Deciphering Molecular Determinants Underlying Penicillium digitatum’s Response to Biological and Chemical Antifungal Agents by Tandem Mass Tag (TMT)-Based High-Resolution LC-MS/MS

Penicillium digitatum is a widespread pathogen responsible for the postharvest decay of citrus, one of the most economically important crops worldwide. Currently, chemical fungicides are still the main strategy to control the green mould disease caused by the fungus. However, the increasing selection and proliferation of fungicide-resistant strains require more efforts to explore new alternatives acting via new or unexplored mechanisms for postharvest disease management. To date, several non-chemical compounds have been investigated for the control of fungal pathogens. In this scenario, understanding the molecular determinants underlying P. digitatum’s response to biological and chemical antifungals may help in the development of safer and more effective non-chemical control methods. In this work, a proteomic approach based on isobaric labelling and a nanoLC tandem mass spectrometry approach was used to investigate molecular changes associated with P. digitatum’s response to treatments with α-sarcin and beetin 27 (BE27), two proteins endowed with antifungal activity. The outcomes of treatments with these biological agents were then compared with those triggered by the commonly used chemical fungicide thiabendazole (TBZ). Our results showed that differentially expressed proteins mainly include cell wall-degrading enzymes, proteins involved in stress response, antioxidant and detoxification mechanisms and metabolic processes such as thiamine biosynthesis. Interestingly, specific modulations in response to protein toxins treatments were observed for a subset of proteins. Deciphering the inhibitory mechanisms of biofungicides and chemical compounds, together with understanding their effects on the fungal physiology, will provide a new direction for improving the efficacy of novel antifungal formulations and developing new control strategies.

Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation

Filovirus family consists of highly pathogenic viruses that have caused fatal outbreaks especially in many African countries. Previously, research focus has been on Ebola, Sudan and Marburg viruses leaving other filoviruses less well studied. Filoviruses, in general, pose a significant global threat since they are highly virulent and potentially transmissible between humans causing sporadic infections and local or widespread epidemics. Filoviruses have the ability to downregulate innate immunity, and especially viral protein 24 (VP24), VP35 and VP40 have variably been shown to interfere with interferon (IFN) gene expression and signaling. Here we systematically analyzed the ability of VP24 proteins of nine filovirus family members to interfere with retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated antigen 5 (MDA5) induced IFN-β and IFN-λ1 promoter activation. All VP24 proteins were localized both in the cell cytoplasm and nucleus in variable amounts. VP24 proteins of Zaire and Sudan ebolaviruses, Lloviu, Taï Forest, Reston, Marburg and Bundibugyo viruses (EBOV, SUDV, LLOV, TAFV, RESTV, MARV and BDBV, respectively) were found to inhibit both RIG-I and MDA5 stimulated IFN-β and IFN-λ1 promoter activation. The inhibition takes place downstream of interferon regulatory factor 3 phosphorylation suggesting the inhibition to occur in the nucleus. VP24 proteins of Mengla (MLAV) or Bombali viruses (BOMV) did not inhibit IFN-β or IFN-λ1 promoter activation. Six ebolavirus VP24s and Lloviu VP24 bound tightly, whereas MARV and MLAV VP24s bound weakly, to importin α5, the subtype that regulates the nuclear import of STAT complexes. MARV and MLAV VP24 binding to importin α5 was very weak. Our data provides new information on the innate immune inhibitory mechanisms of filovirus VP24 proteins, which may contribute to the pathogenesis of filovirus infections.

Competition, Conflict and Change of Mind: A Role of GABAergic Inhibition in the Primary Motor Cortex

Deciding between different voluntary movements implies a continuous control of the competition between potential actions. Many theories postulate a leading role of prefrontal cortices in this executive function, but strong evidence exists that a motor region like the primary motor cortex (M1) is also involved, possibly via inhibitory mechanisms. This was already shown during the pre-movement decision period, but not after movement onset. For this pilot experiment we designed a new task compatible with the dynamics of post-onset control to study the silent period (SP) duration, a pause in electromyographic activity after single-pulse transcranial magnetic stimulation that reflects inhibitory mechanisms. A careful analysis of the SP during the ongoing movement indicates a gradual increase in inhibitory mechanisms with the level of competition, consistent with an increase in mutual inhibition between alternative movement options. However, we also observed a decreased SP duration for high-competition trials associated with change-of-mind inflections in their trajectories. Our results suggest a new post-onset adaptive process that consists in a transient reduction of GABAergic inhibition within M1 for highly conflicting situations. We propose that this reduced inhibition softens the competition between concurrent motor options, thereby favoring response vacillation, an adaptive strategy that proved successful at improving behavioral performance.

Effects of imidazolium- and ammonium-based ionic liquids on clay swelling: experimental and simulation approach

Water-based fracturing fluids without an inhibitor promote clay swelling, which eventually creates wellbore instability. Several ionic liquids (ILs) have been studied as swelling inhibitors in recent years. The cations of the ILs are crucial to the inhibitory mechanisms that take place during hydraulic fracturing. Individual studies were carried out on several ILs with various cations, with the most frequently found being ammonium and imidazolium cations. As a result, the goal of this study is to compare these two cations to find an effective swelling inhibitor. A comparison and evaluation of the clay swelling inhibitory properties of tetramethylammonium chloride (TMACl) and 1-ethyl-3-methylimidazolium chloride (EMIMCl) were conducted in this work. Their results were also compared to a conventional inhibitor, potassium chloride (KCl), to see which performed better. The linear swelling test and the rheology test were used to determine the inhibitory performance of these compounds. Zeta potential measurements, Fourier-transform infrared spectroscopy, and contact angle measurements were carried out to experimentally explain the inhibitory mechanisms. In addition, the COSMO-RS simulation was conducted to explain the inhibitory processes and provide support for the experimental findings. The findings of the linear swelling test revealed that the swelling was reduced by 23.40% and 15.66%, respectively, after the application of TMACl and EMIMCl. The adsorption of ILs on the negatively charged clay surfaces, neutralizing the charges, as well as the lowering of the surface hydrophilicity, aided in the improvement of the swelling inhibition performance.

