DirectMS1Quant: Ultrafast Quantitative Proteomics with MS/MS-Free Mass Spectrometry.

Recently, we presented the DirectMS1 method of ultrafast proteome-wide analysis based on minute-long LC gradients and MS1-only mass spectra acquisition. Currently, the method provides the depth of human cell proteome coverage of 2500 proteins at a 1% false discovery rate (FDR) when using 5 min LC gradients and 7.3 min runtime in total. While the standard MS/MS approaches provide 4000-5000 protein identifications within a couple of hours of instrumentation time, we advocate here that the higher number of identified proteins does not always translate into better quantitation quality of the proteome analysis. To further elaborate on this issue, we performed a one-on-one comparison of quantitation results obtained using DirectMS1 with three popular MS/MS-based quantitation methods: label-free (LFQ) and tandem mass tag quantitation (TMT), both based on data-dependent acquisition (DDA) and data-independent acquisition (DIA). For comparison, we performed a series of proteome-wide analyses of well-characterized (ground truth) and biologically relevant samples, including a mix of UPS1 proteins spiked at different concentrations into an Echerichia coli digest used as a background and a set of glioblastoma cell lines. MS1-only data was analyzed using a novel quantitation workflow called DirectMS1Quant developed in this work. The results obtained in this study demonstrated comparable quantitation efficiency of 5 min DirectMS1 with both TMT and DIA methods, yet the latter two utilized a 10-20-fold longer instrumentation time.

Upregulation of NETO2 gene in colorectal cancer.

BACKGROUND: Neuropilin and tolloid-like 2 (NETO2) is a single-pass transmembrane protein that has been shown primarily implicated in neuron-specific processes. Upregulation of NETO2 gene was also detected in several cancer types. In colorectal cancer (CRC), it was associated with tumor progression, invasion, and metastasis, and seems to be involved in epithelial-mesenchymal transition (EMT). However, the mechanism of NETO2 action is still poorly understood. RESULTS: We have revealed significant increase in the expression of NETO2 gene and deregulation of eight EMT-related genes in CRC. Four of them were upregulated (TWIST1, SNAIL1, LEF1, and FOXA2); the mRNA levels of other genes (FOXA1, BMP2, BMP5, and SMAD7) were decreased. Expression of NETO2 gene was weakly correlated with that of genes involved in the EMT process. CONCLUSIONS: We found considerable NETO2 upregulation, but no significant correlation between the expression of NETO2 and EMT-related genes in CRC. Thus, NETO2 may be involved in CRC progression, but is not directly associated with EMT.

Polyamine Catabolism Revisited: Acetylpolyamine Oxidase Plays a Minor Role due to Low Expression.

Biogenic polyamines are ubiquitous compounds. Dysregulation of their metabolism is associated with the development of various pathologies, including cancer, hyperproliferative diseases, and infections. The canonical pathway of polyamine catabolism includes acetylation of spermine and spermidine and subsequent acetylpolyamine oxidase (PAOX)-mediated oxidation of acetylpolyamines (back-conversion) or their direct efflux from the cell. PAOX is considered to catalyze a non-rate-limiting catabolic step. Here, we show that PAOX transcription levels are extremely low in various tumor- and non-tumor cell lines and, in most cases, do not change in response to altered polyamine metabolism. Its enzymatic activity is undetectable in the majority of cell lines except for neuroblastoma and low passage glioblastoma cell lines. Treatment of A549 cells with N1,N11-diethylnorspermine leads to PAOX induction, but its contribution to polyamine catabolism remains moderate. We also describe two alternative enzyme isoforms and show that isoform 4 has diminished oxidase activity and isoform 2 is inactive. PAOX overexpression correlates with the resistance of cancer cells to genotoxic antitumor drugs, indicating that PAOX may be a useful therapeutic target. Finally, PAOX is dispensable for the replication of various viruses. These data suggest that a decrease in polyamine levels is achieved predominantly by the secretion of acetylated spermine and spermidine rather than by back-conversion.

Recent Developments in Glioblastoma Therapy: Oncolytic Viruses and Emerging Future Strategies.

