Unexpected host dependency of Antarctic Nanohaloarchaeota.

In hypersaline environments, Nanohaloarchaeota (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaeota [DPANN] superphylum) are thought to be free-living microorganisms. We report cultivation of 2 strains of Antarctic Nanohaloarchaeota and show that they require the haloarchaeon Halorubrum lacusprofundi for growth. By performing growth using enrichments and fluorescence-activated cell sorting, we demonstrated successful cultivation of Candidatus Nanohaloarchaeum antarcticus, purification of Ca. Nha. antarcticus away from other species, and growth and verification of Ca. Nha. antarcticus with Hrr. lacusprofundi; these findings are analogous to those required for fulfilling Koch's postulates. We use fluorescent in situ hybridization and transmission electron microscopy to assess cell structures and interactions; metagenomics to characterize enrichment taxa, generate metagenome assembled genomes, and interrogate Antarctic communities; and proteomics to assess metabolic pathways and speculate about the roles of certain proteins. Metagenome analysis indicates the presence of a single species, which is endemic to Antarctic hypersaline systems that support the growth of haloarchaea. The presence of unusually large proteins predicted to function in attachment and invasion of hosts plus the absence of key biosynthetic pathways (e.g., lipids) in metagenome assembled genomes of globally distributed Nanohaloarchaeota indicate that all members of the lineage have evolved as symbionts. Our work expands the range of archaeal symbiotic lifestyles and provides a genetically tractable model system for advancing understanding of the factors controlling microbial symbiotic relationships.

Extending the Depth of Human Plasma Proteome Coverage Using Simple Fractionation Techniques.

Human plasma is one of the most widely used tissues in clinical analysis, and plasma-based biomarkers are used for monitoring patient health status and/or response to medical treatment to avoid unnecessary invasive biopsy. Data-driven plasma proteomics has suffered from a lack of throughput and detection sensitivity, largely due to the complexity of the plasma proteome and in particular the enormous quantitative dynamic range, estimated to be between 9 and 13 orders of magnitude between the lowest and the highest abundance protein. A major challenge is to identify workflows that can achieve depth of plasma proteome coverage while minimizing the complexity of the sample workup and maximizing the sample throughput. In this study, we have performed intensive depletion of high-abundant plasma proteins or enrichment of low-abundant proteins using the Agilent multiple affinity removal liquid chromatography (LC) column-Human 6 (Hu6), the Agilent multiple affinity removal LC column-Human 14 (Hu14), and ProteoMiner followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and C18 prefractionation techniques. We compared the performance of each of these fractionation approaches to identify the method that satisfies requirements for analysis of clinical samples and to include good plasma proteome coverage in combination with reasonable sample output. In this study, we report that one-dimensional (1D) gel-based prefractionation allows parallel sample processing and no loss of proteome coverage, compared with serial chromatographic separation, and significantly accelerates analysis time, particularly important for large clinical projects. Furthermore, we show that a variety of methodologies can achieve similarly high plasma proteome coverage, allowing flexibility in method selection based on project-specific needs. These considerations are important in the effort to accelerate plasma proteomics research so as to provide efficient, reliable, and accurate diagnoses, population-based health screening, clinical research studies, and other clinical work.

Extracellular signal-regulated kinase-1 phosphorylates early growth response-1 at serine 26.

Egr-1, an immediate-early gene product and master regulator was originally described as a phosphoprotein following its discovery in the 1980s. However specific residue(s) phosphorylated in Egr-1 remain elusive. Here we phosphorylated recombinant Egr-1 in vitro with ERK1 prior to mass spectrometry, which identified phosphorylation of Ser12 and Ser26 with the latter ∼12 times more abundant than Ser12. Phosphorylation of wild-type recombinant Egr-1 (as compared with Ser26>Ala26 mutant Egr-1) revealed that Ser26 accounts for the majority of phosphorylation of Egr-1 by ERK1. N-FGSFPH(pS)PTMDNYC-C was used as an antigen to generate mouse monoclonal antibodies (pS26 MAb). pS26 MAb recognised ERK1-phosphorylated Egr-1 but not Egr-1 bearing a point mutation at Ser26. pS26 MAb recognised inducible ∼75 kDa and 100 kDa species in nuclear extracts of cells exposed to FGF-2. Peptide blocking revealed both inducible species were phosphosite-specific. Immunoprecipitation of nuclear extracts of cells exposed to FGF-2 with pS26 MAb followed by SDS-PAGE and mass spectrometry identified Egr-1 sequences corresponding to the ∼75 kDa species but not ∼100 kDa species. This study identifies a specific amino acid phosphorylated in endogenous Egr-1.

