Transcriptome analysis reveals a common adaptive transcriptional response of Candida glabrata to diverse environmental stresses.

Candida glabrata, an opportunistic fungal pathogen, causes superficial and life-threatening infections in humans. In the host microenvironment, C. glabrata encounters a variety of stresses, and its ability to cope with these stresses is crucial for its pathogenesis. To gain insights into how C. glabrata adapts to adverse environmental conditions, we examined its transcriptional landscape under heat, osmotic, cell wall, oxidative, and genotoxic stresses using RNA sequencing and reveal that C. glabrata displays a diverse transcriptional response involving ∼75% of its genome for adaptation to different environmental stresses. C. glabrata mounts a central common adaptation response wherein ∼25% of all genes (n = 1370) are regulated in a similar fashion at different environmental stresses. Elevated cellular translation and diminished mitochondrial activity-associated transcriptional signature characterize the common adaptation response. Transcriptional regulatory association networks of common adaptation response genes revealed a set of 29 transcription factors acting as potential activators and repressors of associated adaptive response genes. Overall, the current work delineates the adaptive responses of C. glabrata to diverse environmental stresses and reports the existence of a common adaptive transcriptional response upon prolonged exposure to environmental stresses.

FungiExpresZ: an intuitive package for fungal gene expression data analysis, visualization and discovery.

Bioinformatics analysis and visualization of high-throughput gene expression data require extensive computer programming skills, posing a bottleneck for many wet-lab scientists. In this work, we present an intuitive user-friendly platform for gene expression data analysis and visualization called FungiExpresZ. FungiExpresZ aims to help wet-lab scientists with little to no knowledge of computer programming to become self-reliant in bioinformatics analysis and generating publication-ready figures. The platform contains many commonly used data analysis tools and an extensive collection of pre-processed public ribonucleic acid sequencing (RNA-seq) datasets of many fungal species, including important human, plant and insect pathogens. Users may analyse their data alone or in combination with public RNA-seq data for an integrated analysis. The FungiExpresZ platform helps wet-lab scientists to overcome their limitations in genomics data analysis and can be applied to analyse data of any organism. FungiExpresZ is available as an online web-based tool (https://cparsania.shinyapps.io/FungiExpresZ/) and an offline R-Shiny package (https://github.com/cparsania/FungiExpresZ).

Mapping the mutual transcriptional responses during Candida albicans and human macrophage interactions by dual RNA-sequencing.

Candida albicans is the leading human fungal pathogen that can cause mucosal and systemic fungal infections. Host phagocytes are the primary immune defense against invading fungal pathogens including C. albicans. To better understand the host-pathogen interaction between C. albicans and host phagocytes, we utilized a human macrophage model of THP-1 macrophages and examined the mutual transcriptomic response of C. albicans and host macrophages by dual RNA-sequencing. Both C. albicans and macrophages displayed marked changes in their transcriptional profiles post 2 h coincubation. We show that C. albicans responds to human macrophages differently than its known response to murine macrophages. C. albicans displays upregulation of its translational machinery and downregulation of glyoxylate and tricarboxylic acid (TCA) cycle upon macrophage phagocytosis. C. albicans triggered strong induction of genes associated with cell surface-mediated signaling and proinflammatory response in THP-1 macrophages. Finally, our data reveal that IL-1ß and TNF signaling are central in mounting a proinflammatory response against C. albicans via MAP kinase, and chemokines and cytokines mediated signaling. Overall, current work uncovers the mutual responses of C. albicans and human macrophages towards each other presenting a better understanding of their interaction during C. albicans infections.

Illuminating the dark protein-protein interactome.

In living systems, a complex network of protein-protein interactions (PPIs) underlies most biochemical events. The human protein-protein interactome has been surveyed using yeast two-hybrid (Y2H)- and mass spectrometry (MS)-based approaches such as affinity purification coupled to MS (AP-MS). Despite decades of systematic investigations and collaborative multi-disciplinary efforts, there is no "gold standard" for documenting PPIs. A surprisingly large fraction of the human interactome remains uncharted, which we refer to as the "dark interactome." In this review, we highlight the complexity of the human interactome and discuss the current status of the human reference interactome maps. We discuss why a large proportion of the human interactome has remained refractory to traditional approaches. We propose an experimental model that can enable the identification of the dark interactome in a cell-type-specific manner. We also propose a framework to implement when embarking on studies designed to rigorously identify and characterize protein interactions.

