Increasing the fungicidal action of Amphotericin B by inhibiting the Nitric Oxide-Dependent tolerance pathway

Amphotericin B (AmB) induces oxidative and nitrosative stresses, characterized by production of reactive oxygen and nitrogen species, in fungi. Yet, how these toxic species contribute to AmB-induced fungal cell death is unclear. We investigated the role of superoxide and nitric oxide radicals in AmB's fungicidal activity in Saccharomyces cerevisiae, using a digital microfluidic platform, which enabled monitoring individual cells at a spatiotemporal resolution, and plating assays. The nitric oxide synthase inhibitor L-NAME was used to interfere with nitric oxide radical production. L-NAME increased and accelerated AmB-induced accumulation of superoxide radicals, membrane permeabilization, and loss of proliferative capacity in S. cerevisiae. In contrast, the nitric oxide donor S-nitrosoglutathione inhibited AmB's action. Hence, superoxide radicals were important for AmB's fungicidal action, whereas nitric oxide radicals mediated tolerance towards AmB. Finally, also the human pathogens Candida albicans and Candida glabrata were more susceptible to AmB in the presence of L-NAME, pointing to the potential of AmB-L-NAME combination therapy to treat fungal infections.Kim Vriens acknowledges the receipt of a predoctoral grant from the Flanders Innovation & Entrepeneurship Agency (IWT-SB 111016); Karin Thevissen acknowledges the receipt of a mandate of Industrial Research Fund (KU Leuven). In addition, the research leading to these results has received funding from the Research Foundation - Flanders (FWO G086114N and G080016N) and the KU Leuven (OT 13/ 058 and IDO 10/012, IOF KP/12/009 Atheromix, IOF KP/ 12/002 Nanodiag). This work was partially developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Belém Sampaio-Marques is supported by the fellowship SFRH/BPD/90533/2012 funded by Fundação para a Ciência e Tecnologia (FCT, Portugal).info:eu-repo/semantics/publishedVersio

The plant decapeptide OSIP108 can alleviate mitochondrial dysfunction induced by cisplatin in human cells

We investigated the effect of the Arabidopsis thaliana-derived decapeptide OSIP108 on human cell tolerance to the chemotherapeutic agent cisplatin (Cp), which induces apoptosis and mitochondrial dysfunction. We found that OSIP108 increases the tolerance of HepG2 cells to Cp and prevents Cp-induced changes in basic cellular metabolism. More specifically, we demonstrate that OSIP108 reduces Cp-induced inhibition of respiration, decreases glycolysis and prevents Cp-uptake in HepG2 cells. Apart from its protective action against Cp in human cells, OSIP108 also increases the yeast Saccharomyces cerevisiae tolerance to Cp. A limited yeast-based study of OSIP108 analogs showed that cyclization does not severely affect its activity, which was further confirmed in HepG2 cells. Furthermore, the similarity in the activity of the D-stereoisomer (mirror image) form of OSIP108 with the L stereoisomer suggests that its mode of action does not involve binding to a stereospecific receptor. In addition, as OSIP108 decreases Cp uptake in HepG2 cells and the anti-Cp activity of OSIP108 analogs without free cysteine is reduced, OSIP108 seems to protect against Cp-induced toxicity only partly via complexation. Taken together, our data indicate that OSIP108 and its cyclic derivatives can protect against Cp-induced toxicity and, thus, show potential as treatment options for mitochondrial dysfunction- and apoptosis-related conditions

Embry-Riddle Fly Paper 1943-01-15

https://commons.erau.edu/fly-paper/1159/thumbnail.jp

GAMER MRI: Gated-attention mechanism ranking of multi-contrast MRI in brain pathology.

