Circular RNAs drive oncogenic chromosomal translocations within the MLL recombinome in leukemia.

The first step of oncogenesis is the acquisition of a repertoire of genetic mutations to initiate and sustain the malignancy. An important example of this initiation phase in acute leukemias is the formation of a potent oncogene by chromosomal translocations between the mixed lineage leukemia (MLL) gene and one of 100 translocation partners, known as the MLL recombinome. Here, we show that circular RNAs (circRNAs)-a family of covalently closed, alternatively spliced RNA molecules-are enriched within the MLL recombinome and can bind DNA, forming circRNA:DNA hybrids (circR loops) at their cognate loci. These circR loops promote transcriptional pausing, proteasome inhibition, chromatin re-organization, and DNA breakage. Importantly, overexpressing circRNAs in mouse leukemia xenograft models results in co-localization of genomic loci, de novo generation of clinically relevant chromosomal translocations mimicking the MLL recombinome, and hastening of disease onset. Our findings provide fundamental insight into the acquisition of chromosomal translocations by endogenous RNA carcinogens in leukemia.

The Nasal Innate Immune Proteome After Saline Irrigation: A Pilot Study in Healthy Individuals.

BACKGROUND: Previous research has shown diminished nasal immune function following nasal saline irrigation (NSI), returning to baseline at 6 hours. The aim of this study was to examine the immune nasal proteome before and after 14 days of nasal irrigation. METHODS: Seventeen healthy volunteers received either isotonic (IsoSal) or low salt (LowNa) NSI. Nasal secretions were collected before and 30 min after NSI at baseline and again after 14 days. Specimens were analyzed using mass spectrometry to detect proteins of relevance to nasal immune function. RESULTS: One thousand eight hundred and sixty-five proteins were identified with significant changes in 71 proteins, of which 23 were identified as part of the innate immune system. Baseline analysis demonstrated an increase of 9 innate proteins after NSI, most after IsoSal. After 14 days, a greater increase in innate peptides was present, with most now in the LowNa group. When NSI solutions were compared, a significant increase in 4 innate proteins, including a 211% in lysozyme, was detected in the LowNa group. CONCLUSION: LowNa NSI demonstrates evidence of improving the innate immune secretions, especially lysozyme, in healthy volunteers.

Purification and Characterization of a Novel Alginate Lyase from a Marine Streptomyces Species Isolated from Seaweed.

Alginate, a natural polysaccharide derived from brown seaweed, is finding multiple applications in biomedicine via its transformation through chemical, physical, and, increasingly, enzymatic processes. In this study a novel alginate lyase, AlyDS44, was purified and characterized from a marine actinobacterium, Streptomyces luridiscabiei, which was isolated from decomposing seaweed. The purified enzyme had a specific activity of 108.6 U/mg, with a molecular weight of 28.6 kDa, and was composed of 260 amino acid residues. AlyDS44 is a bifunctional alginate lyase, active on both polyguluronate and polymannuronate, though it preferentially degrades polyguluronate. The optimal pH of this enzyme is 8.5 and the optimal temperature is 45 °C. It is a salt-tolerant alginate lyase with an optimal activity at 0.6 M NaCl. Metal ions Mn2+, Co2+, and Fe2+ increased the alginate degrading activity, but it was inhibited in the presence of Zn2+ and Cu2+. The highly conserved regions of its amino acid sequences indicated that AlyDS44 belongs to the polysaccharide lyase family 7. The main breakdown products of the enzyme on alginate were disaccharides, trisaccharides, and tetrasaccharides, which demonstrated that this enzyme acted as an endo-type alginate lyase. AlyDS44 is a novel enzyme, with the potential for efficient production of alginate oligosaccharides with low degrees of polymerization.

