Somatic Proteins Research Articles

Proteomics analysis reveals the differential protein expression of female and male adult Toxocara canis using Orbitrap Astral analyzer.

Toxocara canis, the most prevalent helminth in dogs and other canines, is one of the socioeconomically important zoonotic parasites, particularly affecting pediatric and adolescent populations in impoverished communities. However, limited information is available regarding the proteomes of female and male adult T. canis. To address this knowledge gap, we performed a comprehensive proteomic analysis to identify the proteins with differential abundance (PDAs) and gender-specifically expressed proteins between the two sexes adult T. canis. The comparative proteomic analysis was carried out by the Orbitrap mass spectrometry (MS) with asymmetric track lossless (Astral) analyzer. The difference analysis was conducted using t-test and the proteins verification was achieved through parallel reaction monitoring (PRM). The potential biological functions of identified adult T. canis proteins and PDAs were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The domain, transcription factor and subcellular localization of the identified proteins and PDAs were analyzed by InterPro, AnimalTFDB 4.0 and Cell-mPLOC 2.0 databases, respectively. A total of 8565 somatic proteins of adult T. canis were identified. Compared to male adult, 682 up-regulated PDAs and 844 down-regulated PDAs were identified in female adult with P-values < 0.05 and |log2FC|> 1, including 139 proteins exclusively expressed in female and 272 proteins exclusively expressed in male. The GO annotation analysis using all PDAs revealed that the main biological processes, cellular components and molecular functions corresponded to aminoglycan metabolic process, extracellular region and protein tyrosine phosphatase activity, respectively. The KEGG analysis using all PDAs showed that the pathways were mainly associated with adipocytokine signaling pathway, proximal tubule bicarbonate reclamation and PPAR signaling pathway. This study reveals the differential protein expression between female and male adult T. canis, providing valuable resource for developing the novel intervention strategies against T. canis infection in humans and animals, especially from the perspective of sexual development and reproduction.

The expression profiles of piRNAs and their interacting Piwi proteins in cellular model of renal development: Focus on Piwil1 in mitosis

Piwi proteins and Piwi interacting RNAs, piRNAs, presented in germline cells play a role in transposon silencing during germline development. In contrast, the role of somatic Piwi proteins and piRNAs still remains obscure. Here, we characterize the expression pattern and distribution of piRNAs in human renal cells in terms of their potential role in kidney development. Further, we show that all PIWI genes are expressed at the RNA level, however, only PIWIL1 gene is detected at the protein level by western blotting in healthy and cancerous renal cells. So far, the expression of human Piwil1 protein has only been shown in testes and cancer cells, but not in healthy somatic cell lines. Since we observe only Piwil1 protein, the regulation of other PIWI genes is probably more intricated, and depends on environmental conditions. Next, we demonstrate that downregulation of Piwil1 protein results in a decrease in the rate of cell proliferation, while no change in the level of apoptotic cells is observed. Confocal microscopy analysis reveals that Piwil1 protein is located in both cellular compartments, cytoplasm and nucleus in renal cells. Interestingly, in nucleus region Piwil1 is observed close to the spindle during all phases of mitosis in all tested cell lines. It strongly indicates that Piwil1 protein plays an essential role in proliferation of somatic cells. Moreover, involvement of Piwil1 in cell division could, at least partly, explain invasion and metastasis of many types of cancer cells with upregulation of PIWIL1 gene expression. It also makes Piwil1 protein as a potential target in the anticancer therapy.

Y-Box-Binding Proteins Have a Dual Impact on Cellular Translation.

Y-box-binding proteins (YB proteins) are multifunctional DNA- and RNA-binding proteins that play an important role in the regulation of gene expression. The high homology of their cold shock domains and the similarity between their long, unstructured C-terminal domains suggest that Y-box-binding proteins may have similar functions in a cell. Here, we consider the functional interchangeability of the somatic YB proteins YB-1 and YB-3. RNA-seq and Ribo-seq are used to track changes in the mRNA abundance or mRNA translation in HEK293T cells solely expressing YB-1, YB-3, or neither of them. We show that YB proteins have a dual effect on translation. Although the expression of YB proteins stimulates global translation, YB-1 and YB-3 inhibit the translation of their direct CLIP-identified mRNA targets. The impact of YB-1 and YB-3 on the translation of their mRNA targets is similar, which suggests that they can substitute each other in inhibiting the translation of their mRNA targets in HEK293T cells.

