-
PLoS Pathogens Mar 2021Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent,...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.
Topics: Antiviral Agents; COVID-19; Cell Culture Techniques; Containment of Biohazards; Genome, Viral; High-Throughput Screening Assays; Humans; SARS-CoV-2; Virus Replication
PubMed: 33711082
DOI: 10.1371/journal.ppat.1009439 -
Molecular Cell May 2021Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8...
Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.
Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagosomes; Autophagy; Autophagy-Related Protein 8 Family; Autophagy-Related Proteins; Cysteine Endopeptidases; Female; HCT116 Cells; HEK293 Cells; HeLa Cells; Humans; Influenza A virus; Macrolides; Male; Mice; Microtubule-Associated Proteins; Monensin; Phagocytosis; Phosphatidylethanolamines; Phosphatidylserines; Protein Processing, Post-Translational; RAW 264.7 Cells; Signal Transduction
PubMed: 33909989
DOI: 10.1016/j.molcel.2021.03.020 -
BioRxiv : the Preprint Server For... Apr 2023Monkeypox virus (MPXV) infection in humans are historically restricted to endemic regions in Africa. However, in 2022, an alarming number of MPXV cases have been...
UNLABELLED
Monkeypox virus (MPXV) infection in humans are historically restricted to endemic regions in Africa. However, in 2022, an alarming number of MPXV cases have been reported globally with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. MPXV vaccines are limited and only two antivirals, tecovirimat and brincidofovir, approved by the United States (US) Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit Orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (Scarlet or GFP) and luciferase (Nluc) reporter genes to identify compounds with anti-Orthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed antiviral activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating the broad-spectrum antiviral activity against Orthopoxviruses and their potential to be used for the antiviral treatment of MPXV, or other Orthopoxvirus, infections.
IMPORTANCE
Despite the eradication of smallpox, some Orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, there is presently limited access to those vaccines. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV, and other potentially zoonotic Orthopoxvirus infections. Here, we show that thirteen compounds, derived from two different libraries, previously found to inhibit several RNA viruses, exhibit also antiviral activity against VACV. Notably, eleven compounds also displayed antiviral activity against MPXV, demonstrating their potential to be incorporated into the therapeutic armamentarium to combat Orthopoxvirus infections.
PubMed: 37131608
DOI: 10.1101/2023.04.19.537483 -
Microbiology Spectrum Aug 2023Monkeypox virus (MPXV) infections in humans have historically been restricted to regions of endemicity in Africa. However, in 2022, an alarming number of MPXV cases were...
Monkeypox virus (MPXV) infections in humans have historically been restricted to regions of endemicity in Africa. However, in 2022, an alarming number of MPXV cases were reported globally, with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. The supply of MPXV vaccines is limited, and only two antivirals, tecovirimat and brincidofovir, approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (mScarlet or green fluorescent protein [GFP]) and luciferase (Nluc) reporter genes to identify compounds with antiorthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed inhibitory activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating their inhibitory activity against two orthopoxviruses. Despite the eradication of smallpox, some orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, access to those vaccines is limited. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV infection and other potentially zoonotic orthopoxvirus infections. Here, we show that 13 compounds, derived from two different libraries, previously found to inhibit several RNA viruses, also inhibit VACV. Notably, 11 compounds also displayed inhibitory activity against MPXV.
Topics: Humans; Mpox (monkeypox); Mycophenolic Acid; Smallpox; Antimycin A; Monensin; Rotenone; Valinomycin; Monkeypox virus; Antiviral Agents
PubMed: 37278625
DOI: 10.1128/spectrum.04745-22 -
Animals : An Open Access Journal From... Oct 2021Two types of modified nano-montmorillonite (MNM) were developed by ion-exchange reactions using two different surfactants; sodium dodecyl sulfate (SDS) and...
