Phenethyl-4-ANPP: A Marginally Active Byproduct Suggesting a Switch in Illicit Fentanyl Synthesis Routes

2021 ◽  
Author(s):  
Marthe M Vandeputte ◽  
Alex J Krotulski ◽  
Fabian Hulpia ◽  
Serge Van Calenbergh ◽  
Christophe P Stove
Keyword(s):  
Valuable Insight ◽  
Unknown Compound ◽  
The Usa ◽  
A Cell ◽  
Synthesis Routes ◽  
And Control ◽  
First Time ◽  
Single Reaction

Abstract Profiling of the illicit fentanyl supply is invaluable from surveillance and intelligence perspectives. An important strategy includes the study of chemical attribution signatures (e.g., trace amounts of synthesis precursors, impurities/byproducts in seized material and metabolites in biological samples). This information provides valuable insight into the employed synthesis routes at the heart of illicit fentanyl manufacture (previously mainly the so-called Janssen or Siegfried methods), allowing to track and ultimately regulate crucial precursors. This report focuses on phenethyl-4-anilino-N-phenethylpiperidine (phenethyl-4-ANPP), a formerly unknown compound that was identified for the first time in a fentanyl powder sample seized in April 2019, followed by its identification in a biological sample in December 2019. Between 2019-Q4 and 2020-Q3, phenethyl-4-ANPP was detected in 25/1,054 fentanyl cases in the USA. There are currently no reports on how this compound may have ended up in illicit drug preparations and whether its presence may have potential in vivo relevance. We propose three possible fentanyl synthesis routes that, when badly executed in a single reaction vessel, may involve the formation of phenethyl-4-ANPP. We hypothesize that the presence of the latter is the result of a shift in fentanyl synthesis routes in an attempt to circumvent restrictions on previously used precursors. Using a cell-based µ-opioid receptor recruitment assay, we show that the extent of MOR activation caused by 100 µM phenethyl-4-ANPP is comparable to that exerted by a roughly 100,000-fold lower concentration of fentanyl (0.001 µM or 0.336 ng/mL). Negligible in vitro opioid activity, combined with its low abundance in fentanyl preparations, most likely renders phenethyl-4-ANPP biologically irrelevant in vivo. However, as clandestine operations are constantly changing shape, monitoring of fentanyl attributions remains pivotal in our understanding and control of illicit fentanyl manufacture and supply.

scholarly journals In Vivo Remodeling of Altered Autophagy-Lysosomal Pathway by a Phosphopeptide in Lupus

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2328
Author(s):  
Fengjuan Wang ◽  
Inmaculada Tasset ◽  
Ana Maria Cuervo ◽  
Sylviane Muller
Keyword(s):  
Metabolic Disorders ◽  
Inflammatory Diseases ◽  
Cellular Mechanism ◽  
Major Protein ◽  
Recovery Effect ◽  
A Cell ◽  
First Time ◽  
Chaperone Mediated Autophagy

The phosphopeptide P140/Lupuzor, which improves the course of lupus disease in mice and patients, targets chaperone-mediated autophagy (CMA), a selective form of autophagy that is abnormally upregulated in lupus-prone MRL/lpr mice. Administered intravenously to diseased mice, P140 reduces the expression level of two major protein players of CMA, LAMP2A and HSPA8, and inhibits CMA in vitro in a cell line that stably expresses a CMA reporter. Here, we aimed to demonstrate that P140 also affects CMA in vivo and to unravel the precise cellular mechanism of how P140 interacts with the CMA process. MRL/lpr mice and CBA/J mice used as control received P140 or control peptides intravenously. Lysosome-enriched fractions of spleen or liver were prepared to examine lysosomal function. Highly purified lysosomes were further isolated and left to incubate with the CMA substrate to study at which cellular step P140 interacts with the CMA process. The data show that P140 effectively regulates CMA in vivo in MRL/lpr mice at the step of substrate lysosomal uptake and restores some alterations of defective lysosomes. For the first time, it is demonstrated that by occluding the intralysosome uptake of CMA substrates, a therapeutic molecule can attenuate excessive CMA activity in a pathological pro-inflammatory context and protect against hyperinflammation. This recovery effect of P140 on hyperactivated CMA is not only important for lupus therapy but potentially also for treating other (auto)inflammatory diseases, including neurologic and metabolic disorders, where CMA modulation would be highly beneficial.

scholarly journals Development of a Cell-Based Assay Probing the Specific Interaction between the Human Immunodeficiency Virus Type 1 Nucleocapsid and Psi RNA In Vivo