Identification of Somatic Proteins in Haemonchus Contortus Infective Larvae (L3) and Adults.

SummaryHaemonchus contortus is considered the most pathogenic nematode in sheep production systems based on grazing. Comparing infective larvae (L3) with adult parasites can lead to the identification of proteins that play an important role in parasite-host interactions. In this study, we report a list of H. contortus somatic proteins and made a comparative analysis of somatic proteins of L3 and adult worms. L3 and adult parasites were subjected to protein extraction and subsequently to peptide fractionation. Peptides were analysed by mass spectrometry and LC-MS/MS data analysis. Data analysis and search on SEQUEST and MASCOT against H. contortus from the WormBase ParaSite database resulted in the identification of 775 unique peptide sequences corresponding to 227 proteins at 1 % FDR. From these, 18 proteins were specific to L3 and 63 to adult parasites. The gene ontology (GO) enrichment analysis of the proteins specific to L3 and adult worms to gain insight into cellular components, molecular functions and biological processes that affect the parasite-host interaction showed some differences between the two parasite stages. The list of proteins found provides a database to identify target proteins that could be useful as biomarkers of the infection or in the generation of anthelmintic drugs that inhibit proteins essential for the establishment of the infection and the survival of adult parasites. They can also serve as new candidates for vaccine research.

Serological diagnostic potential of the 38-72 kDa somatic antigen of Gastrothylax crumenifer in buffaloes using the Indirect Enzyme-Linked Immunosorbent Assay

Paramphistomosis, caused by digenetic trematodes of the superfamily Paramphistomoidea, causes heavy economic losses in terms of reduced fertility, milk and meat production in the livestock industry. In the present study, somatic and excretory secretory antigens isolated from 500 live Gastrothylax crumenifer were assessed for their diagnostic potential by using an antibody detection enzyme immunoassay. Prior to the enzyme immunoassay, the somatic and excretory/secretory (ES) antigens of G. crumenifer were subjected to SDS-PAGE and Western blot (WB) for the detection of immunogenic proteins. Indirect ELISA analysis was performed on sera from buffaloes naturally infected with G. crumenifer, with control sera of buffaloes infected with Gigantocotyle explanatum, Fasciola spp., Cotylophoron /Paramphistomum spp. The SDS-PAGE results of the somatic products of G. crumenifer identified proteins were between 10-123 kDa, showing a maximum abundance of 10, 15, 25-28, 36, 38-72, 95-123 kDa proteins. The most abundant proteins recorded in ES product were ≥95, 72, 55 and 40 kDa. The antigenic analysis of somatic proteins using WB revealed reactive polypeptides of a size between 55-70 kDa, while metabolic extracts did not show reactivity with naturally infected buffalo sera. The sensitivity and specificity of the ELISA test for 38-72 kDa somatic antigens were 85.71% and 89.74%, respectively. The cross-reactivity with other trematode sera was 16-20%. Antibodies were tested against the 38-72 kDa somatic antigens, and 19.69% (39/198) of buffaloes were found positive, while 12.1% (24/198) presented infection in the fecal/postmortem examination. The study confirmed that ELISA established for the 38-72 kDa somatic antigens of G. crumenifer had diagnostic potential against paramphistome infections.

High Resolution Proteomic Analysis of Subcellular Fractionated Boar Spermatozoa Provides Comprehensive Insights Into Perinuclear Theca-Residing Proteins.

The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus, yet little is known regarding the overall proteomic composition of the PT. Here, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa following the implementation of a long-established subcellular fractionation protocol designed to increase the detection of low abundance proteins. A total of 1,802 proteins were identified, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire annotated proteome of the Sus scrofa species so far. In the PT structure itself, we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data revealed that the PT is replete in proteins critical for sperm-egg fusion and egg activation, including: Izumo family members 1–4 (IZUMO1-4) and phosphoinositide specific phospholipase ζ (PLCZ1). Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. This is particularly intriguing as it is currently held that the ER structure is lost during testicular sperm maturation. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. It remains to be explored whether proteasome subunits within the PT actively assist in the protein degradation of paternal cell structures post-fertilization and how aberrations in PT protein content may delay embryonic development.

A Preliminary Study on the Antigenic Proteins of Linguatula serrata Nymphal Stage in Sheep with Visceral Linguatulosis.