Two types of modified nano-montmorillonite (MNM) were developed by ion-exchange reactions using two different surfactants; sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CETAB), to prepare MNM and MNM, respectively. Both MNM types were on the nano-scale and had higher cation-exchange capacity values than NM clay. The MNM had the highest zeta potential (-27 mV) compared with the other clays. Effects of MNM types on ruminal batch culture fermentation, nutrient degradability, and methane (CH) emission compared with monensin were evaluated using a semi-automatic gas production system. The experimental treatments were the control (0 supplementations), monensin (40 mg/kg DM), and NM (5 g NM/kg DM), and two levels of MNM and MNM were supplemented at 0.05 (low) and 0.5 (high) g/kg DM to the control basal feed substrate. Among the experimental treatments, the high dose of both MNM types reduced ( < 0.01) CH production and ammonia concentrations compared with the control, while only MNM treatment tended to increase ( = 0.08) the truly degraded organic matter compared with monensin. All MNM treatments increased ( < 0.01) acetate molar proportions compared with monensin. The high MNM increased ( < 0.01) the ruminal batch culture pH compared with the control and monensin. The MNM supplemented at 0.5 g/kg DM is the most efficient additive to reduce CH emission with the advantage of enhancing the nutrient degradability of the experimental feed substrate. These results indicated that MNM could modulate the ruminal fermentation pattern in a dose- and type-dependent manner.
PubMed: 34680023
DOI: 10.3390/ani11103005 -
Translational Animal Science Oct 2022Monensin and virginiamycin are included in beef cattle finishing diets as prophylaxis to minimize the incidence of ruminal acidosis and liver abscesses. Due to different...
Monensin and virginiamycin are included in beef cattle finishing diets as prophylaxis to minimize the incidence of ruminal acidosis and liver abscesses. Due to different and probably complementary modes of action, this study aimed to determine the effects of a combination of monensin and virginiamycin, both included in the diet at recommended doses, on ruminal health, the occurrence of liver abscesses, and growth performance of feedlot-finished cattle. One hundred and forty-four steers (6 animals/pen) were fed 1 of 3 corn-based finishing diets containing 30 mg of monensin (), 25 mg of virginiamycin (), or 30 and 25 mg of monensin and virginiamycin (), respectively, per kilogram of dry matter. Ruminal pH probes were inserted into two animals per pen and set to record pH every 10 min. On d 100, animals were slaughtered, and rumens and livers were recovered, on which occurrence and degree of ruminal damage, prevalence and number of liver abscesses, and liver scores (A-: livers with no more than two small abscesses; A+: livers with at least one large abscess or more than four medium abscesses; A: any other abscessed liver) were determined. Simultaneous inclusion of monensin and virginiamycin resulted in a 4.3% decrease ( < 0.04) in dry matter intake (; 8.8, 9.2, and 9.2 ± 0.19 kg/d for MN + VM, MN, and VM-fed animals, respectively) and similar ( > 0.13) average daily body weight gain (; 1.49 ± 0.021 kg/d) and hot carcass weight (; 269 ± 1.7 kg), compared with feeding diets containing one additive or the other. Therefore, in terms of ADG, a 9.4% improvement ( < 0.01) in feed efficiency was observed in MN + VM-fed animals. Backfat thickness (5.6 ± 0.08 mm) and ribeye area (69.9 ± 0.53 cm) remained unaffected ( ≥ 0.74), as well as the minimum (4.98 ± 0.047), mean (6.11 ± 0.037), and maximum ruminal pH (7.23 ± 0.033) values and the time (125 ± 22.3 min/d), area (57.67 ± 12.383 pH × h), and episodes (22 ± 3.8 bouts) of pH below 5.6 ( ≥ 0.12). Overall, prevalence (24 ± 3.4%) and the number of liver abscesses (1.6 ± 0.14 abscesses/abscessed liver), liver scores (20 ± 3.1% of A- and 4 ± 1.8% of A livers), and prevalence (67 ± 3.5%) and degree of damage to the ruminal epithelium (2.5 ± 0.22% affected surface) were similar ( ≥ 0.18) across treatments; however, the occurrence of ruminal lesions tended ( ≤ 0.07) to be associated with that of liver abscesses and reduced ADG when feeding monensin alone.
PubMed: 36601062
DOI: 10.1093/tas/txac154 -
Ecotoxicology and Environmental Safety Oct 2022Antibiotic residue has become an emerging environmental contaminant, while the toxicological effects and underlying mechanisms caused by the co-exposure to multiple...
Antibiotic residue has become an emerging environmental contaminant, while the toxicological effects and underlying mechanisms caused by the co-exposure to multiple veterinary antibiotics were rarely studied. In this study, male Sprague Dawley rats were exposed to monensin (M) (1, 2, 10 mg/(kg·body weight (BW)) combined with sulfamethazine (S) (60, 120, 600 mg/(kg·BW)) or single drugs for 28 consecutive days. The body weight, hematological and blood biochemical parameters, organ coefficients, and histopathology were analyzed to discover their combined toxicity effect. Transcriptomic analysis was used to reveal the possible mechanisms of their joint toxicity. Compared with the control group, the weight gain rate was significantly reduced in the H-M+S and H-S, and alkaline phosphatase in H-M+S was significantly increased. Furthermore, relative liver and kidneys weight was significantly increased, and the liver of H-M+S showed more severe lesions in histopathological analysis. For H-M+S, H-M and H-S, transcriptomic results showed that 344, 246, and 99 genes were differentially expressed, respectively. The Gene Ontology terms mainly differ in sterol biosynthetic process and steroid hydroxylase activity. The Kyoto Encyclopedia of Genes and Genome pathways showed abnormal retinol metabolism, metabolism of xenobiotics by cytochrome P450, and drug metabolism-cytochrome 450; the common 30 genes were screened from the network of protein-protein interaction. The results showed that mixed contamination of M and S produces hepatotoxicity by interfering with linoleic acid metabolism, retinol metabolism and CYP450 enzyme-dominated drug metabolism. Further analysis showed that Cyp1a2, Cyp2c61, Ugt1a3, and Ugt1a5 might be the key genes. These findings could provide more evidence for investigating the toxic effects and metabolism of mixed antibiotics contamination in mammals.
Topics: Alkaline Phosphatase; Animals; Anti-Bacterial Agents; Body Weight; Cytochrome P-450 CYP1A2; Cytochrome P-450 Enzyme System; Linoleic Acid; Liver; Male; Mammals; Monensin; Rats; Rats, Sprague-Dawley; Steroid Hydroxylases; Sterols; Sulfamethazine; Transcriptome; Vitamin A; Xenobiotics
PubMed: 36155339
DOI: 10.1016/j.ecoenv.2022.114110 -
Scientific Reports Nov 2022Asthma is a common respiratory disease associated with airway hyperresponsiveness (AHR), airway inflammation and mast cell (MC) accumulation in the lung. Monensin, an...
Asthma is a common respiratory disease associated with airway hyperresponsiveness (AHR), airway inflammation and mast cell (MC) accumulation in the lung. Monensin, an ionophoric antibiotic, has been shown to induce apoptosis of human MCs. The aim of this study was to define the effect of monensin on MC responses, e.g., antigen induced bronchoconstriction, and on asthmatic features in models of allergic asthma. Tracheal segments from house dust mite (HDM) extract sensitized guinea pigs were isolated and exposed to monensin, followed by histological staining to quantify MCs. Both guinea pig tracheal and human bronchi were used for pharmacological studies in tissue bath systems to investigate the monensin effect on tissue viability and antigen induced bronchoconstriction. Further, an HDM-induced guinea pig asthma model was utilized to investigate the effect of monensin on AHR and airway inflammation. Monensin decreased MC number, caused MC death, and blocked the HDM or anti-IgE induced bronchoconstriction in guinea pig and human airways. In the guinea pig asthma model, HDM-induced AHR, airway inflammation and MC hyperplasia could be inhibited by repeated administration of monensin. This study indicates that monensin is an effective tool to reduce MC number and MCs are crucial for the development of asthma-like features.