Sucrose Metabolism in Haloarchaea: Reassessment Using Genomics, Proteomics, and Metagenomics.

The canonical pathway for sucrose metabolism in haloarchaea utilizes a modified Embden-Meyerhof-Parnas pathway (EMP), in which ketohexokinase and 1-phosphofructokinase phosphorylate fructose released from sucrose hydrolysis. However, our survey of haloarchaeal genomes determined that ketohexokinase and 1-phosphofructokinase genes were not present in all species known to utilize fructose and sucrose, thereby indicating that alternative mechanisms exist for fructose metabolism. A fructokinase gene was identified in the majority of fructose- and sucrose-utilizing species, whereas only a small number possessed a ketohexokinase gene. Analysis of a range of hypersaline metagenomes revealed that haloarchaeal fructokinase genes were far more abundant (37 times) than haloarchaeal ketohexokinase genes. We used proteomic analysis of Halohasta litchfieldiae (which encodes fructokinase) and identified changes in protein abundance that relate to growth on sucrose. Proteins inferred to be involved in sucrose metabolism included fructokinase, a carbohydrate primary transporter, a putative sucrose hydrolase, and two uncharacterized carbohydrate-related proteins encoded in the same gene cluster as fructokinase and the transporter. Homologs of these proteins were present in the genomes of all haloarchaea that use sugars for growth. Enzymes involved in the semiphosphorylative Entner-Doudoroff pathway also had higher abundances in sucrose-grown H. litchfieldiae cells, consistent with this pathway functioning in the catabolism of the glucose moiety of sucrose. The study revises the current understanding of fundamental pathways for sugar utilization in haloarchaea and proposes alternatives to the modified EMP pathway used by haloarchaea for sucrose and fructose utilization.IMPORTANCE Our ability to infer the function that microorganisms perform in the environment is predicated on assumptions about metabolic capacity. When genomic or metagenomic data are used, metabolic capacity is inferred from genetic potential. Here, we investigate the pathways by which haloarchaea utilize sucrose. The canonical haloarchaeal pathway for fructose metabolism involving ketohexokinase occurs only in a small proportion of haloarchaeal genomes and is underrepresented in metagenomes. Instead, fructokinase genes are present in the majority of genomes/metagenomes. In addition to genomic and metagenomic analyses, we used proteomic analysis of Halohasta litchfieldiae (which encodes fructokinase but lacks ketohexokinase) and identified changes in protein abundance that related to growth on sucrose. In this way, we identified novel proteins implicated in sucrose metabolism in haloarchaea, comprising a transporter and various catabolic enzymes (including proteins that are annotated as hypothetical).

Proteomic Adaptation of Australian Epidemic Bordetella pertussis.

Bordetella pertussis causes whooping cough. The predominant strains in Australia changed to single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele ptxP3/pertactin gene allele prn2) from cluster II (non-ptxP3/non-prn2). Cluster I was mostly responsible for the 2008-2012 Australian epidemic and was found to have higher fitness compared to cluster II using an in vivo mouse competition assay, regardless of host's immunization status. This study aimed to identify proteomic differences that explain higher fitness in cluster I using isobaric tags for relative and absolute quantification (iTRAQ), and high-resolution multiple reaction monitoring (MRM-hr). A few key differences in the whole cell and secretome were identified between the cluster I and II strains tested. In the whole cell, nine proteins were upregulated (>1.2 fold change, q < 0.05) and three were downregulated (<0.8 fold change, q < 0.05) in cluster I. One downregulated protein was BP1569, a TLR2 agonist for Th1 immunity. In the secretome, 12 proteins were upregulated and 1 was downregulated which was Bsp22, a type III secretion system (T3SS) protein. Furthermore, there was a trend of downregulation in three T3SS effectors and other virulence factors. Three proteins were upregulated in both whole cell and supernatant: BP0200, molybdate ABC transporter (ModB), and tracheal colonization factor A (TcfA). Important expression differences in lipoprotein, T3SS, and transport proteins between the cluster I and II strains were identified. These differences may affect immune evasion, virulence and metabolism, and play a role in increased fitness of cluster I.