Insight into Yeast–Mycotoxin Relations

Fungal mycotoxins are secondary metabolites that can be present in green forage, hay, or silage. Consumption of contaminated plants or agricultural products can cause various animal and human diseases, which is why problems associated with mycotoxins have received particular attention. In addition, public pressure to produce healthy food and feed is also increasing. As the results of several surveys indicate that yeasts can decrease toxic effects by binding or converting secondary metabolites or control growth of harmful fungi, this article provides an overview of the yeast species that can have great potential in detoxification. The most important antagonistic yeast species against toxigenic fungi are described and the mode of their inhibitory mechanisms is also discussed. We provide an insight into toxin binding and biotransformation capacities of yeasts and examples of their use in silo. Issues requiring further study are also mentioned.

A systems approach to investigate GPCR-mediated Ras signaling network in chemoattractant sensing

A GPCR-mediated signaling network enables a chemotactic cell to generate adaptative Ras signaling in response to a large range of concentrations of a chemoattractant. To explore potential regulatory mechanisms of GPCR-controlled Ras signaling in chemosensing, we applied a software package, Simmune, to construct detailed spatiotemporal models simulating responses of the cAR1-mediated Ras signaling network. We first determined dynamics of G-protein activation and Ras signaling in Dictyostelium cells in response to cAMP stimulations using live-cell imaging and then constructed computation models by incorporating potential mechanisms. Using simulations, we validated the dynamics of signaling events and predicted the dynamic profiles of those events in the cAR1-mediated Ras signaling networks with defective Ras inhibitory mechanisms, such as without RasGAP, with RasGAP overexpression, or RasGAP hyperactivation. We described a method of using Simmune to construct spatiotemporal models of a signaling network and run computational simulations without writing mathematical equations. This approach will help biologists to develop and analyze computational models that parallel live-cell experiments.

Polyphenols as Potential Inhibitors of SARS-CoV-2 RNA Dependent RNA Polymerase (RdRp)

An increasing number of studies have demonstrated the antiviral nature of polyphenols, and many polyphenols have been proposed to inhibit SARS-CoV or SARS-CoV-2. Our previous study revealed the inhibitory mechanisms of polyphenols against DNA polymerase α and HIV reverse transcriptase to show that polyphenols can block DNA elongation by competing with the incoming NTPs. Here we applied computational approaches to examine if some polyphenols can also inhibit RNA polymerase (RdRp) in SARS-CoV-2, and we identified some better candidates than remdesivir, the FDA-approved drug against RdRp, in terms of estimated binding affinities. The proposed compounds will be further examined to develop new treatments for COVID-19.

Function and Inhibitory Mechanisms of Multidrug Efflux Pumps

Multidrug efflux pumps are inner membrane transporters that export multiple antibiotics from the inside to the outside of bacterial cells, contributing to bacterial multidrug resistance (MDR). Postgenomic analysis has demonstrated that numerous multidrug efflux pumps exist in bacteria. Also, the co-crystal structural analysis of multidrug efflux pumps revealed the drug recognition and export mechanisms, and the inhibitory mechanisms of the pumps. A single multidrug efflux pump can export multiple antibiotics; hence, developing efflux pump inhibitors is crucial in overcoming infectious diseases caused by multidrug-resistant bacteria. This review article describes the role of multidrug efflux pumps in MDR, and their physiological functions and inhibitory mechanisms.

Inhibition of Aspergillus flavus growth and aflatoxin B1 production by natamycin

Aspergillus flavus causes huge crop losses, reduces crop quality and has adverse effects on human and animal health. A large amount of food contaminated with aflatoxin can greatly increase the risk of liver cancer. Therefore, prevention and control of aflatoxin production have aroused attention of research in various countries. Natamycin extracted from Streptomyces spp. has been widely used in production practice due to its good specificity and safety. Here, we found that natamycin could significantly inhibit fungal growth, conidia germination, ergosterol and AFB1 production by A. flavus in a dose-dependent manner. Scanning electron microscope analysis indicated that the number of conidia was decreased, the outer wall of conidia was destroyed, and the mycelia were shrivelled and tangled by natamycin. RNA-Seq data indicated that natamycin inhibited fungal growth and conidia development of A. flavus by significantly down-regulating some genes involved in ergosterol biosynthesis, such as Erg13, HMG1 and HMG2. It inhibited conidia germination by significantly down-regulating some genes related to conidia development, such as FluG and VosA. After natamycin exposure, the decreased ratio of aflS/aflR caused by the down-regulation of all the structural genes, which subsequently resulted in the suppression of AFB1 production. In conclusion, this study served to reveal the inhibitory mechanisms of natamycin on fungal growth and AFB1 biosynthesis in A. flavus and to provide solid evidence for its application in controlling AFB1 contamination.