Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygen-controlled fermenter

<p>Abstract</p> <p>Background</p> <p><it>Aspergillus fumigatus </it>is a mold responsible for the majority of cases of aspergillosis in humans. To survive in the human body, <it>A. fumigatus </it>must adapt to microenvironments that are often characterized by low nutrient and oxygen availability. Recent research suggests that the ability of <it>A. fumigatus </it>and other pathogenic fungi to adapt to hypoxia contributes to their virulence. However, molecular mechanisms of <it>A. fumigatus </it>hypoxia adaptation are poorly understood. Thus, to better understand how <it>A. fumigatus </it>adapts to hypoxic microenvironments found <it>in vivo </it>during human fungal pathogenesis, the dynamic changes of the fungal transcriptome and proteome in hypoxia were investigated over a period of 24 hours utilizing an oxygen-controlled fermenter system.</p> <p>Results</p> <p>Significant increases in transcripts associated with iron and sterol metabolism, the cell wall, the GABA shunt, and transcriptional regulators were observed in response to hypoxia. A concomitant reduction in transcripts was observed with ribosome and terpenoid backbone biosynthesis, TCA cycle, amino acid metabolism and RNA degradation. Analysis of changes in transcription factor mRNA abundance shows that hypoxia induces significant positive and negative changes that may be important for regulating the hypoxia response in this pathogenic mold. Growth in hypoxia resulted in changes in the protein levels of several glycolytic enzymes, but these changes were not always reflected by the corresponding transcriptional profiling data. However, a good correlation overall (R²= 0.2, p < 0.05) existed between the transcriptomic and proteomics datasets for all time points. The lack of correlation between some transcript levels and their subsequent protein levels suggests another regulatory layer of the hypoxia response in <it>A. fumigatus</it>.</p> <p>Conclusions</p> <p>Taken together, our data suggest a robust cellular response that is likely regulated both at the transcriptional and post-transcriptional level in response to hypoxia by the human pathogenic mold <it>A. fumigatus</it>. As with other pathogenic fungi, the induction of glycolysis and transcriptional down-regulation of the TCA cycle and oxidative phosphorylation appear to major components of the hypoxia response in this pathogenic mold. In addition, a significant induction of the transcripts involved in ergosterol biosynthesis is consistent with previous observations in the pathogenic yeasts <it>Candida albicans </it>and <it>Cryptococcus neoformans </it>indicating conservation of this response to hypoxia in pathogenic fungi. Because ergosterol biosynthesis enzymes also require iron as a co-factor, the increase in iron uptake transcripts is consistent with an increased need for iron under hypoxia. However, unlike <it>C. albicans </it>and <it>C. neoformans</it>, the GABA shunt appears to play an important role in reducing NADH levels in response to hypoxia in <it>A. fumigatus </it>and it will be intriguing to determine whether this is critical for fungal virulence. Overall, regulatory mechanisms of the <it>A. fumigatus </it>hypoxia response appear to involve both transcriptional and post-transcriptional control of transcript and protein levels and thus provide candidate genes for future analysis of their role in hypoxia adaptation and fungal virulence.</p

Queen Conch (Strombus gigas) Testis Regresses during the Reproductive Season at Nearshore Sites in the Florida Keys

BACKGROUND: Queen conch (Strombus gigas) reproduction is inhibited in nearshore areas of the Florida Keys, relative to the offshore environment where conchs reproduce successfully. Nearshore reproductive failure is possibly a result of exposure to environmental factors, including heavy metals, which are likely to accumulate close to shore. Metals such as Cu and Zn are detrimental to reproduction in many mollusks. METHODOLOGY/PRINCIPAL FINDINGS: Histology shows gonadal atrophy in nearshore conchs as compared to reproductively healthy offshore conchs. In order to determine molecular mechanisms leading to tissue changes and reproductive failure, a microarray was developed. A normalized cDNA library for queen conch was constructed and sequenced using the 454 Life Sciences GS-FLX pyrosequencer, producing 27,723 assembled contigs and 7,740 annotated transcript sequences. The resulting sequences were used to design the microarray. Microarray analysis of conch testis indicated differential regulation of 255 genes (p<0.01) in nearshore conch, relative to offshore. Changes in expression for three of four transcripts of interest were confirmed using real-time reverse transcription polymerase chain reaction. Gene Ontology enrichment analysis indicated changes in biological processes: respiratory chain (GO:0015992), spermatogenesis (GO:0007283), small GTPase-mediated signal transduction (GO:0007264), and others. Inductively coupled plasma-mass spectrometry analysis indicated that Zn and possibly Cu were elevated in some nearshore conch tissues. CONCLUSIONS/SIGNIFICANCE: Congruence between testis histology and microarray data suggests that nearshore conch testes regress during the reproductive season, while offshore conch testes develop normally. Possible mechanisms underlying the testis regression observed in queen conch in the nearshore Florida Keys include a disruption of small GTPase (Ras)-mediated signaling in testis development. Additionally, elevated tissue levels of Cu (34.77 ng/mg in testis) and Zn (831.85 ng/mg in digestive gland, 83.96 ng/mg in testis) nearshore are similar to reported levels resulting in reproductive inhibition in other gastropods, indicating that these metals possibly contribute to NS conch reproductive failure

