Lung Cancer Proteogenomics: Shaping the Future of Clinical Investigation.

BACKGROUND: Lung cancer is associated with a high incidence of mortality worldwide. Molecular mechanisms governing the disease have been explored by genomic studies; however, several aspects remain elusive. The integration of genomic profiling with in-depth proteomic profiling has introduced a new dimension to lung cancer research, termed proteogenomics. The aim of this review article was to investigate proteogenomic approaches in lung cancer, focusing on how elucidation of proteogenomic features can evoke tangible clinical outcomes. METHODS: A strict methodological approach was adopted for study selection and key article features included molecular attributes, tumor biomarkers, and major hallmarks involved in oncogenesis. RESULTS: As a consensus, in all studies it becomes evident that proteogenomics is anticipated to fill significant knowledge gaps and assist in the discovery of novel treatment options. Genomic profiling unravels patient driver mutations, and exploration of downstream effects uncovers great variability in transcript and protein correlation. Also, emphasis is placed on defining proteogenomic traits of tumors of major histological classes, generating a diverse portrait of predictive markers and druggable targets. CONCLUSIONS: An up-to-date synthesis of landmark lung cancer proteogenomic studies is herein provided, underpinning the importance of proteogenomics in the landscape of personalized medicine for combating lung cancer.

RNA binding protein AUF1/HNRNPD regulates nuclear export, stability and translation of SNCA transcripts.

Alpha-synuclein (SNCA) accumulation plays a central role in the pathogenesis of Parkinson's disease. Determining and interfering with the mechanisms that control SNCA expression is one approach to limiting disease progression. Currently, most of our understanding of SNCA regulation is protein-based. Post-transcriptional mechanisms directly regulating SNCA mRNA expression via its 3' untranslated region (3'UTR) were investigated here. Mass spectrometry of proteins pulled down from murine brain lysates using a biotinylated SNCA 3'UTR revealed multiple RNA-binding proteins, of which HNRNPD/AUF1 was chosen for further analysis. AUF1 bound both proximal and distal regions of the SNCA 3'UTR, but not the 5'UTR or CDS. In the nucleus, AUF1 attenuated SNCA pre-mRNA maturation and was indispensable for the export of SNCA transcripts. AUF1 destabilized SNCA transcripts in the cytosol, primarily those with shorter 3'UTRs, independently of microRNAs by recruiting the CNOT1-CNOT7 deadenylase complex to trim the polyA tail. Furthermore, AUF1 inhibited SNCA mRNA binding to ribosomes. These data identify AUF1 as a multi-tasking protein regulating maturation, nucleocytoplasmic shuttling, stability and translation of SNCA transcripts.

Egg White and Yolk Protein Atlas: New Protein Insights of a Global Landmark Food.

(1) Background: The chicken egg is an animal product of great agronomic interest. The egg white and yolk constitute high-quality protein sources for humans with high digestibility and well-balanced amino acid profiles. Despite the egg white and yolk protein's undisputed value, research to unravel their full proteome content and its properties is still ongoing. We aimed to exhaustively analyze the proteome of egg white and yolk by applying intrinsic proteomics and bioinformatics approaches in order to unravel the full protein potential of this landmark food. (2) Methods: A total of 45 freshly laid, unfertilized, chicken eggs were subjected to nanoLC-MS/MS Orbitrap analysis following a peptide pre-fractionation step. A comprehensive bioinformatics processing step was undertaken towards elucidating potential activities and roles of identified molecules. In parallel, the literature was mined concerning all reported egg white and yolk protein identifications. (3) Results: Our analysis revealed 371 and 428 new proteins, reported for the first time to be present in the egg white and yolk, respectively. From the bioactivity standpoint, egg white and yolk proteins showed high enrichment for antioxidant and anti-inflammatory processes, while exerting high relevance for the apoptosis and focal adhesion pathways. (4) Conclusions: Egg white and yolk proteins exert diverse and multifaceted properties. A total of 799 proteins were reported for the first time as being part of the egg and yolk. Our novel protein data enriched those already published in the literature and the first ever chicken egg white and yolk Protein Atlas, comprising 1392 protein entries, was generated. This dataset will provide a cornerstone reference for future studies involving egg proteins.

Proteomics Evaluation of Semen of Clinically Healthy Beagle-Breed Dogs.

