocellular carcinoma via phosphorylation regulation of alternative splicing protein SRSF3

Cell Death and Disease ◽

10.1038/s41419-021-04013-y ◽

2021 ◽

Vol 12 (8) ◽

Author(s):

Dawei Chen ◽

Zhenguo Zhao ◽

Lu Chen ◽

Qinghua Li ◽

Jixue Zou ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Alternative Splicing ◽

Protein Function ◽

Vital Role ◽

Normal Tissues ◽

Mechanistic Analysis ◽

Highly Correlated ◽

Splicing Patterns

AbstractEmerging evidence has demonstrated that alternative splicing has a vital role in regulating protein function, but how alternative splicing factors can be regulated remains unclear. We showed that the PPM1G, a protein phosphatase, regulated the phosphorylation of SRSF3 in hepatocellular carcinoma (HCC) and contributed to the proliferation, invasion, and metastasis of HCC. PPM1G was highly expressed in HCC tissues compared to adjacent normal tissues, and higher levels of PPM1G were observed in adverse staged HCCs. The higher levels of PPM1G were highly correlated with poor prognosis, which was further validated in the TCGA cohort. The knockdown of PPM1G inhibited the cell growth and invasion of HCC cell lines. Further studies showed that the knockdown of PPM1G inhibited tumor growth in vivo. The mechanistic analysis showed that the PPM1G interacted with proteins related to alternative splicing, including SRSF3. Overexpression of PPM1G promoted the dephosphorylation of SRSF3 and changed the alternative splicing patterns of genes related to the cell cycle, the transcriptional regulation in HCC cells. In addition, we also demonstrated that the promoter of PPM1G was activated by multiple transcription factors and co-activators, including MYC/MAX and EP300, MED1, and ELF1. Our study highlighted the essential role of PPM1G in HCC and shed new light on unveiling the regulation of alternative splicing in malignant transformation.

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Requirement for PKC Epsilon in Kras-Driven Lung Tumorigenesis

10.1101/2020.06.26.173690 ◽

2020 ◽

Author(s):

Rachana Garg ◽

Mariana Cooke ◽

Shaofei Wang ◽

Fernando Benavides ◽

Martin C. Abba ◽

...

Keyword(s):

Lung Cancer ◽

Lung Adenocarcinoma ◽

Cancer Therapeutics ◽

Rna Seq ◽

Lung Tumorigenesis ◽

Mechanistic Analysis ◽

Oncogenic Driver ◽

Histological Form

ABSTRACTNon-small cell lung cancer (NSCLC), the most frequent subtype of lung cancer, remains a highly lethal malignancy and one of the leading causes of cancer deaths worldwide. Mutant KRAS is the prevailing oncogenic driver of lung adenocarcinoma, the most common histological form of NSCLC. In this study, we examined the role of PKCε, an oncogenic kinase highly expressed in NSCLC and other cancers, in KRAS-driven tumorigenesis. Notably, database analysis revealed an association between PKCε expression and poor outcome in lung adenocarcinoma patients specifically having KRAS mutation. By generating a PKCε-deficient, conditionally activatable allele of oncogenic Kras (LSL-KrasG12D;PKCε−/− mice) we were able to demonstrate the requirement of PKCε for Kras-driven lung tumorigenesis in vivo, which is consistent with the impaired transformed growth observed in PKCε-deficient KRAS-dependent NSCLC cells. Moreover, PKCε-knockout mice were found to be less susceptible to lung tumorigenesis induced by benzo[a]pyrene, a carcinogen that induces mutations in Kras. Mechanistic analysis using RNA-Seq revealed little overlapping for PKCε and KRAS in the control of genes/biological pathways relevant in NSCLC, suggesting that a permissive role of PKCε in KRAS-driven lung tumorigenesis may involve non-redundant mechanisms. Our results thus highlight the relevance and potential of targeting PKCε for lung cancer therapeutics.