Network hygiene, incentives, and regulation: Deployment of source address validation in the internet

The Spoofer project has collected data on the deployment and characteristics of IP source address validation on the Internet since 2005. Data from the project comes from participants who install an active probing client that runs in the background. The client automatically runs tests both periodically and when it detects a new network attachment point. We analyze the rich dataset of Spoofer tests in multiple dimensions: across time, networks, autonomous systems, countries, and by Internet protocol version. In our data for the year ending August 2019, at least a quarter of tested ASes did not filter packets with spoofed source addresses leaving their networks. We show that routers performing Network Address Translation do not always filter spoofed packets, as 6.4% of IPv4/24 tested in the year ending August 2019 did not filter. Worse, at least two thirds of tested ASes did not filter packets entering their networks with source addresses claiming to be from within their network that arrived from outside their network. We explore several approaches to encouraging remediation and the challenges of evaluating their impact. While we have been able to remediate 352 IPv4/24, we have found an order of magnitude more IPv4/24 that remains unremediated, despite myriad remediation strategies, with 21% unremediated for more than six months. Our analysis provides the most complete and confident picture of the Internet's susceptibility to date of this long-standing vulnerability. Although there is no simple solution to address the remaining long-tail of unremediated networks, we conclude with a discussion of possible non-technical interventions, and demonstrate how the platform can support evaluation of the impact of such interventions over time

Oxygen Abundances in Two Metal-Poor Subgiants from the Analysis of the 6300 A Forbidden O I Line

Recent LTE analyses (Israelian et al. 1998 and Bosegaard et al. 1999) of the OH bands in the optical-ultraviolet spectra of nearby metal-poor subdwarfs indicate that oxygen abundances are generally higher than those previously determined. The difference increases with decreasing metallicity and reaches delta([O/Fe]) ~ +0.6 dex as [Fe/H] approaches -3.0. Employing high resolution (R = 50000), high S/N (~ 250) echelle spectra of the two stars found by Israelian et al. (1998) to have the highest [O/Fe]-ratios, viz, BD +23 3130 and BD +37 1458, we conducted abundance analyses based on about 60 Fe I and 7-9 Fe II lines. We determined from Kurucz LTE models the values of the stellar parameters, as well as abundances of Na, Ni, and the traditional alpha-elements, independent of the calibration of color vs $T_{eff}$ scales. We determined oxygen abundances from spectral synthesis of the stronger line (6300 A) of the [O I] doublet. The syntheses of the [O I] line lead to smaller values of [O/Fe], consistent with those found earlier among halo field and globular cluster giants. We obtain [O/Fe] = +0.35 +/- 0.2 for BD +23 3130 and +0.50 +/- 0.2 for BD +37 1458. In the former, the [O I] line is very weak (~ 1 mA), so that the quoted [O/Fe] value may in reality be an upper limit. Therefore in these two stars a discrepancy exists between the [O/Fe]- ratios derived from [O I] and the OH feature, and the origin of this difference remains unclear. Until the matter is clarified, we suggest it is premature to conclude that the ab initio oxygen abundances of old, metal-poor stars need to be revised drastically upward.Comment: 38 pages, 5 tables, 14 figures To appear in July 1999 AJ Updated April 16, 1999. Fixed typo

Expression Profiling the Temperature-Dependent Amphibian Response to Infection by Batrachochytrium dendrobatidis

Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd). However, all species are not equally at risk of infection, and risk is further modified by environmental variables, specifically temperature. In order to understand how, and when, hosts mount a response to Bd we analysed infection dynamics and patterns of gene expression in the model amphibian species Silurana (Xenopus) tropicalis. Mathematical modelling of infection dynamics demonstrate the existence of a temperature-dependent protective response that is largely independent of the intrinsic growth-rate of Bd. Using temporal expression-profiling by microarrays and qRT-PCR, we characterise this response in the main amphibian lymphoid tissue, the spleen. We demonstrate that clearance of Bd at the host-optimal temperature is not clearly associated with an adaptive immune response, but rather is correlated with the induction of components of host innate immunity including the expression of genes that are associated with the production of the antimicrobial skin peptide preprocareulein (PPCP) as well as inflammatory responses. We find that adaptive immunity appears to be lacking at host-optimal temperatures. This suggests that either Bd does not stimulate, or suppresses, adaptive immunity, or that trade-offs exist between innate and adaptive limbs of the amphibian immune system. At cold temperatures, S. tropicalis loses the ability to mount a PPCP-based innate response, and instead manifests a more pronounced inflammatory reaction that is characterised by the production of proteases and higher pathogen burdens. This study demonstrates the temperature-dependency of the amphibian response to infection by Bd and indicates the influence that changing climates may exert on the ectothermic host response to pathogens

The Current State of Performance Appraisal Research and Practice: Concerns, Directions, and Implications