2007 ◽  
Vol 81 (11) ◽  
pp. 6151-6155 ◽  
Author(s):  
Soo In Jang ◽  
Young Ho Kim ◽  
Soon Young Paik ◽  
Ji Chang You
Keyword(s):  
Human Immunodeficiency Virus ◽  
Specific Interaction ◽  
Deletion Mapping ◽  
Packaging Signal ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
First Time

ABSTRACT Here, we describe a cell-based in vivo assay that probes the specific interaction between nucleocapsid (NC) protein and Psi (Ψ) RNA, the human immunodeficiency virus (HIV) packaging signal. The results demonstrate for the first time a specific NC-Ψ interaction within living cells. The specificity and applicability of the assay were confirmed by mutational studies of NC and deletion-mapping analyses of Ψ-RNA as well as by testing the in vivo NC-binding effects of NC-aptamer RNAs identified previously in vitro. This assay system would facilitate further detailed studies of the NC-Ψ interaction in vivo and the screening of various anti-HIV molecules targeting NC and the specific interaction.

scholarly journals Interaction between innate immunity and Ro52-induced antibody causes Sjögren's syndrome-like disorder in mice

2015 ◽  
Vol 75 (3) ◽  
pp. 617-622 ◽  
Author(s):  
Barbara M Szczerba ◽  
Paulina Kaplonek ◽  
Nina Wolska ◽  
Anna Podsiadlowska ◽  
Paulina D Rybakowska ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Sjögren’S Syndrome ◽  
Sjögren's Syndrome ◽  
Sjogren’S Syndrome ◽  
Sjogren's Syndrome ◽  
And Control ◽  
First Time ◽  
Sjögren‘S Syndrome

ObjectivesAutoantibodies reactive with Ro52 are often found in sera of patients with Sjögren's syndrome (SS). This study was undertaken to investigate the role of Ro52-induced immune responses in pathogenesis of SS.MethodsNew Zealand Mixed (NZM) 2758 mice were immunised with Ro52 in alum adjuvant. Control mice were immunised either with maltose-binding protein or injected with alum alone. Mice were monitored for anti-Ro52 antibody, sialoadenitis and pilocarpine-induced salivation. Antibody binding to salivary gland (SG) cells was analysed in vivo and in vitro by immunofluorescence. Sera from immunised mice were passively transferred into untreated or alum injected NZM2758 mice.ResultsBy day 30 post-immunisation, Ro52 immunised mice generated immunoprecipitating anti-Ro52 antibodies and they had the maximum drop in saliva production. Both Ro52 immunised and control mice showed evidence of mild sialoadenitis. However, only Ro52 immunised mice had antibody deposition in their SG. Passive transfer of Ro52-immune sera induced SG dysfunction in recipient mice, only if the recipients were primed with alum. In vitro, antibodies from Ro52-immune sera were internalised by a SG cell line and this uptake was inhibited by cytochalasin D treatment.ConclusionsOur data show for the first time that antibodies induced by Ro52 are capable of inducing SG dysfunction, and that this phenomenon is dependent on the activation of innate immunity. The mouse model described in this study implies that autoantibody deposition in the SG might be an important step in the induction of xerostomia and pathogenesis of SS.

scholarly journals MIF is among the proinflammatory cytokines increased by LPS in the human trophoblast line

2016 ◽  
Vol 68 (4) ◽  
pp. 715-722 ◽  
Author(s):  
Milica Jovanovic-Krivokuca ◽  
Ivana Stefanoska ◽  
Abu Rabi ◽  
Aleksandra Vilotic ◽  
Milos Petronijevic ◽  
...  
Keyword(s):  
Gelatin Zymography ◽  
Strong Immune Response ◽  
Human Trophoblast ◽  
E Coli ◽  
Cell Wall Constituent ◽  
A Cell ◽  
First Time ◽  
Trophoblast Cell Line

Infection is increasingly considered to contribute to pathological conditions in pregnancy. The placenta acts as a protective immunological fetomaternal barrier which recognizes microbes by pattern recognition receptors on the trophoblast. Lipopolysaccharide (LPS) is a cell wall constituent of Gram-negative bacteria that elicits a strong immune response. In this study, LPS from E. coli was used to treat the HTR-8/SVneo trophoblast cell line and examine its influence on cytokines IL-6, IL-8 and MIF using real-time PCR, metalloproteinases (MMP)-2 and -9 by gelatin zymography, and Western analysis of integrin subunits ?1 and ?1, all known to contribute to migration of human trophoblasts in vitro. The results described herein for the first time, show that MIF mRNA and secreted MIF protein were significantly elevated (2.5-3- and 2-fold, respectively) in LPS-treated cells. MMP-2 and MMP-9 levels were increased, as well as cell migration, as judged by a wound-healing test, however, no changes in the studied integrin subunits, cell viability or cell numbers were observed. The data obtained furthers our understanding of LPS actions on the trophoblast in vitro, additionally implicate MIF, and suggest that infection in vivo could indeed alter the functional characteristics of the trophoblast.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

