Combining compositional data sets introduces error in covariance network reconstruction.

Microbial communities are diverse biological systems that include taxa from across multiple kingdoms of life. Notably, interactions between bacteria and fungi play a significant role in determining community structure. However, these statistical associations across kingdoms are more difficult to infer than intra-kingdom associations due to the nature of the data involved using standard network inference techniques. We quantify the challenges of cross-kingdom network inference from both theoretical and practical points of view using synthetic and real-world microbiome data. We detail the theoretical issue presented by combining compositional data sets drawn from the same environment, e.g. 16S and ITS sequencing of a single set of samples, and we survey common network inference techniques for their ability to handle this error. We then test these techniques for the accuracy and usefulness of their intra- and inter-kingdom associations by inferring networks from a set of simulated samples for which a ground-truth set of associations is known. We show that while the two methods mitigate the error of cross-kingdom inference, there is little difference between techniques for key practical applications including identification of strong correlations and identification of possible keystone taxa (i.e. hub nodes in the network). Furthermore, we identify a signature of the error caused by transkingdom network inference and demonstrate that it appears in networks constructed using real-world environmental microbiome data.

Associated bacterial communities, confrontation studies, and comparative genomics reveal important interactions between Morchella with Pseudomonas spp.

Members of the fungal genus Morchella are widely known for their important ecological roles and significant economic value. In this study, we used amplicon and genome sequencing to characterize bacterial communities associated with sexual fruiting bodies from wild specimens, as well as vegetative mycelium and sclerotia obtained from Morchella isolates grown in vitro. These investigations included diverse representatives from both Elata and Esculenta Morchella clades. Unique bacterial community compositions were observed across the various structures examined, both within and across individual Morchella isolates or specimens. However, specific bacterial taxa were frequently detected in association with certain structures, providing support for an associated core bacterial community. Bacteria from the genus Pseudomonas and Ralstonia constituted the core bacterial associates of Morchella mycelia and sclerotia, while other genera (e.g., Pedobacter spp., Deviosa spp., and Bradyrhizobium spp.) constituted the core bacterial community of fruiting bodies. Furthermore, the importance of Pseudomonas as a key member of the bacteriome was supported by the isolation of several Pseudomonas strains from mycelia during in vitro cultivation. Four of the six mycelial-derived Pseudomonas isolates shared 16S rDNA sequence identity with amplicon sequences recovered directly from the examined fungal structures. Distinct interaction phenotypes (antagonistic or neutral) were observed in confrontation assays between these bacteria and various Morchella isolates. Genome sequences obtained from these Pseudomonas isolates revealed intriguing differences in gene content and annotated functions, specifically with respect to toxin-antitoxin systems, cell adhesion, chitinases, and insecticidal toxins. These genetic differences correlated with the interaction phenotypes. This study provides evidence that Pseudomonas spp. are frequently associated with Morchella and these associations may greatly impact fungal physiology.

The endohyphal microbiome: current progress and challenges for scaling down integrative multi-omic microbiome research.

As microbiome research has progressed, it has become clear that most, if not all, eukaryotic organisms are hosts to microbiomes composed of prokaryotes, other eukaryotes, and viruses. Fungi have only recently been considered holobionts with their own microbiomes, as filamentous fungi have been found to harbor bacteria (including cyanobacteria), mycoviruses, other fungi, and whole algal cells within their hyphae. Constituents of this complex endohyphal microbiome have been interrogated using multi-omic approaches. However, a lack of tools, techniques, and standardization for integrative multi-omics for small-scale microbiomes (e.g., intracellular microbiomes) has limited progress towards investigating and understanding the total diversity of the endohyphal microbiome and its functional impacts on fungal hosts. Understanding microbiome impacts on fungal hosts will advance explorations of how "microbiomes within microbiomes" affect broader microbial community dynamics and ecological functions. Progress to date as well as ongoing challenges of performing integrative multi-omics on the endohyphal microbiome is discussed herein. Addressing the challenges associated with the sample extraction, sample preparation, multi-omic data generation, and multi-omic data analysis and integration will help advance current knowledge of the endohyphal microbiome and provide a road map for shrinking microbiome investigations to smaller scales. Video Abstract.

Importance of appropriate genome information for the design of mating type primers in black and yellow morel populations.