Topics: Guinea Pigs; Humans; Animals; Mast Cells; Pyroglyphidae; Monensin; Asthma; Allergens; Inflammation; Disease Models, Animal
PubMed: 36344588
DOI: 10.1038/s41598-022-23486-1 -
Antibiotics (Basel, Switzerland) Jun 2023Neuroblastoma is the most common pediatric solid tumor originating from the neural crest. New treatment options are needed to improve treatment outcomes and the survival...
Neuroblastoma is the most common pediatric solid tumor originating from the neural crest. New treatment options are needed to improve treatment outcomes and the survival of patients with neuroblastoma. Monensin is an ionophore antibiotic with antiparasitic, antibacterial, and anticancer properties isolated from . The aim of this study was to investigate the therapeutic effects of single and combined monensin and rapamycin treatments on mTOR (mammalian target of rapamycin) signaling pathway-mediated apoptosis and tumor growth in an SH-SY5Y neuroblastoma cell xenograft model. Control, monensin, rapamycin, and monensin + rapamycin groups were formed in the xenograft neuroblastoma model obtained from CD1 nude mice, and tumor volumes and animal weights were recorded throughout the treatment. In xenograft neuroblastoma tumor tissues, apoptosis was determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) and cleaved-caspase 3 immunohistochemistry, and PI3K (phosphoinositide-3-kinase)/AKT/mTOR expression was determined by the immunohistochemistry and immunofluorescence methods. The combination of monensin and rapamycin was to reduce the growth of xenograft neuroblastoma tumor tissues, trigger apoptosis, and suppress the expression of PI3K/AKT/mTOR. A significant increase in apoptotic cell rate was demonstrated in the combination group, supported by cleaved-caspase 3 immunohistochemistry results. In addition, it was reported that the combination treatment regime triggered apoptosis by reducing the expression of phosphorylated PI3K/AKT/mTOR. Our preclinical results may be a precursor to develop new therapeutic approaches to treat neuroblastoma.
PubMed: 37370314
DOI: 10.3390/antibiotics12060995 -
[Electrophysiological Effects of Ionophore-induced Increases in Intracellular Na in Cardiomyocytes].Yakugaku Zasshi : Journal of the... 2021Na ionophores increase intracellular Na ([Na]i). Membrane potentials and currents were measured using microelectrode and whole-cell patch-clamp techniques. Monensin... (Review)
Review
Na ionophores increase intracellular Na ([Na]i). Membrane potentials and currents were measured using microelectrode and whole-cell patch-clamp techniques. Monensin (10-3×10 M) reduced the slope of the pacemaker potentials and shortened the action potential duration (APD) in sino-atrial nodal and Purkinje cells. Monensin (10 M) shortened the APD and reduced the amplitude of the plateau phase in ventricular myocytes. Monensin decreased the hyperpolarization-activated inward current (I), and it increased the transient outward potassium current (I) in Purkinje cells. In addition, monensin decreased the sodium current (I), shifting the inactivation curve to the hyperpolarized direction. Moreover, monensin decreased the L-type calcium current (I) in ventricular myocytes. The Na-Ca exchange current (I) was augmented particularly in the reverse mode, and the Na-K pump current (I) was also activated by monensin in cardiomyocytes. The ATP-activated potassium current (I) could be induced by monensin. Notably, the inward rectifying K current (I), and the slow delayed outward K current (I) were not affected evidently by monensin. Collectively, alteration of [Na]i can influence the activities of various ion channels and transporters. Thus, the significance of altered [Na]i should be taken into consideration in the action of drugs affecting [Na]i such as digitalis, Na channel blockers, and Na channel activating agents.
Topics: Action Potentials; Animals; Electrophysiological Phenomena; Humans; Monensin; Myocytes, Cardiac; Patch-Clamp Techniques; Potassium; Purkinje Cells; Rabbits; Sinoatrial Node; Sodium; Sodium Ionophores
PubMed: 33952755
DOI: 10.1248/yakushi.20-00235