The objectives of the present work were to evaluate the semen of dogs by means of proteomics methods and to compare with proteomics results of the blood of the animals, in order to increase available knowledge on the topic and present relevant reference values for semen samples. Semen samples were collected from five Beagle-breed dogs. Reproductive assessment of the animals by means of clinical, ultrasonographic and seminological examinations confirmed their reproductive health. The sperm-rich fraction and the prostatic fraction of semen were processed for proteomics evaluation. LC-MS/MS analysis was performed by means of a LTQ Orbitrap Elite system. The technology combines high separation capacity and strong qualitative ability of proteins in biological samples that require deep proteome coverage. Protein classification was performed based on their functional annotations using Gene Ontology (GO). In blood plasma, semen sperm-rich fraction, and semen prostatic fraction, 59, 42 and 43 proteins, respectively, were detected. Two proteins were identified simultaneously in plasma and the semen sperm-rich fraction, 11 proteins in plasma and the semen prostatic fraction, and three proteins in the semen sperm-rich and prostatic fractions. In semen samples, most proteins were related to cell organization and biogenesis, metabolic processes or transport of ions and molecules. Most proteins were located in the cell membrane, the cytosol or the nucleus. Finally, most proteins performed functions related to binding or enzyme regulation. There were no differences between the semen sperm-rich fraction and prostatic fractions in terms of the clustering of proteins. In conclusion, a baseline reference for proteins in the semen of Beagle-breed dogs is provided. These proteins are involved mostly in supporting spermatozoan maturation, survival and motility, enhancing the reproductive performance of male animals. There appears potential for the proteomics examination of semen to become a tool in semen evaluation. This analysis may potentially identify biomarkers for reproductive disorders. This can be particularly useful in stud animals, also given its advantage as a non-invasive method.

Serum Proteome Signatures of Anti-SARS-CoV-2 Vaccinated Healthcare Workers in Greece Associated with Their Prior Infection Status.

Over the course of the pandemic, proteomics, being in the frontline of anti-COVID-19 research, has massively contributed to the investigation of molecular pathogenic properties of the virus. However, data on the proteome on anti-SARS-CoV-2 vaccinated individuals remain scarce. This study aimed to identify the serum proteome characteristics of anti-SARS-CoV-2 vaccinated individuals who had previously contracted the virus and comparatively assess them against those of virus-naïve vaccine recipients. Blood samples of n = 252 individuals, out of whom n = 35 had been previously infected, were collected in the "G. Gennimatas" General Hospital of Thessaloniki, from 4 January 2021 to 31 August 2021. All participants received the BNT162b2 mRNA COVID-19 vaccine (Pfizer/BioNTech). A label-free quantitative proteomics LC-MS/MS approach was undertaken, and the identified proteins were analyzed using the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes) databases as well as processed by bioinformatics tools. Titers of total RBD-specific IgGs against SARS-CoV-2 were also determined using the SARS-CoV-2 IgG II Quant assay. A total of 47 proteins were significantly differentially expressed, the majority of which were down-regulated in sera of previously infected patients compared to virus-naïve controls. Several pathways were affected supporting the crucial role of the humoral immune response in the protection against SARS-CoV-2 infection provided by COVID-19 vaccination. Overall, our comprehensive proteome profiling analysis contributes novel knowledge of the mechanisms of immune response induced by anti-SARS-CoV-2 vaccination and identified protein signatures reflecting the immune status of vaccine recipients.

Radiomics/Radiogenomics in Lung Cancer: Basic Principles and Initial Clinical Results.

Lung cancer is the leading cause of cancer-related deaths worldwide, and elucidation of its complicated pathobiology has been traditionally targeted by studies incorporating genomic as well other high-throughput approaches. Recently, a collection of methods used for cancer imaging, supplemented by quantitative aspects leading towards imaging biomarker assessment termed "radiomics", has introduced a novel dimension in cancer research. Integration of genomics and radiomics approaches, where identifying the biological basis of imaging phenotypes is feasible due to the establishment of associations between molecular features at the genomic-transcriptomic-proteomic level and radiological features, has recently emerged termed radiogenomics. This review article aims to briefly describe the main aspects of radiogenomics, while discussing its basic limitations related to lung cancer clinical applications for clinicians, researchers and patients.

High-throughput Proteomic Profiling of Male Breast Cancer Tissue.