A quantitative proteomics approach identifies ETV6 and IKZF1 as new regulators of an ERG-driven transcriptional network.

Aberrant stem cell-like gene regulatory networks are a feature of leukaemogenesis. The ETS-related gene (ERG), an important regulator of normal haematopoiesis, is also highly expressed in T-ALL and acute myeloid leukaemia (AML). However, the transcriptional regulation of ERG in leukaemic cells remains poorly understood. In order to discover transcriptional regulators of ERG, we employed a quantitative mass spectrometry-based method to identify factors binding the 321 bp ERG +85 stem cell enhancer region in MOLT-4 T-ALL and KG-1 AML cells. Using this approach, we identified a number of known binders of the +85 enhancer in leukaemic cells along with previously unknown binders, including ETV6 and IKZF1. We confirmed that ETV6 and IKZF1 were also bound at the +85 enhancer in both leukaemic cells and in healthy human CD34+ haematopoietic stem and progenitor cells. Knockdown experiments confirmed that ETV6 and IKZF1 are transcriptional regulators not just of ERG, but also of a number of genes regulated by a densely interconnected network of seven transcription factors. At last, we show that ETV6 and IKZF1 expression levels are positively correlated with expression of a number of heptad genes in AML and high expression of all nine genes confers poorer overall prognosis.

Quantitative Performance Evaluator for Proteomics (QPEP): Web-based Application for Reproducible Evaluation of Proteomics Preprocessing Methods.

Tandem mass spectrometry is one of the most popular techniques for quantitation of proteomes. There exists a large variety of options in each stage of data preprocessing that impact the bias and variance of the summarized protein-level values. Using a newly released data set satisfying a replicated Latin squares design, a diverse set of performance metrics has been developed and implemented in a web-based application, Quantitative Performance Evaluator for Proteomics (QPEP). QPEP has the flexibility to allow users to apply their own method to preprocess this data set and share the results, allowing direct and straightforward comparison of new methodologies. Application of these new metrics to three case studies highlights that (i) the summarization of peptides to proteins is robust to the choice of peptide summary used, (ii) the differences between iTRAQ labels are stronger than the differences between experimental runs, and (iii) the commercial software ProteinPilot performs equivalently well at between-sample normalization to more complicated methods developed by academics. Importantly, finding (ii) underscores the benefits of using the principles of randomization and blocking to avoid the experimental measurements being confounded by technical factors. Data are available via ProteomeXchange with identifier PXD003608.

Cold adaptation of the Antarctic haloarchaea Halohasta litchfieldiae and Halorubrum lacusprofundi.

Halohasta litchfieldiae represents ∼ 44% and Halorubrum lacusprofundi ∼ 10% of the hypersaline, perennially cold (≥ -20°C) Deep Lake community in Antarctica. We used proteomics and microscopy to define physiological responses of these haloarchaea to growth at high (30°C) and low (10 and 4°C) temperatures. The proteomic data indicate that both species responded to low temperature by modifying their cell envelope including protein N-glycosylation, maintaining osmotic balance and translation initiation, and modifying RNA turnover and tRNA modification. Distinctions between the two species included DNA protection and repair strategies (e.g. roles of UspA and Rad50), and metabolism of glycerol and pyruvate. For Hrr. lacusprofundi, low temperature led to the formation of polyhydroxyalkanoate-like granules, with granule formation occurring by an unknown mechanism. Hrr. lacusprofundi also formed biofilms and synthesized high levels of Hsp20 chaperones. Hht. litchfieldiae was characterized by an active CRISPR system, and elevated levels of the core gene expression machinery, which contrasted markedly to the decreased levels of Hrr. lacusprofundi. These findings greatly expand the understanding of cellular mechanisms of cold adaptation in psychrophilic archaea, and provide insight into how Hht. litchfieldiae gains dominance in Deep Lake.

Specific global responses to N and Fe nutrition in toxic and non-toxic Microcystis aeruginosa.

