50 research outputs found
Rational Design of a Two-Photon Fluorescent Probe for Human Cytochrome P450 3A and the Visualization of Mechanism-Based Inactivation
Mechanism-based inactivation (MBI) can mediate adverse reactions and hepatotoxicity from drugs, which is a result of their conversion into highly reactive metabolites catalyzed by enzymes such as cytochrome P450 3A (CYP3A). In the present research, we optimized the key interaction domain of the fluorophore with the target protein to develop a two-photon fluorescent probe for CYP3A that is involved in the metabolism of more than half of all clinical drugs. The developed BN-1 probe exhibited appropriate selectivity and sensitivity for the semi-quantitative detection and imaging of endogenous CYP3A activity in various living systems, thereby providing a high-throughput screening system enabling evaluation of MBI-associated hepatotoxicity by CYP3A. Using BN-1 as a fluorescent molecular tool facilitates the efficient discovery and characterization of CYP3A-induced MBI in natural systems.</p
Inhibition of synovitis and joint destruction by a new single domain antibody specific for cyclophilin A in two different mouse models of rheumatoid arthritis
POIROT: Aligning Attack Behavior with Kernel Audit Records for Cyber Threat Hunting
Cyber threat intelligence (CTI) is being used to search for indicators of
attacks that might have compromised an enterprise network for a long time
without being discovered. To have a more effective analysis, CTI open standards
have incorporated descriptive relationships showing how the indicators or
observables are related to each other. However, these relationships are either
completely overlooked in information gathering or not used for threat hunting.
In this paper, we propose a system, called POIROT, which uses these
correlations to uncover the steps of a successful attack campaign. We use
kernel audits as a reliable source that covers all causal relations and
information flows among system entities and model threat hunting as an inexact
graph pattern matching problem. Our technical approach is based on a novel
similarity metric which assesses an alignment between a query graph constructed
out of CTI correlations and a provenance graph constructed out of kernel audit
log records. We evaluate POIROT on publicly released real-world incident
reports as well as reports of an adversarial engagement designed by DARPA,
including ten distinct attack campaigns against different OS platforms such as
Linux, FreeBSD, and Windows. Our evaluation results show that POIROT is capable
of searching inside graphs containing millions of nodes and pinpoint the
attacks in a few minutes, and the results serve to illustrate that CTI
correlations could be used as robust and reliable artifacts for threat hunting.Comment: The final version of this paper is going to appear in the ACM SIGSAC
Conference on Computer and Communications Security (CCS'19), November 11-15,
2019, London, United Kingdo
Molecular Design Strategy to Construct the Near-Infrared Fluorescent Probe for Selectively Sensing Human Cytochrome P450 2J2
Cytochrome P450 2J2 (CYP2J2), a key enzyme responsible for oxidative metabolism of various xenobiotics and endogenous compounds, participates in a diverse array of physiological and pathological processes in humans. Its biological role in tumorigenesis and cancer diagnosis remains poorly understood, owing to the lack of molecular tools suitable for real-time monitoring CYP2J2 in complex biological systems. Using molecular design principles we were able to modify the distance between the catalytic unit and metabolic recognition moiety, allowing us to develop a CYP2J2 selective fluorescent probe using a near-infrared fluorophore (E)-2-(2-(6-hydroxy-2, 3-dihydro-1H-xanthen-4-yl)vinyl)-3,3- dimethyl-1-propyl-3H-indol-1-ium iodide (HXPI). To improve the reactivity and isoform specificity, a self-immolative linker was introduced to the HXPI derivatives in order to better fit the narrow substrate channel of CYP2J2, the modification effectively shortened the spatial distance between the metabolic moiety (O-alkyl group) and catalytic center of