Genotypic, proteomic, and phenotypic approaches to decipher the response to caspofungin and calcineurin inhibitors in clinical isolates of echinocandin-resistant Candida glabrata.

BACKGROUND: Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets. OBJECTIVES: We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates. METHODS: Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival. RESULTS: F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin-calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity. CONCLUSIONS: Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.

Design and evaluation of synthetic bacterial consortia for optimized phenanthrene degradation through the integration of genomics and shotgun proteomics.

Two synthetic bacterial consortia (SC) composed of bacterial strains Sphingobium sp. (AM), Klebsiella aerogenes (B), Pseudomonas sp. (Bc-h and T), Burkholderia sp. (Bk) and Inquilinus limosus (Inq) isolated from a natural phenanthrene (PHN)-degrading consortium (CON) were developed and evaluated as an alternative approach to PHN biodegradation in bioremediation processes. A metabolic network showing the potential role of strains was reconstructed by in silico study of the six genomes and classification of dioxygenase enzymes using RHObase and AromaDeg databases. Network analysis suggested that AM and Bk were responsible for PHN initial attack, while Inq, B, T and Bc-h would degrade PHN metabolites. The predicted roles were further confirmed by physiological, RT-qPCR and metaproteomic assays. SC-1 with AM as the sole PHN degrader was the most efficient. The ecological roles inferred in this study can be applied to optimize the design of bacterial consortia and tackle the biodegradation of complex environmental pollutants.

Assessing interactions, predicting function, and increasing degradation potential of a PAH-degrading bacterial consortium by effect of an inoculant strain.

A natural phenanthrene-degrading consortium CON was inoculated with an exogenous strain Sphingobium sp. (ex Sp. paucimobilis) 20006FA yielding the consortium called I-CON, in order to study ecological interactions into the bacterial community. DGGE and proteomic profiles and analyses by HTS (High-Throughput Sequencing) technologies demonstrated inoculant establishment and changes on CON composition. Inoculation increased degradation efficiency in I-CON and prevented intermediate HNA accumulation. This could be explained not only by the inoculation, but also by enrichment in Achromobacter genus at expense of a decrease in Klebsiella genus. After inoculation, cooperation between Sphingobium and Achromobacter genera were improved, thereby, some competition could have been generated, and as a consequence, species in minor proportion (cheaters), as Inquilinus sp. and Luteibacter sp., were not detected. Sequences of Sphingobium (corresponding to the inoculated strain) did not vary. PICRUSt predicted a network with bacterial phylotypes connected with enzymes, showing functional redundancy in the phenanthrene pathway, with exception of the first enzymes biphenyl-2,3-diol 1,2-dioxygenase and protocatechuate 4,5-dioxygenase that were only encoded in Sphingobium sp. This is the first report where a natural consortium that has been characterized by HTS technologies is inoculated with an exogenous strain in order to study competitiveness and interactions.

PDGF-BB Preserves Mitochondrial Morphology, Attenuates ROS Production, and Upregulates Neuroglobin in an Astrocytic Model Under Rotenone Insult.

Platelet-derived growth factor, subtype BB (PDGF-BB) is a mitogenic growth factor produced in different cell types such as platelets, fibroblasts, neurons, and astrocytes. Previous reports have shown that different PDGF isoforms exert a neuroprotective effect in neurons and astrocytes against multiple degenerative insults. Previously, we showed that pretreatment with PDGF-BB for 24 h increased cell viability, preserved nuclear morphology and mitochondrial membrane potential following stimulation with rotenone, and reduced free radical production nearly to control conditions. In the present study, we explored the potential mechanisms associated with PDGF-BB protection against oxidative damage. Our results showed that PDGF-BB protected astrocytic cells through multiple responses, including decrease in the expression of cytoskeleton proteins, attenuated free radicals (reactive oxygen species (ROS)) production, preservation of mitochondrial ultrastructure, and improved expression of neuroglobin (Ngb1). In summary, these findings point out that PDGF-BB protects astrocytic cells by a reduction in ROS production and activation of antioxidant mechanisms.

L-Type Calcium Channels Modulation by Estradiol.

Voltage-gated calcium channels are key regulators of brain function, and their dysfunction has been associated with multiple conditions and neurodegenerative diseases because they couple membrane depolarization to the influx of calcium-and other processes such as gene expression-in excitable cells. L-type calcium channels, one of the three major classes and probably the best characterized of the voltage-gated calcium channels, act as an essential calcium binding proteins with a significant biological relevance. It is well known that estradiol can activate rapidly brain signaling pathways and modulatory/regulatory proteins through non-genomic (or non-transcriptional) mechanisms, which lead to an increase of intracellular calcium that activate multiple kinases and signaling cascades, in the same way as L-type calcium channels responses. In this context, estrogens-L-type calcium channels signaling raises intracellular calcium levels and activates the same signaling cascades in the brain probably through estrogen receptor-independent modulatory mechanisms. In this review, we discuss the available literature on this area, which seems to suggest that estradiol exerts dual effects/modulation on these channels in a concentration-dependent manner (as a potentiator of these channels in pM concentrations and as an inhibitor in nM concentrations). Indeed, estradiol may orchestrate multiple neurotrophic responses, which open a new avenue for the development of novel estrogen-based therapies to alleviate different neuropathologies. We also highlight that it is essential to determine through computational and/or experimental approaches the interaction between estradiol and L-type calcium channels to assist these developments, which is an interesting area of research that deserves a closer look in future biomedical research.

Growth Factors and Astrocytes Metabolism: Possible Roles for Platelet Derived Growth Factor.

Astrocytes exert multiple functions in the brain such as the development of blood-brain barrier characteristics, the promotion of neurovascular coupling, attraction of cells through the release of chemokines, clearance of toxic substances and generation of antioxidant molecules and growth factors. In this aspect, astrocytes secrete several growth factors (BDNF, GDNF, NGF, and others) that are fundamental for cell viability, oxidant protection, genetic expression and modulation of metabolic functions. The platelet derived growth factor (PDGF), which is expressed by many SNC cells, including astrocytes, is an important molecule that has shown neuroprotective potential, improvement of wound healing, regulation of calcium metabolism and mitochondrial function. Here we explore some of these astrocyte-driven functions of growth factors and their possible therapeutic uses in the context of neurodegeneration.