The bloom-forming cyanobacteria species Microcystis aeruginosa includes toxic and non-toxic (microcystin-producing) strains. Certain stress conditions stimulate synthesis of microcystin (MCYST) and enhance the binding of the MCYST molecule to proteins. In this quantitative proteomic study, we compared the response of a wild-type toxic strain PCC 7806, an mcyH(-) knockout non-toxic strain, and a naturally occurring non-toxic strain, PCC 7005, after 8 days in low iron (Fe) and nitrogen (N) starvation in order to assess the benefit of MCYST synthesis in non-optimal conditions. Fe limitation increased MCYST synthesis and caused an accumulation of phycobilisome proteins and the ferric iron transporter FutA only in the toxic PCC 7806 but not the non-toxic strains. In N starvation, photosynthetic, C and N metabolism proteins were more abundant in the non-toxic strains, as were chaperones and proteases. Significant interaction between nutrient availability and toxicity existed for thioredoxin peroxidase and several thioredoxin-regulated proteins. We propose a competition of MCYST for binding sites in thioredoxin-regulated proteins during oxidative stress (low Fe) but not in growth-limiting conditions (low N). This then leads to differences in the regulation of C:N metabolism in toxic and non-toxic M. aeruginosa in nutrient-replete and nutrient-limited conditions.

Amyloid Plaque in the Human Brain Can Decompose from Aß(1-40/1-42) by Spontaneous Nonenzymatic Processes.

The degradation of long-lived proteins in the body is an important aspect of aging, and much of the breakdown is due to the intrinsic instability of particular amino acids. In this study, peptides were examined to discover if spontaneous nonenzymatic reactions could be responsible for the composition of Alzheimer's (AD) plaque in the human brain. The great majority of AD plaque consists of N-terminally truncated versions of Aß(1-40/1-42), with the most abundant peptide commencing with Glu (residue 3 in Aß1-40/1-42) that is present as pyroGlu. Several Asp residues are racemized in Aß plaque, with residue 1 being predominantly l-isoAsp and peptide bond cleavage next to Ser 8 is also evident. In peptides, loss of the two N-terminal amino acids as a diketopiperazine was demonstrated at pH 7. For the Aß N-terminal hexapeptide, AspAlaGluPheArgHis, this resulted in the removal of AspAla diketopiperazine and the generation of Glu as the new N-terminal residue. The Glu cyclized readily to pyroGlu. This pathway was altered significantly by zinc, which promoted pyroGlu formation but decreased AspAla diketopiperazine release. Zinc also facilitated cleavage on the N-terminal side of Ser 8. Racemization of the original N-terminal Asp to l-isoAsp was also detected and loss of one amino acid from the N-terminus. These data are therefore entirely consistent with plaque in the human brain forming from deposition of Aß(1-40/1-42) and, over time, decomposing spontaneously. Since amyloid plaque is present in the human brain for years prior to the onset of AD, gradual spontaneous changes to the polypeptides within it will alter its properties and those of the oligomers that can diffuse from it. Such incremental changes in composition may therefore contribute to the origin of AD-associated cytotoxicity.

Ecophysiological Distinctions of Haloarchaea from a Hypersaline Antarctic Lake as Determined by Metaproteomics.

UNLABELLED: Deep Lake in the Vestfold Hills is hypersaline and the coldest system in Antarctica known to support microbial growth (temperatures as low as -20°C). It represents a strong experimental model because the lake supports a low-complexity community of haloarchaea, with the three most abundant species totaling ∼72%. Moreover, the dominant haloarchaea are cultivatable, and their genomes are sequenced. Here we use metaproteomics linked to metagenome data and the genome sequences of the isolates to characterize the main pathways, trophic strategies, and interactions associated with resource utilization. The dominance of the most abundant member, Halohasta litchfieldiae, appears to be predicated on competitive utilization of substrates (e.g., starch, glycerol, and dihydroxyacetone) produced by Dunaliella, the lake's primary producer, while also possessing diverse mechanisms for acquiring nitrogen and phosphorus. The second most abundant member, strain DL31, is proficient in degrading complex proteinaceous matter. Hht. litchfieldiae and DL31 are inferred to release labile substrates that are utilized by Halorubrum lacusprofundi, the third most abundant haloarchaeon in Deep Lake. The study also linked genome variation to specific protein variants or distinct genetic capacities, thereby identifying strain-level variation indicative of specialization. Overall, metaproteomics revealed that rather than functional differences occurring at different lake depths or through size partitioning, the main lake genera possess major trophic distinctions, and phylotypes (e.g., strains of Hht. litchfieldiae) exhibit a more subtle level of specialization. This study highlights the extent to which the lake supports a relatively uniform distribution of taxa that collectively possess the genetic capacity to effectively exploit available nutrients throughout the lake. IMPORTANCE: Life on Earth has evolved to colonize a broad range of temperatures, but most of the biosphere (∼85%) exists at low temperatures (≤5°C). By performing unique roles in biogeochemical cycles, environmental microorganisms perform functions that are critical for the rest of life on Earth to survive. Cold environments therefore make a particularly important contribution to maintaining healthy, stable ecosystems. Here we describe the main physiological traits of the dominant microorganisms that inhabit Deep Lake in Antarctica, the coldest aquatic environment known to support life. The hypersaline system enables the growth of halophilic members of the Archaea: haloarchaea. By analyzing proteins of samples collected from the water column, we determined the functions that the haloarchaea were likely to perform. This study showed that the dominant haloarchaea possessed distinct lifestyles yet formed a uniform community throughout the lake that was collectively adept at using available light energy and diverse organic substrates for growth.

