Identification of Daboia siamensis venome using integrated multi-omics data.

Snakebite, classified by World Health Organization as a neglected tropical disease, causes more than 100,000 deaths and 2 million injuries per year. Currently, available antivenoms do not bind with strong specificity to target toxins, which means that severe complications can still occur despite treatment. Moreover, the cost of antivenom is expensive. Knowledge of venom compositions is fundamental for producing a specific antivenom that has high effectiveness, low side effects, and ease of manufacture. With advances in mass spectrometry techniques, venom proteomes can now be analyzed in great depth at high efficiency. However, these techniques require genomic and transcriptomic data for interpreting mass spectrometry data. This study aims to establish and incorporate genomics, transcriptomics, and proteomics data to study venomics of a venomous snake, Daboia siamensis. Multiple proteins that have not been reported as venom components of this snake such as hyaluronidase-1, phospholipase B, and waprin were discovered. Thus, multi-omics data are advantageous for venomics studies. These findings will be valuable not only for antivenom production but also for the development of novel therapeutics.

Specific Interaction of DDX6 with an RNA Hairpin in the 3' UTR of the Dengue Virus Genome Mediates G1 Phase Arrest.

The extent to which viral genomic RNAs interact with host factors and contribute to host response and disease pathogenesis is not well known. Here, we report that the human RNA helicase DDX6 specifically binds to the viral most conserved RNA hairpin in the A3 element in the dengue 3' UTR, with nanomolar affinities. DDX6 CLIP confirmed the interaction in HuH-7 cells infected by dengue virus serotype 2. This interaction requires three conserved residues-Lys307, Lys367, and Arg369-as well as the unstructured extension in the C-terminal domain of DDX6. Interestingly, alanine substitution of these three basic residues resulted in RNA-independent ATPase activity, suggesting a mechanism by which RNA-binding and ATPase activities are coupled in DEAD box helicases. Furthermore, we applied a cross-omics gene enrichment approach to suggest that DDX6 is functionally related to cell cycle regulation and viral pathogenicity. Indeed, infected cells exhibited cell cycle arrest in G1 phase and a decrease in the early S phase. Exogenous expression of intact DDX6, but not A3-binding-deficient mutants, alleviated these effects by rescue of the DNA preinitiation complex expression. Disruption of the DDX6-binding site was found in dengue and Zika live-attenuated vaccine strains. Our results suggested that dengue virus has evolved an RNA aptamer against DDX6 to alter host cell states and defined DDX6 as a new regulator of G1/S transition. IMPORTANCE Dengue virus (DENV) is transmitted by mosquitoes to humans, infecting 390 million individuals per year globally. About 20% of infected patients shows a spectrum of clinical manifestation, ranging from a mild flu-like syndrome, to dengue fever, to life-threatening severe dengue diseases, including dengue hemorrhagic fever and dengue shock syndrome. There is currently no specific treatment for dengue diseases, and the molecular mechanism underlying dengue pathogenesis remains poorly understood. In this study, we combined biochemical, bioinformatics, high-content analysis and RNA sequencing approaches to characterize a highly conserved interface of the RNA genome of DENV with a human factor named DDX6 in infected cells. The significance of our research is in identifying the mechanism for a viral strategy to alter host cell fates, which conceivably allows us to generate a model for live-attenuated vaccine and the design of new therapeutic reagent for dengue diseases.

Deep Proteomic Deconvolution of Interferons and HBV Transfection Effects on a Hepatoblastoma Cell Line.

