False discovery rate: the Achilles' heel of proteogenomics.

Proteogenomics refers to the integrated analysis of the genome and proteome that leverages mass-spectrometry (MS)-based proteomics data to improve genome annotations, understand gene expression control through proteoforms and find sequence variants to develop novel insights for disease classification and therapeutic strategies. However, proteogenomic studies often suffer from reduced sensitivity and specificity due to inflated database size. To control the error rates, proteogenomics depends on the target-decoy search strategy, the de-facto method for false discovery rate (FDR) estimation in proteomics. The proteogenomic databases constructed from three- or six-frame nucleotide database translation not only increase the search space and compute-time but also violate the equivalence of target and decoy databases. These searches result in poorer separation between target and decoy scores, leading to stringent FDR thresholds. Understanding these factors and applying modified strategies such as two-pass database search or peptide-class-specific FDR can result in a better interpretation of MS data without introducing additional statistical biases. Based on these considerations, a user can interpret the proteogenomics results appropriately and control false positives and negatives in a more informed manner. In this review, first, we briefly discuss the proteogenomic workflows and limitations in database construction, followed by various considerations that can influence potential novel discoveries in a proteogenomic study. We conclude with suggestions to counter these challenges for better proteogenomic data interpretation.

Glucose and glutamine drive hepatitis E virus replication.

Viruses have undergone evolutionary adaptations to tune their utilization of carbon sources, enabling them to extract specific cellular substrates necessary for their replication. The lack of a reliable cell culture system and a small-animal model has hampered our understanding of the molecular mechanism of replication of hepatitis E virus (HEV) genotype 1. Our recent identification of a replicative ensemble of mutant HEV RNA libraries has allowed us to study the metabolic prerequisites for HEV replication. Initial assessments revealed increased glucose and glutamine utilization during HEV replication. Inhibition of glycolysis and glycolysis + glutaminolysis reduced the levels of HEV replication to similar levels. An integrated analysis of protein-metabolite pathways suggests that HEV replication markedly alters glycolysis, the TCA cycle, and glutamine-associated metabolic pathways. Cells supporting HEV replication showed a requirement for fructose-6-phosphate and glutamine utilization through the hexosamine biosynthetic pathway (HBP), stimulating HSP70 expression to facilitate virus replication. Observations of mannose utilization and glutamine dependence suggest a crucial role of the HBP in supporting HEV replication. Inhibition of glycolysis and HSP70 activity or knockdown of glutamine fructose-6-phosphate amidotransferase expression led to a substantial reduction in HEV RNA and ORF2 expression accompanied by a significant decrease in HSP70 levels. This study demonstrates that glucose and glutamine play critical roles in facilitating HEV replication.

Risk assessment of trace elements in vegetables grown in river Yamuna floodplain in Delhi.

Urban agriculture is common in fertile river floodplains of many developing countries. However, there is a risk of contamination in highly polluted regions. This study quantifies health risks associated with the consumption of vegetables grown in the floodplain of the urban river 'Yamuna' in the highly polluted yet data-scarce megacity Delhi, India. Six trace elements are analyzed in five kinds of vegetable samples. Soil samples from the cultivation area are also analyzed for elemental contamination. Ni, Mn, and Co are observed to be higher in leafy vegetables than others. Fruit and inflorescence vegetables are found to have higher concentrations of Cr, Pb, and Zn as compared to root vegetables. Transfer Factor indicates that Cr and Co have the highest and least mobility, respectively. Vegetable Pollution Index indicates that contamination levels follow as Cr > Ni > Pb > Zn. Higher Metal Pollution Index of leafy and inflorescence vegetables than root and fruit vegetables indicate that atmospheric deposition is the predominant source. Principal Component Analysis indicates that Pb and Cr have similar sources and patterns in accumulation. Among the analyzed vegetables, radish may pose a non-carcinogenic risk to the age group of 1-5 year. Carcinogenic risk is found to be potentially high due to Ni and Cr accumulation. Consumption of leafy vegetables was found to have relatively less risk than other vegetables due to lower Cr accumulation. Remediation of Cr and Ni in floodplain soil and regular monitoring of elemental contamination is a priority.

Interleukin-35 Mitigates ox-LDL-Induced Proatherogenic Effects via Modulating miRNAs Associated with Coronary Artery Disease (CAD).

