Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments

Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range of Deltaproteobacteria diversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seep sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. Many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1, Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involving Epsilon-, Delta-, and Gammaproteobacteria in methane seep ecosystems

Rapid identification of mutations in GJC2 in primary lymphoedema using whole exome sequencing combined with linkage analysis with delineation of the phenotype.

Background: Primary lymphoedema describes a chronic, frequently progressive, failure of lymphatic drainage. This disorder is frequently genetic in origin, and a multigenerational family in which eight individuals developed postnatal lymphoedema of all four limbs was ascertained from the joint Lymphoedema/Genetic clinic at St George's Hospital. Methods: Linkage analysis was used to determine a locus, and exome sequencing was employed to look for causative variants. Results: Linkage analysis revealed cosegregation of a 16.1 Mb haplotype on chromosome 1q42 that contained 173 known or predicted genes. Whole exome sequencing in a single affected individual was undertaken, and the search for the causative variant was focused to within the linkage interval. This approach revealed two novel non-synonymous single nucleotide substitutions within the chromosome 1 locus, in NVL and GJC2. NVL and GJC2 were sequenced in an additional cohort of individuals with a similar phenotype and non-synonymous variants were found in GJC2 in four additional families. Conclusion: This report demonstrates the power of exome sequencing efficiently applied to a traditional positional cloning pipeline in disease gene discovery, and suggests that the phenotype produced by GJC2 mutations is predominantly one of 4 limb lymphoedema

A novel small molecule that selectively inhibits glioblastoma cells expressing EGFRvIII

BACKGROUND: Mutations of the epidermal growth factor receptor (EGFR) are a possible molecular target for cancer therapy. EGFR is frequently amplified in glioblastomas and 30 to 40% of glioblastomas also express the deletion mutation EGFRvIII. This frequent oncogenic mutation provides an opportunity for identifying new anti-glioblastoma therapies. In this study, we sought small molecule inhibitors specific for cancer cells expressing EGFRvIII, using isogenic parental cells without EGFRvIII as a control. RESULTS: A screen of the NCI small molecule diversity set identified one compound, NSC-154829, which consistently inhibited growth of different human glioblastoma cells expressing EGFRvIII, but permitted normal growth of matched control cells. NSC-154829 had no previously established medicinal use, but has a purine-like structural component. Further experiments showed this compound increased apoptosis in cells with EGFRvIII, and moderately affected the expression of p21, independent of any changes in p53 levels or in Akt phosphorylation. CONCLUSION: These initial results suggest that NSC-154829 or a closely related structure might be further investigated for its potential as an anti-glioblastoma drug, although its precise molecular mechanism is still undefined

Time-series transcriptomics from cold, oxic subseafloor crustal fluids reveals a motile, mixotrophic microbial community

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Seyler, L. M., Trembath-Reichert, E., Tully, B. J., & Huber, J. A. Time-series transcriptomics from cold, oxic subseafloor crustal fluids reveals a motile, mixotrophic microbial community. Isme Journal, (2020), doi:10.1038/s41396-020-00843-4.The oceanic crustal aquifer is one of the largest habitable volumes on Earth, and it harbors a reservoir of microbial life that influences global-scale biogeochemical cycles. Here, we use time series metagenomic and metatranscriptomic data from a low-temperature, ridge flank environment representative of the majority of global hydrothermal fluid circulation in the ocean to reconstruct microbial metabolic potential, transcript abundance, and community dynamics. We also present metagenome-assembled genomes from recently collected fluids that are furthest removed from drilling disturbances. Our results suggest that the microbial community in the North Pond aquifer plays an important role in the oxidation of organic carbon within the crust. This community is motile and metabolically flexible, with the ability to use both autotrophic and organotrophic pathways, as well as function under low oxygen conditions by using alternative electron acceptors such as nitrate and thiosulfate. Anaerobic processes are most abundant in subseafloor horizons deepest in the aquifer, furthest from connectivity with the deep ocean, and there was little overlap in the active microbial populations between sampling horizons. This work highlights the heterogeneity of microbial life in the subseafloor aquifer and provides new insights into biogeochemical cycling in ocean crust.The Gordon and Betty Moore Foundation sponsored most of the observatory components at North Pond through grant GBMF1609. This work was supported by NSF OCE-1062006, OCE-1745589 and OCE-1635208 to J.A.H. E.T.R. was supported by a NASA Postdoctoral Fellowship with the NASA Astrobiology Institute and a L’Oréal USA For Women in Science Fellowship. The Center for Dark Energy Biosphere Investigations (C-DEBI OCE-0939564) also supported the participation of J.A.H. and B.T. This is C-DEBI contribution number 548

Overcoming data scarcity of Twitter: using tweets as bootstrap with application to autism-related topic content analysis

Notwithstanding recent work which has demonstrated the potential of using Twitter messages for content-specific data mining and analysis, the depth of such analysis is inherently limited by the scarcity of data imposed by the 140 character tweet limit. In this paper we describe a novel approach for targeted knowledge exploration which uses tweet content analysis as a preliminary step. This step is used to bootstrap more sophisticated data collection from directly related but much richer content sources. In particular we demonstrate that valuable information can be collected by following URLs included in tweets. We automatically extract content from the corresponding web pages and treating each web page as a document linked to the original tweet show how a temporal topic model based on a hierarchical Dirichlet process can be used to track the evolution of a complex topic structure of a Twitter community. Using autism-related tweets we demonstrate that our method is capable of capturing a much more meaningful picture of information exchange than user-chosen hashtags.Comment: IEEE/ACM International Conference on Advances in Social Networks Analysis and Mining, 201

