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Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

BACKGROUND: Short-chain fatty acids (SCFAs) produced by the gut microbiota have beneficial anti-inflammatory and gut homeostasis effects and prevent type 1 diabetes (T1D) in mice. Reduced SCFA production indicates a loss of beneficial bacteria, commonly associated with chronic autoimmune and inflammatory diseases, including T1D and type 2 diabetes. Here, we addressed whether a metabolite-based dietary supplement has an impact on humans with T1D. We conducted a single-arm pilot-and-feasibility trial with high-amylose maize-resistant starch modified with acetate and butyrate (HAMSAB) to assess safety, while monitoring changes in the gut microbiota in alignment with modulation of the immune system status. RESULTS: HAMSAB supplement was administered for 6 weeks with follow-up at 12 weeks in adults with long-standing T1D. Increased concentrations of SCFA acetate, propionate, and butyrate in stools and plasma were in concert with a shift in the composition and function of the gut microbiota. While glucose control and insulin requirements did not change, subjects with the highest SCFA concentrations exhibited the best glycemic control. Bifidobacterium longum, Bifidobacterium adolescentis, and vitamin B7 production correlated with lower HbA1c and basal insulin requirements. Circulating B and T cells developed a more regulatory phenotype post-intervention. CONCLUSION: Changes in gut microbiota composition, function, and immune profile following 6 weeks of HAMSAB supplementation were associated with increased SCFAs in stools and plasma. The persistence of these effects suggests that targeting dietary SCFAs may be a mechanism to alter immune profiles, promote immune tolerance, and improve glycemic control for the treatment of T1D. TRIAL REGISTRATION: ACTRN12618001391268. Registered 20 August 2018, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=375792 Video Abstract.

Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA-RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA-RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA-mRNA interactions are recovered and we find that an mRNA encoding a long 3' untranslated region (UTR) (termed vigR 3'UTR) functions as a regulatory 'hub' within the RNA-RNA interaction network. We demonstrate that the vigR 3'UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA-mRNA base-pairing. Deletion of the vigR 3'UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3'UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk ALL subtype with high rates of relapse and poor patient outcome. Activating mutations affecting components of the JAK-STAT signaling pathway occur in the majority of Ph-like ALL cases. The use of JAK inhibitors represents a potential treatment option for Ph-like ALL, although we and others have shown that CRLF2-rearranged Ph-like ALL responds poorly to single-agent JAK inhibitors in the preclinical setting. Therefore, the aim of this study was to identify effective combination treatments against CRLF2-rearranged Ph-like ALL, and to elucidate the underlying mechanisms of synergy. We carried out a series of high-throughput combination drug screenings and found that ruxolitinib exerted synergy with standard-of-care drugs used in the treatment of ALL. In addition, we investigated the molecular effects of ruxolitinib on Ph-like ALL by combining mass spectrometry phosphoproteomics with gene expression analysis. Based on these findings, we conducted preclinical in vivo drug testing and demonstrated that ruxolitinib enhanced the in vivo efficacy of an induction-type regimen consisting of vincristine, dexamethasone, and l-asparaginase in 2/3 CRLF2-rearranged Ph-like ALL xenografts. Overall, our findings support evaluating the addition of ruxolitinib to conventional induction regimens for the treatment of CRLF2-rearranged Ph-like ALL.

Background: Inhibition of hepatocyte growth factor (HGF)/c-MET pathway, a major mediator of pancreatic stellate cell (PSC)−PC cell interactions, retards local and distant cancer progression. This study examines the use of this treatment in preventing PC progression after resection. We further investigate the postulated existence of circulating PSCs (cPSCs) as a mediator of metastatic PC. Methods: Two orthotopic PC mouse models, produced by implantation of a mixture of luciferase-tagged human pancreatic cancer cells (AsPC-1), and human PSCs were used. Model 1 mice underwent distal pancreatectomy 3-weeks post-implantation (n = 62). One-week post-resection, mice were randomised to four treatments of 8 weeks: (i) IgG, (ii) gemcitabine (G), (iii) HGF/c-MET inhibition (HiCi) and (iv) HiCi + G. Tumour burden was assessed longitudinally by bioluminescence. Circulating tumour cells and cPSCs were enriched by filtration. Tumours of Model 2 mice progressed for 8 weeks prior to the collection of primary tumour, metastases and blood for single-cell RNA-sequencing (scRNA-seq). Results: HiCi treatments: (1) reduced both the risk and rate of disease progression after resection; (2) demonstrated an anti-angiogenic effect on immunohistochemistry; (3) reduced cPSC counts. cPSCs were identified using immunocytochemistry (α-smooth muscle actin+, pan-cytokeratin−, CD45−), and by specific PSC markers. scRNA-seq confirmed the existence of cPSCs and identified potential genes associated with development into cPSCs. Conclusions: This study is the first to demonstrate the efficacy of adjuvant HGF/c-Met inhibition for PC and provides the first confirmation of the existence of circulating PSCs.