Morels are highly prized edible fungi where sexual reproduction is essential for fruiting-body production. As a result, a comprehensive understanding of their sexual reproduction is of great interest. Central to this is the identification of the reproductive strategies used by morels. Sexual reproduction in fungi is controlled by mating-type (MAT) genes and morels are thought to be mainly heterothallic with two idiomorphs, MAT1-1 and MAT1-2. Genomic sequencing of black (Elata clade) and yellow (Esculenta clade) morel species has led to the development of PCR primers designed to amplify genes from the two idiomorphs for rapid genotyping of isolates from these two clades. To evaluate the design and theoretical performance of these primers we performed a thorough bioinformatic investigation, including the detection of the MAT region in publicly available Morchella genomes and in-silico PCR analyses. All examined genomes, including those used for primer design, appeared to be heterothallic. This indicates an inherent fault in the original primer design which utilized a single Morchella genome, as the use of two genomes with complementary mating types would be required to design accurate primers for both idiomorphs. Furthermore, potential off-targets were identified for some of the previously published primer sets, but verification was challenging due to lack of adequate genomic information and detailed methodologies for primer design. Examinations of the black morel specific primer pairs (MAT11L/R and MAT22L/R) indicated the MAT22 primers would correctly target and amplify the MAT1-2 idiomorph, but the MAT11 primers appear to be capable of amplifying incorrect off-targets within the genome. The yellow morel primer pairs (EMAT1-1 L/R and EMAT1-2 L/R) appear to have reporting errors, as the published primer sequences are dissimilar with reported amplicon sequences and the EMAT1-2 primers appear to amplify the RNA polymerase II subunit (RPB2) gene. The lack of the reference genome used in primer design and descriptive methodology made it challenging to fully assess the apparent issues with the primers for this clade. In conclusion, additional work is still required for the generation of reliable primers to investigate mating types in morels and to assess their performance on different clades and across multiple geographical regions.

Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria.

Several bacteria have long been known to interact intimately with fungi, but molecular approaches have only recently uncovered how cosmopolitan these interactions are in nature. Currently, bacterial-fungal interactions (BFI) are inferred based on patterns of co-occurrence in amplicon sequencing investigations. However, determining the nature of these interactions, whether the bacteria are internally or externally associated, remains a grand challenge in BFI research. Fluorescence in situ hybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing intra-hyphal targets, such as mitochondrial organelles or, as in this study, bacteria. We evaluate the challenges and employable strategies to resolve intra-hyphal BFI to address pertinent criteria in BFI research, such as culturing media, spatial distribution of bacteria, and abundance of bacterial 16S rRNA copies for fluorescent labeling. While these experimental factors influence labeling and detection of endobacteria, we demonstrate how to overcome these challenges thorough permeabilization, appropriate media choice, and targeted amplification using hybridization chain reaction FISH. Such microscopy imaging approaches can now be utilized by the broader research community to complement sequence-based investigations and provide more conclusive evidence on the nature of specific bacterial-fungal relationships.

Widespread bacterial diversity within the bacteriome of fungi.

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

Multiple Cases of Bacterial Sequence Erroneously Incorporated Into Publicly Available Chloroplast Genomes.

Public sequencing databases are invaluable resources to biological researchers, but assessing data veracity as well as the curation and maintenance of such large collections of data can be challenging. Genomes of eukaryotic organelles, such as chloroplasts and other plastids, are particularly susceptible to assembly errors and misrepresentations in these databases due to their close evolutionary relationships with bacteria, which may co-occur within the same environment, as can be the case when sequencing plants. Here, based on sequence similarities with bacterial genomes, we identified several suspicious chloroplast assemblies present in the National Institutes of Health (NIH) Reference Sequence (RefSeq) collection. Investigations into these chloroplast assemblies reveal examples of erroneous integration of bacterial sequences into chloroplast ribosomal RNA (rRNA) loci, often within the rRNA genes, presumably due to the high similarity between plastid and bacterial rRNAs. The bacterial lineages identified within the examined chloroplasts as the most likely source of contamination are either known associates of plants, or co-occur in the same environmental niches as the examined plants. Modifications to the methods used to process untargeted 'raw' shotgun sequencing data from whole genome sequencing efforts, such as the identification and removal of bacterial reads prior to plastome assembly, could eliminate similar errors in the future.

Genomic Analysis of Diverse Members of the Fungal Genus Monosporascus Reveals Novel Lineages, Unique Genome Content and a Potential Bacterial Associate.