PURPOSE: Recent emergence of miRNAs as important regulators of processes involving lesion formation and regression has highlighted miRNAs as potent therapeutic targets for the treatment of atherosclerosis. Few studies have reported the atheroprotective role of IL-35, a novel immunosuppressive and anti-inflammatory cytokine; however, miRNA-dependent regulation underlying the anti-atherosclerotic potential of IL-35 remains elusive. METHODS: THP-1 macrophages were incubated with human recombinant IL-35 (rIL-35) either in the presence or absence of ox-LDL. qRT-PCR was conducted to validate the expression levels of previously identified miRNAs including miR-197-5p, miR-4442, miR-324-3p, miR-6879-5p, and miR-6069 that were differentially expressed in peripheral blood mononuclear cells of coronary artery disease (CAD) patients vs. controls. Additionally, bioinformatic analysis was performed to predict miRNA-associated targets and their corresponding functional significance in CAD. RESULTS: Exogenous IL-35 significantly decreased the average area of ox-LDL-stimulated macrophages, indicating the inhibitory effect of IL-35 on lipid-laden foam cell formation. Furthermore, rIL-35 treatment alleviated the ox-LDL-mediated atherogenic effects by modulating the expression levels of aforementioned CAD-associated miRNAs in the cultured macrophages. Moreover, functional enrichment analysis of these miRNA-related targets revealed their role in the molecular processes affecting different stages of atheroslerotic plaque development, such as macrophage polarization, T cell suppression, lipoprotein metabolism, foam cell formation, and iNOS-mediated inflammation. CONCLUSION: Our observations uncover the novel role of IL-35 as an epigenetic modifier as it influences the expression level of miRNAs implicated in the pathogenesis of atherosclerosis. Thus, IL-35 cytokine therapy-mediated miRNA targeting could be an effective therapeutic strategy against the development of early atheromas in asymptomatic high-risk CAD patients.

Dissipation kinetics, risk assessment, and waiting period of spiromesifen on chili fruits using gas chromatography-electron capture detector.

A supervised field trial was designed in Rajasthan Agricultural Research Institute, Durgapura, Jaipur, Rajasthan, to assess the dissipation and persistence of spiromesifen in chili fruits. Spiromesifen (22.9% suspension concentrate) was sprayed two times at an interval of 10 days at the recommended dose (96 g. a.i. ha-1 ) and double the recommended dose (192 g. a.i. ha-1 ) with four replications. Sampling was done according to the planned interval of days after the second spray. Extraction and cleanup were performed using the modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method and the spiromesifen residue was analyzed by GC-electron capture detector and confirmation performed using GC-MS. The average initial deposit of spiromesifen was 1.207 mg kg-1 and 1.948 mg kg-1 at the recommended and double the recommended dose, respectively. The half-life values of spiromesifen ranged between 2.7 and 3.2 days at the recommended and double the recommended dose. The safe waiting period was calculated for the respective doses and it was concluded that an average of 7 days is safe for picking. The FSSAI (Food Safety and Standards Authority of India) have set the maximum residue limit of 0.1 mg kg-1 for spiromesifen in green chili. The theoretical maximum residue contribution value of spiromesifen was lower than the maximum permissible intake at both the applications on the 0th day. Hence, there will be no adverse effects on human health after consumption of green chilies.

The effect of different decontamination processes on the residues of fipronil and its metabolites in chili fruits (Capsicum annuum L.).

Fipronil is a broad-spectrum phenyl pyrazole insecticide that has a high degree of environmental toxicity. Commonly available chilies in the market are treated with fipronil insecticides. Demand for insecticide-free chili has thus been increasing globally. This needs various sustainable and economical methods to remove insecticides from chilies. The present study examined the effectiveness of several cleaning methods to remove pesticide residues in chili fruits. A supervised field trial was conducted in randomized block design at Rajasthan Agricultural Research Institute, Durgapura, Jaipur, India. Chili samples were subjected to seven different household methods. The samples were extracted using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. The residues were analyzed using a gas chromatograph-electron capture detector and confirmed by GC-MS. Of the seven methods, the acetic acid treatment removes the maximum residue effect of fipronil and its metabolites (desulfinyl [MB046513]), sulfide (MB045950), and sulfone (MB046136) on chili fruits. By contrast, the tap water treatment was the least effective. The Food Safety and Standards Authority of India (FSSAI) have set the maximum residue limit value of 0.001 mg kg-1 for fipronil on green chili.