Proteomic analysis of nasal mucus samples of healthy patients and patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) has a complex and multifactorial pathogenesis with a heterogeneous inflammatory profile. Proteomic analysis of nasal mucus may enable further understanding of protein abundances and biologic processes present in CRS and its endotypes compared with in healthy patients. OBJECTIVE: Our aim was to determine differences in the nasal mucus proteome of healthy patients and patients with CRS. METHODS: Nasal mucus was obtained from healthy patients, patients with CRS without nasal polyps (CRSsNP), and patients with CRS with nasal polyps (CRSwNP) before surgery. Gel electrophoresis was performed to fractionate the complex protein extracts before mass spectrometry analysis. Gene set enrichment analysis was performed on differentially expressed proteins. RESULTS: A total of 33 patients were included in this study (12 healthy, 10 with CRSsNP, and 11 with CRSwNP). In all, 1142 proteins were identified in mucus samples from healthy patients, 761 in mucus samples from patients with CRSsNP, and 998 in mucus samples from patients with CRSwNP. Dysfunction in immunologic pathways, reduced cellular signaling, and increased cellular metabolism with associated tissue remodeling pathways were present in patients with CRS compared with in healthy patients. CONCLUSION: Significant downregulation of mucosal immunity and antioxidant pathways with increased tissue modeling processes may account for the clinical manifestations of CRS. Ultimately, the differing proteome and biologic processes provide further insight into CRS pathogenesis and its endotypes.

Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Adverse events associated with peanut oral immunotherapy in children - a systematic review and meta-analysis.

While peanut oral immunotherapy (POIT) represents a promising treatment for peanut allergies in children, safety concerns remain a common barrier to widespread adoption. We aimed to systematically assess available evidence to determine the risk and frequency of adverse events occurring during POIT, and examine study-level characteristics associated with their occurrence and severity. A systematic search of MEDLINE, EMBASE, and Web of Science was conducted through April 2019. Controlled and non-controlled studies evaluating POIT were eligible. Twenty-seven studies, involving 1488 subjects, were included. Adverse events to POIT were common and led to treatment discontinuation in 6.6% of children (95% CI 4.4-9.0; 27 studies, I2 = 48.7%). Adverse events requiring treatment with epinephrine occurred among 7.6% (4.5-11.4; 26 studies, I2 = 75.5%) of participants, at a rate of 2.0 per 10,000 doses (0.8-3.7; 15 studies, I2 = 64.4). Use of a rush treatment phase and targeting a higher maintenance dose were associated with a higher risk and frequency of epinephrine use, while using co-treatments in addition to POIT was associated with a lower risk of treatment discontinuation due to adverse events. While adverse events to POIT are common, this study provides promising explorative evidence that certain modifications to existing treatment protocols could significantly improve treatment outcomes.

Proteomic profiling of precipitated Clostridioides difficile toxin A and B antibodies.

Clostridioides difficile infection is the leading cause of nosocomial diarrhoea globally. Immune responses to toxins produced by C. difficile are important in disease progression and outcome. Here, we analysed the anti-toxin A and anti-toxin B serum antibody proteomes following natural infection or vaccination with a C. difficile toxoid A/toxoid B vaccine using a modified miniaturised proteomic approach based on de novo mass spectrometric sequencing. Analysis of immunoglobulin variable region (IgV) subfamily expression in immunoprecipitated toxin A and toxin B antibodies from four and seven participants of a vaccine trial, respectively, revealed a polyclonal proteome with restricted IGHV, IGKV and IGLV subfamily usage. No dominant IGHV subfamily was observed in the toxin A response, however the dominant anti-toxin B heavy (H)-chain was encoded by IGHV3-23. Light (L)-chain usage was convergent for both anti-toxin A and anti-toxin B proteomes with IGKV3-11, 3-15, 3-20 and 4-1 shared among all subjects in both cohorts. Peptide mapping of common IgV families showed extensive public and private amino acid substitutions. The cohort responses to toxin A and toxin B showed limited similarity in shared IGHV subfamilies. L-chain subfamily usage was more similar in the anti-toxin A and anti-toxin B responses, however the mutational signatures for each subfamily were toxin-dependent. Samples taken both post vaccination (n = 5) or at baseline, indicating previous exposure (n = 2), showed similar anti-toxin B IgV subfamily usage and mutational profiles. In summary, this study provides the first sequence-based proteomic analysis of the antibody response to the major disease-mediating toxins of C. difficile, toxin A and toxin B, and demonstrates that despite the potential for extreme diversity, the immunoglobulin repertoire can raise convergent responses to specific pathogens whether through natural infection or following vaccination.

Early treatment with minocycline following stroke in rats improves functional recovery and differentially modifies responses of peri-infarct microglia and astrocytes.