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An Integration of Network Pharmacology and Experimental Verification to Investigate the Mechanism of Guizhi to Treat Nephrotic Syndrome

Frontiers in Pharmacology ◽

10.3389/fphar.2021.755421 ◽

2021 ◽

Vol 12 ◽

Author(s):

Dan He ◽

Qiang Li ◽

Guangli Du ◽

Guofeng Meng ◽

Jijia Sun ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Molecular Docking ◽

Protein Expression ◽

Signaling Pathway ◽

P38 Mapk ◽

Experimental Verification ◽

Active Component ◽

Mapk Signaling

Background: Guizhi has the pharmacological activity of anti-inflammatory. However, the effect mechanism of Guizhi against nephrotic syndrome (NS) remains unclear. A network pharmacological approach with experimental verification in vitro and in vivo was performed to investigate the potential mechanisms of Guizhi to treat NS.Methods: Active compounds and potential targets of Guizhi, as well as the related targets of NS were obtained from the public databases. The intersecting targets of Guizhi and NS were obtained through Venny 2.1.0. The key targets and signaling pathways were determined by protein-protein interaction (PPI), genes ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis. And the overall network was constructed with Cytoscape. Molecular docking verification was carried out by AutoDock Vina. Finally, in vitro and in vivo experiments were performed to verify the mechanism of Guizhi to treat NS.Results: 63 intersecting targets were obtained, and the top five key targets mainly involed in NF- Kappa B and MAPK signaling pathway. In the overall network, cinnamaldehyde (CA) was the top one active compound with the highest degree value. The molecular docking showed that the top five key targets were of good binding activity with the active components of Guizhi. To in vitro experiment, CA, the main active component of Guizhi, inhibited the secretion of IL-1β, IL-6, TNF-α in LPS challenged RAW264.7 cells, and down regulated the protein expression of p-NF-κB p65 and p-p38 MAPK in LPS challenged RAW264.7 cells. In vitro experiment showed that, 24 urinary protein and renal function were increased in ADR group. To western blot, CA down regulated the protein expression of p-p38 MAPK in rats of adriamycin-induced nephropathy.Conclusion: CA might be the main active component of Guizhi to treat NS, and the underlying mechanism might mainly be achieved by inhibiting MAPK signaling pathway.

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Unstressing intemperate models: how cold stress undermines mouse modeling

Journal of Experimental Medicine ◽

10.1084/jem.20120988 ◽

2012 ◽

Vol 209 (6) ◽

pp. 1069-1074 ◽

Cited By ~ 102

Author(s):

Christopher L. Karp

Keyword(s):

Cold Stress ◽

Biomedical Research ◽

Current Model ◽

Environmental Variable ◽

Model System ◽

Laboratory Mice ◽

Therapeutic Approaches ◽

Mechanistic Analysis ◽

Mouse Modeling

Mus musculus enjoys pride of place at the center of contemporary biomedical research. Despite being the current model system of choice for in vivo mechanistic analysis, mice have clear limitations. The literature is littered with examples of therapeutic approaches that showed promise in mouse models but failed in clinical trials. More generally, mice often provide poor mimics of the human diseases being modeled. Available data suggest that the cold stress to which laboratory mice are ubiquitously subjected profoundly affects mouse physiology in ways that impair the modeling of human homeostasis and disease. Experimental attention to this key, albeit largely ignored, environmental variable is likely to have a broad transformative effect on biomedical research.