Persistence and dissipation kinetics of novaluron 9.45% + lambda-cyhalothrin 1.9% ZC insecticides in tomato crop under semi-arid region.

In recent decades, fate studies of pesticides have been a topic of interest worldwide due to human health concerns tomato, contain abundant nutritional phytochemicals and lycopene which is known for antioxidant. Tomato is susceptible to many pest, so to overcome from these pests many insecticides are used, leaving residual effects on the crop. So to find out the persistence, the present study was carried out to investigate the residual levels and dissipation behaviour of novaluron 9.45% + lambda-cyhalothrin 1.9% ZC in tomato crop during Rabi session of 2017-18 in randomized block design. The first spray of insecticide was done at fruit formation stage and second spray at 10-day interval at recommended dose @43.31 g a.i. ha-1 and double of recommended dose @86.62 g a.i. ha-1. The residue of novaluron determined by HPLC (high-performance liquid chromatography) on 0 day (two hours after spraying) was 0.154 ppm at lower dose and 0.234 ppm at higher dose. The residue of lambda-cyhalothrin determined by GC ECD (gas chromatography electron capture detector) at 0 day (two hours after spraying) was 0.451 ppm at lower dose and 0.849 ppm at higher dose. The deposition of novaluron 9.45% + lambda-cyhalothrin 1.9% ZC was gradually decreased with increasing days after spraying (DAS). The mean initial deposition of the pesticide novaluron and lambda-cyhalothrin was recorded as 0.154 mg/kg, 0.451 mg/kg, respectively, at the recommended dose @43.31 g a.i. ha-1 while at double of recommended dose @86.62 g a.i. ha-1 novaluron and lambda-cyhalothrin, the mean initial deposition of 0.234 mg/kg and 0.849 mg/kg was recorded, respectively. The residue of the novaluron and lambda-cyhalothrin was at BDL (below determination level) (0.01 and 0.05 ppm) on 5th and 7th day, respectively, at lower dose (x), whereas at higher dose (2x) it was below determination level on 7th and 10th day, respectively. In soil samples, the residue levels were at below the determination level (0.01 mg/kg) for novaluron and (0.05 mg/kg) for lambda-cyhalothrin at both doses. The half-life DT50 of novaluron and lambda-cyhalothrin in the tomato fruit was found to be 2 days at recommended dose (X) @43.31 g a.i. ha-1 for both the pesticide and at double of the recommended dose @86.62 g a.i. ha-1 it was 3 and 2 days, respectively.

Diffusion Monte Carlo evaluation of disiloxane linearisation barrier.

The disiloxane molecule is a prime example of silicate compounds containing the Si-O-Si bridge. The molecule is of significant interest within the field of quantum chemistry, owing to the difficulty in theoretically predicting its properties. Herein, the linearisation barrier of disiloxane is investigated using a fixed-node diffusion Monte Carlo (FNDMC) approach, which is one of the most reliable ab initio methods in accounting for the electronic correlation. Calculations utilizing the density functional theory (DFT) and the coupled cluster method with single and double substitutions, including noniterative triples (CCSD(T)) are carried out alongside FNDMC for comparison. It is concluded that FNDMC successfully predicts the disiloxane linearisation barrier and does not depend on the completeness of the basis-set as much as DFT or CCSD(T), thus establishing its suitability.

Proteomic landscape of Japanese encephalitis virus-infected fibroblasts.

Advances in proteomics have enabled a comprehensive understanding of host-pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS-STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird's-eye view into how fibroblast protein composition is rewired following JEV infection.

Parosteal Lipoma of the Proximal Phalanx of Hand.

Parosteal lipomas are rare benign tumors accounting for less than 0.1% of all primary bone tumors. Only 3 cases of parosteal lipoma have been previously described affecting the distal and middle phalanges. We describe a case of parosteal lipoma in a 45-year-old man involving the proximal phalanx of the right middle finger. The tumor was marginally excised with the osseous attachment. There was no clinical or radiological recurrence at a follow-up of 2 years, with full range of movement at the proximal interphalangeal joint.

Serum protein signature of coronary artery disease in type 2 diabetes mellitus.