Scant information is available on the immunological aspect of Linguatula serrata causing linguatulosis in humans and animals. The present study aimed to analyze the content of crude somatic extracts and excretory-secretory products of L. serrata nymphs to detect the immune response of sheep and immunogenic proteins of the parasite. After collecting the nymphs, somatic extracts were prepared by sonication. Excretory secretory products were prepared by the incubation of nymphs in RPMI medium at 37°C with 5% CO2. Somatic and excretory-secretory proteins were isolated using SDS-PAGE. The immunogenic properties of the resulting proteins were determined using immunoblotting and positive sera from sheep infected with visceral linguatulosis. The total content of somatic extracts and excretory-secretory products of L. serrata nymphs analyzed by SDS-PAGE (12% gel) revealed two protein patterns with more than 18 and 9 strong bands, respectively. Immunoblots using sera samples of sheep infected with the parasite, somatic extracts and excretory-secretory products demonstrated 12 and 3 antigenic proteins with molecular weights mostly in the range of 24-100kDa and an antigen more than 180kDa. Three common immunodominant antigenic proteins with molecular weights of 38 and 57, as well as an antigen of more than 180 kDa, were detected in the somatic extracts and excretory-secretory products of L. serrata nymphs in sheep with visceral linguatulosis. These antigens can be considetered prime candidates for future serodiagnosis and immunoprotective studies of the parasite.

The efficacy of specific immunocorrection drugs against ovine oestrosis

The purpose of the research is to characterize the effect of specific immunocorrection drugs on the survival rate of the sheep botfly larvae and the immune response of the host organism. Materials and methods. Three variants of laboratory samples of specific immunocorrection drugs against sheep oestrosis have been developed based on water-soluble proteins of Oestrus ovis L. and Lucilia serricata Mg. larvae. The studies were carried out on 12 test and 3 control groups of lambs born in the current year artificially infected with 80 larvae of the sheep botfly. Fourteen days before infection, the test lambs were immunized with the prepared drugs. The drugs were injected subcutaneously in various doses (2–6 ml) and repeatedly (1–3) or by a single intranasal irrigation at a dose of 10 ml per animal. The efficacy was evaluated for the early (July – September) and summer-spring (August – April) periods of parasitism. In the test and control animals, we determined specific antibodies (JgG), stable E-rosette-forming cells (sE-RFC) and antigen-reactive (AR-RFC) T-lymphocytes. Results and discussion. The tested specific immunocorrection drugs based on somatic proteins of the sheep botfly and the sheep green bottle larvae have a pronounced protective effect against ovine oestrosis. In experiments, being affected by various drug variants, 44.6–99.1% of the sheep botfly larvae died in the early period of parasitism, and 22.6–88.1% of the sheep botfly larvae died in the summer-spring period. Subcutaneously injected drugs stimulate the produced specific antibodies and activate the effector function of the T-cell immune system to a greater extent than intranasal irrigation.

Chromatin characteristics of spermatogenesis in the spider crab Lissa chiragra (Fabricius, 1775) (Majoidea, Epialtidae)

Abstract This article uses the male spider crab Lissa chiragra, collected from Ras el Tin beach on the Mediterranean Sea at Alexandria, Egypt between January and December 2017, as a model to isolate the histone variants, H2A, H2B, H3 and H4 from spermatozoal nuclei to detect the presence of their genes and the chromatin-associated proteins. The spermatozoal chromatin in Brachyura is electron dense. During spermiogenesis somatic histones and sperm nuclear basic proteins (SNBP’s) are replaced partially or totally by protamines or keratin proteins, which are rich in arginine and oxidized cysteine. Histones, which are part of the nucleosome, are classified according to their functional and structural criteria into H2A, H2B, H3 and H4. Amino acids of the purified chromatin associated proteins are analysed with high resolution liquid chromatography. The low condensation of the nucleus of the spermatozoa is due to a low proportion and modification of the chromatin-associated histones. The DNA is organized into nucleosomes but not in higher order structures due to acetylation of histone H4, but rather via regions of histone-free DNA.

PIWI proteomics identifies Atari and Pasilla as piRNA biogenesis factors in Aedes mosquitoes.