BACKGROUND: Altered neuronal connectivity in peri-infarct tissue is an important contributor to both the spontaneous recovery of neurological function that commonly develops after stroke and improvements in recovery that have been induced by experimental treatments in animal models. Microglia and astrocytes are primary determinants of the environment in peri-infarct tissue and hence strongly influence the potential for neuronal plasticity. However, the specific roles of these cells and the timing of critical changes in their function are not well understood. Minocycline can protect against ischemic damage and promote recovery. These effects are usually attributed, at least partially, to the ability of this drug to suppress microglial activation. This study tested the ability of minocycline treatment early after stroke to modify reactive responses in microglia and astrocytes and improve recovery. METHODS: Stroke was induced by photothrombosis in the forelimb sensorimotor cortex of Sprague-Dawley rats. Minocycline was administered for 2 days after stroke induction and the effects on forelimb function assessed up to 28 days. The responses of peri-infarct Iba1-positive cells and astrocytes were evaluated using immunohistochemistry and Western blots. RESULTS: Initial characterization showed that the numbers of Iba1-positive microglia and macrophages decreased in peri-infarct tissue at 24 h then increased markedly over the next few days. Morphological changes characteristic of activation were readily apparent by 3 h and increased by 24 h. Minocycline treatment improved the rate of recovery of motor function as measured by a forelimb placing test but did not alter infarct volume. At 3 days, there were only minor effects on core features of peri-infarct microglial reactivity including the morphological changes and increased density of Iba1-positive cells. The treatment caused a decrease of 57% in the small subpopulation of cells that expressed CD68, a marker of phagocytosis. At 7 days, the expression of glial fibrillary acidic protein and vimentin was markedly increased by minocycline treatment, indicating enhanced reactive astrogliosis. CONCLUSIONS: Early post-stroke treatment with minocycline improved recovery but had little effect on key features of microglial activation. Both the decrease in CD68-positive cells and the increased activation of astrogliosis could influence neuronal plasticity and contribute to the improved recovery.

A comparison of LKB1/AMPK/mTOR metabolic axis response to global ischaemia in brain, heart, liver and kidney in a rat model of cardiac arrest.

BACKGROUND: Cellular energy failure in high metabolic rate organs is one of the underlying causes for many disorders such as neurodegenerative diseases, cardiomyopathies, liver and renal failures. In the past decade, numerous studies have discovered the cellular axis of LKB1/AMPK/mTOR as an essential modulator of cell homeostasis in response to energy stress. Through regulating adaptive mechanisms, this axis adjusts the energy availability to its demand by a systematized control on metabolism. Energy stress, however, could be sensed at different levels in various tissues, leading to applying different strategies in response to hypoxic insults. METHODS: Here the immediate strategies of high metabolic rate organs to time-dependent short episodes of ischaemia were studied by using a rat model (n = 6/group) of cardiac arrest (CA) (15 and 30 s, 1, 2, 4 and 8 min CA). Using western blot analysis, we examined the responses of LKB1/AMPK/mTOR pathway in brain, heart, liver and kidney from 15 s up to 8 min of global ischaemia. The ratio of ADP/ATP was assessed in all ischemic and control groups, using ApoSENSOR bioluminescent assay kit. RESULTS: Brain, followed by kidney showed the early dephosphorylation response in AMPK (Thr172) and LKB1 (Ser431); in the absence of ATP decline (ADP/ATP elevation). Dephosphorylation of AMPK was followed by rephosphorylation and hyperphosphorylation, which was associated with a significant ATP decline. While heart's activity of AMPK and LKB1 remained at the same level during short episodes of ischaemia, liver's LKB1 was dephosphorylated after 2 min. AMPK response to ischaemia in liver was mainly based on an early alternative and a late constant hyperphosphorylation. No significant changes was observed in mTOR activity in all groups. CONCLUSION: Together our results suggest that early AMPK dephosphorylation followed by late hyperphosphorylation is the strategy of brain and kidney in response to ischaemia. While the liver seemed to get benefit of its AMPK system in early ischameia, possibly to stabilize ATP, the level of LKB1/AMPK activity in heart remained unchanged in short ischaemic episodes up to 8 min. Further researches must be conducted to elucidate the molecular mechanism underlying LKB1/AMPK response to oxygen supply.

