The structure of associations: Method insights from analyzing 28 clinical isolates of Cryptococcus neoformans.

Clinical isolates of a fungal pathogen from a single region or country often exhibit structural clonality or phylogenetic clustering at the sequence or MLST level; such population structure can persist also in larger samples. In efforts to improve causal understanding of pathogenesis at the molecular level, genome-wide association screening methods initially designed for other kingdoms have been applied to fungi. The example of a Colombian dataset of 28 clinical Cryptococcus neoformans VNI isolates indicates where the output from standard pipelines may need to be analyzed in new ways in order to efficiently extract hypotheses for experiments from fungal genotype-phenotype data.

Collections of clinical isolates of a human fungal pathogen can consist of clusters of genetically similar isolates. Such clustering complicates the screening for genetic associations with clinically relevant traits. We propose new methods, illustrating them for the fungus causing cryptococcosis.

Deciphering the Association among Pathogenicity, Production and Polymorphisms of Capsule/Melanin in Clinical Isolates of Cryptococcus neoformans var. grubii VNI.

BACKGROUND: Cryptococcus neoformans is an opportunistic fungal pathogen that can cause meningitis in immunocompromised individuals. The objective of this work was to study the relationship between the phenotypes and genotypes of isolates of clinical origin from different cities in Colombia. METHODS: Genome classification of 29 clinical isolates of C. neoformans var. grubii was performed using multilocus sequence typing (MLST), and genomic sequencing was used to genotype protein-coding genes. Pathogenicity was assessed in a larval model, and melanin production and capsule size were evaluated in vitro and in vivo. RESULTS: Eleven MLST sequence types (STs) were found, the most frequent being ST69 (n = 9), ST2, ST93, and ST377 (each with n = 4). In the 29 isolates, different levels of pigmentation, capsule size and pathogenicity were observed. Isolates classified as highly pathogenic showed a tendency to exhibit larger increases in capsule size. In the analysis of polymorphisms, 48 non-synonymous variants located in the predicted functional domains of 39 genes were found to be associated with capsule size change, melanin, or pathogenicity. CONCLUSIONS: No clear patterns were found in the analysis of the phenotype and genotype of Cryptococcus. However, the data suggest that the increase in capsule size is a key variable for the differentiation of pathogenic isolates, regardless of the method used for its induction.

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.

Genetic structure along an altitudinal gradient in Lippia origanoides, a promising aromatic plant species restricted to semiarid areas in northern South America.

The genetic diversity and population structure of Lippia origanoides, a species of the Verbenaceae family that shows promise as a crop plant, was investigated along an altitudinal gradient in the basin of the Chicamocha River in northeastern Colombia. The economic importance of the species, quality of its essential oils, and the fact that it is restricted to some few semiarid areas in northern South America may put the species at risk in a scenario of uncontrolled harvest of natural populations. Lippia origanoides was sampled along an altitudinal gradient from 365 to 2595 m.a.s.l. throughout Chicamocha River Canyon, a semiarid area in northeastern Colombia. Genetic diversity was assessed by means of AFLP markers. The number of AFLP loci (355) and the number of individuals sampled (173) were sufficient to reliably identify four populations at contrasting altitudes (F(ST) = 0.18, P-value < 0.0000), two populations in the lower basin, one population in the medium basin, and one population in the upper basin, with a low level of admixture between them. In average, genetic diversity within populations was relatively high (Ht = 0.32; I = 0.48); nevertheless, diversity was significantly reduced at higher altitude, a pattern that may be consistent with a scenario of range expansion toward higher elevations in an environment with more extreme conditions. The differences in altitude among the basins in the Chicamocha River seem to be relevant in determining the genetic structure of this species.