The genus Monosporascus represents an enigmatic group of fungi important in agriculture and widely distributed in natural arid ecosystems. Of the nine described species, two (M. cannonballus and M. eutypoides) are important pathogens on the roots of members of Cucurbitaceae in agricultural settings. The remaining seven species are capable of colonizing roots from a diverse host range without causing obvious disease symptoms. Recent molecular and culture studies have shown that members of the genus are nearly ubiquitous as root endophytes in arid environments of the Southwestern United States. Isolates have been obtained from apparently healthy roots of grasses, shrubs and herbaceous plants located in central New Mexico and other regions of the Southwest. Phylogenetic and genomic analyses reveal substantial diversity in these isolates. The New Mexico isolates include close relatives of M. cannonballus and M. ibericus, as well as isolates that represent previously unrecognized lineages. To explore evolutionary relationships within the genus and gain insights into potential ecological functions, we sequenced and assembled the genomes of three M. cannonballus isolates, one M. ibericus isolate, and six diverse New Mexico isolates. The assembled genomes were significantly larger than what is typical for the Sordariomycetes despite having predicted gene numbers similar to other members of the class. Differences in predicted genome content and organization were observed between endophytic and pathogenic lineages of Monosporascus Several Monosporascus isolates appear to form associations with members of the bacterial genus Ralstonia (Burkholdariaceae).

Accuracy of partial biopsies in the management of cutaneous melanoma.

BACKGROUND: The recommended method for histopathological diagnosis of cutaneous melanoma is excisional biopsy, although partial biopsies (shave and punch) are often used. Following a partial biopsy, treatment guidelines recommend a narrow excisional biopsy to plan definitive management. There is limited evidence on the benefits of direct wide local excision (WLE) following diagnostic partial biopsies. METHODS: Retrospective cohort study of cutaneous melanoma cases, from two tertiary referral centres from January 2013 to December 2015. Demographic and histopathological data, including tumour thickness (T-stage) from initial biopsy and subsequent excisions, were collected. Logistic regression was used to examine histopathological T-staging between biopsy and subsequent excisions (upstaging). RESULTS: 2304 melanomas (2157 patients) were identified; 455 shave, 308 punch, 14 incisional and 1527 excisional biopsies. Out of 1527, 5 (<1%) excisional biopsies were upstaged from original biopsy T-stage to final WLE; compared to 28/455 (6%) for shave, 45/308 (15%) for punch and 2/14 (14%) for incisional biopsies. Histopathology upstaging were increased with punch (OR, 52.1; 95% CI, 20.5-132.4. P < 0.001) and shave biopsy (OR, 20.0; 95% CI, 7.7-52.0. P < 0.001) compared to excisional biopsy. Upstaging rates of 9.4% for desmoplastic (OR, 6.9; 95% CI, 2.4-19.7. P < 0.001) and 21.9% for acral lentiginous (OR, 18.4; 95% CI, 6.9-49.2. P < 0.001) melanomas were elevated compared to 1.4% for superficial spreading melanoma. CONCLUSIONS: In most cases, partial biopsy (particularly shave biopsy) can provide sufficient information to plan for definitive surgical melanoma management. Punch and incisional biopsies have elevated upstaging rates, a consideration in planning therapy. Partial biopsies of desmoplastic or acral lentiginous melanomas have high rates of upstaging and should have a complete excision prior to definitive treatment.

Diverse members of the Xylariales lack canonical mating-type regions.

A survey of genomes reported here for 10 isolates of Monosporascus species and an additional 25 genomes from other members of the Xylariales (representing 15 genera) available in public databases indicated that genes typically associated with MAT1-1 (mat A) or MAT1-2 (mat a) mating types are absent or have diverged greatly relative to counterparts in other Pezizomycotina. This was particularly surprising for isolates known to be homothallic, given that homothallic members of the Pezizomycotina typically possess a MAT1-1-1 (mat A-1) gene and one or both of two other closely-linked mating-type genes, MAT1-1-2 (mat A-2) and MAT1-1-3 (mat A-3), in addition to MAT1-2-1 (mat a-1). We failed to detect candidate genes for either MAT1-1-1 or MAT1-1-2 in any member of the Xylariales. Genes related to MAT1-2-1 and MAT1-1-3 are present in the genomes examined, but most appear to be orthologs of MATA_HMG (high-mobility group) genes with non-mating-type functions rather than orthologs of mating-type genes. Several MATA_HMG genes were found in genome positions that suggest they are derived from mating-type genes, but these genes are highly divergent relative to known MAT1-2-1 and MAT1-1-3 genes. The genomes examined represent substantial diversity within the order and include M. cannonballus, M. ibericus, Xylaria hypoxylon, X. striata, Daldinia eschscholzii, Eutypa lata, Rosellinia necatrix, Microdochium bolleyi and several others. We employed a number of avenues to search for homologs, including multiple BLAST approaches and examination of annotated genes adjacent to genes known to flank mating regions in other members of the Ascomycota. The results suggest that the mating regions have been lost from, or altered dramatically in, the Xylariales genomes examined and that mating and sexual development in these fungi are controlled differently than has been reported for members of the Pezizomycotina studied to date.