BACKGROUND/AIM: Until now, little emphasis has been placed on the protein expression profile of male breast cancer (MBC) tumors, due to the rarity of the disease. The present study aimed to identify a proteomic pattern that is characteristic for malignant male breast tissue epithelium. MATERIALS AND METHODS: The protein content of four male breast tumors and corresponding adjacent healthy (control) tissues was analyzed by high-throughput nano-liquid chromatography-MS/MS technology. RESULTS: A total of 2,352 proteins were identified, that correspond to 1,249 single gene products, with diverse biological roles. Of those, a panel of 119 differentially expressed tissue proteins was identified in MBC samples compared to controls; 90 were found to be over-expressed in MBC tissues, while 29 were down-regulated. Concurrently, 844 proteins were detected only in MBC tumors and 197 were expressed exclusively in control mammary samples. CONCLUSION: Differential proteomic expression was found in MBC tissue, leading to improved understanding of MBC pathology and highlighting the need for personalized management of male patients.

On the Evidence Supporting That AN11127 Encodes an Aspergillus Nidulans Sec12 Orthologous Protein. Reply to Bravo-Plaza et al. Comment on "Dimou et al. Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans. J. Fungi 2021, 7, 560".

Prof. Peñalva and co-workers provided evidence that AN11127 is related by sequence and function to Sec12 [...].

Proteomic data of donkey's milk.

Donkey's milk has been recognized as milk of high biological value and it also has the closest composition to human milk. However, the total protein content of donkey's milk has not been adequately identified. The aim of this analysis is to investigate the proteomic content of that milk. Specific commercially available only milk was analyzed by ``shotgun'' proteomic methods to identify the proteins it contained in as much detail as possible. The application of the above approach resulted in the identification of a total of 633 different proteins, which were grouped based on their molecular function and their biological process. Furthermore, the proteins visualized graphically according to the GeneOntology (GO) system. The identified proteins confirm the high nutritional value of the donkey milk, governing future steps in optimizing its characteristic and uses.

Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans.

Solute and ion transporters are proteins essential for cell nutrition, detoxification, signaling, homeostasis and drug resistance. Being polytopic transmembrane proteins, they are co-translationally inserted and folded into the endoplasmic reticulum (ER) of eukaryotic cells and subsequently sorted to their final membrane destination via vesicular secretion. During their trafficking and in response to physiological/stress signals or prolonged activity, transporters undergo multiple quality control processes and regulated turnover. Consequently, transporters interact dynamically and transiently with multiple proteins. To further dissect the trafficking and turnover mechanisms underlying transporter subcellular biology, we herein describe a novel mass spectrometry-based proteomic protocol adapted to conditions allowing for maximal identification of proteins related to N source uptake in A. nidulans. Our analysis led to identification of 5690 proteins, which to our knowledge constitutes the largest protein dataset identified by omics-based approaches in Aspergilli. Importantly, we detected possibly all major proteins involved in basic cellular functions, giving particular emphasis to factors essential for membrane cargo trafficking and turnover. Our protocol is easily reproducible and highly efficient for unearthing the full A. nidulans proteome. The protein list delivered herein will form the basis for downstream systematic approaches and identification of protein-protein interactions in living fungal cells.

From Proteomic Mapping to Invasion-Metastasis-Cascade Systemic Biomarkering and Targeted Drugging of Mutant BRAF-Dependent Human Cutaneous Melanomagenesis.

Melanoma is classified among the most notoriously aggressive human cancers. Despite the recent progress, due to its propensity for metastasis and resistance to therapy, novel biomarkers and oncogenic molecular drivers need to be promptly identified for metastatic melanoma. Hence, by employing nano liquid chromatography-tandem mass spectrometry deep proteomics technology, advanced bioinformatics algorithms, immunofluorescence, western blotting, wound healing protocols, molecular modeling programs, and MTT assays, we comparatively examined the respective proteomic contents of WM115 primary (n = 3955 proteins) and WM266-4 metastatic (n = 6681 proteins) melanoma cells. It proved that WM115 and WM266-4 cells have engaged hybrid epithelial-to-mesenchymal transition/mesenchymal-to-epithelial transition states, with TGF-ß controlling their motility in vitro. They are characterized by different signatures of SOX-dependent neural crest-like stemness and distinct architectures of the cytoskeleton network. Multiple signaling pathways have already been activated from the primary melanoma stage, whereas HIF1α, the major hypoxia-inducible factor, can be exclusively observed in metastatic melanoma cells. Invasion-metastasis cascade-specific sub-routines of activated Caspase-3-triggered apoptosis and LC3B-II-dependent constitutive autophagy were also unveiled. Importantly, WM115 and WM266-4 cells exhibited diverse drug response profiles, with epirubicin holding considerable promise as a beneficial drug for metastatic melanoma clinical management. It is the proteome navigation that enables systemic biomarkering and targeted drugging to open new therapeutic windows for advanced disease.

