Development of a novel mycobiome diagnostic for fungal infection.

BACKGROUND: Amplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target. METHODS: Novel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed. RESULTS: The Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data. CONCLUSIONS: This study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.

Evaluation of a pharmacist-managed anemia service in pregnant patients.

PURPOSE: Studies have shown the positive impact pharmacist-managed services have on patient care. However, little information is available on services for pregnant patients. This study contributes to the current literature by providing data on the impact of a pharmacist-managed service on outcomes in pregnant patients with iron-deficiency anemia. METHODS: This was a retrospective, data-only, multicenter study comparing pregnant patients with iron-deficiency anemia managed by a pharmacist (n = 100) to those who received standard care (n = 100). During the study period, patients were 16 years of age or older and pregnant with concurrent iron-deficiency anemia. The percentage of patients with a hemoglobin level above goal (>11.0 g/dL) at delivery, the mean hemoglobin level at delivery, and the type of iron supplementation were compared between the groups. RESULTS: The percentage of patients with a hemoglobin level of greater than 11.0 g/dL at delivery was 87% for the pharmacist-managed group compared to 71% for the group receiving standard care (P < 0.01). The mean hemoglobin level at delivery was significantly higher in the pharmacist-managed group than in the group receiving standard care, at 12.1 g/dL vs 11.6 g/dL, respectively (P < 0.1). There was no difference between the groups in the percentage of patients receiving blood transfusions (4% vs 3%; P = 0.56). The percentage of patients who received intravenous iron was significantly lower in the pharmacist-managed group than in the group receiving standard care (8% vs 21%; P < 0.01). Most patients in the pharmacist-managed group received oral ferrous sulfate (81%). CONCLUSION: This study showed that including pharmacists in the management of pregnant patients with iron-deficiency anemia significantly improved patient hemoglobin levels at delivery.

Disruption of the Aspergillus fumigatus RNA interference machinery alters the conidial transcriptome.

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

Influence of Protein Glycosylation on Campylobacter fetus Physiology.

Campylobacter fetus is commonly associated with venereal disease and abortions in cattle and sheep, and can also cause intestinal or systemic infections in humans that are immunocompromised, elderly, or exposed to infected livestock. It is also believed that C. fetus infection can result from the consumption or handling of contaminated food products, but C. fetus is rarely detected in food since isolation methods are not suited for its detection and the physiology of the organism makes culturing difficult. In the related species, Campylobacter jejuni, the ability to colonize the host has been linked to N-linked protein glycosylation with quantitative proteomics demonstrating that glycosylation is interconnected with cell physiology. Using label-free quantitative (LFQ) proteomics, we found more than 100 proteins significantly altered in expression in two C. fetus subsp. fetus protein glycosylation (pgl) mutants (pglX and pglJ) compared to the wild-type. Significant increases in the expression of the (NiFe)-hydrogenase HynABC, catalyzing H2-oxidation for energy harvesting, correlated with significantly increased levels of cellular nickel, improved growth in H2 and increased hydrogenase activity, suggesting that N-glycosylation in C. fetus is involved in regulating the HynABC hydrogenase and nickel homeostasis. To further elucidate the function of the C. fetus pgl pathway and its enzymes, heterologous expression in Escherichia coli followed by mutational and functional analyses revealed that PglX and PglY are novel glycosyltransferases involved in extending the C. fetus hexasaccharide beyond the conserved core, while PglJ and PglA have similar activities to their homologs in C. jejuni. In addition, the pgl mutants displayed decreased motility and ethidium bromide efflux and showed an increased sensitivity to antibiotics. This work not only provides insight into the unique protein N-glycosylation pathway of C. fetus, but also expands our knowledge on the influence of protein N-glycosylation on Campylobacter cell physiology.

Chicken, beams, and Campylobacter: rapid differentiation of foodborne bacteria via vibrational spectroscopy and MALDI-mass spectrometry.

Campylobacter species are one of the main causes of food poisoning worldwide. Despite the availability of established culturing and molecular techniques, due to the fastidious nature of these microorganisms, simultaneous detection and species differentiation still remains challenging. This study focused on the differentiation of eleven Campylobacter strains from six species, using Fourier transform infrared (FT-IR) and Raman spectroscopies, together with matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS), as physicochemical approaches for generating biochemical fingerprints. Cluster analysis of data from each of the three analytical approaches provided clear differentiation of each Campylobacter species, which was generally in agreement with a phylogenetic tree based on 16S rRNA gene sequences. Notably, although C. fetus subspecies fetus and venerealis are phylogenetically very closely related, using FT-IR and MALDI-TOF-MS data these subspecies were readily differentiated based on differences in the lipid (2920 and 2851 cm(-1)) and fingerprint regions (1500-500 cm(-1)) of the FT-IR spectra, and the 500-2000 m/z region of the MALDI-TOF-MS data. A finding that was further investigated with targeted lipidomics using liquid chromatography-mass spectrometry (LC-MS). Our results demonstrate that such metabolomics approaches combined with molecular biology techniques may provide critical information and knowledge related to the risk factors, virulence, and understanding of the distribution and transmission routes associated with different strains of foodborne Campylobacter spp.

Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis.

MicroRNAs (miRs) are a novel class of small RNA molecules, the dysregulation of which can contribute to cancer. A combinatorial approach was used to identify miRs that promote prostate cancer progression in a unique set of prostate cancer cell lines, which originate from the parental p69 cell line and extend to a highly tumorigenic/metastatic M12 subline. Together, these cell lines are thought to mimic prostate cancer progression in vivo. Previous network analysis and miR arrays suggested that the loss of hsa-miR-125b together with the overexpression of hsa-miR-22 could contribute to prostate tumorigenesis. The dysregulation of these two miRs was confirmed in human prostate tumor samples as compared to adjacent benign glandular epithelium collected through laser capture microdissection from radical prostatectomies. In fact, alterations in hsa-miR-125b expression appeared to be an early event in tumorigenesis. Reverse phase microarray proteomic analysis revealed ErbB2/3 and downstream members of the PI3K/AKT and MAPK/ERK pathways as well as PTEN to be protein targets differentially expressed in the M12 tumor cell compared to its parental p69 cell. Relevant luciferase+3'-UTR expression studies confirmed a direct interaction between hsa-miR-125b and ErbB2 and between hsa-miR-22 and PTEN. Restoration of hsa-miR-125b or inhibition of hsa-miR-22 expression via an antagomiR resulted in an alteration of M12 tumor cell behavior in vitro. Thus, the dual action of hsa-miR-125b as a tumor suppressor and hsa-miR-22 as an oncomiR contributed to prostate tumorigenesis by modulations in PI3K/AKT and MAPK/ERK signaling pathways, key pathways known to influence prostate cancer progression.

A networks method for ranking microRNA dysregulation in cancer.

BACKGROUND: Despite the lack of agreement on their exact roles, it is known that miRNAs contribute to cancer progression. Many studies utilize methods to detect differential regulation of miRNA expression. It is prohibitively expensive to examine all potentially dysregulated miRNAs and traditionally, researchers have focused their efforts on the most extremely dysregulated miRNAs. These methods may overlook the contribution of less differentially expressed but more functionally relevant miRNAs. The purpose of this study was to outline a method that not only utilizes differential expression but ranks miRNAs based on the functional relevance of their targets. This work uses a networks based approach to determine the sum node degree for all experimentally verified miRNA targets to identify potential regulators of prostate cancer initiation, progression and metastasis. RESULTS: Here, we present a method for identifying functionally relevant miRNAs that contribute to prostate cancer development. This paper shows that miRNAs preferentially regulate highly connected, central proteins within a protein-protein interaction network. Known targets of miRNAs differentially regulated during prostate cancer progression are enriched in pathways with known involvement in tumorigenesis. To demonstrate the applicability of our method, we utilized a unique model of prostate cancer progression to identify five miRNAs that may contribute to the oncogenic state of the cell. Three of these miRNAs have been shown by other studies to have a role in cancer but their exact role in prostate cancer remains undefined. CONCLUSION: Developing methods to determine which miRNAs to carry forward into biological and biochemical analyses is important as traditional approaches often overlook miRNAs that contribute to oncogenesis. Our method applied to a model of prostate cancer progression was able to identify miRNAs with roles in prostate cancer development.

microRNA dysregulation in prostate cancer: network analysis reveals preferential regulation of highly connected nodes.

microRNAs (miRNAs) are small RNAs shown to contribute to a number of cellular processes including cell growth, differentiation, and apoptosis. MiRNAs regulate gene expression of their targets post-transcriptionally by binding to messenger RNA (mRNA), causing translational inhibition or mRNA degradation. Dysregulation of miRNA expression can promote cancer formation and progression. Research has largely focused on the function and expression of single miRNAs. However, complex physiological processes require the interaction, regulation and coordination of many molecules including miRNAs and proteins. Highly connected molecules often serve important roles in the cell. A protein-protein interaction network of established miRNA targets confirmed these proteins to be highly connected and essential to the cell, affecting tumorigenesis, cell growth/proliferation, cellular death, cell assembly, and maintenance pathways. This analysis showed that miRNAs contribute to the overall health of the prostate, and their aberrant expression destabilized homeostatic balance. This integrative network approach can reveal important miRNAs and proteins in prostate cancer that will be useful to identify specific disease biomarkers, which may be used as targets for therapeutics or drugs in themselves.