Transcriptional profiling of eosinophil subsets in interleukin-5 transgenic mice.

Eosinophils are important in fighting parasitic infections and are implicated in the pathogenesis of asthma and allergy. IL-5 is a critical regulator of eosinophil development, controlling proliferation, differentiation, and maturation of the lineage. Mice that constitutively express IL-5 have in excess of 10-fold more eosinophils in the hematopoietic organs than their wild type (WT) counterparts. We have identified that much of this expansion is in a population of Siglec-F high eosinophils, which are rare in WT mice. In this study, we assessed transcription in myeloid progenitors, eosinophil precursors, and Siglec-F medium and Siglec-F high eosinophils from IL-5 transgenic mice and in doing so have created a useful resource for eosinophil biologists. We have then utilized these populations to construct an eosinophil trajectory based on gene expression and to identify gene sets that are associated with eosinophil lineage progression. Cell cycle genes were significantly associated with the trajectory, and we experimentally demonstrate an increasing trend toward quiescence along the trajectory. Additionally, we found gene expression changes associated with constitutive IL-5 signaling in eosinophil progenitors, many of which were not observed in eosinophils.

Low-dose rituximab and concurrent adjuvant therapy for pemphigus: Protocol and single-centre long-term review of nine patients.

Pemphigus is an autoimmune B-cell mediated blistering disease associated with significant morbidity and mortality. Rituximab has proven effective for the treatment of steroid-refractory pemphigus, although there is controversy over the optimum dosing protocol. Additionally, effective disease control often requires long-term immunosuppression, even in disease-free periods. We present a case series of a single-centre long-term follow up of nine patients with pemphigus, treated with two 500-mg doses of rituximab separated by 14 days along with concurrent adjuvant therapy. In all these patients, low-dose rituximab resulted in B-cell depletion, along with a reduction in blistering disease. Three of these patients required repeat dosing cycles due to either relapsed disease or incomplete disease control following the first dosing cycle, and have remained disease free up to 154 weeks thus far. Six patients developed minor infections during the course of their treatment, but no major complications were observed.

Diagnostic Impact and Cost-effectiveness of Whole-Exome Sequencing for Ambulant Children With Suspected Monogenic Conditions.

Importance: Optimal use of whole-exome sequencing (WES) in the pediatric setting requires an understanding of who should be considered for testing and when it should be performed to maximize clinical utility and cost-effectiveness. Objectives: To investigate the impact of WES in sequencing-naive children suspected of having a monogenic disorder and evaluate its cost-effectiveness if WES had been available at different time points in their diagnostic trajectory. Design, Setting, and Participants: This prospective study was part of the Melbourne Genomics Health Alliance demonstration project. At the ambulatory outpatient clinics of the Victorian Clinical Genetics Services at the Royal Children's Hospital, Melbourne, Australia, children older than 2 years suspected of having a monogenic disorder were prospectively recruited from May 1 through November 30, 2015, by clinical geneticists after referral from general and subspecialist pediatricians. All children had nondiagnostic microarrays and no prior single-gene or panel sequencing. Exposures: All children underwent singleton WES with targeted phenotype-driven analysis. Main Outcomes and Measures: The study examined the clinical utility of a molecular diagnosis and the cost-effectiveness of alternative diagnostic trajectories, depending on timing of WES. Results: Of 61 children originally assessed, 44 (21 [48%] male and 23 [52%] female) aged 2 to 18 years (mean age at initial presentation, 28 months; range, 0-121 months) were recruited, and a diagnosis was achieved in 23 (52%) by singleton WES. The diagnoses were unexpected in 8 of 23 (35%), and clinical management was altered in 6 of 23 (26%). The mean duration of the diagnostic odyssey was 6 years, with each child having a mean of 19 tests and 4 clinical genetics and 4 nongenetics specialist consultations, and 26 (59%) underwent a procedure while under general anesthetic for diagnostic purposes. Economic analyses of the diagnostic trajectory identified that WES performed at initial tertiary presentation resulted in an incremental cost savings of A$9020 (US$6838) per additional diagnosis (95% CI, A$4304-A$15 404 [US$3263-US$11 678]) compared with the standard diagnostic pathway. Even if WES were performed at the first genetics appointment, there would be an incremental cost savings of A$5461 (US$4140) (95% CI, A$1433-A$10 557 [US$1086- US$8004]) per additional diagnosis compared with the standard diagnostic pathway. Conclusions and Relevance: Singleton WES in children with suspected monogenic conditions has high diagnostic yield, and cost-effectiveness is maximized by early application in the diagnostic pathway. Pediatricians should consider early referral of children with undiagnosed syndromes to clinical geneticists.