CYP2J2. After screening a panel of O-alkylated HXPI derivatives, BnXPI displayed the best combination of specificity, sensitivity and applicability for detecting CYP2J2 in vitro and in vivo. Upon O-demethylation by CYP2J2, a self-immolative reaction occurred spontaneously via 1,6-elimination of p-hydroxybenzyl resulting in the release of HXPI. Allowing BnXPI to be successfully used to monitor CYP2J2 activity in real-time for various living systems including cells, tumor tissues, and tumor-bearing animals. In summary, our practical strategy could help the development of a highly specific and broadly applicable tool for monitoring CYP2J2, which offers great promise for exploring the biological functions of CYP2J2 in tumorigenesis.</p
Targeting P-Glycoprotein: Nelfinavir Reverses Adriamycin Resistance in K562/ADR Cells
Background/Aims: The emergence of multidrug resistance (MDR) caused by P-glycoprotein (P-gp) overexpression is a serious obstacle to the treatment of chronic myelocytic leukemia. In recent years, some clinical trials have shown that nelfinavir (NFV), a traditional anti-HIV drug, has anti-cancer effects. Some researchers have also shown NFV might be a potential P-gp inhibitor. This study is aimed at investigating whether nelfinavir can act as an MDR-reversal drug and to clarify its molecular mechanism as well. Methods: K562 and K562/ADR cell lines were applied in the study. Cytotoxicity was detected by CCK-8 reagents. Cell apoptosis was detected by flow cytometry and inverted fluorescence microscopy to detect the binding of apoptotic dyes to cells. Western blot was used to detect the expression of proteins. Drug-protein molecular docking simulation by using Sybyl-x 2.0 software. Results: Non-toxic concentrations of NFV (1.25–5 μM) could reverse Adriamycin (ADR), colchicine, paclitaxel, and imatinib resistance of K562/ADR cells, with reversal indexes of up to 10.8, 7.4, 57, and 9.3, respectively. NFV inhibited P-gp efflux function, as evidenced by the significant increase in the intracellular accumulation of ADR and Rho-123, without affecting P-gp protein and mRNA expression levels. Further ATP content detection and molecular docking simulations showed that NFV could decrease intracellular ATP content and has a high affinity with the active functional regions of P-gp, respectively. When co-administered with ADR, NFV increased intracellular reactive oxygen species as well as blocked the ERK/Akt signaling pathway, leading to cell apoptosis. Conclusion: NFV inhibited P-gp function, decreased intracellular ATP content, and promoted cell apoptosis in K562/ADR cells, thereby reversing MDR. These findings encourage further animal and clinical MDR studies with a combination therapy consisting of NFV and chemotherapeutic drugs
ROS-dependent catalytic mechanism of melatonin metabolism and its application in the measurement of reactive oxygen
Melatonin (Mel) is an endogenous active molecule whose metabolism progress significantly influences its bioactivity. However, the detailed metabolic pathway of Mel in the pathological state has not yet been fully illustrated. In this study, 16 metabolites of Mel in cancer cells and human liver microsomes were identified, of which seven novel metabolites were newly discovered. Among them, 2-hydroxymelatonin (2-O-Mel), as the major metabolite in cancer cells, was revealed for the first time, which was different from the metabolite found in the human liver. Furthermore, CYP1A1/1A2- and reactive oxygen species (ROS)-mediated 2-hydroxylation reactions of Mel were verified to be the two metabolic pathways in the liver and cancer cells, respectively. ROS-dependent formation of 2-O-Mel was the major pathway in cancer cells. Furthermore, the underlying catalytic mechanism of Mel to 2-O-Mel in the presence of ROS was fully elucidated using computational chemistry analysis. Therefore, the generation of 2-O-Mel from Mel could serve as another index for the endogenous reactive oxygen level. Finally, based on the ROS-dependent production of 2-O-Mel, Mel was successfully used for detecting the oxygen-carrying capacity of hemoglobin in human blood. Our investigation further enriched the metabolic pathway of Mel, especially for the ROS-dependent formation of 2-O-Mel that serves as a diagnostic and therapeutic target for the rational use of Mel in clinics