Glioblastoma is the most aggressive form of malignant brain tumor. Standard treatment protocols and traditional immunotherapy are poorly effective as they do not significantly increase the long-term survival of glioblastoma patients. Oncolytic viruses (OVs) may be an effective alternative approach. Combining OVs with some modern treatment options may also provide significant benefits for glioblastoma patients. Here we review virotherapy for glioblastomas and describe several OVs and their combination with other therapies. The personalized use of OVs and their combination with other treatment options would become a significant area of research aiming to develop the most effective treatment regimens for glioblastomas.

2-Deoxyglucose, an Inhibitor of Glycolysis, Enhances the Oncolytic Effect of Coxsackievirus.

Glioblastoma multiforme (GBM) is one of the most common types of brain tumor. Despite intensive research, patients with GBM have a poor prognosis due to a very high rate of relapse and significant side effects of the treatment, with a median survival of 14.6 months. Oncolytic viruses are considered a promising strategy to eliminate GBM and other types of cancer, and several viruses have already been introduced into clinical practice. However, identification of the factors that underly the sensitivity of tumor species to oncolytic viruses or that modulate their clinical efficacy remains an important target. Here, we show that Coxsackievirus B5 (CVB5) demonstrates high oncolytic potential towards GBM primary cell species and cell lines. Moreover, 2-deoxyglucose (2DG), an inhibitor of glycolysis, potentiates the cytopathic effects of CVB5 in most of the cancer cell lines tested. The cells in which the inhibition of glycolysis enhanced oncolysis are characterized by high mitochondrial respiratory activity and glycolytic capacity, as determined by Seahorse analysis. Thus, 2-deoxyglucose and other analogs should be considered as adjuvants for oncolytic therapy of glioblastoma multiforme.

De Novo Transcriptome Profiling of Brain Tissue from the Annual Killifish Nothobranchius guentheri.

Nothobranchius is a genus of small annual killifish found in Africa. Due to the relatively short lifespan, as well as easy breeding and care, Nothobranchius fish are becoming widely used as a vertebrate model system. Studying the genome and transcriptome of these fish is essential for advancing the field. In this study, we performed de novo transcriptome assembly of brain tissues from Nothobranchius guentheri using Trinity. Annotation of 104,271 potential genes (with transcripts longer than 500 bp) was carried out; for 24,967 genes (53,654 transcripts), in which at least one GO annotation was derived. We also analyzed the effect of a long-term food supplement with Torin 2, second-generation ATP-competitive inhibitor of mTOR, on the gene expression changes in brain tissue of adult N. guentheri. Overall, 1491 genes in females and 249 genes in males were differently expressed under Torin 2-supplemented diet. According to the Gene Set Enrichment Analysis (GSEA), the majority of identified genes were predominantly involved in the regulation of metabolic process, dendritic spine maintenance, circadian rhythms, retrotransposition, and immune response. Thus, we have provided the first transcriptome assembly and assessed the differential gene expression in response to exposure to Torin 2, which allow a better understanding of molecular changes in the brain tissues of adult fish in the mTOR pathway inhibition.

Cultivation of Cells in a Physiological Plasmax Medium Increases Mitochondrial Respiratory Capacity and Reduces Replication Levels of RNA Viruses.

Changes in metabolic pathways are often associated with the development of various pathologies including cancer, inflammatory diseases, obesity and metabolic syndrome. Identification of the particular metabolic events that are dysregulated may yield strategies for pharmacologic intervention. However, such studies are hampered by the use of classic cell media that do not reflect the metabolite composition that exists in blood plasma and which cause non-physiological adaptations in cultured cells. In recent years two groups presented media that aim to reflect the composition of human plasma, namely human plasma-like medium (HPLM) and Plasmax. Here we describe that, in four different mammalian cell lines, Plasmax enhances mitochondrial respiration. This is associated with the formation of vast mitochondrial networks and enhanced production of reactive oxygen species (ROS). Interestingly, cells cultivated in Plasmax displayed significantly less lysosomes than when any standard media were used. Finally, cells cultivated in Plasmax support replication of various RNA viruses, such as hepatitis C virus (HCV) influenza A virus (IAV), severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and several others, albeit at lower levels and with delayed kinetics. In conclusion, studies of metabolism in the context of viral infections, especially those concerning mitochondria, lysosomes, or redox systems, should be performed in Plasmax medium.