As in most arthropods, the PIWI-interacting RNA (piRNA) pathway in the vector mosquito Aedes aegypti is active in diverse biological processes in both soma and germline. To gain insights into piRNA biogenesis and effector complexes, we mapped the interactomes of the somatic PIWI proteins Ago3, Piwi4, Piwi5, and Piwi6 and identify numerous specific interactors as well as cofactors associated with multiple PIWI proteins. We describe the Piwi5 interactor AAEL014965, the direct ortholog of the Drosophila splicing factor pasilla. We find that Ae. aegypti Pasilla encodes a nuclear isoform and a cytoplasmic isoform, the latter of which is required for efficient piRNA production. In addition, we characterize a splice variant of the Tudor protein AAEL008101/Atari that associates with Ago3 and forms a scaffold for PIWI proteins and target RNAs to promote ping-pong amplification of piRNAs. Our study provides a useful resource for follow-up studies of somatic piRNA biogenesis, mechanism, and function in Aedes mosquitoes.

Identification of Somatic Antigens of Adult Fasciola gigantica Isolated from Bali Cattle

In most tropical countries, such as Indonesia, fasciolosis is generally caused by Fasciola gigantica known as tropical liver fluke. However, most fasciolosis serodiagnostic tests have been developed solely for diagnosing fasciolosis caused by Fasciola hepatica (non-tropical liver fluke), and very few have been specifically designed for F. gigantica. The aim of this study was to determine the profile of antigenic proteins from the somatic extract of F. gigantica isolated from Bali cattle (Bos javanicus). The liver flukes were collected from a slaughtering house in Mataram, Indonesia. The somatic extracts were prepared by homogenizing in buffers containing 0.05 M NaCl, 0.02 M PMSF, and 0.05% Triton X-100. The characterization of the somatic extract proteins was performed using one-dimension gel electrophoresis and followed by Western blotting to determine the profile of its antigenic proteins. There were 14 bands of the somatic extracts with an estimated molecular weight ranging from 8 to105 8 kDa shown on the gel electrophoresis. The results of the Western blot show that there were five prominent protein bands. Three out of five prominent antigenic proteins with molecular weights of 8, 27, and 33 kDa are promising to enrich the existence of antigens that have immunodiagnostic value for fasciolosis. Therefore, further studies are required to examine more deeply the potency of those three antigenic somatic proteins of F. gigantica.

Identification and Immunological Characterization of Somatic Proteins from Adults of Toxocara cati by Proteomics Technique.

Background:Toxocara cati is considered as one of the main etiological agents of toxocariasis with global and regional importance. As there is no information on proteomics of T. cati, herein, we reported the results obtained by proteomic analysis of somatic proteins extract, using a mass spectrometry (LC–MS/MS) approach.Methods:Somatic extract fractions were separated by two-dimensional SDS-PAGE and were electro blotted on to PVDF membranes for immunoblot analysis, then collected the immunogenic spots which response of antibodies of the paratenic hosts (mice) to the antigens (Mashhad, 2017), and analyzed by LC–MS/MS. The LC-MS/MS data were analyzed by Mascot database, Taxonomy Toxocara, and common contaminants, in Omics Center, Biotechnology Medical University of Graz (Austria, 2018).Result:The protein spots were isolated between 15–140 kDa ranges using 3–10 non-linear IPG strips and Brilliant Blue Coomassie. Ten proteins were characterized as immunogenic proteins, seven of them were identified and three of them were unknown proteins.Conclusion:This study provided additional information about the somatic antigens of T. cati, which can lead to the development of new strategies for novel immuno-modulators, drug targets, subunit vaccines and immunodiagnostic kits for toxocariasis.

Abstract 1067: Proteogenomic discovery of novel tumor proteins as neoantigens for personalized T cell immunotherapy in pediatric medulloblastoma

Abstract Medulloblastoma is a common childhood brain tumor for which current therapies fail to cure high-risk subgroups, including metastatic and recurrent disease. Toxicity limits further intensification of conventional chemo-radiation therapy and most survivors endure significant lifelong sequelae from treatment. There is an urgent need to develop new tools to combat this cancer without increasing the late effects burden. Neoantigens exclusively expressed on tumor cells, are one of the main targets of an effective antitumor T-cell response. The ideal target antigen is abundantly expressed by tumor cells but not by normal tissues, in order to limit off-target effects. Tumors translate a host of unique transcripts that are the result of aberrations in tumor DNA and the unmasking of alternative or novel exons. We used a novel proteogenomic approach to identify tumor-restricted peptides and used them to select and expand T cells capable of mounting a tumor-specific cytotoxic immune response, with no off-target effects for patients with MB. Using RNA-seq and WGS data, we created personalized custom searchable databases containing predicted novel proteins from somatic mutations, novel isoforms and fusion proteins from 46 medulloblastoma tumors. By searching this database with raw mass spectrometry data from the paired medulloblastoma tumor, we have identified tens of peptides arising from the translation of tumor-specific transcripts; these peptides are potential neoantigens. Our data indicates that in cases of tumors with low mutation rates, such as pediatric brain tumors, novel isoforms are the main source of neoantigens. In a pilot study, we tested these peptides for their ability to select and expand polyclonal populations of T cells from a patient whose tumor was the source of the peptides. The immunogenicity of each individual peptide was then determined. Flow cytometry cellular characterization reveals populations of both CD4+ and CD8+ cells with an activation profile marked by IFN-γ and TNF-α production. Inhibitory co-receptor profiles were also characterized for these cells. Using cytotoxicity assays, we demonstrated that tumor specific T cells can eliminate neoantigen bearing tumor cells. These findings demonstrate an initial proof of principle that proteogenomics can be used to identify immunogenic tumor specific peptides and lay the groundwork for a personalized, targeted T cell therapy for children with high risk medulloblastoma. Citation Format: Samuel Rivero-Hinojosa, Melanie Grant, Aswini Panigrahi, Huizhen Zhang, Veronika Caisova, Catherine M. Bollard, Brian R. Rood. Proteogenomic discovery of novel tumor proteins as neoantigens for personalized T cell immunotherapy in pediatric medulloblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1067.

Proteomics and bioinformatics analysis of Fasciola hepatica somatic proteome in different growth phases.

Fasciola hepatica (F. hepatica) is a well-known zoonotic parasite that is crucial for economic and public health worldwide. Quantitative proteomics studies have been performed on proteins expressed by F. hepatica to investigate the differential expression of proteomes in different growth phases. And the screening of several marker proteins for use as early diagnostic antigens is essential. In this study, high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted to analyze the differences in the expression of F. hepatica somatic proteins in different growth phases. Furthermore, gene ontology (GO) functional annotation, KEGG metabolic pathway, and clustering analyses were also performed. LC-MS/MS identified 629, 2286, 2254, and 2192 proteins in metacercariae, juvenile flukes 28dpi, immature flukes 59dpi, and adult phases, respectively. GO analysis revealed that differentially expressed proteins (DEPs) were mainly involved in transport, localization, metabolism, enzyme regulation, protein folding and binding, and nucleoside and nucleotide binding. The DEPs were enriched in cells, intracellular components, organelles, cytoplasm, vesicles, and membranes. KEGG pathway annotation results showed that the DEPs were involved in metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and other processes. These findings provide a theoretical basis for vaccine development and establishing early diagnostic methods in the future.Electronic supplementary materialThe online version of this article (10.1007/s00436-020-06833-x) contains supplementary material, which is available to authorized users.

BRD4 regulates key transcription factors that drive epithelial-mesenchymal transition in castration-resistant prostate cancer.

BackgroundAndrogen deprivation therapies for the hormone-dependent stages of prostate cancer have become so effective that new forms of chemoresistant tumors are emerging in clinical practice, and require new targeted therapies in the metastatic setting. Yet there are important gaps in our understanding of the relevant transcriptional networks driving this process. Progression from localized to metastatic castration resistant prostate cancer (mCRPC) occurs as a result of accumulated resistance mechanisms that develop upon sustained androgen receptor (AR) suppression. Critical to this progression is the plastic nature by which prostate tumor cells transition from epithelial to mesenchymal states (EMT).MethodsHere, using prostate cancer cell lines with different AR composition, we systematically manipulated somatic proteins of the Bromodomain and ExtraTerminal (BET) family (BRD2, BRD3 and BRD4) to determine which BET proteins influence EMT. We used the TCGA repository to correlate the expression of individual BET genes with key EMT genes and determined biochemical recurrence in 414 patients and progression free survival in 488 patients.ResultsWe found that only BRD4 – and not BRD2 or BRD3 – regulates the expression of SNAI1 and SNAI2, and that the downregulation of these EMT transcription factors significantly increases E-cadherin expression. Furthermore, of the BET genes, only BRD4 correlates with survival outcomes in prostate cancer patients. Moreover, selective degradation of BRD4 protein with MZ1 ablates EMT (transcriptionally and morphologically) induced by TGFß signaling.ConclusionsMany relapsed/refractory tumors share a neuroendocrine transcriptional signature that had been relatively rare until highly successful antiandrogen drugs like abiraterone and enzalutamide came into widespread use. New therapeutic targets must therefore be developed. Our results identify key EMT genes regulated by BRD4, and offers a novel druggable target to treat mCRPC. BRD4-selective protein degraders offer a promising next generation approach to treat the emerging forms of chemoresistance in advanced prostate cancer.

Object-Based Analyses in FIJI/ImageJ to Measure Local RNA Translation Sites in Neurites in Response to Aβ1-42 Oligomers.

Subcellular protein delivery is especially important in signal transduction and cell behavior, and is typically achieved by localization signals within the protein. However, protein delivery can also rely on localization of mRNAs that are translated at target sites. Although once considered heretical, RNA localization has proven to be highly conserved in eukaryotes. RNA localization and localized translation are especially relevant in polarized cells like neurons where neurites extend dozens to hundreds of centimeters away from the soma. Local translation confers dendrites and axons the capacity to respond to their environment in an acute manner without fully relying on somatic signals. The relevance of local protein synthesis in neuron development, maintenance and disease has not been fully acknowledged until recent years, partly due to the limited amount of locally produced proteins. For instance, in hippocampal neurons levels of newly synthesized somatic proteins can be more than 20–30 times greater than translation levels of neuritic proteins. Thus local translation events can be easily overlooked under the microscope. Here we describe an object-based analysis used to visualize and quantify local RNA translation sites in neurites. Newly synthesized proteins are tagged with puromycin and endogenous RNAs labeled with SYTO. After imaging, signals corresponding to neuritic RNAs and proteins are filtered with a Laplacian operator to enhance the edges. Resulting pixels are converted into objects and selected by automatic masking followed by signal smoothing. Objects corresponding to RNA or protein and colocalized objects (RNA and protein) are quantified along individual neurites. Colocalization between RNA and protein in neurites correspond to newly synthesized proteins arising from localized RNAs and represent localized translation sites. To test the validity of our analyses we have compared control neurons to Aβ1–42-treated neurons. Aβ is involved in the pathology of Alzheimer’s disease and was previously reported to induce local translation in axons and dendrites which in turn contributes to the disease. We have observed that Aβ increases the synthesis of neuritic proteins as well as the fraction of translating RNAs in distal sites of the neurite, suggesting an induction of local protein synthesis. Our results thus confirm previous reports and validate our quantification method.

Liquid biopsy for cancer management: a revolutionary but still limited new tool for precision medicine.

The term liquid biopsy is used in contraposition to the traditional "solid" tissue biopsy. In the oncology field it has opened a new plethora of clinical opportunities as tumor-derived material is shedded into the different biofluids from where it can be isolated and analyzed. Common biofluids include blood, urine, saliva, cerebrospinal fluid (CSF), pleural effusion or bile. Starting from these biological specimens several analytes can be isolated, among which we will review the most widely used: circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA), proteins, metabolites, and exosomes. Regarding the nature of the biomarkers it will depend on the analyte, the type of tumor and the clinical application of the liquid biopsy and it includes, somatic point mutations, deletions, amplifications, gene-fusions, DNA-methylated marks, tumor-specific miRNAs, proteins or metabolites. Here we review the characteristics of the analytes and the methodologies used for their isolation. We also describe the applications of the liquid biopsy in the management of patients with cancer, from the early detection of cancers to treatment guidance in patients with advanced tumors. Finally, we also discuss some current limitations and still open questions.

TCR-mimic antibody-drug conjugates targeting intracellular tumor-specific mutant antigen KRAS G12V mutation

Limited clinical application of antibody-drug conjugates (ADCs) targeting tumor associated antigens (TAAs) is usually caused by on-target off-tumor side effect. Tumor-specific mutant antigens (TSMAs) only expressed in tumor cells which are ideal targets for ADCs. In addition, intracellular somatic mutant proteins can be presented on the cell surface by human leukocyte antigen class I (HLA I)molecules forming tumor-specific peptide/HLA I complexes. KRAS G12V mutation frequently occurred in varied cancer and was verified as a promising target for cancer therapy. In this study, we generated two TCR-mimic antibody-drug conjugates (TCRm-ADCs), 2E8-MMAE and 2A5-MMAE, targeting KRAS G12V/HLA-A*0201 complex, which mediated specific antitumor activity in vitro and in vivo without obvious toxicity. Our findings are the first time validate the strategy of TCRm-ADCs targeting intracellular TSMAs, which improves the safety of antibody-based drugs and provides novel strategy for precision medicine in cancer therapy.