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Synergistic activity of an OmpA inhibitor and colistin against colistin-resistant Acinetobacter baumannii: mechanistic analysis and in vivo efficacy

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dky343 ◽

2018 ◽

Cited By ~ 1

Author(s):

Raquel Parra-Millán ◽

Xavier Vila-Farrés ◽

Rafael Ayerbe-Algaba ◽

Monica Varese ◽

Viviana Sánchez-Encinales ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Synergistic Activity ◽

In Vivo Efficacy ◽

Mechanistic Analysis

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Lentiviral Nef Proteins Utilize PAK2-Mediated Deregulation of Cofilin as a General Strategy To Interfere with Actin Remodeling

Journal of Virology ◽

10.1128/jvi.02467-09 ◽

2010 ◽

Vol 84 (8) ◽

pp. 3935-3948 ◽

Cited By ~ 43

Author(s):

Bettina Stolp ◽

Libin Abraham ◽

Jochen M. Rudolph ◽

Oliver T. Fackler

Keyword(s):

Host Cells ◽

General Strategy ◽

Actin Remodeling ◽

Mechanistic Analysis ◽

Immunodeficiency Virus ◽

Hiv Type 1 ◽

Hiv 1

ABSTRACT Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1SF2 (HIV-1SF2) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo.

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Long-term culture expanded alveolar macrophages restore full epigenetic identity in vivo

10.21203/rs.3.rs-756350/v1 ◽

2021 ◽

Author(s):

Michael Sieweke ◽

Sethuraman Subramanian ◽

Clara Busch ◽

Kaaweh Molawi ◽

Laufey Geirsdottir ◽

...

Keyword(s):

Gene Expression ◽

Alveolar Macrophages ◽

Therapeutic Potential ◽

Ex Vivo ◽

Open Chromatin ◽

Mechanistic Analysis ◽

Long Term Changes ◽

Cellular Identity

Abstract Alveolar macrophages (AM) are tissue resident macrophages of the lung that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. Here we show that long-term ex vivo expanded mouse AM (exAM) maintain core AM gene expression but show culture adaptations related to adhesion, metabolism and proliferation. Strikingly, even after several months in culture exAM reacquired full transcriptional and epigenetic identity upon transplantation into the lung and could self-maintain in the natural niche long-term. Changes in open chromatin regions (OCR) observed in culture were fully reversible in transplanted exAM (texAM) and resulted in a gene expression profile indistinguishable from resident AM. Our results demonstrate that long-term proliferation of AM in culture does not compromise cellular identity in vivo. The demonstrated robustness of exAM identity provides new opportunities for mechanistic analysis and highlights the therapeutic potential of ex vivo expanded macrophages.

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AR facilitates YAP-TEAD interaction with the AM promoter to enhance mast cell infiltration into cutaneous neurofibroma

Scientific Reports ◽

10.1038/s41598-019-56022-9 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Jing Jia ◽

Haibao Zhang ◽

Hongke Zhang ◽

Wenbo Liu ◽

Huicong Du ◽

...

Keyword(s):

Mast Cell ◽

Cell Infiltration ◽

Clinical Samples ◽

Cell Recruitment ◽

Cell Accumulation ◽

Mechanistic Analysis ◽

Mast Cell Density ◽

Disease Initiation

AbstractAbundant mast cell infiltration and disease initiation at puberty are hallmark features of cutaneous neurofibroma (cNF). However, the association between mast cell infiltration and steroid hormones in cNF remains unclear. Here, we determined that androgen receptor (AR) expression is positively associated with mast cell density in cNF tissues. Moreover, both in vitro cell experiments and in vivo mouse models verified that activated AR promoted mast cell infiltration and that AR inhibition reduced mast cell infiltration. Analyses in cell models and xenograft tumours both demonstrated that AR upregulated Yes associate protein 1 (YAP)-adrenomedullin (AM) signalling. Clinical samples from cNF patients further verified that AR was positively related to YAP and AM. Mechanistic analysis revealed that AR accelerates AM transcription via enhancing YAP- TEA domain transcription factor (TEAD) binding to the AM promoter. Consequently, the upregulated AM enhanced mast cell recruitment. Interruption of the YAP-TEAD interaction or inhibition of AM could impair mast cell accumulation induced by active AR, which indicated that this newly found signalling pathway may provide novel targets for cNF treatment.

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