Multi-Omics Analysis of Glioblastoma Cells' Sensitivity to Oncolytic Viruses.

Oncolytic viruses have gained momentum in the last decades as a promising tool for cancer treatment. Despite the progress, only a fraction of patients show a positive response to viral therapy. One of the key variable factors contributing to therapy outcomes is interferon-dependent antiviral mechanisms in tumor cells. Here, we evaluated this factor using patient-derived glioblastoma multiforme (GBM) cultures. Cell response to the type I interferons' (IFNs) stimulation was characterized at mRNA and protein levels. Omics analysis revealed that GBM cells overexpress interferon-stimulated genes (ISGs) and upregulate their proteins, similar to the normal cells. A conserved molecular pattern unambiguously differentiates between the preserved and defective responses. Comparing ISGs' portraits with titration-based measurements of cell sensitivity to a panel of viruses, the "strength" of IFN-induced resistance acquired by GBM cells was ranked. The study demonstrates that suppressing a single ISG and encoding an essential antiviral protein, does not necessarily increase sensitivity to viruses. Conversely, silencing IFIT3 and PLSCR1 genes in tumor cells can negatively affect the internalization of vesicular stomatitis and Newcastle disease viruses. We present evidence of a complex relationship between the interferon response genes and other factors affecting the sensitivity of tumor cells to viruses.

NETO2 Is Deregulated in Breast, Prostate, and Colorectal Cancer and Participates in Cellular Signaling.

The NETO2 gene (neuropilin and tolloid-like 2) encodes a protein that acts as an accessory subunit of kainate receptors and is predominantly expressed in the brain. Upregulation of NETO2 has been observed in several tumors; however, its role in tumorigenesis remains unclear. In this study, we investigated NETO2 expression in breast, prostate, and colorectal cancer using quantitative PCR (qPCR), as well as the effect of shRNA-mediated NETO2 silencing on transcriptome changes in colorectal cancer cells. In the investigated tumors, we observed both increased and decreased NETO2 mRNA levels, presenting no correlation with the main clinicopathological characteristics. In HCT116 cells, NETO2 knockdown resulted in the differential expression of 17 genes and 2 long non-coding RNAs (lncRNAs), associated with the upregulation of circadian rhythm and downregulation of several cancer-associated pathways, including Wnt, transforming growth factor (TGF)-ß, Janus kinase (JAK)-signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathways. Furthermore, we demonstrated the possibility to utilize a novel model organism, short-lived fish Nothobranchius furzeri, for evaluating NETO2 functions. The ortholog neto2b in N. furzeri demonstrated a high similarity in nucleotide and amino acid sequences with human NETO2, as well as was characterized by stable expression in various fish tissues. Collectively, our findings demonstrate the deregulation of NETO2 in the breast, prostate, and colorectal cancer and its participation in the tumor development primarily through cellular signaling.

Multiple paragangliomas: a case report.

BACKGROUND: Carotid and vagal paragangliomas (CPGLs and VPGLs) are rare neoplasms that arise from the paraganglia located at the bifurcation of carotid arteries and vagal trunk, respectively. Both tumors can occur jointly as multiple paragangliomas accounting for approximately 10 to 20% of all head and neck paragangliomas. However, molecular and genetic mechanisms underlying the pathogenesis of multiple paragangliomas remain elusive. CASE PRESENTATION: We report a case of multiple paragangliomas in a patient, manifesting as bilateral CPGL and unilateral VPGL. Tumors were revealed via computed tomography and ultrasound study and were resected in two subsequent surgeries. Both CPGLs and VPGL were subjected to immunostaining for succinate dehydrogenase (SDH) subunits and exome analysis. A likely pathogenic germline variant in the SDHD gene was indicated, while likely pathogenic somatic variants differed among the tumors. CONCLUSIONS: The identified germline variant in the SDHD gene seems to be a driver in the development of multiple paragangliomas. However, different spectra of somatic variants identified in each tumor indicate individual molecular mechanisms underlying their pathogenesis.

Exome analysis of carotid body tumor.

