Cellular Modifications Research Articles

Time courses of post-injury mitochondrial oxidative damage and respiratory dysfunction and neuronal cytoskeletal degradation in a rat model of focal traumatic brain injury

Traumatic brain injury (TBI) results in rapid reactive oxygen species (ROS) production and oxidative damage to essential brain cellular components leading to neuronal dysfunction and cell death. It is increasingly appreciated that a major player in TBI-induced oxidative damage is the reactive nitrogen species (RNS) peroxynitrite (PN) which is produced in large part in injured brain mitochondria. Once formed, PN decomposes into highly reactive free radicals that trigger membrane lipid peroxidation (LP) of polyunsaturated fatty acids (e.g. arachidonic acid) and protein nitration (3-nitrotyrosine, 3-NT) in mitochondria and other cellular membranes causing various functional impairments to mitochondrial oxidative phosphorylation and calcium (Ca2+) buffering capacity. The LP also results in the formation of neurotoxic reactive aldehyde byproducts including 4-hydroxynonenal (4-HNE) and propenal (acrolein) which exacerbates ROS/RNS production and oxidative protein damage in the injured brain. Ultimately, this results in intracellular Ca2+ overload that activates proteolytic degradation of α-spectrin, a neuronal cytoskeletal protein. Therefore, the aim of this study was to establish the temporal evolution of mitochondrial dysfunction, oxidative damage and cytoskeletal degradation in the brain following a severe controlled cortical impact (CCI) TBI in young male adult rats. In mitochondria isolated from an 8 mm diameter cortical punch including the 5 mm wide impact site and their respiratory function studied ex vivo, we observed an initial decrease in complex I and II mitochondrial bioenergetics within 3 h (h). For complex I bioenergetics, this partially recovered by 12–16 h, whereas for complex II respiration the recovery was complete by 12 h. During the first 24 h, there was no evidence of an injury-induced increase in LP or protein nitration in mitochondrial or cellular homogenates. However, beginning at 24 h, there was a gradual secondary decline in complex I and II respiration that peaked at 72 h. post-TBI that coincided with progressive peroxidation of mitochondrial and cellular lipids, protein nitration and protein modification by 4-HNE and acrolein. The oxidative damage and respiratory failure paralleled an increase in Ca2+-induced proteolytic degradation of the neuronal cytoskeletal protein α-spectrin indicating a failure of intracellular Ca2+ homeostasis. These findings of a surprisingly delayed peak in secondary injury, suggest that the therapeutic window and needed treatment duration for certain antioxidant treatment strategies following CCI-TBI in rodents may be longer than previously believed.

Forecasting of Interregional Migration Flows

The article explores the problem of interregional migration flows modelling. As a rule, the existing models of interregional migration flows use the aggregated data and do not take into consideration the fact that a decision to relocate is formed and taken at the micro-level, at the level of households. The purpose of the present research is to develop the forecasting method for interregional migration flows of the region taking into consideration the behaviour of households at the micro-level. The research tests the hypothesis that the modelling of household behaviours at the local level as regards taking a decision to relocate to other regions, which takes into account the existing interactions with relatives and other communities, allows to obtain adequate forecasts of interregional migration flows. To forecast the interregional migration flows of the region, we develop a methodological approach and software solution, based on cellular automaton model modification proposed in the current work and on econometric models of birth and death processes, which have been tested on the Primorsky Krai data. The authors’ model of the cellular automaton is a kind of the combined probabilistic cellular automaton in which the condition of each cell changes depending on a condition of four closest neighbours (von Neumann vicinity) and four cells chosen in a random way. The article builds a mid-term forecast of Primorsky Krai interregional migration flows. The research demonstrates the possibility and reasonability of modelling the interregional migration flows by cellular automatons. The highly perspective direction of the research is the modelling of other macroeconomic processes based on modelling by cellular automatons of a behaviour of households, companies and other economic entities at the local level.