Interferons are commonly utilized in the treatment of chronic hepatitis B virus (HBV) infection but are not effective for all patients. A deep understanding of the limitations of interferon treatment requires delineation of its activity at multiple "omic" levels. While myriad studies have characterized the transcriptomic effects of interferon treatment, surprisingly, few have examined interferon-induced effects at the proteomic level. To remedy this paucity, we stimulated HepG2 cells with both IFN-α and IFN-λ and performed proteomic analysis versus unstimulated cells. Alongside, we examined the effects of HBV transfection in the same cell line, reasoning that parallel IFN and HBV analysis might allow determination of cases where HBV transfection counters the effects of interferons. More than 6000 proteins were identified, with multiple replicates allowing for differential expression analysis at high confidence. Drawing on a compendium of transcriptomic data, as well as proteomic half-life data, we suggest means by which transcriptomic results diverge from our proteomic results. We also invoke a recent multiomic study of HBV-related hepatocarcinoma (HCC), showing that despite HBV's role in initiating HCC, the regulated proteomic landscapes of HBV transfection and HCC do not strongly align. Special focus is applied to the proteasome, with numerous components divergently altered under IFN and HBV-transfection conditions. We also examine alterations of other protein groups relevant to HLA complex peptide display, unveiling intriguing alterations in a number of ubiquitin ligases. Finally, we invoke genome-scale metabolic modeling to predict relevant alterations to the metabolic landscape under experimental conditions. Our data should be useful as a resource for interferon and HBV researchers.

Recent developments in neoantigen-based cancer vaccines.

Cynics point out that a cure for cancer has been "around the corner" for the last 50 years. Nevertheless, the recent convergence of deep DNA, RNA, and proteomic technologies with enhanced understanding of the nuances of the adaptive immune system has generated great optimism amongst researchers. The extraordinary heterogeneity of various cancers, once thought to be a major therapeutic hurdle, may now be bypassed via "personalized" vaccine treatments. Specifically, these treatments involve the identification of MHC-bound peptides that are unique to a patient's cancer (neoantigens), followed by immunization with peptides, RNA, or DNA that encodes these neoantigens via various delivery systems, thus amplifying the immune system's response to the particular cancer. Such approaches have shown dramatic results in animal studies. Not surprisingly, then, the field of neoantigen-based immunotherapy has advanced at a spectacular rate, necessitating that interested individuals stay apprised of recent developments. Following an introduction to the subject, we thus focus on aspects that are particularly fast-moving; the cellular sources of neoantigens, which are surprisingly diverse, the tools that are used for their identification, and the status of the numerous clinical trials that are now being conducted.

Bilateral ureteral obstruction is rapidly accompanied by ER stress and activation of autophagic degradation of IMCD proteins, including AQP2.

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including "cell-cell adhesion," "cell-cell adherens junction," "mitochondrial inner membrane," "endoplasmic reticulum chaperone complex," and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

PTM-Logo: a program for generation of sequence logos based on position-specific background amino-acid probabilities.

SUMMARY: Identification of the amino-acid motifs in proteins that are targeted for post-translational modifications (PTMs) is of great importance in understanding regulatory networks. Information about targeted motifs can be derived from mass spectrometry data that identify peptides containing specific PTMs such as phosphorylation, ubiquitylation and acetylation. Comparison of input data against a standardized 'background' set allows identification of over- and under-represented amino acids surrounding the modified site. Conventionally, calculation of targeted motifs assumes a random background distribution of amino acids surrounding the modified position. However, we show that probabilities of amino acids depend on (i) the type of the modification and (ii) their positions relative to the modified site. Thus, software that identifies such over- and under-represented amino acids should make appropriate adjustments for these effects. Here we present a new program, PTM-Logo, that generates representations of these amino acid preferences ('logos') based on position-specific amino-acid probability backgrounds calculated either from user-input data or curated databases. AVAILABILITY AND IMPLEMENTATION: PTM-Logo is freely available online at http://sysbio.chula.ac.th/PTMLogo/ or https://hpcwebapps.cit.nih.gov/PTMLogo/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation.

For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision-making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA-centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro-derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self-regulation of viral replication is revealed.

Proteomic identification of predictive biomarkers for malignant transformation in complete hydatidiform moles.