Accumulating evidence supports the high prevalence of co-infections among Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) patients, and their potential to worsen the clinical outcome of COVID-19. However, there are few data on Southern Hemisphere populations, and most studies to date have investigated a narrow spectrum of viruses using targeted qRT-PCR. Here we assessed respiratory viral co-infections among SARS-CoV-2 patients in Australia, through respiratory virome characterization. Nasopharyngeal swabs of 92 SARS-CoV-2-positive cases were sequenced using pan-viral hybrid-capture and the Twist Respiratory Virus Panel. In total, 8% of cases were co-infected, with rhinovirus (6%) or influenzavirus (2%). Twist capture also achieved near-complete sequencing (> 90% coverage, > tenfold depth) of the SARS-CoV-2 genome in 95% of specimens with Ct < 30. Our results highlight the importance of assessing all pathogens in symptomatic patients, and the dual-functionality of Twist hybrid-capture, for SARS-CoV-2 whole-genome sequencing without amplicon generation and the simultaneous identification of viral co-infections with ease.

In the original version of this Article, a relevant paper on the detection of PLLP in human corneal epithelium during KC pathogenesis was not cited. This article is now cited as Ref 56 and discussed. As a result, in the Conclusion, "Notch1 and PLLP have not previously been linked to KC pathogenesis. PLLP has not been previously reported to be expressed in human corneal epithelium, although its roles in other tissues suggests it could play a critical role in normal corneal epithelial cellular activities, and interact with the Notch1 signaling pathway." now reads: "Notch1 has not previously been linked to KC pathogenesis. PLLP was reported to be upregulated in KC corneal epithelium compared to normal through proteomic analysis56. Both previous finding and our paper suggested an abnormal expression of PLLP in KC, however our study showed PLLP was down-regulated in KC. The difference can be attributed to the sample types and preparation. Myopia rather than normal corneal samples were used as control in this study. We measured and compared PLLP expression in each sample, whereas the previous study used pooled sample56 and therefore may mask the individual differences. The role of PLLP in human corneal epithelium is unclear, however findings from studies in other tissues suggest that it could be important in maintaining normal corneal epithelial cellular activities and interact with the Notch 1 signalling pathway." Subsequent references in the Article have been renumbered accordingly. The original Article has been corrected.

Crosstalk exists when two or more post-translational modifications, nearby in sequence or 3D space, affect each other or a protein's interactions. Saccharomyces cerevisiae protein Npl3p has six repeats of sequence SRGG, in a disordered domain, which can carry arginine methylation and serine phosphorylation. Crosstalk of the modifications controls Npl3p interactions with nuclear import, export, and other proteins. Here, we asked whether repeated SRGG motifs existed in other S. cerevisiae proteins and whether they serve a related function. Two other proteins had multiple SRGG motifs: Nop1p (fibrillarin) and Gar1p, both nucleolar proteins, which had nine and four motifs, respectively. For Nop1p, we first showed it to be extensively methylated in vivo. We then showed that the Nop1p SRGG motif is subjected to methylation by Hmt1p, phosphorylation by Sky1p, and Glc7p dephosphorylation and that there is crosstalk whereby phosphorylation blocks methylation. This is consistent with our recent motif analysis of Hmt1p, which revealed a negative specificity for acidic residues at −1 and −2 positions. On knockout of HMT1, Nop1p-GFP localization was not typically nucleolar. Conditional two-hybrid analysis, of Nop1p with C/D box small ribonuclear proteins Nop56p and Nop58p, suggested this may be associated with decreased protein-protein interactions on loss of arginine methylation. The effect of SRGG phosphorylation on the interactions of Nop1p remains unknown yet was predicted to cause a structural disorder-to-order transition in the Nop1p N-terminal domain. The SRGG motif is one of very few examples of modification crosstalk that has related functions in multiple proteins from the same species.