Proteomic analysis of influenza haemagglutinin-specific antibodies following vaccination reveals convergent immunoglobulin variable region signatures.

Analysis of the anti-haemagglutinin serum antibody proteome from six H1N1pdm09 influenza A vaccinated subjects demonstrated restricted IgG1 heavy chain species encoded by IGHV5-51 and IGHV3-7 gene families in 2 subjects and either IGHV5-51 or IGHV3-7 in 4 individuals. All subjects exhibited a dominant IGKV3-20 light chain, however 5 subjects also exhibited IGKV3-11 and IGKV4-1 families. Sequences were closely aligned with the matched germline sequence, with few shared mutations. This study illustrates the feasibility of using a proteomic approach to determine the expressed V region signatures of serum antibodies induced by vaccination.

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins.

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1-/- cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions.

Proteomic analysis reveals downregulation of housekeeping proteins in the diabetic vascular proteome.

AIMS: Type 2 diabetes (T2D) increases the risk of death associated with cardiovascular complications. However, a complete understanding of protein changes within the diabetic vasculature is still lacking. METHODS: Herein, we utilized mass spectrometry to perform vascular and urinary proteome analysis using a rat model of high-fat feeding and low-dose streptozotocin to simulate late-stage T2D. The purpose of this study was to identify aortic and urine proteins that are differentially expressed in normal and T2D rats. RESULTS: High-fat feeding and low-dose streptozotocin resulted in hyperglycemia, hypoinsulinemia and high levels of circulating free fatty acids. Using a shotgun proteomic approach, high-mobility-group protein B1 and spondin-1 were significantly increased in T2D aorta compared to control aorta, suggesting vascular inflammation and smooth muscle proliferation, respectively. However, the majority of differentially expressed aortic proteins were downregulated in T2D, including proteins associated with coagulation, cell differentiation and redox homeostasis. Strikingly, we report a significant downregulation of commonly used cytoskeletal housekeeping proteins in T2D aorta. Urine from T2D rats displayed increased expression of proteins involved in inflammation and oxidative stress and decreased expression of proteins associated with lipid metabolism and cell adhesion. A number of differentially expressed proteins in urine of T2D rats have previously been reported in human T2D, thereby supporting this animal model as a good representation of human T2D. CONCLUSIONS: Our data offer new information regarding key pathways that could be therapeutically targeted to combat the cardiovascular complications of T2D.

Huntingtin-associated protein-1 is a synapsin I-binding protein regulating synaptic vesicle exocytosis and synapsin I trafficking.

Huntingtin-associated protein-1 (HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle (SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1(-/-) cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1(-/-) brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. The Proposed mechanism of synapsin I transport mediated by HAP1 in neurons. HAP1 interacts with synapsin I, regulating the trafficking of synapsin I containing vesicles and/or transport packets, possibly through its engagement of microtubule motors. The absence of HAP1 reduces synapsin I transport and neuronal exocytosis. These findings provide insights into the processes of neuronal trafficking and synaptic signaling.

Proteomic analysis of hemolymph from poly(I:C)-stimulated Crassostrea gigas.

Synthetic double stranded RNA (Poly(I:C)) injection of Crassostrea gigas results in a systemic antiviral response involving many evolutionary conserved antiviral effectors (ISGs). Compared to mammals, the timing of C. gigas ISG expression to viral or poly(I:C) injection is delayed (>12 h p.i.). It could be interpreted that a cytokine is responsible for the systemic, but delayed expression of C. gigas ISGs. We therefore analysed the acellular fraction of C. gigas hemolymph by two-dimensional electrophoresis (2-DE) to identify hemolymph proteins induced by poly(I:C). Poly(I:C) injection increased the relative intensity of four protein spots. These protein spots were identified by tandem mass spectrometry (LC-MS/MS) as a small heat shock protein (sHSP), poly(I:C)-inducible protein 1 (PIP1) and two isoforms of C1q-domain containing protein (C1qDC). RT-qPCR analysis confirmed that the genes encoding these proteins are induced in hemocytes of C. gigas injected with poly(I:C) (p < 0.05). Proteomic data from this experiment corroborates previous microarray and whole transcriptome studies that have reported up-regulation of C1qDC and sHSP during mass mortality events among farmed oysters.