During the last decade, a multitude of novel quantitative and semiquantitative MRI techniques have provided new information about the pathophysiology of neurological diseases. Yet, selection of the most relevant contrasts for a given pathology remains challenging. In this work, we developed and validated a method, Gated-Attention MEchanism Ranking of multi-contrast MRI in brain pathology (GAMER MRI), to rank the relative importance of MR measures in the classification of well understood ischemic stroke lesions. Subsequently, we applied this method to the classification of multiple sclerosis (MS) lesions, where the relative importance of MR measures is less understood. GAMER MRI was developed based on the gated attention mechanism, which computes attention weights (AWs) as proxies of importance of hidden features in the classification. In the first two experiments, we used Trace-weighted (Trace), apparent diffusion coefficient (ADC), Fluid-Attenuated Inversion Recovery (FLAIR), and T1-weighted (T1w) images acquired in 904 acute/subacute ischemic stroke patients and in 6,230 healthy controls and patients with other brain pathologies to assess if GAMER MRI could produce clinically meaningful importance orders in two different classification scenarios. In the first experiment, GAMER MRI with a pretrained convolutional neural network (CNN) was used in conjunction with Trace, ADC, and FLAIR to distinguish patients with ischemic stroke from those with other pathologies and healthy controls. In the second experiment, GAMER MRI with a patch-based CNN used Trace, ADC and T1w to differentiate acute ischemic stroke lesions from healthy tissue. The last experiment explored the performance of patch-based CNN with GAMER MRI in ranking the importance of quantitative MRI measures to distinguish two groups of lesions with different pathological characteristics and unknown quantitative MR features. Specifically, GAMER MRI was applied to assess the relative importance of the myelin water fraction (MWF), quantitative susceptibility mapping (QSM), T1 relaxometry map (qT1), and neurite density index (NDI) in distinguishing 750 juxtacortical lesions from 242 periventricular lesions in 47 MS patients. Pair-wise permutation t-tests were used to evaluate the differences between the AWs obtained for each quantitative measure. In the first experiment, we achieved a mean test AUC of 0.881 and the obtained AWs of FLAIR and the sum of AWs of Trace and ADC were 0.11 and 0.89, respectively, as expected based on previous knowledge. In the second experiment, we achieved a mean test F1 score of 0.895 and a mean AW of Trace = 0.49, of ADC = 0.28, and of T1w = 0.23, thereby confirming the findings of the first experiment. In the third experiment, MS lesion classification achieved test balanced accuracy = 0.777, sensitivity = 0.739, and specificity = 0.814. The mean AWs of T1map, MWF, NDI, and QSM were 0.29, 0.26, 0.24, and 0.22 (p < 0.001), respectively. This work demonstrates that the proposed GAMER MRI might be a useful method to assess the relative importance of MRI measures in neurological diseases with focal pathology. Moreover, the obtained AWs may in fact help to choose the best combination of MR contrasts for a specific classification problem

Recommended Implementation of Quantitative Susceptibility Mapping for Clinical Research in The Brain: A Consensus of the ISMRM Electro-Magnetic Tissue Properties Study Group

This article provides recommendations for implementing quantitative susceptibility mapping (QSM) for clinical brain research. It is a consensus of the ISMRM Electro-Magnetic Tissue Properties Study Group. While QSM technical development continues to advance rapidly, the current QSM methods have been demonstrated to be repeatable and reproducible for generating quantitative tissue magnetic susceptibility maps in the brain. However, the many QSM approaches available give rise to the need in the neuroimaging community for guidelines on implementation. This article describes relevant considerations and provides specific implementation recommendations for all steps in QSM data acquisition, processing, analysis, and presentation in scientific publications. We recommend that data be acquired using a monopolar 3D multi-echo GRE sequence, that phase images be saved and exported in DICOM format and unwrapped using an exact unwrapping approach. Multi-echo images should be combined before background removal, and a brain mask created using a brain extraction tool with the incorporation of phase-quality-based masking. Background fields should be removed within the brain mask using a technique based on SHARP or PDF, and the optimization approach to dipole inversion should be employed with a sparsity-based regularization. Susceptibility values should be measured relative to a specified reference, including the common reference region of whole brain as a region of interest in the analysis, and QSM results should be reported with - as a minimum - the acquisition and processing specifications listed in the last section of the article. These recommendations should facilitate clinical QSM research and lead to increased harmonization in data acquisition, analysis, and reporting

Identification of survival-promoting OSIP108 peptide variants and their internalization in human cells

The plant-derived decapeptide OSIP108 increases tolerance of yeast and human cells to apoptosis-inducing agents, such as copper and cisplatin. We performed a whole amino acid scan of OSIP108 and conducted structure-activity relationship studies on the induction of cisplatin tolerance (CT) in yeast. The use of cisplatin as apoptosis-inducing trigger in this study should be considered as a tool to better understand the survival-promoting nature of OSIP108 and not for purposes related to anti-cancer treatment. We found that charged residues (Arg, His, Lys, Glu or Asp) or a Pro on positions 4–7 improved OSIP108 activity by 10% or more. The variant OSIP108[G7P] induced the most pronounced tolerance to toxic concentrations of copper and cisplatin in yeast and/or HepG2 cells. Both OSIP108 and OSIP108[G7P] were shown to internalize equally into HeLa cells, but at a higher rate than the inactive OSIP108[E10A], suggesting that the peptides can internalize into cells and that OSIP108 activity is dependent on subsequent intracellular interactions. In conclusion, our studies demonstrated that tolerance/survival-promoting properties of OSIP108 can be significantly improved by single amino acid substitutions, and that these properties are dependent on (an) intracellular target(s), yet to be determined

Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies

Sphingolipid accumulation causes mitochondrial dysregulation and cell death

Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2Δ cells depends on nutritional status. Abnormal Ras activation in csg2Δ cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2Δ cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways