An entomopathogenic fungus exploits its host humoral antibacterial immunity to minimize bacterial competition in the hemolymph.

BACKGROUND: The insect hemolymph (blood-equivalent fluid), composed of a large number of hemocytes (blood cells) and a variety of soluble immune effectors, is hostile for pathogens including fungi. In order to survive in the insect hemocoel (body cavity), the entomopathogenic fungus (EPF) has evolved two classical coping strategies, namely evasion and suppression of the host immune reactions. However, it remains unclear whether EPF has other ways of coping with host immunity. RESULTS: In this study, we demonstrated that Metarhizium rileyi (an EPF) infection by injection of blastospores into the hemocoel enhanced the plasma antibacterial activity of cotton bollworm (Helicoverpa armigera), which was partially due to the enhanced expression of antimicrobial peptides (AMPs). The early stage of M. rileyi infection induced the translocation of gut bacteria into the hemocoel, where they were subsequently cleared due to the enhanced plasma antibacterial activity. Further, we showed that the enhanced plasma antibacterial activity and AMP expression were attributable to M. rileyi but not the invasive gut bacteria (opportunistic bacteria). Elevated ecdysone (major steroid hormone in insects) levels in the hemolymph at 48 h post-M. rileyi infection might contribute to the enhanced expression of AMPs. The fungus-elicited AMPs, such as cecropin 3 or lebocin, exhibited potent inhibitory activity against the opportunistic bacteria but not against hyphal bodies. In addition, the opportunistic bacteria competed with hyphal bodies for amino acid nutrients. CONCLUSIONS: M. rileyi infection induced the translocation of gut bacteria, and then the fungi activated and exploited its host humoral antibacterial immunity to eliminate opportunistic bacteria, preventing them from competing for nutrients in the hemolymph. Unlike the classical strategies, EPF utilizes to evade or suppress host immunity, our findings reveal a novel strategy of interaction between EPF and host immunity. Video Abstract.

The treatment of SARS-CoV2 with antivirals and mitigation of the cytokine storm syndrome: the role of gene expression.

In the absence of a vaccine, the treatment of SARS-CoV2 has focused on eliminating the virus with antivirals or mitigating the cytokine storm syndrome (CSS) that leads to the most common cause of death: respiratory failure. Herein we discuss the mechanisms of antiviral treatments for SARS-CoV2 and treatment strategies for the CSS. Antivirals that have shown in vitro activity against SARS-CoV2, or the closely related SARS-CoV1 and MERS-CoV, are compared on the enzymatic level and by potency in cells. For treatment of the CSS, we discuss medications that reduce the effects or expression of cytokines involved in the CSS with an emphasis on those that reduce IL-6 because of its central role in the development of the CSS. We show that some of the medications covered influence the activity or expression of enzymes involved in epigenetic processes and specifically those that add or remove modifications to histones or DNA. Where available, the latest clinical data showing the efficacy of the medications is presented. With respect to their mechanisms, we explain why some medications are successful, why others have failed, and why some untested medications may yet prove useful.

The Energy Density and Treatment Times Are the Main Factors That Affect the Efficacy of Long-Pulsed 1,064-nm Nd:YAG Laser Treatment for Onychomycosis Caused by Trichophyton rubrum.

BACKGROUND: No optimal regimen exists for the LPNYL (long-pulsed 1,064-nm neodymium:yttrium-aluminum-garnet laser) for treating onychomycosis. OBJECTIVE: To establish an optimal LPNYL treatment regimen for onychomycosis caused by Trichophyton rubrum (OCTr). PATIENTS AND METHODS: First, 511 infected nails of 177 patients were treated using LPNYL with orthogonally designed regimens according to various energy densities, spot sizes, pulse widths, and treatment times. The optimal treatment regimen was established by multivariate analysis. Next, 69 patients with 221 infected nails were randomized to receive oral itraconazole (drug group) and the optimal regimen of LPNYL treatment (laser group). The clinical efficacy (CE) and mycological efficacy (ME) were evaluated at 6 and 12 months following the start of treatment, and adverse reactions were recorded in both groups. RESULTS: Both CE and ME were significantly correlated with the energy density (p < 0.05) and treatment times (p < 0.05), but not with the spot size (0.071 < p < 0.083) or pulse width (0.051 < p < 0.060), at 6 or 12 months. There were no significant differences at 6 or 12 months (p > 0.05), and no significant difference was observed in CE at 12 months between the two groups (p > 0.05). At 6 months, the CE in the laser group was significantly higher than that in the drug group (p < 0.001). CONCLUSIONS: LPNYL is effective and safe for treating OCTr. The energy density and treatment times are the main factors that affect the efficacy. The optimal regimen for LPNYL is an energy density of 45 J/cm2, pulse width of 35 ms, spot size of 4 mm, frequency of 1 Hz, and 6 treatments with 1-week intervals. Laser treatment has rapid clinical recovery.