Identification of a novel oligomerization disrupting mutation in CRYΑA associated with congenital cataract in a South Australian family.

Congenital cataract is a heterogeneous disorder causing severe visual impairment in affected children. We screened four South Australian families with autosomal dominant congenital cataract for mutations in 10 crystallin genes known to cause congenital cataract. We identified a novel segregating heterozygous mutation, c.62G>A (p.R21Q), in the CRYΑA gene in one family. Western blotting of proteins freshly extracted from cataractous lens material of the proband demonstrated a marked reduction in the amount of the high-molecular-weight oligomers seen in the lens material of an unaffected individual. We conclude that the p.R21Q mutation, which is located in the highly conserved and structurally significant N-terminal region of the protein, is responsible for the cataract phenotype observed in the family as this mutation likely reduces the formation of the functional oligomeric alpha-crystallin.

Preliminary characterisation of two early meiotic wheat proteins after identification through 2D gel electrophoresis proteomics.

Various genetic-based approaches including mutant population screens, microarray analyses, cloning and transgenesis have broadened our knowledge of gene function during meiosis in plants. Nonetheless, these genetic tools are not without inherent limitations. One alternative approach to studying plant meiosis, especially in polyploids such as Triticum aestivum L. (bread wheat), is proteomics. However, protein-based approaches using proteomics have seldom been described, with only two attempts at studying early plant meiosis reported. Here, we report the investigation of early bread wheat meiosis using proteomics. Five differentially expressed protein spots were identified using 2D gel electrophoresis (2DGE) on protein extracts from four pooled stages of meiosis and three genotypes (Chinese Spring wild-type, ph1b and ph2a wheat mutant lines). Tandem mass spectrometry (MS/MS) identification of peptides from these protein spots led to the isolation and characterisation of the full-length clones of a wheat Speckle-type POZ protein, an SF21-like protein and HSP70, and a partial coding sequence of a hexose transporter. Significantly, the putative functions of the Speckle-type POZ protein and HSP70 were confirmed using in vitro DNA binding assays. Through the use of a 2DGE proteomics approach, we show that proteomics is a viable alternative to genetic-based approaches when studying meiosis in wheat. More significantly, we report a potential role for a Speckle-type POZ protein and a HSP70 in chromosome pairing during the early stages of meiosis in bread wheat.

Comparison of the specific incorporation of intracrystalline proteins into urinary calcium oxalate monohydrate and dihydrate crystals.

The aim of this study was to compare the comprehensive intracrystalline protein profiles of calcium oxalate monohydrate (COM) and dihydrate (COD) crystals precipitated from the same human urine samples. Three separate batches of COM and COD crystals were precipitated from pooled healthy human urine by the addition of sodium oxalate at calcium concentrations of 2 and 8 mM, respectively. Proteins in whole extracts of demineralised COM and COD crystals, as well as in spots excised from 2D-PAGE gels of the extracts, were identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). The number and type of individual proteins differed between COM and COD: 14 substantive proteins were found inside COM crystal extracts and 34 inside COD, with 9 proteins occurring in both crystal types. Numerous keratins were detected. However, in line with consensus in the proteomics literature, as well as a lack of published evidence linking them to urolithiasis, they were excluded as contaminants, leaving very few consistently detected proteins. On the basis of their known association with stone disease or identification in multiple runs, the principal proteins in COM crystal extracts were prothrombin fragment 1, protein S100A9, and IGkappaV1-5, while those in extracts of COD crystals included osteopontin, IGkappaV1-5, protein S100A9, annexin A1, HMW kininogen-1, and inter-alpha-inhibitor (IalphaI). In general, proteins incorporated into both hydromorphs were acidic (pI<6), smaller than 55 kDa, and calcium binders. We concluded that the incorporation of proteins into urinary COM and COD crystals is selective and that only a few of the urinary proteins associated with the two hydromorphs are likely to play any significant role in stone pathogenesis.

Dominantly-inherited adult-onset leukodystrophy with palatal tremor caused by a mutation in the glial fibrillary acidic protein gene.

We report on a pedigree of dominantly-inherited, adult-onset Alexander disease caused by the glial fibrillary acidic protein (GFAP) gene mutation, R416W. This pedigree highlights the importance of genetic analysis of the GFAP gene in leukodystrophy with palatal tremor.