Saccharomyces cerevisiae has the most comprehensively characterized protein-protein interaction network, or interactome, of any eukaryote. This has predominantly been generated through multiple, systematic studies of protein-protein interactions by two-hybrid techniques and of affinity-purified protein complexes. A pressing question is to understand how large-scale cross-linking mass spectrometry (XL-MS) can confirm and extend this interactome. Here, intact yeast nuclei were subject to cross-linking with disuccinimidyl sulfoxide (DSSO) and analyzed using hybrid MS2-MS3 methods. XlinkX identified a total of 2,052 unique residue pair cross-links at 1% FDR. Intraprotein cross-links were found to provide extensive structural constraint data, with almost all intralinks that mapped to known structures and slightly fewer of those mapping to homology models being within 30 Å. Intralinks provided structural information for a further 366 proteins. A method for optimizing interprotein cross-link score cut-offs was developed, through use of extensive known yeast interactions. Its application led to a high confidence, yeast nuclear interactome. Strikingly, almost half of the interactions were not previously detected by two-hybrid or AP-MS techniques. Multiple lines of evidence existed for many such interactions, whether through literature or ortholog interaction data, through multiple unique interlinks between proteins, and/or through replicates. We conclude that XL-MS is a powerful means to measure interactions, that complements two-hybrid and affinity-purification techniques.

Co-fractionation MS (CF-MS) is a technique with potential to characterize endogenous and unmanipulated protein complexes on an unprecedented scale. However this potential has been offset by a lack of guidelines for best-practice CF-MS data collection and analysis. To obtain such guidelines, this study thoroughly evaluates novel and published Saccharomyces cerevisiae CF-MS data sets using very high proteome coverage libraries of yeast gold standard complexes. A new method for identifying gold standard complexes in CF-MS data, Reference Complex Profiling, and the Extending ‘Guilt-by-Association’ by Degree (EGAD) R package are used for these evaluations, which are verified with concurrent analyses of published human data. By evaluating data collection designs, which involve fractionation of cell lysates, it is found that near-maximum recall of complexes can be achieved with fewer samples than published studies. Distributing sample collection across orthogonal fractionation methods, rather than a single high resolution data set, leads to particularly efficient recall. By evaluating 17 different similarity scoring metrics, which are central to CF-MS data analysis, it is found that two metrics rarely used in past CF-MS studies – Spearman and Kendall correlations – and the recently introduced Co-apex metric frequently maximize recall, whereas a popular metric—Euclidean distance—delivers poor recall. The common practice of integrating external genomic data into CF-MS data analysis is also evaluated, revealing that this practice may improve the precision and recall of known complexes but is generally unsuitable for predicting novel complexes in model organisms. If studying nonmodel organisms using orthologous genomic data, it is found that particular subsets of fractionation profiles (e.g. the lowest abundance quartile) should be excluded to minimize false discovery. These assessments are summarized in a series of universally applicable guidelines for precise, sensitive and

Purpose: RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are common in some hematologic cancers but are relatively uncommon in acute myeloid leukemia (AML, < 20% of patients). We examined whether RNA splicing differences exist in AML, even in the absence of splicing factor mutations. Experimental Design: We developed a bioinformatics pipeline to study alternative RNA splicing in RNA-sequencing data from large cohorts of patients with AML. Results: We have identified recurrent differential alternative splicing between patients with poor and good prognosis. These splicing events occurred even in patients without any discernible splicing factor mutations. Alternative splicing recurrently occurred in genes with specific molecular functions, primarily related to protein translation. Developing tools to predict the functional impact of alternative splicing on the translated protein, we discovered that approximately 45% of the splicing events directly affected highly conserved protein domains. Several splicing factors were themselves misspliced and the splicing of their target transcripts were altered. Studying differential gene expression in the same patients, we identified that alternative splicing of protein translation genes in ELNAdv patients resulted in the induction of an integrated stress response and upregulation of inflammation-related genes. Finally, using machine learning techniques, we identified a splicing signature of four genes which refine the accuracy of existing risk prognosis schemes and validated it in a completely independent cohort. Conclusions: Our discoveries therefore identify aberrant alternative splicing as a molecular feature of adverse AML with clinical relevance.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.