Transcriptomic and proteomic analyses reveal key innate immune signatures in the host response to the gastrointestinal pathogen Campylobacter concisus.

Pathogenic species within the genus Campylobacter are responsible for a considerable burden on global health. Campylobacter concisus is an emergent pathogen that plays a role in acute and chronic gastrointestinal disease. Despite ongoing research on Campylobacter virulence mechanisms, little is known regarding the immunological profile of the host response to Campylobacter infection. In this study, we describe a comprehensive global profile of innate immune responses to C. concisus infection in differentiated THP-1 macrophages infected with an adherent and invasive strain of C. concisus. Using RNA sequencing (RNA-seq), quantitative PCR (qPCR), mass spectrometry, and confocal microscopy, we observed differential expression of pattern recognition receptors and robust upregulation of DNA- and RNA-sensing molecules. In particular, we observed IFI16 inflammasome assembly in C. concisus-infected macrophages. Global profiling of the transcriptome revealed the significant regulation of a total of 8,343 transcripts upon infection with C. concisus, which included the activation of key inflammatory pathways involving CREB1, NF-κB, STAT, and interferon regulatory factor signaling. Thirteen microRNAs and 333 noncoding RNAs were significantly regulated upon infection, including MIR221, which has been associated with colorectal carcinogenesis. This study represents a major advance in our understanding of host recognition and innate immune responses to infection by C. concisus.

Proteomic analysis of livers from fat-fed mice deficient in either PKCδ or PKCε identifies Htatip2 as a regulator of lipid metabolism.

Insulin resistance contributes to the development of Type 2 diabetes, and is associated with lipid oversupply. Deletion of isoforms of the lipid-activated protein kinase C (PKC) family, PKCδ or PKCε, improves insulin action in fat-fed mice, but differentially affects hepatic lipid metabolism. To investigate the mechanisms involved, we employed an in vivo adaptation of SILAC to examine the effects of a fat diet together with deletion of PKCδ or PKCε on the expression of liver proteins. We identified a total of 3359 and 3488 proteins from the PKCδ and PKCε knockout study groups, respectively, and showed that several enzymes of lipid metabolism were affected by the fat diet. In fat-fed mice, 23 proteins showed changes upon PKCδ deletion while 19 proteins were affected by PKCε deletion. Enzymes of retinol metabolism were affected by the absence of either PKC. Pathway analysis indicated that monosaccharide metabolism was affected only upon PKCδ deletion, while isoprenoid biosynthesis was affected in a PKCε-specific manner. Certain proteins were regulated inversely, including HIV-1 tat interactive protein 2 (Htatip2). Overexpression or knockdown of Htatip2 in hepatocytes affected fatty acid storage and oxidation, consistent with a novel role in mediating the differential effects of PKC isoforms on lipid metabolism. All MS data have been deposited in the ProteomeXchange with identifier PXD000971 (http://proteomecentral.proteomexchange.org/dataset/PXD000971).

Glycosylation in a mammalian expression system is critical for the production of functionally active leukocyte immunoglobulin-like receptor A3 protein.