BACKGROUND: Coronary artery disease (CAD) is the leading cause of morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). The purpose of the present study was to discriminate the Indian CAD patients with or without T2DM by using multiple pathophysiological biomarkers. METHODS: Using sensitive multiplex protein assays, we assessed 46 protein markers including cytokines/chemokines, metabolic hormones, adipokines and apolipoproteins for evaluating different pathophysiological conditions of control, T2DM, CAD and T2DM with CAD patients (T2DM_CAD). Network analysis was performed to create protein-protein interaction networks by using significantly (p < 0.05) altered protein markers in each disease using STRING 10.5 database. We used two supervised analysis methods i.e., between class analysis (BCA) and principal component analysis (PCA) to reveals distinct biomarkers profiles. Further, random forest classification (RF) was used to classify the diseases by the panel of markers. RESULTS: Our two supervised analysis methods BCA and PCA revealed a distinct biomarker profiles and high degree of variability in the marker profiles for T2DM_CAD and CAD. Thereafter, the present study identified multiple potential biomarkers to differentiate T2DM, CAD, and T2DM_CAD patients based on their relative abundance in serum. RF classified T2DM based on the abundance patterns of nine markers i.e., IL-1ß, GM-CSF, glucagon, PAI-I, rantes, IP-10, resistin, GIP and Apo-B; CAD by 14 markers i.e., resistin, PDGF-BB, PAI-1, lipocalin-2, leptin, IL-13, eotaxin, GM-CSF, Apo-E, ghrelin, adipsin, GIP, Apo-CII and IP-10; and T2DM _CAD by 12 markers i.e., insulin, resistin, PAI-1, adiponectin, lipocalin-2, GM-CSF, adipsin, leptin, Apo-AII, rantes, IL-6 and ghrelin with respect to the control subjects. Using network analysis, we have identified several cellular network proteins like PTPN1, AKT1, INSR, LEPR, IRS1, IRS2, IL1R2, IL6R, PCSK9 and MYD88, which are responsible for regulating inflammation, insulin resistance, and atherosclerosis. CONCLUSION: We have identified three distinct sets of serum markers for diabetes, CAD and diabetes associated with CAD in Indian patients using nonparametric-based machine learning approach. These multiple marker classifiers may be useful for monitoring progression from a healthy person to T2DM and T2DM to T2DM_CAD. However, these findings need to be further confirmed in the future studies with large number of samples.

Integrated Transcriptomic-Proteomic Analysis Using a Proteogenomic Workflow Refines Rat Genome Annotation.

Proteogenomic re-annotation and mRNA splicing information can lead to the discovery of various protein forms for eukaryotic model organisms like rat. However, detection of novel proteoforms using mass spectrometry proteomics data remains a formidable challenge. We developed EuGenoSuite, an open source multiple algorithmic proteomic search tool and utilized it in our in-house integrated transcriptomic-proteomic pipeline to facilitate automated proteogenomic analysis. Using four proteogenomic pipelines (integrated transcriptomic-proteomic, Peppy, Enosi, and ProteoAnnotator) on publicly available RNA-sequence and MS proteomics data, we discovered 363 novel peptides in rat brain microglia representing novel proteoforms for 249 gene loci in the rat genome. These novel peptides aided in the discovery of novel exons, translation of annotated untranslated regions, pseudogenes, and splice variants for various loci; many of which have known disease associations, including neurological disorders like schizophrenia, amyotrophic lateral sclerosis, etc. Novel isoforms were also discovered for genes implicated in cardiovascular diseases and breast cancer for which rats are considered model organisms. Our integrative multi-omics data analysis not only enables the discovery of new proteoforms but also generates an improved reference for human disease studies in the rat model.

Dendrimeric amide- and carbamate-linked lysine-based efficient molecular transporters.

Amide- and carbamate-linked dendrimeric oligomers are reported as molecular transporters. They effectively complex with pDNA and transport it into cells at an efficiency superior to Lipofectamine, when complexation is carried out by incubation overnight. The carbamate-linked K2C is superior to amide-linked K2A; their pDNA complexes have very low associated cytotoxicity.

Clinicopathologic Analysis of Stable and Unstable Vitiligo: A Study of 66 Cases.

Vitiligo is an acquired skin disorder characterized by milky-white macules and absence of functioning melanocytes. The cornerstone of its management is the correct categorization of a case into its 2 broad types-stable and unstable vitiligo. This distinction is at present based mainly on clinical criteria because the histopathological features are not fully established. This study was thus undertaken to examine histopathological features of vitiligo and to come up with a reliable and systematic approach toward this diagnostic challenge. All patients presenting with clinical features of vitiligo at our institution were included in the study. A 3-mm punch biopsy was taken from 3 sites-lesional, perilesional, and normal skin. Histopathological examination was primarily focused on evaluating 5 histopathological variables-spongiosis, epidermal lymphocytes, basal cell vacuolation, dermal lymphocytes, and melanophages. A total number of 66 patients were included in the study. There were 30 patients in stable and 36 in unstable vitiligo groups. It was observed that all 5 histopathological pattens were associated with unstable vitiligo. All the cases were then scored using a scoring system devised by the authors and the scores obtained were correlated with clinical categorization. It was observed that while there is a definite overlap in histological findings in the 2 groups, adoption of a systematic reporting system brings more consistency and objectivity in the diagnosis. The authors have recommended diagnoses that should be reported for the various scores. This in turn will help us to more reliably and confidently manage these patients.

Proteogenomic analysis of Bradyrhizobium japonicum USDA110 using GenoSuite, an automated multi-algorithmic pipeline.

We present GenoSuite, an integrated proteogenomic pipeline to validate, refine and discover protein coding genes using high-throughput mass spectrometry (MS) data from prokaryotes. To demonstrate the effectiveness of GenoSuite, we analyzed proteomics data of Bradyrhizobium japonicum (USDA110), a model organism to study agriculturally important rhizobium-legume symbiosis. Our analysis confirmed 31% of known genes, refined 49 gene models for their translation initiation site (TIS) and discovered 59 novel protein coding genes. Notably, a novel protein which redefined the boundary of a crucial cytochrome P450 system related operon was discovered, known to be highly expressed in the anaerobic symbiotic bacteroids. A focused analysis on N-terminally acetylated peptides indicated downstream TIS for gene blr0594. Finally, ortho-proteogenomic analysis revealed three novel genes in recently sequenced B. japonicum USDA6(T) genome. The discovery of large number of missing genes and correction of gene models have expanded the proteomic landscape of B. japonicum and presents an unparalleled utility of proteogenomic analyses and versatility of GenoSuite for annotating prokaryotic genomes including pathogens.

Discovery of rare protein-coding genes in model methylotroph Methylobacterium extorquens AM1.

Proteogenomics involves the use of MS to refine annotation of protein-coding genes and discover genes in a genome. We carried out comprehensive proteogenomic analysis of Methylobacterium extorquens AM1 (ME-AM1) from publicly available proteomics data with a motive to improve annotation for methylotrophs; organisms capable of surviving in reduced carbon compounds such as methanol. Besides identifying 2482(50%) proteins, 29 new genes were discovered and 66 annotated gene models were revised in ME-AM1 genome. One such novel gene is identified with 75 peptides, lacks homolog in other methylobacteria but has glycosyl transferase and lipopolysaccharide biosynthesis protein domains, indicating its potential role in outer membrane synthesis. Many novel genes are present only in ME-AM1 among methylobacteria. Distant homologs of these genes in unrelated taxonomic classes and low GC-content of few genes suggest lateral gene transfer as a potential mode of their origin. Annotations of methylotrophy related genes were also improved by the discovery of a short gene in methylotrophy gene island and redefining a gene important for pyrroquinoline quinone synthesis, essential for methylotrophy. The combined use of proteogenomics and rigorous bioinformatics analysis greatly enhanced the annotation of protein-coding genes in model methylotroph ME-AM1 genome.

ProteoStats--a library for estimating false discovery rates in proteomics pipelines.

SUMMARY: Statistical validation of peptide assignments from a large-scale shotgun proteomics experiment is a critical step, and various methods for evaluating significance based on decoy database search are in practice. False discovery rate (FDR) estimation of peptide assignments assesses global significance and corrects for multiple comparisons. Various approaches have been proposed for FDR estimation but unavailability of standard tools or libraries leads to development of many in-house scripts followed by manual steps that are error-prone and low-throughput. The ProteoStats library provides an open-source framework for developers with many FDR estimation and visualization features for several popular search algorithms. It also provides accurate q-values, which can be easily integrated in any proteomics pipeline to provide automated, accurate, high-throughput statistical validation and minimize manual errors. AVAILABILITY: https://sourceforge.net/projects/mssuite/files/ProteoStats/. CONTACT: ddash@igib.res.in or aky.compbio@gmail.com or amit.yadav@igib.in. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.