The Experience of Greece as a Model to Contain COVID-19 Infection Spread.

The severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) emerged in late 2019 and has caused a pandemic known as corona virus disease 2019 (COVID-19), responsible for the death of more than 2 million people worldwide. The outbreak of COVID-19 has posed an unprecedented threat on human lives and public safety. The aim of this review is to describe key aspects of the bio-pathology of the novel disease, and discuss aspects of its spread, as well as targeted protective strategies that can help shape the outcome of the present and future health crises. Greece is used as a model to inhibit SARS-COV-2 spread, since it is one of the countries with the lowest fatality rates among nations of the European Union (E.U.), following two consecutive waves of COVID-19 pandemic. Furthermore, niche research technological approaches and scientific recommendations that emerged during the COVID-19 era are discussed.

Proteomics of pediatric ependymomas: a review.

Ependymomas, affecting both children and adults, are neuroepithelial tumors occurring throughout all compartments of the central nervous system. Pediatric ependymomas arise almost exclusively intracranially and are associated with a poor 10-year overall survival of around 60%. During the last years, the application of multi-omics technologies on the study and understanding of neuro-cancer diseases has become a standard; in this regard, application of these approaches on ependymomas has gained noticeable momentum. The objective of this review article was to summarize all knowledge generated by the application of modern omics approaches with regard to pediatric ependymal tumors, aiming at elucidating molecular mechanisms of oncogenesis as well as identification of pathway strategies that will help in therapeutic intervention.

Normal Mouse Brain Proteome II: Analysis of Brain Regions by High-resolution Mass Spectrometry.

BACKGROUND/AIM: Proteomics technologies provide fundamental insights into the high organizational complexity and diversity of the central nervous system. In the present study, high-resolution mass spectrometry (MS) was applied in order to identify whole-proteome content of anatomically distinct and functionally specific mouse brain regions. MATERIALS AND METHODS: Brains from eight 8-week-old C57BL/6N normal male mice were separated into seven anatomically district regions. The protein content of each region was analyzed by high-throughput nano-liquid chromatography-MS/MS Orbitrap elite technology. RESULTS: A total of 16,574 proteins were identified: 2,795 in cerebral cortex, 2,311 in olfactory bulb, 2,246 in hippocampus, 2,247 in hypothalamus, 2,250 in mid brain, 2,334 in cerebellum and 2,391 in medulla. Of these proteins, 534 were uniquely expressed in cerebral cortex, 323 in olfactory bulb, 230 in hippocampus, 272 in hypothalamus, 1,326 in mid brain, 320 in cerebellum and 268 in medulla. CONCLUSION: These data represent the most comprehensive proteomic map of the normal mouse brain and they might further be used in studies related to brain diseases, including cancer and neurodegenerative diseases.

Proteomic mapping of Drosophila transgenic elav.L-GAL4/+ brain as a tool to illuminate neuropathology mechanisms.

Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.

Detailed data from experimentally-induced mastitis in ewes, with the aim to evaluate cathelicidin-1 in milk.

Bacteriological, cytological and proteomics data have been obtained from ewes in two experiments, after intramammary challenge with Mannheimia haemolytica or Staphylococcus chromogenes. Animals were sampled before and sequentially after challenge. Conventional techniques were employed for bacterial isolation and somatic cell counting in milk samples; milk whey samples were subjected to proteomics evaluation by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. There was a correlation between leucocyte content and cathelicidin-1 spot densities in milk samples, although the protein was detected in milk earlier than the increase in leucocyte content. There was also a significant association between presence of mastitis in a mammary gland and detection of cathelicidin-1 in the respective milk sample; the degree of association was greater during the first 24 h post-inoculation. The data are further discussed in the research article "Detection of cathelicidin-1 in the milk as an early indicator of mastitis in ewes" [1].