Analysis of the Dynamics of the Electric Capacitance of a Cell Monolayer at the Late Stages of Caco-2 cell Differentiation

2019 ◽  
Vol 35 (6) ◽  
pp. 87-90
Author(s):  
S.V. Nikulin ◽  
V.A. Petrov ◽  
D.A. Sakharov
Keyword(s):  
Impedance Spectroscopy ◽  
Cell Monolayer ◽  
Microfluidic Chips ◽  
Russian Science ◽  
Electric Capacitance ◽  
A Cell ◽  
Static Conditions ◽  
Late Stages

The real-time monitoring of electric capacitance (impedance spectroscopy) allowed obtaining evidence that structures which look like intestinal villi can be formed during the cultivation under static conditions as well as during the cultivation in microfluidic chips. It was shown in this work via transcriptome analysis that the Hh signaling pathway is involved in the formation of villus-like structures in vitro, which was previously shown for their formation in vivo. impedance spectroscopy, intestine, villi, electric capacitance, Hh The study was funded by the Russian Science Foundation (Project 16-19-10597).

scholarly journals Stage-dependent effect of deferoxamine on growth of Plasmodium falciparum in vitro

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1250-1255 ◽  
Author(s):  
S Whitehead ◽  
TE Peto
Keyword(s):  
Plasmodium Falciparum ◽  
Growth Inhibition ◽  
Antimalarial Activity ◽  
Clinical Malaria ◽  
Inhibitory Effect ◽  
Parasite Development ◽  
And Control ◽  
Speed Of Onset

Abstract Deferoxamine (DF) has antimalarial activity that can be demonstrated in vitro and in vivo. This study is designed to examine the speed of onset and stage dependency of growth inhibition by DF and to determine whether its antimalarial activity is cytostatic or cytocidal. Growth inhibition was assessed by suppression of hypoxanthine incorporation and differences in morphologic appearance between treated and control parasites. Using synchronized in vitro cultures of Plasmodium falciparum, growth inhibition by DF was detected within a single parasite cycle. Ring and nonpigmented trophozoite stages were sensitive to the inhibitory effect of DF but cytostatic antimalarial activity was suggested by evidence of parasite recovery in later cycles. However, profound growth inhibition, with no evidence of subsequent recovery, occurred when pigmented trophozoites and early schizonts were exposed to DF. At this stage in parasite development, the activity of DF was cytocidal and furthermore, the critical period of exposure may be as short as 6 hours. These observations suggest that iron chelators may have a role in the treatment of clinical malaria.

scholarly journals Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 819
Author(s):  
Nicolai Rügen ◽  
Timothy P. Jenkins ◽  
Natalie Wielsch ◽  
Heiko Vogel ◽  
Benjamin-Florian Hempel ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Galleria Mellonella ◽  
Systemic Reactions ◽  
Venom Composition ◽  
Assassin Bug ◽  
Molecular Components ◽  
First Time ◽  
Tissue Digestion

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

scholarly journals Fe3O4@Pt nanoparticles to enable combinational electrodynamic/chemodynamic therapy

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Tong Chen ◽  
Qiang Chu ◽  
Mengyang Li ◽  
Gaorong Han ◽  
Xiang Li
Keyword(s):  
Fenton Reaction ◽  
Pt Nanoparticles ◽  
Therapeutic Modality ◽  
Ros Generation ◽  
Tumor Treatment ◽  
Superior Efficacy ◽  
Platinum Nanocrystals ◽  
First Time

AbstractElectrodynamic therapy (EDT) has recently emerged as a potential external field responsive approach for tumor treatment. While it presents a number of clear superiorities, EDT inherits the intrinsic challenges of current reactive oxygen species (ROS) based therapeutic treatments owing to the complex tumor microenvironment, including glutathione (GSH) overexpression, acidity and others. Herein for the first time, iron oxide nanoparticles are decorated using platinum nanocrystals (Fe3O4@Pt NPs) to integrate the current EDT with chemodynamic phenomenon and GSH depletion. Fe3O4@Pt NPs can effectively induce ROS generation based on the catalytic reaction on the surface of Pt nanoparticles triggered by electric field (E), and meanwhile it may catalyze intracellular H2O2 into ROS via Fenton reaction. In addition, Fe3+ ions released from Fe3O4@Pt NPs under the acidic condition in tumor cells consume GSH in a rapid fashion, inhibiting ROS clearance to enhance its antitumor efficacy. As a result, considerable in vitro and in vivo tumor inhibition phenomena are observed. This study has demonstrated an alternative concept of combinational therapeutic modality with superior efficacy.

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