INTRODUCTION: Protein expression in cells are associated with oncogenesis. This study aims to explore proteomic profiles and discover potential biomarkers that can predict malignant transformation of hydatidiform mole. METHODS: Retrospective analysis was done in 14 cases of remission hydatidiform mole and 14 cases of hydatidiform mole who later developed malignancy (GTN group). Molar tissues were retrieved from -70 °C frozen tissue. Subsequently, a large-scale proteomic analysis was performed to identify proteins and compare their abundance levels in the preserved molar tissues from these two groups using a dimethyl-labeling technique coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 2,153 proteins were identified from all samples. 22 and 10 proteins were significantly up-regulated and down-regulated, respectively, in the GTN group compared with the mole group. These altered proteins were found in several biological groups such as cell-cell adhesion, secreted proteins, and ribonucleoproteins. Several hormone-related proteins were among the most up-regulated proteins in the GTN group including choriogonadotropin subunit beta (ß-hCG) and alpha (α-hCG), growth/differentiation factor 15, as well as both pregnancy-specific beta-1-glycoproteins 2 and 3. In contrast, protein S100-A11 and l-lactate dehydrogenase A chain, were down-regulated in molar tissue from most patients in the GTN group. DISCUSSION: This study identified a set of differentially expressed proteins in molar tissues that could potentially be further examined as predictive biomarkers for the malignant transformation of CHMs. A molar proteome database was constructed and can be accessible online at http://sysbio.chula.ac.th/Database/GTD_DB/Supplementary_Data.xlsx.

Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells.

Interferon lambda (IFN-λ) is a relatively unexplored, yet promising antiviral agent. IFN-λ has recently been tested in clinical trials of chronic hepatitis B virus infection (CHB), with the advantage that side effects may be limited compared with IFN-α, as IFN-λ receptors are found only in epithelial cells. To date, IFN-λ's downstream signaling pathway remains largely unelucidated, particularly via proteomics methods. Here, we report that IFN-λ3 inhibits HBV replication in HepG2.2.15 cells, reducing levels of both HBV transcripts and intracellular HBV DNA. Quantitative proteomic analysis of HBV-transfected cells was performed following 24-hour IFN-λ3 treatment, with parallel IFN-α2a and PBS treatments for comparison using a dimethyl labeling method. The depth of the study allowed us to map the induction of antiviral proteins to multiple points of the viral life cycle, as well as facilitating the identification of antiviral proteins not previously known to be elicited upon HBV infection (e.g. IFITM3, XRN2, and NT5C3A). This study also shows up-regulation of many effectors involved in antigen processing/presentation indicating that this cytokine exerted immunomodulatory effects through several essential molecules for these processes. Interestingly, the 2 subunits of the immunoproteasome cap (PSME1 and PSME2) were up-regulated whereas cap components of the constitutive proteasome were down-regulated upon both IFN treatments, suggesting coordinated modulation toward the antigen processing/presentation mode. Furthermore, in addition to confirming canonical activation of interferon-stimulated gene (ISG) transcription through the JAK-STAT pathway, we reveal that IFN-λ3 restored levels of RIG-I and RIG-G, proteins known to be suppressed by HBV. Enrichment analysis demonstrated that several biological processes including RNA metabolism, translation, and ER-targeting were differentially regulated upon treatment with IFN-λ3 versus IFN-α2a. Our proteomic data suggests that IFN-λ3 regulates an array of cellular processes to control HBV replication.

The model of circulating immune complexes and interleukin-6 improves the prediction of disease activity in systemic lupus erythematosus.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease resulting in autoantibody production, immune complex deposition, and complement activation. The standard biomarkers such as anti-dsDNA and complements (C3 and C4) do not always correlate with active clinical SLE. The heterogeneity of SLE patients may require additional biomarkers to designate disease activity. Ninety SLE patients participated in this study. Evaluation of disease activity was achieved with the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) and modified SLEDAI-2K. The measured serum biomarkers were anti-dsDNA, C3, C4, ESR, interleukin-6 (IL-6), and circulating immune complexes (CIC). IL-6, ESR and CIC significantly increased in active clinical SLE. Complement, anti-dsDNA, ESR and CIC correlated with SLEDAI-2K while only anti-dsDNA, CIC, ESR and IL-6 correlated with modified SLEDAI-2K. A combination of biomarkers gave a higher odds ratio (OR) than any single biomarker. A combination of IL-6 or CIC exhibited the highest OR (OR = 7.27, 95%CI (1.99-26.63), p = 0.003) while either complement or anti-dsDNA showed a moderate odds ratio (OR = 3.14, 95%CI (1.16-8.48), p = 0.024) of predicting clinical active SLE. The combination of CIC and IL-6 strongly predicts active clinical SLE. CIC and IL-6 can be used in addition to standard biomarkers to determine SLE activity.