Evaluating biases in filter-based aerosol absorption measurements using photoacoustic spectroscopy

This is the final version. Available from Copernicus Publications via the DOI in this record.Biases in absorption coefficients measured using a filter-based absorption photometer (Tricolor Absorption Photometer, or TAP) at wavelengths of 467, 528 and 652 nm are evaluated by comparing to measurements made using photoacoustic spectroscopy (PAS). We report comparisons for ambient sampling covering a range of aerosol types including urban, fresh biomass burning and aged biomass burning. Data are also used to evaluate the performance of three different TAP correction schemes. We found that photoacoustic and filter-based measurements were well correlated, but filter-based measurements generally overestimated absorption by up to 45 %. Biases varied with wavelength and depended on the correction scheme applied. Optimal agreement to PAS data was achieved by processing the filterbased measurements using the recently developed correction scheme of Müller et al. (2014), which consistently reduced biases to 0 %–18% at all wavelengths. The biases were found to be a function of the ratio of organic aerosol mass to light-absorbing carbon mass, although applying the Müller et al. (2014) correction scheme to filter-based absorption measurements reduced the biases and the strength of this correlation significantly. Filter-based absorption measurement biases led to aerosol single-scattering albedos that were biased low by values in the range 0.00–0.07 and absorption Ångström exponents (AAEs) that were in error by (0.03–0.54). The discrepancy between the filter-based and PAS absorption measurements is lower than reported in some earlier studies and points to a strong dependence of filterbased measurement accuracy on aerosol source type.Natural Environment Research CouncilNatural Environment Research Council/Met OfficeResearch Council of Norway (ACBC and NetBC grants)Royal Society of Chemistry (Analytical Chemistry Trust Fund, Tom West Fellowship

Activity and interactions of methane seep microorganisms assessed by parallel transcription and FISH-NanoSIMS analyses

To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5′-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no ^(15)NH^+_4 assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active ‘free-living’ population, and are not dependent on methane or ANME activity. We investigated the possibility of N_2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. ^(15)N_2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, ^(15)N_2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced ^(15)N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations

Familial Infiltrative Fibromatosis (Desmoid Tumours) (MIM135290) Caused by a Recurrent 3′ APC Gene Mutation

Desmoid tumours are generally very rare but occur about 100 times more frequently in the colorectal cancer predisposition syndrome familial adenomatous polyposis (MIM 175100), being represented in about 10% of patients. In addition to desmoid disease occurring in familial adenomatous polyposis (FAP) there exist familial infiltrative fibromatosis (MIM 135290) kindreds where there is no evidence of FAP. Previously we have described a kindred with familial infiltrative fibromatosis (FIF) in which desmoid tumours were associated with nonpolyposis colorectal cancer. FAP is caused by mutations in the APC gene and various genotype-phenotype relationships have been defined including reports that colorectal polyposis is less severe with mutations 5′ to codon 157 and that the risk of desmoid tumours is high in FAP patients with APC gene mutations between codons 1444 and 1598. There is relatively little information on the phenotype of APC gene mutations 3′ to codon 1598; however, one large family has been reported with a mutation at codon 1987 which presents with a highly variable phenotype which includes desmoid disease. We screened our original FIF kindred and three further families with a similar phenotype for mutations in the APC gene. A 4 bp frameshift deletion in codon 1962 was identified in the original FIF kindred and two further apparently unrelated families. Haplotype analysis suggests a common origin for the APC mutation in all three families. Affected individuals had no evidence of congenital hypertrophy of the retinal pigment epithelium. Colorectal polyposis was variable, and most affected patients had either none or a few late onset polyps. These findings demonstrate (i) that FAP and FIF are allelic, and (ii) that APC gene mutations which truncate the APC protein distal to the beta-catenin binding domain are associated with desmoid tumours, absent CHRPE and variable but attenuated polyposis expressio

Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension

BACKGROUND: Inflammation is a feature of pulmonary arterial hypertension (PAH), and increased circulating levels of cytokines are reported in patients with PAH. However, to date, no information exists on the significance of elevated cytokines or their potential as biomarkers. We sought to determine the levels of a range of cytokines in PAH and to examine their impact on survival and relationship to hemodynamic indexes. METHODS AND RESULTS: We measured levels of serum cytokines (tumor necrosis factor-alpha, interferon-gamma and interleukin-1beta, -2, -4, -5, -6, -8, -10, -12p70, and -13) using ELISAs in idiopathic and heritable PAH patients (n=60). Concurrent clinical data included hemodynamics, 6-minute walk distance, and survival time from sampling to death or transplantation. Healthy volunteers served as control subjects (n=21). PAH patients had significantly higher levels of interleukin-1beta, -2, -4, -6, -8, -10, and -12p70 and tumor necrosis factor-alpha compared with healthy control subjects. Kaplan-Meier analysis showed that levels of interleukin-6, 8, 10, and 12p70 predicted survival in patients. For example, 5-year survival with interleukin-6 levels of >9 pg/mL was 30% compared with 63% for patients with levels < or = 9 pg/mL (P=0.008). In this PAH cohort, cytokine levels were superior to traditional markers of prognosis such as 6-minute walk distance and hemodynamics. CONCLUSIONS: This study illustrates dysregulation of a broad range of inflammatory mediators in idiopathic and familial PAH and demonstrates that cytokine levels have a previously unrecognized impact on patient survival. They may prove to be useful biomarkers and provide insight into the contribution of inflammation in PAH