Background: Microbial exposures in utero and early life shape the infant microbiome, which can profoundly impact on health. Compared to the bacterial microbiome, very little is known about the virome. We set out to characterize longitudinal changes in the gut virome of healthy infants born to mothers with or without type 1 diabetes using comprehensive virome capture sequencing. Methods: Healthy infants were selected from Environmental Determinants of Islet Autoimmunity (ENDIA), a prospective cohort of Australian children with a first‐degree relative with type 1 diabetes, followed from pregnancy. Fecal specimens were collected three‐monthly in the first year of life. Results: Among 25 infants (44% born to mothers with type 1 diabetes) at least one virus was detected in 65% (65/100) of samples and 96% (24/25) of infants during the first year of life. In total, 26 genera of viruses were identified and >150 viruses were differentially abundant between the gut of infants with a mother with type 1 diabetes vs without. Positivity for any virus was associated with maternal type 1 diabetes and older infant age. Enterovirus was associated with older infant age and maternal smoking. Conclusions: We demonstrate a distinct gut virome profile in infants of mothers with type 1 diabetes, which may influence health outcomes later in life. Higher prevalence and greater number of viruses observed compared to previous studies suggests significant underrepresentation in existing virome datasets, arising most likely from less sensitive techniques used in data acquisition.

Post-translational modifications (PTMs) of proteins act as key regulators of protein activity, including the regulation of protein-protein interactions (PPIs). However, exploring functional links between PTMs and PPIs can be difficult. PTMOracle is a Cytoscape app that facilitates the co-visualization and co-analysis of PTMs in the context of PPI networks. PTMOracle also allows extensive data to be integrated and co-analyzed, allowing the role of domains, motifs, and disordered regions to be considered. Here, we describe several PTMOracle protocols investigating complex PTM-associated relationships and their role in PPIs. This is assisted by OraclePainter for coloring proteins by the modifications present and visualizing these in the context of networks, by OracleTools for cross-matching PTMs with sequence feature for all nodes in the network, and by OracleResults for exploring specific proteins and visualizing their PTMs in the context of protein sequences. This unit aims to demonstrate how PTMOracle can be used to systematically explore network visualizations and generate testable hypotheses regarding the functional role of PTMs in PPIs, and how the results can be analyzed to better understand the regulatory role of PTMs in PPIs. © 2019 by John Wiley & Sons, Inc.

High-resolution MS/MS spectra of peptides can be deisotoped to identify monoisotopic masses of peptide fragments. The use of such masses should improve protein identification rates. However, deisotoping is not universally used and its benefits have not been fully explored. Here, MS2-Deisotoper, a tool for use prior to database search, is used to identify monoisotopic peaks in centroided MS/MS spectra. MS2-Deisotoper works by comparing the mass and relative intensity of each peptide fragment peak to every other peak of greater mass, and by applying a set of rules concerning mass and intensity differences. After comprehensive parameter optimization, it is shown that MS2-Deisotoper can improve the number of peptide spectrum matches (PSMs) identified by up to 8.2% and proteins by up to 2.8%. It is effective with SILAC and non-SILAC MS/MS data. The identification of unique peptide sequences is also improved, increasing the number of human proteoforms by 3.7%. Detailed investigation of results shows that deisotoping increases Mascot ion scores, improves FDR estimation for PSMs, and leads to greater protein sequence coverage. At a peptide level, it is found that the efficacy of deisotoping is affected by peptide mass and charge. MS2-Deisotoper can be used via a user interface or as a command-line tool.

The field of proteomics is increasingly concerned with the diversity and functional relevance of protein modifications. Differential ion mobility spectrometry (DMS) is emerging as a tool to detect and quantify additional peptide and protein species that are difficult to analyse with conventional instrumental methods. In this review, recent advances in DMS are discussed, with a focus on the different types of DMS instruments now available to researchers in proteomics. Furthermore, the combination of DMS with mass spectrometry (MS) for increased proteome coverage and the targeted analysis of modification patterns on single proteins is highlighted. Frontier areas of research, such as the analysis of intact proteoforms, and future directions for the implementation of DMS in proteomics are also discussed.