AbDesigner3D: a structure-guided tool for peptide-based antibody production.

Summary: We present AbDesigner3D, a new tool for identification of optimal immunizing peptides for antibody production using a peptide-based strategy. AbDesigner3D integrates 3D structural data from the Protein Data Bank (PDB) with UniProt data, which includes basic sequence data, post-translational modification sites, SNP occurrences and more. Other features, such as uniqueness and conservation scores, are calculated based on sequences from UniProt. The 3D visualization capabilities allow an intuitive interface, while an abundance of quantitative output simplifies the process of comparing immunogen peptides. Important quantitative features added in this tool include calculation and display of accessible surface area (ASA) and protein-protein interacting residues (PPIR). The specialized data visualization features of AbDesigner3D will greatly assist users to optimize their choice of immunizing peptides. Availability and implementation: AbDesigner3D is freely available at http://sysbio.chula.ac.th/AbDesigner3D or https://hpcwebapps.cit.nih.gov/AbDesigner3D/. Supplementary information: Supplementary data are available at Bioinformatics online.

Identification of a Conserved RNA-dependent RNA Polymerase (RdRp)-RNA Interface Required for Flaviviral Replication.

Dengue virus, an ∼10.7-kb positive-sense RNA virus, is the most common arthropod-communicated pathogen in the world. Despite dengue's clear epidemiological importance, mechanisms for its replication remain elusive. Here, we probed the entire dengue genome for interactions with viral RNA-dependent RNA polymerase (RdRp), and we identified the dominant interaction as a loop-forming ACAG motif in the 3' positive-stranded terminus, complicating the prevailing model of replication. A subset of interactions coincides with known flaviviral recombination sites inside the viral protein-coding region. Specific recognition of the RNA element occurs via an arginine patch in the C-terminal thumb domain of RdRp. We also show that the highly conserved nature of the consensus RNA motif may relate to its tolerance to various mutations in the interacting region of RdRp. Disruption of the interaction resulted in loss of viral replication ability in cells. This unique RdRp-RNA interface is found throughout flaviviruses, implying possibilities for broad disease interventions.

Development of viable TAP-tagged dengue virus for investigation of host-virus interactions in viral replication.

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The viral replication machinery containing the core non-structural protein 5 (NS5) is implicated in severe dengue symptoms but molecular details remain obscure. To date, studies seeking to catalogue and characterize interaction networks between viral NS5 and host proteins have been limited to the yeast two-hybrid system, computational prediction and co-immunoprecipitation (IP) of ectopically expressed NS5. However, these traditional approaches do not reproduce a natural course of infection in which a number of DENV NS proteins colocalize and tightly associate during the replication process. Here, we demonstrate the development of a recombinant DENV that harbours a TAP tag in NS5 to study host-virus interactions in vivo. We show that our engineered DENV was infective in several human cell lines and that the tags were stable over multiple viral passages, suggesting negligible structural and functional disturbance of NS5. We further provide proof-of-concept for the use of rationally tagged virus by revealing a high confidence NS5 interaction network in human hepatic cells. Our analysis uncovered previously unrecognized hnRNP complexes and several low-abundance fatty acid metabolism genes, which have been implicated in the viral life cycle. This study sets a new standard for investigation of host-flavivirus interactions.

Structural platform for the autolytic activity of an intact NS2B-NS3 protease complex from dengue virus.