On the surface, it is not readily apparent how some performance appraisal research issues inform performance appraisal practice. Because performance appraisal is an applied topic, it is useful to periodically consider the current state of performance research and its relation to performance appraisal practice. This review examines the performance appraisal literature published in both academic and practitioner outlets between 1985 and 1990, briefly discusses the current state of performance appraisal practice, highlights the juxtaposition of research and practice, and suggests directions for further research

Global Genetic Structure and Molecular Epidemiology of Encapsulated Haemophilus influenzae

A collection of 2,209 isolates of six polysaccharide capsule types of Haemophilus influenzoe, including 1,975 serotype b isolates recovered in 30 countries was characterized for electrophoretically demonstrable allele profiles at 17 metabolic enzyme loci. Two hundred eighty distinct multilocus genotypes were distinguished, and cluster analysis revealed two primary phylogenetic divisions. The population structure of encapsulated H. influenzae is clonal. Currently, most of the invasive disease worldwide is caused by serotype b strains of nine clones, Strains producing serotype c, e, and f capsules belong to single divisions and have no close genetic relationships to strains of other serotypes, Serotype a and b strains occur in both primary phylogenetic divisions, probably as a result of transfer and recombination of serotype-specific sequences of the cap region between clonal lineages. A close genetic relatedness between serotype d isolates and some strains of serotypes a and b was identified, There are strong patterns of geographic variation, on an intercontinental scale, in both the extent of genetic diversity and the clonal composition of populations of encapsulated strains, The analysis suggests that the present distribution of clones is, in part, related to patterns of racial or ethnic differentiation and historical demographic movements of the human host population

An airway epithelial IL-17A response signature identifies a steroid-unresponsive COPD patient subgroup

BACKGROUND. Chronic obstructive pulmonary disease (COPD) is a heterogeneous smoking-related disease characterized by airway obstruction and inflammation. This inflammation may persist even after smoking cessation and responds variably to corticosteroids. Personalizing treatment to biologically similar "molecular phenotypes" may improve therapeutic efficacy in a COPD. IL-17A is involved in neutrophilic inflammation and corticosteroid resistance, and thus may be particularly important in a COPD molecular phenotype. METHODS. We generated a gene expression signature of IL-17A response in bronchial airway epithelial brushings from smokers with and without COPD (n = 238) , and validated it using data from 2 randomized trials of IL-17 blockade in psoriasis. This IL-17 signature was related to clinical and pathologic characteristics in 2 additional human studies of COPD: (a) SPIROMICS (n = 47), which included former and current smokers with COPD, and (b) GLUCOLD (n = 79), in which COPD participants were randomized to placebo or corticosteroids. RESULTS. The IL-17 signature was associated with an inflammatory profile characteristic of an IL-17 response, including increased airway neutrophils and macrophages. In SPIROMICS the signature was associated with increased airway obstruction and functional small airways disease on quantitative chest CT. In GLUCOLD the signature was associated with decreased response to corticosteroids, irrespective of airway eosinophilic or type 2 inflammation. CONCLUSION. These data suggest that a gene signature of IL-17 airway epithelial response distinguishes a biologically, radiographically, and clinically distinct COPD subgroup that may benefit from personalized therapy

Manipulating charge transfer excited state relaxation and spin crossover in iron coordination complexes with ligand substitution

Developing light-harvesting and photocatalytic molecules made with iron could provide a cost effective, scalable, and environmentally benign path for solar energy conversion. To date these developments have been limited by the sub-picosecond metal-to-ligand charge transfer (MLCT) electronic excited state lifetime of iron based complexes due to spin crossover-the extremely fast intersystem crossing and internal conversion to high spin metal-centered excited states. We revitalize a 30 year old synthetic strategy for extending the MLCT excited state lifetimes of iron complexes by making mixed ligand iron complexes with four cyanide (CN-;) ligands and one 2,2′-bipyridine (bpy) ligand. This enables MLCT excited state and metal-centered excited state energies to be manipulated with partial independence and provides a path to suppressing spin crossover. We have combined X-ray Free-Electron Laser (XFEL) Kβ hard X-ray fluorescence spectroscopy with femtosecond time-resolved UV-visible absorption spectroscopy to characterize the electronic excited state dynamics initiated by MLCT excitation of [Fe(CN)4(bpy)]2-. The two experimental techniques are highly complementary; the time-resolved UV-visible measurement probes allowed electronic transitions between valence states making it sensitive to ligand-centered electronic states such as MLCT states, whereas the Kβ fluorescence spectroscopy provides a sensitive measure of changes in the Fe spin state characteristic of metal-centered excited states. We conclude that the MLCT excited state of [Fe(CN)4(bpy)]2- decays with roughly a 20 ps lifetime without undergoing spin crossover, exceeding the MLCT excited state lifetime of [Fe(2,2′-bipyridine)3]2+ by more than two orders of magnitude

ESRRA-C11orf20 Is a Recurrent Gene Fusion in Serous Ovarian Carcinoma

Many ovarian cancers have a chromosomal rearrangement that fuses two neighboring genes, ESRRA and c11orf20. Similar rearrangements may be common, important features of cancer genomes that have largely escaped detection