Fungal infections are an increasing cause of morbidity and mortality. Current antifungal drugs are limited in spectrum, few new drugs are in development, and resistance is an increasing issue. Drug synergy can enhance available drugs and extend their lifetime, however, few synergistic combinations are in clinical use and mechanistic data on how combinations work is lacking. The multifunctional glycoprotein lactoferrin (LF) acts synergistically with amphotericin B (AMB) in a range of fungal species. Whole LF binds and sequesters iron, and LF can also be digested enzymatically to produce cationic peptides with distinct antimicrobial functions. To understand how LF synergizes AMB, we previously undertook a transcriptomic analysis in Saccharomyces and found a paradoxical down-regulation of iron and stress response, suggesting stress pathway interference was dysregulating an appropriate response, resulting in cell death. To extend this to a fungal pathogen, we here perform the same analysis in Cryptococcus neoformans. While both fungi responded to AMB in a similar way, the addition of LF produced remarkably contrasting results, with the Cryptococcus transcriptome enriched for processes relating to cellular stress, up-regulation of endoplasmic-reticulum-associated protein degradation (ERAD), stress granule disassembly and protein folding, endoplasmic reticulum-Golgi-vacuole trafficking and autophagy, suggesting an overall disruption of protein and lipid biosynthesis. These studies demonstrate that the mechanism of LF-mediated synergy is species-specific, possibly due to differences in the way LF peptides are generated, bind to and enter cells and act on intracellular targets, illustrating how very different cellular processes can underlie what appears to be a similar phenotypic response.

Enteroviruses (EVs) are prime candidate environmental triggers of islet autoimmunity (IA), with potential as vaccine targets for type 1 diabetes prevention. However, the use of targeted virus detection methods and the selective focus on EVs by most studies increases the risk for substantial investigation bias and an overestimated association between EV and type 1 diabetes. Here we performed comprehensive virome-capture sequencing to examine all known vertebrate-infecting viruses without bias in 182 specimens (faeces and plasma) collected before or at seroconversion from 45 case children with IA and 48 matched controls. From >2.6 billion reads, 28 genera of viruses were detected and 62% of children (58/93) were positive for ≥1 vertebrate-infecting virus. We identified 129 viruses as differentially abundant between the gut of cases and controls, including 5 EV-A types significantly more abundant in the cases. Our findings further support EV’s hypothesised contribution to IA and corroborate the proposal that viral load may be an important parameter in disease pathogenesis. Furthermore, our data indicate a previously unrecognised association of IA with higher EV-A abundance in the gut of children and provide a catalog of viruses to be interrogated further to determine a causal link between virus infection and type 1 diabetes.

Development of mammalian ovarian follicles is promoted by the combined action of endocrine cues and paracrine factors. Follicle stimulating hormone (FSH), through the action of cAMP drives follicular growth and development. The oocyte secretes powerful growth factors such as bone morphogenetic protein 15 (BMP15) to regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation through the activation of SMAD1/5/8. This study investigated the role of the cAMP signalling pathway on SMAD1/5/8 action in human granulosa cells. Cyclic AMP enhanced BMP15-induction of a SMAD1/5/8-specific BRE reporter. Moreover, in the absence of BMP ligand, cAMP also activated SMAD1/5/8-induced BRE activity. Cyclic AMP increased canonical downstream targets of BMP signalling such as inhibitor of differentiation (ID) mRNA expression. The observed effects were not mediated by secretion of BMPs as cAMP did not promote BMP ligand mRNA expression and a BMP extracellular antagonist, the BMP type II receptor ectodomain, did not affect cAMP-induced ID mRNA expression. Finally, the ERK1/2 pathway was shown to be required for the maintenance of cAMP-induced SMAD1/5/8 activity. Together our results suggest a novel and non-canonical pathway for cAMP signalling in human granulosa cells. Cyclic AMP appears to promote SMAD1/5/8 pathway activity intracellularly and has the ability to activate canonical SMAD1/5/8 downstream targets. Our results add another layer of complexity to the interactions between endocrine signalling and oocyte-secreted BMP ligands during folliculogenesis. Given the importance of both cAMP and SMAD1/5/8 pathways in follicular development, these interactions are likely required for the fine-tuning of oocyte paracrine signalling by endocrine stimuli.