Dengue virus completes its protein synthesis inside human cells on the endoplasmic reticulum membrane by processing the single-chain polyprotein precursor into 10 functional proteins. This vital process relies on the two-component virus-encoded protease complex; nonstructural protein 3 (NS3) possesses the proteolytic activity in its N-terminus, and NS2B acts as a fundamental activator and membrane-anchoring subunit. The membrane-associated NS2B-NS3 complex has essentially not yet been isolated or studied. We describe here a useful protocol for the preparation of the full-length NS2B-NS3 complex from dengue serotype 2 virus by utilizing a Mistic-fusion expression cassette in Escherichia coli. The protease complex was successfully solubilized and stabilized from the bacterial membrane and purified with the use of fos-choline-14 detergent. The detergent-solubilized protease complex retained autolytic activity and, intriguingly, exists as a robust trimer, implying a molecular assembly in the membrane. We further conducted a random mutagenesis study to efficiently scan for entire residues and motifs contributing to autocleavage and provide evidence of the importance of the two distal ß-hairpins in the activity of the viral protease. Our results provide the first comprehensive view of an active dengue protease in the membrane-bound form.

Open-label, long-term safety study of cevimeline in the treatment of postirradiation xerostomia.

PURPOSE: To assess the safety of long-term cevimeline treatment of radiation-induced xerostomia in patients with head-and-neck cancer; and to assess the efficacy of cevimeline in these patients. METHODS AND MATERIALS: A total of 255 adults with head-and-neck cancer who had received more than 40 Gy of radiation 4 months or more before entry and had clinically significant salivary gland dysfunction received cevimeline hydrochloride 45 mg t.i.d. orally for 52 weeks. Adverse events (AEs), their severity, and their relationship to the study medication were assessed by each investigator. The efficacy assessment was based on subjects' global evaluation of oral dryness on a scale of 0 (none) to 3 (severe). RESULTS: Overall, 175 subjects (68.6%) experienced expected treatment-related AEs, most mild to moderate. The most frequent was increased sweating (47.5%), followed by dyspepsia (9.4%), nausea (8.2%), and diarrhea (6.3%). Fifteen subjects (5.9%) experienced Grade 3 treatment-related AEs, of which the most frequent was increased sweating. Eighteen subjects (7.1%) reported at least one serious AE, and 45 subjects (17.6%) discontinued study medication because of an AE. The global efficacy evaluation at the last study visit showed that cevimeline improved dry mouth in most subjects (59.2%). Significant improvement was seen at each study visit in the mean change from baseline of the numeric global evaluation score (p < 0.0001). CONCLUSIONS: Cevimeline 45 mg t.i.d. was generally well tolerated over a period of 52 weeks in subjects with xerostomia secondary to radiotherapy for cancer in the head-and-neck region.

Cevimeline for the treatment of postirradiation xerostomia in patients with head and neck cancer.

PURPOSE: To study the efficacy and safety of cevimeline in two double-blind trials (Studies 003 and 004) enrolling patients with head and neck cancer in whom xerostomia developed after radiotherapy. METHODS AND MATERIALS: Subjects were randomly assigned to receive cevimeline, 30 mg three times daily, or placebo for 12 weeks, with the possibility of dose escalation to 45 mg three times daily at 6 weeks. The primary efficacy endpoint was the patient's final global evaluation of oral dryness; change in unstimulated salivary flow was a secondary endpoint. RESULTS: Five hundred seventy subjects (284 in Study 003 and 286 in Study 004) were randomized. Significantly more cevimeline-treated subjects than placebo recipients (47.4% vs. 33.3%, p = 0.0162) in Study 003 reported improvement in dry mouth in the final global evaluation of oral dryness. No significant difference between groups in the final global evaluation was seen in Study 004, in which a high placebo response rate of 47.6% was observed. In both studies, cevimeline-treated subjects had significantly greater increases in the objective measure of unstimulated salivary flow than placebo recipients (p = 0.0093 [Study 003] and p = 0.0215 [Study 004]), whereas no significant differences in stimulated salivary flow were observed. The most frequent adverse event was increased sweating. CONCLUSION: Cevimeline was well tolerated by patients with xerostomia after radiotherapy for head and neck cancer, and oral administration of 30-45 mg of cevimeline three times daily increased unstimulated salivary flow.