Salmonid alphavirus infection causes skin dysbiosis in Atlantic salmon (Salmo salar L.) post-smolts.

Interactions among host, microbiota and viral pathogens are complex and poorly understood. The goal of the present study is to assess the changes in the skin microbial community of Atlantic salmon (Salmo salar L.) in response to experimental infection with salmonid alphavirus (SAV). The salmon skin microbial community was determined using 16S rDNA pyrosequencing in five different experimental groups: control, 7 days after infection with low-dose SAV, 14 days after infection with low-dose SAV, 7 days after infection with high-dose SAV, and 14 days after infection with high-dose SAV. Both infection treatment and time after infection were strong predictors of the skin microbial community composition. Skin samples from SAV3 infected fish showed an unbalanced microbiota characterized by a decreased abundance of Proteobacteria such as Oleispira sp. and increased abundances of opportunistic taxa including Flavobacteriaceae, Streptococcaceae and Tenacibaculum sp. These results demonstrate that viral infections can result in skin dysbiosis likely rendering the host more susceptible to secondary bacterial infections.

Consensus statement for the treatment of infantile haemangiomas with propranolol.

Although most infantile haemangiomas do not require treatment due to a natural history of spontaneous involution, some require early intervention. The Australasian Vascular Anomalies Network and the Australasian Paediatric Dermatology Network have developed a consensus statement for the treatment of infantile haemangiomas with oral propranolol. Infants with haemangiomas that are life threatening, at risk of ulceration, or at risk of causing a significant functional impairment, psychological impact or physical deformity should be treated early with oral propranolol. Oral propranolol is safe and effective and in most healthy infants oral propranolol can be started in an outpatient setting.

Granular parakeratosis induced by benzalkonium chloride exposure from laundry rinse aids.

Benzalkonium chloride is a quaternary ammonium cationic detergent present in a number of household products, which can act as a major skin irritant. We present the case of six children who developed granular parakeratosis after exposure to benzalkonium chloride in laundry rinse aids, presenting as a brightly erythematous, tender but minimally pruritic, intertriginous eruption followed by superficial desquamation. The eruptions resolved over 3-4 weeks after cessation of exposure.

Haemopedia: An Expression Atlas of Murine Hematopoietic Cells.

Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

Embryonic stem cell-derived glial precursors as a vehicle for sulfamidase production in the MPS-IIIA mouse brain.

Pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, have generated much excitement about their prospects for use in cell transplantation therapies. This is largely attributable to their virtually unlimited growth potential, their ability to be precisely genetically altered in culture, and their utility for forming differentiated cell populations with potential clinical applications. Lysosomal storage diseases such as Sanfilippo syndrome (MPS-IIIA) represent ideal candidate diseases for the evaluation of cell therapies in the central nervous system (CNS). These diseases exhibit widespread pathology yet result from a single gene deficiency, in the case of Sanfilippo syndrome the lysosomal enzyme sulfamidase. The aim of this study was to investigate mouse embryonic stem (ES) cell-derived glial precursor cells as a vehicle for sulfamidase delivery in the MPS-IIIA mouse brain. In this study we have created a mouse ES cell line genetically modified to stably express and secrete high levels of human sulfamidase and a protocol for the in vitro derivation of large numbers glial precursors from ES cells. Differentiation of sulfamidase-expressing ES cells resulted in cell populations with sustained secretion of high levels of sulfamidase, comprised primarily of glial precursor cells with minor contaminants of other neural cell phenotypes but not residual pluripotent cells. CNS implantation studies demonstrated that ES cell-derived glial precursor cells formed using this differentiation method were able to engraft and survive for at least 12 weeks following implantation. The percentage of engraftment was quantified in different regions of the brain in 2-, 4-, and 8-week-old normal and MPS-IIIA mice. No teratomas were observed in any of the cell-transplanted animals. The results of this study support the further investigation of sulfamidase-expressing glial precursor cells as a vehicle for delivery of deficient enzyme into the CNS of MPS-IIIA mice.