Background & Aims: Fecal microbiota transplantation (FMT) can induce remission in patients with ulcerative colitis (UC). In a randomized controlled trial of FMT in patients with active UC, we aimed to identify bacterial taxonomic and functional factors associated with response to therapy. Methods: We performed a double-blind trial of 81 patients with active UC randomly assigned to groups that received an initial colonoscopic infusion and then intensive multidonor FMT or placebo enemas, 5 d/wk for 8 weeks. Patients in the FMT group received blended homogenized stool from 3–7 unrelated donors. Patients in the placebo group were eligible to receive open-label FMT after the double-blind study period. We collected 314 fecal samples from the patients at screening, every 4 weeks during treatment, and 8 weeks after the blinded or open-label FMT therapy. We also collected 160 large-bowel biopsy samples from the patients at study entry, at completion of 8 weeks of blinded therapy, and at the end of open-label FMT, if applicable. We analyzed 105 fecal samples from the 14 individual donors (n = 55), who in turn contributed to 21 multidonor batches (n = 50). Bacteria in colonic and fecal samples were analyzed by both 16S ribosomal RNA gene and transcript amplicon sequencing; 285 fecal samples were analyzed by shotgun metagenomics, and 60 fecal samples were analyzed for metabolome features. Results: FMT increased microbial diversity and altered composition, based on analyses of colon and fecal samples collected before vs after FMT. Diversity was greater in fecal and colon samples collected before and after FMT treatment from patients who achieved remission compared with patients who did not. Patients in remission after FMT had enrichment of Eubacterium hallii and Roseburia inulivorans compared with patients who did not achieve remission after FMT and had increased levels of short-chain fatty acid biosynthesis and secondary bile acids. Patients who did not achieve remission had en

EMBL Australia Bioinformatics Resource (EMBL-ABR) is a developing national research infrastructure, providing bioinformatics resources and support to life science and biomedical researchers in Australia. EMBL-ABR comprises 10 geographically distributed national nodes with one coordinating hub, with current funding provided through Bioplatforms Australia and the University of Melbourne for its initial 2-year development phase. The EMBL-ABR mission is to: (1) increase Australia's capacity in bioinformatics and data sciences; (2) contribute to the development of training in bioinformatics skills; (3) showcase Australian data sets at an international level and (4) enable engagement in international programs. The activities of EMBL-ABR are focussed in six key areas, aligning with comparable international initiatives such as ELIXIR, CyVerse and NIH Commons. These key areas-Tools, Data, Standards, Platforms, Compute and Training-are described in this article.

BACKGROUND: The importance of gut bacteria in human physiology, immune regulation, and disease pathogenesis is well established. In contrast, the composition and dynamics of the gut virome are largely unknown; particularly lacking are studies in pregnancy. We used comprehensive virome capture sequencing to characterize the gut virome of pregnant women with and without type 1 diabetes (T1D), longitudinally followed in the Environmental Determinants of Islet Autoimmunity study. METHODS: In total, 61 pregnant women (35 with T1D and 26 without) from Australia were examined. Nucleic acid was extracted from serial fecal specimens obtained at prenatal visits, and viral genomes were sequenced by virome capture enrichment. The frequency, richness, and abundance of viruses were compared between women with and without T1D. RESULTS: Two viruses were more prevalent in pregnant women with T1D: picobirnaviruses (odds ratio [OR], 4.2; 95% confidence interval [CI], 1.0-17.1; P = .046) and tobamoviruses (OR, 3.2; 95% CI, 1.1-9.3; P = .037). The abundance of 77 viruses significantly differed between the 2 maternal groups (≥2-fold difference; P < .02), including 8 Enterovirus B types present at a higher abundance in women with T1D. CONCLUSIONS: These findings provide novel insight into the composition of the gut virome during pregnancy and demonstrate a distinct profile of viruses in women with T1D.

Keratoconus is a common degenerative corneal disease that can lead to significant visual morbidity, and both genetic and environmental factors have been implicated in its pathogenesis. We compared the transcriptome of keratoconus and control epithelium using RNA-Seq. Epithelial tissues were obtained prior to surgery from keratoconus and myopia control patients, undergoing collagen cross-linking and photorefractive keratectomy, respectively. We identified major differences in keratoconus linked to cell-cell communication, cell signalling and cellular metabolism. The genes associated with the Hedgehog, Wnt and Notch1 signaling pathways were down-regulated in keratoconus. We also identified plasmolipin and Notch1 as being significantly reduced in keratoconus for both gene and protein expression (p < 0.05). Plasmolipin is a novel protein identified in human corneal epithelium, and has been demonstrated to have a key role in epithelial cell differentiation in other tissues. This study shows altered gene and protein expression of these three proteins in keratoconus, and further studies are clearly warranted to confirm the functional role of these proteins in the pathogenesis of keratoconus.