The leukocyte immunoglobulin-like receptor (LILR) A3 is a member of the highly homologous activating and inhibitory receptors expressed on leukocytes. LILRA3 is a soluble receptor of unknown functions but is predicted to act as a broad antagonist to other membrane-bound LILRs. Functions of LILRA3 are unclear primarily because of the lack of high quality functional recombinant protein and insufficient knowledge regarding its ligand(s). Here, we expressed and characterized recombinant LILRA3 (rLILRA3) proteins produced in 293T cells, Escherichia coli, and Pichia pastoris. We found that the purified rLILRA3 produced in the mammalian system was the same size as a 70-kDa native macrophage LILRA3. This is 20 kDa larger than the calculated size, suggesting significant post-translational modifications. In contrast, rLILRA3 produced in E. coli was similar in size to the unprocessed protein, but yeast-produced protein was 2-4 times larger than the unprocessed protein. Treatment with peptide-N-glycosidase F reduced the size of the mammalian cell- and yeast-produced rLILRA3 to 50 kDa, suggesting that most modifications are due to glycosylation. Consistent with this, mass spectrometric analysis of the mammalian rLILRA3 revealed canonical N-glycosylation at the predicted Asn(140), Asn(281), Asn(302), Asn(341), and Asn(431) sites. Functionally, only mammalian cell-expressed rLILRA3 bound onto the surface of monocytes with high affinity, and importantly, only this significantly abrogated LPS-induced TNFα production by monocytes. Binding to monocytes was partially blocked by ß-lactose, indicating that optimally glycosylated LILRA3 might be critical for ligand binding and function. Overall, our data demonstrated for the first time that LILRA3 is a potential new anti-inflammatory protein, and optimal glycosylation is required for its functions.

Safety and tolerability of an intratumorally injected DNAzyme, Dz13, in patients with nodular basal-cell carcinoma: a phase 1 first-in-human trial (DISCOVER).

BACKGROUND: The nuclear transcription factor c-Jun is preferentially expressed in basal-cell carcinoma. Dz13 is a deoxyribozyme that targets JUN messenger RNA and has inhibited the growth of a range of tumours in mice. We did a phase 1 study to assess safety and tolerability in human beings. METHODS: Adults with nodular basal-cell carcinoma were recruited from Royal Prince Alfred Hospital, Sydney, Australia, between September, 2010, and October, 2011. Patients were assigned to receive one intratumoral injected dose of 10, 30, or 100 µg Dz13, in a 50 µL volume of lipid carrier, and were assessed for adverse effects in the first 24 h then at 7, 14, and 28 days after injection. Treated tumours were surgically excised 14 days after injection and compared with the baseline biopsy samples for expression of c-Jun and tumorigenesis markers. FINDINGS: Nine patients were recruited, of whom three received each dose of Dz13. All patients completed the study with no drug-related serious adverse events. No systemic Dz13 exposure was detected. c-Jun expression was reduced in the excised tumours of all nine (100%) patients, compared with baseline, and histological tumour depth had decreased in five (56%) of nine. Proportions of cells positive for caspases 3, 8, and 9 and P53 were increased, but those of cells positive for Bcl-2 and MMP-9 were decreased. Infiltration by inflammatory and immune cells was stimulated. INTERPRETATION: Dz13 was safe and well tolerated after single intratumoral injections at all doses. FUNDING: Cancer Institute NSW, Cancer Council Australia, and National Health and Medical Research Council.

Plasma protein profiling of Mild Cognitive Impairment and Alzheimer's disease using iTRAQ quantitative proteomics.

BACKGROUND: With the promise of disease modifying treatments, there is a need for more specific diagnosis and prognosis of Alzheimer's disease (AD) and mild cognitive impairment (MCI). Plasma biomarkers are likely to be utilised to increase diagnostic accuracy and specificity of AD and cognitive decline. METHODS: Isobaric tags (iTRAQ) and proteomic methods were used to identify potential plasma biomarkers of MCI and AD. Relative protein expression level changes were quantified in plasma of 411 cognitively normal subjects, 19 AD patients and 261 MCI patients. Plasma was pooled into 4 groups including normal control, AD, amnestic single and multiple domain MCI (aMCI), and nonamnestic single and multiple domain MCI (nMCI). Western-blotting was used to validate iTRAQ data. Integrated function and protein interactions were explored using WEB based bioinformatics tools (DAVID v6.7 and STRING v9.0). RESULTS: In at least two iTRAQ replicate experiments, 30 proteins were significantly dysregulated in MCI and AD plasma, relative to controls. These proteins included ApoA1, ApoB100, complement C3, C4b-binding protein, afamin, vitamin D-binding protein precursor, isoform 1 of Gelsolin actin regulator, Ig mµ chain C region (IGHM), histidine-rich glycoprotein and fibrinogen ß and γ chains. Western-blotting confirmed that afamin was decreased and IGHM was increased in MCI and AD groups. Bioinformatics results indicated that these dysregulated proteins represented a diversity of biological processes, including acute inflammatory response, cholesterol transport and blood coagulation. CONCLUSION: These findings demonstrate that expression level changes in multiple proteins are observed in MCI and AD plasma. Some of these, such as afamin and IGHM, may be candidate biomarkers for AD and the predementia condition of MCI.