Molecular Design Strategy to Construct the Near-Infrared Fluorescent Probe for Selectively Sensing Human Cytochrome P450 2J2
Cytochrome P450 2J2 (CYP2J2), a key
enzyme responsible for oxidative
metabolism of various xenobiotics and endogenous compounds, participates
in a diverse array of physiological and pathological processes in
humans. Its biological role in tumorigenesis and cancer diagnosis
remains poorly understood, owing to the lack of molecular tools suitable
for real-time monitoring CYP2J2 in complex biological systems. Using
molecular design principles, we were able to modify the distance between
the catalytic unit and metabolic recognition moiety, allowing us to
develop a CYP2J2 selective fluorescent probe using a near-infrared
fluorophore (E)-2-(2-(6-hydroxy-2, 3-dihydro-1H-xanthen-4-yl)Âvinyl)-3,3-dimethyl-1-propyl-3H-indol-1-ium iodide (HXPI). To improve the reactivity
and isoform specificity, a self-immolative linker was introduced to
the HXPI derivatives in order to better fit the narrow
substrate channel of CYP2J2, the modification effectively shortened
the spatial distance between the metabolic moiety (O-alkyl group) and catalytic center of CYP2J2. After screening a panel
of O-alkylated HXPI derivatives, BnXPI displayed the best combination of specificity, sensitivity
and applicability for detecting CYP2J2 in vitro and in vivo. Upon O-demethylation by CYP2J2,
a self-immolative reaction occurred spontaneously via 1,6-elimination
of p-hydroxybenzyl resulting in the release of HXPI. Allowing BnXPI to be successfully used
to monitor CYP2J2 activity in real-time for various living systems
including cells, tumor tissues, and tumor-bearing animals. In summary,
our practical strategy could help the development of a highly specific
and broadly applicable tool for monitoring CYP2J2, which offers great
promise for exploring the biological functions of CYP2J2 in tumorigenesis
M-CSF Induces Monocyte Survival by Activating NF-κB p65 Phosphorylation at Ser276 via Protein Kinase C
Macrophage colony-stimulating factor (M-CSF) promotes mononuclear phagocyte survival and proliferation. The transcription factor Nuclear Factor-kappaB (NF-κB) is a key regulator of genes involved in M-CSF-induced mononuclear phagocyte survival and this study focused at identifying the mechanism of NF-κB transcriptional activation. Here, we demonstrate that M-CSF stimulated NF-κB transcriptional activity in human monocyte-derived macrophages (MDMs) and the murine macrophage cell line RAW 264.7. The general protein kinase C (PKC) inhibitor Ro-31-8220, the conventional PKCα/β inhibitor Gö-6976, overexpression of dominant negative PKCα constructs and PKCα siRNA reduced NF-κB activity in response to M-CSF. Interestingly, Ro-31-8220 reduced Ser276 phosphorylation of NF-κBp65 leading to decreased M-CSF-induced monocyte survival. In this report, we identify conventional PKCs, including PKCα as important upstream kinases for M-CSF-induced NF-κB transcriptional activation, NF-κB-regulated gene expression, NF-κB p65 Ser276 phosphorylation, and macrophage survival. Lastly, we find that NF-κB p65 Ser276 plays an important role in basal and M-CSF-stimulated NF-κB activation in human mononuclear phagocytes
Research of Car Navigation Data Retrieval System
In recent years, the automotive industry has become a pillar industry of the world, the role played by the automobile in the daily life and work has become increasingly prominent. In the car navigation retrieval system, whether from the perspective of retrieval speed, screen display, or memory management, it can not meet the needs of users. In the design, the retrieval algorithm along the road of facilities retrieval can not only improve the retrieval system's modular structure, but also introduce the idea of thread pool. The whole system is divided into four modules; B-Tree + phenotype data structures being used can improve the search speed; a new road merging algorithm being proposed can improve the retrieval performance