Hmt1p is the predominant arginine methyltransferase in Saccharomyces cerevisiae. Its substrate proteins are involved in transcription, transcriptional regulation, nucleocytoplasmic transport and RNA splicing. Hmt1p-catalyzed methylation can also modulate protein-protein interactions. Hmt1p is conserved from unicellular eukaryotes through to mammals where its ortholog, PRMT1, is lethal upon knockout. In yeast, however, the effect of knockout on the transcriptome and proteome has not been described. Transcriptome analysis revealed downregulation of phosphate-responsive genes in hmt1, including acid phosphatases PHO5, PHO11, and PHO12, phosphate transporters PHO84 and PHO89 and the vacuolar transporter chaperone VTC3. Analysis of the hmt1 proteome revealed decreased abundance of phosphate-associated proteins including phosphate transporter Pho84p, vacuolar alkaline phosphatase Pho8p, acid phosphatase Pho3p and subunits of the vacuolar transporter chaperone complex Vtc1p, Vtc3p and Vtc4p. Consistent with this, phosphate homeostasis was dysregulated in hmt1 cells, showing decreased extracellular phosphatase levels and decreased total Pi in phosphate-depleted medium. In vitro, we showed that transcription factor Pho4p can be methylated at Arg-241, which could explain phosphate dysregulation in hmt1 if interplay exists with phosphorylation at Ser-242 or Ser-243, or if Arg-241 methylation affects the capacity of Pho4p to homodimerize or interact with Pho2p. However, the Arg-241 methylation site was not validated in vivo and the localization of a Pho4p-GFP fusion in hmt1 was not different from wild type. To our knowledge, this is the first study to reveal an association between Hmt1p and phosphate homeostasis and one which suggests a regulatory link between S-adenosyl methionine and intracellular phosphate.

Protein methylation is one of the major post-translational modifications (PTMs) in the cell. In Saccharomyces cerevisiae, over 20 protein methyltransferases (MTases) and their respective substrates have been identified. However, the way in which these MTases are modified and potentially subject to regulation remains poorly understood. Here, we investigated six overexpressed S. cerevisiae protein MTases (Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1) to identify PTMs of potential functional relevance. We identified 48 PTM sites across the six MTases, including phosphorylation, acetylation and methylation. Forty-two sites are novel. We contextualized the PTM sites in structural models of the MTases and revealed that many fell in catalytic pockets or enzyme–substrate interfaces. These may regulate MTase activity. Finally, we compared PTMs on Hmt1 with those on its human homologs PRMT1, PRMT3, CARM1, PRMT6 and PRMT8. This revealed that several PTMs are conserved from yeast to human, whereas others are only found in Hmt1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD006767.

Eukaryotic elongation factor 1A (eEF1A) is an essential and highly conserved protein involved in diverse cellular processes, including translation, cytoskeleton organisation, nuclear export, and proteasomal degradation. Recently, nine novel and site-specific methyltransferases were discovered that target eEF1A, five in yeast and four in human, making it the eukaryotic protein with the highest number of independent methyltransferases. Some of these methyltransferases show striking evolutionary conservation. Yet, they come from diverse methyltransferase families, indicating they confer competitive advantage through independent origins. As might be expected, the first functional studies of specific methylation sites found them to have distinct effects, notably on eEF1A-related processes of translation and tRNA aminoacylation. Further functional studies of sites will likely reveal other unique roles for this interesting modification.

This study investigated the enzyme-substrate interaction between Saccharomyces cerevisiae arginine methyltransferase Hmt1p and nucleolar protein Npl3p, using chemical cross linking/mass spectrometry (XL/MS). We show that XL/MS can capture transient interprotein interactions that occur during the process of methylation, involving a disordered region in Npl3p with tandem SRGG repeats, and we confirm that Hmt1p and Npl3p exist as homomultimers. Additionally, the study investigated the interdependencies between variables of an XL/MS experiment that lead to the identification of identical or different cross-linked peptides. We report that there are substantial benefits, in terms of biologically relevant cross-links identified, that result from the use of two mass-spectrometry-cleavable cross-linkers [disuccinimido sulfoxide (DSSO) and disuccinimido dibutyric urea (DSBU)], two fragmentation approaches [collision-induced dissociation and electron-transfer dissociation (CID+ETD)] and stepped high-energy collision dissociation (HCD)], and two programs (MeroX and XlinkX). We also show that there are specific combinations of XL/MS methods that are more successful than others for the two proteins investigated here; these are explored in detail in the text. Data are available via ProteomeXchange with identifier PXD008348.