Abstract A23: Proteogenomic discovery of novel tumor proteins as neoantigens for personalized T-cell immunotherapy in pediatric medulloblastoma

Abstract Medulloblastoma is a common childhood brain tumor for which current therapies fail to cure high-risk subgroups, including metastatic and recurrent disease. Toxicity limits further intensification of conventional chemo-radiation therapy, and most survivors endure significant lifelong sequelae from treatment. There is an urgent need to develop new tools to combat this cancer without increasing the late effects burden. Neoantigens, which are exclusively expressed on tumor cells, are one of the main targets of an effective antitumor T-cell response. The ideal target antigen is abundantly expressed by tumor cells but not by normal tissues, in order to limit off-target effects. Tumors translate a host of highly novel transcripts that are the result of aberrations in tumor DNA and the unmasking of alternative or novel exons. We used a novel proteogenomic approach to identify tumor-restricted peptides and used them to select and expand T cells capable of mounting a tumor-specific cytotoxic immune response, with no off-target effects for patients with MB. Using RNA-seq and WGS data, we created personalized custom searchable databases containing predicted novel proteins from somatic mutations, novel isoforms, and fusion proteins from 56 medulloblastoma tumors. By searching this database with raw mass spectrometry data from the paired medulloblastoma tumor, we have identified tens of peptides arising from the translation of tumor-specific transcripts; these peptides are potential neoantigens. Our data indicate that in cases of tumors with low mutation rates, such as pediatric brain tumors, novel isoforms are the main source of neoantigens. In a pilot study, we have tested these peptides for their ability to select and expand polyclonal populations of T cells from the same patient whose tumor was the source of the peptides. The immunogenicity of each individual peptide was then determined. Flow cytometry cellular characterization reveals populations of both CD4+ and CD8+ cells with an activation profile marked by IFN-γ and TNF-α production. Immunosuppressive marker profiles have also been characterized for these cells. Using cytotoxicity assays, we demonstrated that tumor-specific T cells can eliminate neoantigen-bearing tumor cells. These findings demonstrate an initial proof of principle that proteogenomics can be used to identify immunogenic tumor specific peptides and lay the groundwork for a personalized, targeted T-cell therapy for children with high-risk medulloblastoma. Citation Format: Samuel Rivero-Hinojosa, Melanie Grant, Aswini Panigrahi, Huizhen Zhang, Veronika Caisova, Catherine Bollard, Brian Rood. Proteogenomic discovery of novel tumor proteins as neoantigens for personalized T-cell immunotherapy in pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A23.

Toxocara "omics" and the promises it holds for medicine and veterinary medicine.

Toxocariasis is one of the most neglected worldwide zoonoses that is caused by larval nematode parasites of the genus Toxocara, Toxocara canis, and to a lesser extent, Toxocara cati, whose migration mechanism is still largely unknown. Fortunately, some advanced tools have been employed, such as genomics, transcriptomics, and proteomics, to better understand the molecular biology and regulatory mechanisms of Toxocara. Using genomics and transcriptomics, we can identify a large number of genes that participate in the development of Toxocara and the interaction of parasites and their hosts and can predict the functions of unknown genes by comparing them with other relevant species. Using proteomics, we can identify somatic proteins and excretory and secretory (ES) proteins that perform specific biological functions in tissue degradation, pathogen invasion, immune evasion or modulation. These "omics" techniques also can contribute enormously to the development of new drugs, vaccines and diagnostic tools for toxocariasis. In a word, by utilizing "omics", we can better understand the Toxocara and toxocariasis. In this review, we summarized the representative achievements in Toxocara and the interaction between Toxocara spp. and their hosts based on expressed sequence tags (ESTs), microarray gene expression, next-generation sequencing (NGS) technologies and liquid chromatography-tandem mass spectrometry (LC-MS/MS), hoping to better understand the molecular biology of Toxocara, and contribute to new progress in the application areas of new drugs, vaccines and diagnostic tool for toxocariasis in the future.