Co-option of an extracellular protease for transcriptional control of nutrient degradation in the fungus Aspergillus nidulans.

Nutrient acquisition is essential for all organisms. Fungi regulate their metabolism according to environmental nutrient availability through elaborate transcription regulatory programs. In filamentous fungi, a highly conserved GATA transcription factor AreA and its co-repressor NmrA govern expression of genes involved in extracellular breakdown, uptake, and metabolism of nitrogen nutrients. Here, we show that the Aspergillus nidulans PnmB protease is a moonlighting protein with extracellular and intracellular functions for nitrogen acquisition and metabolism. PnmB serves not only as a secreted protease to degrade extracellular nutrients, but also as an intracellular protease to control the turnover of the co-repressor NmrA, accelerating AreA transcriptional activation upon nitrogen starvation. PnmB expression is controlled by AreA, which activates a positive feedback regulatory loop. Hence, we uncover a regulatory mechanism in the well-established controls determining the response to nitrogen starvation, revealing functional evolution of a protease gene for transcriptional regulation and extracellular nutrient breakdown.

Transcriptomic analysis reveals global and temporal transcription changes during Candida glabrata adaptation to an oxidative environment.

The ability to survive host-elicited oxidative stress is critical for microbial pathogens to cause infection. The human fungal pathogen C.glabrata can tolerate high levels of oxidative stress and proliferate inside phagocytes. Previous studies had successfully identified a transcription response to oxidative stress including induction of a core set of detoxification genes. However, the findings only represent an early snapshot of a highly dynamic process lacking temporal resolution. Here, we compare the transcriptome of C. glabrata at various points after exposure to hydrogen peroxide in order to study its adaptation to an oxidative environment. Our results reveal global and temporal gene expression changes during an immediate response; up-regulating genes related to peroxide detoxification, while down-regulating genes essential for growth. As cells adapt to the oxidative environment, a dramatic transcriptome reprogramming occurred to restore key cellular functions, protein homeostasis and biosynthesis of trehalose, carbohydrate, fatty acid and ergosterol. Interestingly, biofilm and drug transporter genes as well as many genes implicated in virulence, were induced during the adaptation stage. Our finding, therefore, suggests a role of oxidative stress adaptation in promoting virulence and drug resistance traits of C. glabrata during infection.

Identification and characterization of a specific 13-miRNA expression signature during follicle activation in the zebrafish ovary.

Ovarian folliculogenesis is always of great interest in reproductive biology. However, the molecular mechanisms that control follicle development, particularly the early phase of follicle activation or recruitment, still remain poorly understood. In an attempt to decipher the gene networks and signaling pathways involved in such transition, we conducted a transcriptomic analysis (RNA-seq) on zebrafish primary growth (PG, stage I; inactive) and previtellogenic (PV, stage II; activated) follicles. A total of 118 unique microRNAs (miRNAs) (11 downregulated and 83 upregulated during PG/PV transition) and 56711 unique messenger RNAs (mRNAs) (1839 downregulated and 7243 upregulated during PG/PV transition) were identified. Real-time quantitative polymerase chain reaction analysis confirmed differential expression of 46 miRNAs from 66 candidates (66.67%). Among which, we chose to focus on 13 miRNAs (let-7a, -7b, -7c-5p, -7d-5p, -7h, -7i; miR-21, -23a-3p, -27c-3p, -107a-3p, -125b-5p, -145-3p, and -202-5p) that exhibited significant differential expression between PG and PV follicles (P ≤ 0.045*). With this 13-miRNA expression signature alone, PG follicles can be well differentiated from PV follicles by hierarchical clustering, suggesting their functional relevance during PG-to-PV transition. By overlaying predicted target genes and the differentially expressed mRNAs revealed by the RNA-seq analysis, especially those showing reciprocal miRNA-mRNA expression patterns, we shortlisted a panel of miRNA downstream targets for luciferase reporter validation. The reporter assay confirmed the interactions of let-7i:: atg4a (P = 0.01*), miR-202-5p::c23h20orf24 (P = 0.0004***), and miR-144-5p::ybx1 (P = 0.003**), implicating these potential miRNA-mRNA gene pairs in follicle activation during folliculogenesis. Our transcriptomic data analyses suggest that miRNA-mediated post-transcriptional control may represent an important mechanism underlying follicle activation.