A simple assay for determination of levetiracetam in rat dried blood spots by LC-MS/MS.

BACKGROUND: A simple LC-MS/MS method was developed and validated for the quantification of levetiracetam (LEV, Keppra®), a broad-spectrum antiepileptic drug (AED) in rat dried blood spots (DBS). LEV was simply extracted with methanol spiked with adenosine (ADE) as IS before LC-MS/MS analysis. The correlation between the DBS and plasma concentrations of LEV was also determined. RESULTS: Linearity was from 0.067-60 µg/ml for LEV in DBS samples. The intra- and inter-day accuracy and precision of the assay met validation acceptance criteria. The developed assay was applied to monitor levetiracetam DBS levels in Sprague-Dawley rats after intravenous administration. DBS concentrations were well correlated to the plasma concentrations (R² = 0.9399), as fraction of LEV bound to blood cells remains very constant (0.466 ± 0.041) over a wide concentration range. CONCLUSION: The study illustrated that DBS could be used as alternative matrix for monitoring LEV in preclinical studies.

Metabolism of fenofibrate in beagle dogs: new metabolites identified and metabolic pathways revealed.

Fenofibrate is a prototypical agonist of peroxisome proliferator-activated receptor alpha (PPARalpha) which is well known to be associated with species related carcinogenesis. Important species differences have been reported in its metabolism and elimination pattern. Its new metabolites have been revealed in Cynomolgus monkeys and Sprague-Dawley rats. However in beagle dogs, several polar metabolites of fenofibrate have not been identified yet. In this study, beagle dogs were orally dosed with fenofibrate mixed with feeds. Urine and plasma samples were collected and subject to LC-MS/MS by comparison with authentic compounds and confirmed using an API 4000 Q-TRAP system. In vitro cultured primary hepatocytes were used to reveal metabolic pathways and confirm the data in vivo. Seven new metabolites of fenofibrate in dogs were identified, and their metabolic pathways were revealed. Fenofibrate in beagle dogs was found to be more prone to be metabolized into other secondary metabolites than fenofibric acid, compared with that in rats.

Quantification of pinosylvin in rat plasma by liquid chromatography-tandem mass spectrometry: application to a pre-clinical pharmacokinetic study.

Pinosylvin (trans-3,5-dihydroxystilbene), a naturally occurring analogue of resveratrol (trans-3,5,4'-trihydoxystilbene), exhibited various beneficial pharmacological activities in pre-clinical studies. To further probe its potential medicinal application, a sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the quantification of pinosylvin in rat plasma. A simple protein precipitation procedure was used for plasma cleanup before analysis by LC-MS/MS with electrospray ionisation and multiple reaction monitoring in its negative ion mode. This LC-MS/MS method demonstrated good selectivity, accuracy (intra- and inter-day analytical recovery within 100±7.7%), precision (intra- and inter-day coefficient of variation<12.0%) and sensitivity (lower limit of detection=1.0ng/mL), with excellent linearity (R(2)>0.99) over the range of 1-1000ng/mL. The pharmacokinetic profiles of pinosylvin were subsequently assessed in Sprague-Dawley rats. Following intravenous administration (5 or 10mg/kg), plasma levels of pinosylvin declined rapidly with a short half-life (t1/2<10min). Upon oral administration at 15mg/kg, pinosylvin could not be quantified in plasma (<1ng/mL) while dose-escalation to 50mg/kg led to a low and erratic plasma exposure with very poor estimated oral bioavailability (F<1%). The short half-life and limited systemic exposure of pinosylvin prompt caution in its therapeutic application and it warrants exploration in developing pinosylvin pro-drug.

Simultaneous analysis of bambuterol and its active metabolite terbutaline enantiomers in rat plasma by chiral liquid chromatography-tandem mass spectrometry.

A chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) simultaneous stereoselective analysis of bambuterol and its active metabolite terbutaline enantiomers in Wistar rat plasma has been developed and validated. All analytes and the internal standard were extracted from rat plasma samples by liquid-liquid extraction, separated on macrocyclic glycopeptide teicoplanin column with mobile phase constituted of 20mM ammonium acetate solution-methanol (10:90, v/v) at a flow-rate of 0.4 mL/min. Detection was performed on an API 3000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The calibration curves in the range 1-800 ng/mL were linear and the accuracy for each analyte was within 8.0%. The intra- and inter-day precision as determined from quality control samples was less than 10.1%. The validated assay was successfully used to determine the enantiomers of bambuterol and terbutaline in rat plasma samples in the pharmacokinetic studies of rac-bambuterol.