BACKGROUND: Carotid body tumor (CBT) is a form of head and neck paragangliomas (HNPGLs) arising at the bifurcation of carotid arteries. Paragangliomas are commonly associated with germline and somatic mutations involving at least one of more than thirty causative genes. However, the specific functionality of a number of these genes involved in the formation of paragangliomas has not yet been fully investigated. METHODS: Exome library preparation was carried out using Nextera® Rapid Capture Exome Kit (Illumina, USA). Sequencing was performed on NextSeq 500 System (Illumina). RESULTS: Exome analysis of 52 CBTs revealed potential driver mutations (PDMs) in 21 genes: ARNT, BAP1, BRAF, BRCA1, BRCA2, CDKN2A, CSDE1, FGFR3, IDH1, KIF1B, KMT2D, MEN1, RET, SDHA, SDHB, SDHC, SDHD, SETD2, TP53BP1, TP53BP2, and TP53I13. In many samples, more than one PDM was identified. There are also 41% of samples in which we did not identify any PDM; in these cases, the formation of CBT was probably caused by the cumulative effect of several not highly pathogenic mutations. Estimation of average mutation load demonstrated 6-8 mutations per megabase (Mb). Genes with the highest mutation rate were identified. CONCLUSIONS: Exome analysis of 52 CBTs for the first time revealed the average mutation load for these tumors and also identified potential driver mutations as well as their frequencies and co-occurrence with the other PDMs.

hsa-miR-4485 regulates mitochondrial functions and inhibits the tumorigenicity of breast cancer cells.

The modulation of mitochondrial functions is important for maintaining cellular homeostasis. Mitochondria essentially depend on the import of RNAs and proteins encoded by the nuclear genome. MicroRNAs encoded in the nucleus can translocate to mitochondria and target the genome, affecting mitochondrial function. Here, we analyzed the role of miR-4485 in the regulation of mitochondrial functions. We showed that miR-4485 translocated to mitochondria where its levels varied in response to different stress conditions. A direct binding of miR-4485 to mitochondrial 16S rRNA was demonstrated. MiR-4485 regulated the processing of pre-rRNA at the 16S rRNA-ND1 junction and the translation of downstream transcripts. MiR-4485 modulated mitochondrial complex I activity, the production of ATP, ROS levels, caspase-3/7 activation, and apoptosis. Transfection of a miR-4485 mimic downregulated the expression of regulatory glycolytic pathway genes and reduced the clonogenic ability of breast cancer cells. Ectopic expression of miR-4485 in MDA-MB-231 breast carcinoma cells decreased the tumorigenicity in a nude mouse xenograft model. Furthermore, levels of both precursor and mature miR-4485 are decreased in tumor tissue of breast cancer patients. We conclude that the mitochondria-targeted miR-4485 may act as a tumor suppressor in breast carcinoma cells by negatively regulating mitochondrial RNA processing and mitochondrial functions.

The Dysregulation of Polyamine Metabolism in Colorectal Cancer Is Associated with Overexpression of c-Myc and C/EBPß rather than Enterotoxigenic Bacteroides fragilis Infection.

Colorectal cancer is one of the most common cancers in the world. It is well known that the chronic inflammation can promote the progression of colorectal cancer (CRC). Recently, a number of studies revealed a potential association between colorectal inflammation, cancer progression, and infection caused by enterotoxigenic Bacteroides fragilis (ETBF). Bacterial enterotoxin activates spermine oxidase (SMO), which produces spermidine and H2O2 as byproducts of polyamine catabolism, which, in turn, enhances inflammation and tissue injury. Using qPCR analysis, we estimated the expression of SMOX gene and ETBF colonization in CRC patients. We found no statistically significant associations between them. Then we selected genes involved in polyamine metabolism, metabolic reprogramming, and inflammation regulation and estimated their expression in CRC. We observed overexpression of SMOX, ODC1, SRM, SMS, MTAP, c-Myc, C/EBPß (CREBP), and other genes. We found that two mediators of metabolic reprogramming, inflammation, and cell proliferation c-Myc and C/EBPß may serve as regulators of polyamine metabolism genes (SMOX, AZIN1, MTAP, SRM, ODC1, AMD1, and AGMAT) as they are overexpressed in tumors, have binding site according to ENCODE ChIP-Seq data, and demonstrate strong coexpression with their targets. Thus, increased polyamine metabolism in CRC could be driven by c-Myc and C/EBPß rather than ETBF infection.