Virophage control of antarctic algal host-virus dynamics.

Viruses are abundant ubiquitous members of microbial communities and in the marine environment affect population structure and nutrient cycling by infecting and lysing primary producers. Antarctic lakes are microbially dominated ecosystems supporting truncated food webs in which viruses exert a major influence on the microbial loop. Here we report the discovery of a virophage (relative of the recently described Sputnik virophage) that preys on phycodnaviruses that infect prasinophytes (phototrophic algae). By performing metaproteogenomic analysis on samples from Organic Lake, a hypersaline meromictic lake in Antarctica, complete virophage and near-complete phycodnavirus genomes were obtained. By introducing the virophage as an additional predator of a predator-prey dynamic model we determined that the virophage stimulates secondary production through the microbial loop by reducing overall mortality of the host and increasing the frequency of blooms during polar summer light periods. Virophages remained abundant in the lake 2 y later and were represented by populations with a high level of major capsid protein sequence variation (25-100% identity). Virophage signatures were also found in neighboring Ace Lake (in abundance) and in two tropical lakes (hypersaline and fresh), an estuary, and an ocean upwelling site. These findings indicate that virophages regulate host-virus interactions, influence overall carbon flux in Organic Lake, and play previously unrecognized roles in diverse aquatic ecosystems.

Identification of the RSX interactome in a marsupial shows functional coherence with the Xist interactome during X inactivation.

The marsupial specific RSX lncRNA is the functional analogue of the eutherian specific XIST, which coordinates X chromosome inactivation. We characterized the RSX interactome in a marsupial representative (the opossum Monodelphis domestica), identifying 135 proteins, of which 54 had orthologues in the XIST interactome. Both interactomes were enriched for biological pathways related to RNA processing, regulation of translation, and epigenetic transcriptional silencing. This represents a remarkable example showcasing the functional coherence of independently evolved lncRNAs in distantly related mammalian lineages.

TOPORS E3 ligase mediates resistance to hypomethylating agent cytotoxicity in acute myeloid leukemia cells.

Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are commonplace. To address this, we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identify TOPORS as a loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable, we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML.

Hsp90 Inhibitor SNX-7081 dysregulates proteins involved with DNA repair and replication and the cell cycle in human chronic lymphocytic leukemia (CLL) cells.

The proteomic effects of the Hsp90 inhibitor, SNX-7081, have been determined on the p53-mutated B-cell chronic lymphocytic leukemia (CLL) cell line, MEC1. Following SNX-7081 treatment (500 nM, 24 h), 51 proteins changed abundance by more than 2-fold (p < 0.05); 7 proteins increased while 44 proteins decreased. Proteins identified as differentially abundant by LC-MS/MS were validated by Western blotting (DDB1, PCNA, MCM2, Hsp90, Hsp70, GRP78, PDIA6, HLA-DR). RT-PCR showed that SNX-7081 unexpectedly modulates a number of these proteins in MEC1 cells at the mRNA level (PCNA, MCM2, Nup155, Hsp70, GRP78, PDIA6, and HLA-DR). Pathway analysis determined that 3 of the differentially abundant proteins (cyclin D1, c-Myc and pRb) were functionally related. p53 levels did not change upon SNX-7081 treatment of p53 wild-type Raji cells or p53-mutated MEC1 and U266 cells, indicating that SNX-7081 has a p53-independent mechanism. The decreases in DDB1, MCM2, c-Myc, and PCNA and increases of pRb and cyclin D1 were confirmed in MEC1, U266, Raji, and p53 null HL60 cells by Western blotting. These data suggest that SNX-7081 arrests the cell cycle and inhibits DNA replication and r epair and provides evidence for the mechanism of the observed synergy between Hsp90 inhibitors and drugs that induce DNA strand breaks.