PBrowse: a web-based platform for real-time collaborative exploration of genomic data.

Genome browsers are widely used for individually exploring various types of genomic data. A handful of genome browsers offer limited tools for collaboration among multiple users. Here, we describe PBrowse, an integrated real-time collaborative genome browser that enables multiple users to simultaneously view and access genomic data, thereby harnessing the wisdom of the crowd. PBrowse is based on the Dalliance genome browser and has a re-designed user and data management system with novel collaborative functionalities, including real-time collaborative view, track comment and an integrated group chat feature. Through the Distributed Annotation Server protocol, PBrowse can easily access a wide range of publicly available genomic data, such as the ENCODE data sets. We argue that PBrowse represents a paradigm shift from using a genome browser as a static data visualization tool to a platform that enables real-time human-human interaction and knowledge exchange in a collaborative setting. PBrowse is available at http://pbrowse.victorchang.edu.au, and its source code is available via an open source BSD 3 license at http://github.com/VCCRI/PBrowse.

De Novo Assembly and Characterization of Stress Transcriptome in a Salinity-Tolerant Variety CS52 of Brassica juncea.

Oilseed mustard, Brassica juncea, exhibits high levels of genetic variability for salinity tolerance. To obtain the global view of transcriptome and investigate the molecular basis of salinity tolerance in a salt-tolerant variety CS52 of B. juncea, we performed transcriptome sequencing of control and salt-stressed seedlings. De novo assembly of 184 million high-quality paired-end reads yielded 42,327 unique transcripts longer than 300 bp with RPKM ≥1. When compared with non-redundant proteins, we could annotate 67% unigenes obtained in our study. Based on the mapping to expressed sequence tags (ESTs), 52.6% unigenes are novel compared to EST data available for B. juncea and constituent genomes. Differential expression analysis revealed altered expression of 1469 unigenes in response to salinity stress. Of these, 587, mainly associated with ROS detoxification, sulfur assimilation and calcium signaling pathways, are up regulated. Notable of these is RSA1 (SHORT ROOT IN SALT MEDIUM 1) INTERACTING TRANSCRIPTION FACTOR 1 (RITF1) homolog up regulated by >100 folds in response to stress. RITF1, encoding a bHLH transcription factor, is a positive regulator of SOS1 and several key genes involved in scavenging of salt stress-induced reactive oxygen species (ROS). Further, we performed comparative expression profiling of key genes implicated in ion homeostasis and sequestration (SOS1, SOS2, SOS3, ENH1, NHX1), calcium sensing pathway (RITF1) and ROS detoxification in contrasting cultivars for salinity tolerance, B. juncea and B. nigra. The results revealed higher transcript accumulation of most of these genes in B. juncea var. CS52 compared to salt-sensitive cultivar even under normal growth conditions. Together, these findings reveal key pathways and signaling components that contribute to salinity tolerance in B. juncea var. CS52.

Molecular insights into the dynamics of pharmacogenetically important N-terminal variants of the human ß2-adrenergic receptor.

The human ß2-adrenergic receptor (ß2AR), a member of the G-protein coupled receptor (GPCR) family, is expressed in bronchial smooth muscle cells. Upon activation by agonists, ß2AR causes bronchodilation and relief in asthma patients. The N-terminal polymorphism of ß2AR at the 16th position, Arg16Gly, has warranted a lot of attention since it is linked to variations in response to albuterol (agonist) treatment. Although the ß2AR is one of the well-studied GPCRs, the N-terminus which harbors this mutation, is absent in all available experimental structures. The goal of this work was to study the molecular level differences between the N-terminal variants using structural modeling and atomistic molecular dynamics simulations. Our simulations reveal that the N-terminal region of the Arg variant shows greater dynamics than the Gly variant, leading to differential placement. Further, the position and dynamics of the N-terminal region, further, affects the ligand binding-site accessibility. Interestingly, long-range effects are also seen at the ligand binding site, which is marginally larger in the Gly as compared to the Arg variant resulting in the preferential docking of albuterol to the Gly variant. This study thus reveals key differences between the variants providing a molecular framework towards understanding the variable drug response in asthma patients.