Detection of Cathelicidin-1 in the Milk as an Early Indicator of Mastitis in Ewes.

The objective of the study was the investigation of the behaviour of cathelicidin-1 in the milk after experimental infection with two prominent bacterial pathogens (experiment 1: Mannheimia haemolytica, experiment 2: M. haemolytica and Staphylococcus chromogenes) as a potential early indicator for diagnosis of mastitis in sheep. In two experiments, after bacterial inoculation into the udder of ewes, bacteriological and cytological examinations of milk samples as well as proteomics examinations [two-dimensional gel electrophoresis analysis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis] were performed sequentially. Cathelicidin-1 was detected and spot densities obtained from PDQuest v.8.0 were recorded. Associations were calculated between cell content and spot densities as well as between presence of mastitis in a mammary gland at a given time-point and detection of cathelicidin-1 in the respective milk sample. All inoculated mammary glands developed mastitis, confirmed by the consistent bacterial isolation from mammary secretion and increased leucocyte content therein. Spot density of cathelicidin-1 in samples from inoculated glands increased 3 h post-inoculation; spot density of cathelicidin-1 in samples from inoculated glands was higher than in samples from uninoculated controls. There was clear evidence of correlation between cell content and cathelicidin-1 spot densities in milk samples. There was significant association between presence of mastitis in the mammary gland and detection of cathelicidin-1 in the respective milk sample; overall accuracy was 0.818-this was significantly greater during the first 24 h post-challenge (0.903) than after the first day (0.704). In conclusion, detection of cathelicidin-1 in milk was significantly associated with presence of mastitis in ewes. The associations were stronger during the first 24 h post-infection than after the first day. Cathelicidin-1 has the advantage that it can be a non-specific biomarker, as simply a "positive" / "negative" assessment would be sufficient.

DNA Replication Inhibitor Geminin and Retinoic Acid Signaling Participate in Complex Interactions Associated With Pluripotency.

BACKGROUND/AIM: Several links between DNA replication, pluripotency and development have been recently identified. The involvement of miRNA in the regulation of cell cycle events and pluripotency factors has also gained attention. MATERIALS AND METHODS: In the present study, we used the g:Profiler platform to analyze transcription factor binding sites, miRNA networks and protein-protein interactions to identify novel links among the aforementioned processes. RESULTS AND CONCLUSION: A complex circuitry between retinoic acid signaling, SWI/SNF components, pluripotency factors including Oct4, Sox2 and Nanog and cell cycle regulators was identified. It is suggested that the DNA replication inhibitor geminin plays a central role in this circuitry.

In Silico Identification of Antimicrobial Peptides in the Proteomes of Goat and Sheep Milk and Feta Cheese.

Milk and dairy products are a major functional food group of growing scientific and commercial interest due to their nutritional value and bioactive "load". A major fraction of the latter is attributed to milk's rich protein content and its biofunctional peptides that occur naturally during digestion. On the basis of the identified proteome datasets of milk whey from sheep and goat breeds in Greece and feta cheese obtained during previous work, we applied an in silico workflow to predict and characterise the antimicrobial peptide content of these proteomes. We utilised existing tools for predicting peptide sequences with antimicrobial traits complemented by in silico protein cleavage modelling to identify frequently occurring antimicrobial peptides (AMPs) in the gastrointestinal (GI) tract in humans. The peptides of interest were finally assessed for their stability with respect to their susceptibility to cleavage by endogenous proteases expressed along the intestinal part of the GI tract and ranked with respect to both their antimicrobial and stability scores.

Proteomics data of ovine mastitis associated with Mannheimia haemolytica.

Proteomics data have been obtained from experimental mastitis in ewes after intramammary challenge with Mannheimia haemolytica. Animals were sampled before and sequentially after challenge; blood plasma and milk whey samples were produced and were subjected to proteomics evaluation by means of two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Full protein maps and differential proteomics in sequential samples from blood plasma and milk whey of experimental ewes were presented. Post-challenge, 33 and 89 proteins were identified with differential abundance in blood plasma and milk whey, respectively. Also, 74 proteins were identified with differential abundance between the inoculated and contralateral glands. The data provide further insight in the pathogenesis of mastitis in sheep and indicate potential biomarkers for the disease. The data are further discussed in the research article "Differential quantitative proteomics study of experimental Mannheimia haemolytica mastitis in sheep" [1].