The study of post-translational methylation is hampered by the fact that large-scale LC-MS/MS experiments produce high methylpeptide false discovery rates (FDRs). The use of heavy-methyl stable isotope labeling by amino acids in cell culture (heavy-methyl SILAC) can drastically reduce these FDRs; however, this approach is limited by a lack of heavy-methyl SILAC compatible software. To fill this gap, we recently developed MethylQuant. Here, using an updated version of MethylQuant, we demonstrate its methylpeptide validation and quantification capabilities and provide guidelines for its best use. Using reference heavy-methyl SILAC data sets, we show that MethylQuant predicts with statistical significance the true or false positive status of methylpeptides in samples of varying complexity, degree of methylpeptide enrichment, and heavy to light mixing ratios. We introduce methylpeptide confidence indicators, MethylQuant Confidence and MethylQuant Score, and demonstrate their strong performance in complex samples characterized by a lack of methylpeptide enrichment. For these challenging data sets, MethylQuant identifies 882 of 1165 true positive methylpeptide spectrum matches (i.e., >75% sensitivity) at high specificity (<2% FDR) and achieves near-perfect specificity at 41% sensitivity. We also demonstrate that MethylQuant produces high accuracy relative quantification data that are tolerant of interference from coeluting peptide ions. Together MethylQuant's capabilities provide a path toward routine, accurate characterizations of the methylproteome using heavy-methyl SILAC.

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as ‘vulnerable’ due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala’s ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala’s survival in the wild.

Background: The esophageal microbiome has been proposed to be involved in a range of diseases including the esophageal adenocarcinoma cascade; however, little is currently known about its function and relationship to the host. Here, the esophageal microbiomes of 106 prospectively recruited patients were assessed using 16S rRNA and 18S rRNA amplicon sequencing as well as shotgun sequencing, and associations with age, gender, proton pump inhibitor use, host genetics, and disease were tested. Results: The esophageal microbiome was found to cluster into functionally distinct community types (esotypes) defined by the relative abundances of Streptococcus and Prevotella. While age was found to be a significant factor driving microbiome composition, bacterial signatures and functions such as enrichment with Gram-negative oral-associated bacteria and microbial lactic acid production were associated with the early stages of the esophageal adenocarcinoma cascade. Non-bacterial microbes such as archaea, Candida spp., and bacteriophages were also identified in low abundance in the esophageal microbiome. Specific host SNPs in NOTCH2, STEAP2-AS1, and NREP were associated with the composition of the esophageal microbiome in our cohort. Conclusions: This study provides the most comprehensive assessment of the esophageal microbiome to date and identifies novel signatures and host markers that can be investigated further in the context of esophageal adenocarcinoma development.

Negative genetic interactions in Saccharomyces cerevisiae have been systematically screened to near-completeness, with >500 000 interactions identified. Nevertheless, the biological basis of these interactions remains poorly understood. To investigate this, we analyzed negative genetic interactions within an integrated biological network, being the union of protein–protein, kinase–substrate, and transcription factor–target gene interactions. Network triplets, containing two genes/proteins that show negative genetic interaction and a third protein from the network, were then analyzed. Strikingly, just six out of 15 possible triplet motif types were present, as compared to randomized networks. These were in three clear groups: protein–protein interactions, signaling, and regulatory triplets where the latter two showed no overlap. In the triplets, negative genetic interactions were associated with paralogs and ohnologs; however, these were very rare. Negative genetic interactions among the six triplet motifs did however show strong dosage constraints, with genes being significantly associated with toxicity on overexpression and periodicity in the cell cycle. Negative genetic interactions overlapped with other interaction types in 37% of cases; these were predominantly associated with protein complexes or signaling events. Finally, we highlight regions of “network vulnerability” containing multiple negative genetic interactions; these could be targeted in fungal species for the regulation of cell growth.

A subset of human follicular helper T cells (TFH) cells expresses CD57 for which no distinct function has been identified. We show that CD57+ TFH cells are universally PD-1hi, but compared to their CD57-PD-1hi counterparts, express little IL-21 or IL-10 among others. Instead, CD57 expression on TFH cells marks cytotoxicity transcriptional signatures that translate into only a weak cytotoxic phenotype. Similarly, circulating PD-1+ CD57+ CD4+ T cells make less cytokine than their CD57-PD-1+ counterparts, but have a prominent cytotoxic phenotype. By analysis of responses to STAT3-dependent cytokines and cells from patients with gain-or loss-of-function STAT3 mutations, we show that CD4+ T cell cytotoxicity is STAT3-dependent. TFH formation also requires STAT3, but paradoxically, once formed, PD-1hi cells become unresponsive to STAT3. These findings suggest that changes in blood and germinal center cytotoxicity might be affected by changes in STAT3 signaling, or modulation of PD-1 by therapy.