Fabrication of nitrogen-doped carbon dots for screening the purine metabolic disorder in human fluids

Fabrication of nitrogen-doped carbon dots (N-CDs) electrode for the screening of purine metabolic disorder was described in this paper. Peroxynitrite is a short-lived oxidant species that is a potent inducer of cell death. Uric acid (UA) can scavenge the peroxynitrite to avoid the formation of nitrotyrosine, which is formed from the reaction between peroxynitrite and tyrosine (Try). Scavenging the peroxynitrite avoids the inactivation of cellular enzymes and modification of the cytoskeleton. Reduced level of UA decreases the ability of the body from preventing the peroxynitrite toxicity. On the other hand, the abnormal level of UA leads to gout and hyperuricemia. Allopurinol (AP) is administered in UA lowering therapy. Thus, the simultaneous determination of UA, Try and AP using N-CDs modified glassy carbon (GC) electrode was demonstrated for the first time. Initially, N-CDs were prepared from L-asparagine by pyrolysis and characterized by different spectroscopic and microscopic techniques. The HR-TEM image shows that the average size of the prepared N-CDs was 1.8±0.03nm. Further, the N-CDs were directly attached on GC electrode by simple immersion, follows Micheal's nucleophilic addition. XPS of N-CDs shows a peak at 285.3eV corresponds to the formation of C–N bond. The GC/N-CDs electrode shows higher electrocatalytic activity towards UA, Tyr and AP by not only shifting their oxidation potentials toward less positive potential but also enhanced their oxidation currents in contrast to bare GC electrode. The GC/N-CDs electrode shows the limit of detection of 13×10−10M (S/N=3) and the sensitivity of 924μAmM−1cm−2 towards the determination of UA. Finally, the N-CDs modified electrode was utilized for the determination of UA, Tyr and AP in human blood serum and urine samples.

HIV signaling through CD4 and CCR5 activates Rho family GTPases that are required for optimal infection of primary CD4+ T cells

BackgroundHIV-1 hijacks host cell machinery to ensure successful replication, including cytoskeletal components for intracellular trafficking, nucleoproteins for pre-integration complex import, and the ESCRT pathway for assembly and budding. It is widely appreciated that cellular post-translational modifications (PTMs) regulate protein activity within cells; however, little is known about how PTMs influence HIV replication. Previously, we reported that blocking deacetylation of tubulin using histone deacetylase inhibitors promoted the kinetics and efficiency of early post-entry viral events. To uncover additional PTMs that modulate entry and early post-entry stages in HIV infection, we employed a flow cytometric approach to assess a panel of small molecule inhibitors on viral fusion and LTR promoter-driven gene expression.ResultsWhile viral fusion was not significantly affected, early post-entry viral events were modulated by drugs targeting multiple processes including histone deacetylation, methylation, and bromodomain inhibition. Most notably, we observed that inhibitors of the Rho GTPase family of cytoskeletal regulators—including RhoA, Cdc42, and Rho-associated kinase signaling pathways—significantly reduced viral infection. Using phosphoproteomics and a biochemical GTPase activation assay, we found that virion-induced signaling via CD4 and CCR5 activated Rho family GTPases including Rac1 and Cdc42 and led to widespread modification of GTPase signaling-associated factors.ConclusionsTogether, these data demonstrate that HIV signaling activates members of the Rho GTPase family of cytoskeletal regulators that are required for optimal HIV infection of primary CD4+ T cells.

Viral Interplay with the Host Sumoylation System.

Viruses have evolved elaborate means to regulate diverse cellular pathways in order to create a cellular environment that facilitates viral survival and reproduction. This includes enhancing viral macromolecular synthesis and assembly, as well as preventing antiviral responses, including intrinsic, innate, and adaptive immunity. There are numerous mechanisms by which viruses mediate their effects on the host cell, and this includes targeting various cellular post-translational modification systems, including sumoylation. The wide-ranging impact of sumoylation on cellular processes such as transcriptional regulation, apoptosis, stress response, and cell cycle control makes it an attractive target for viral dysregulation. To date, proteins from both RNA and DNA virus families have been shown to be modified by SUMO conjugation, and this modification appears critical for viral protein function. More interestingly, members of the several viral families have been shown to modulate sumoylation, including papillomaviruses, adenoviruses, herpesviruses, orthomyxoviruses, filoviruses, and picornaviruses. This chapter will focus on mechanisms by which sumoylation both impacts human viruses and is used by viruses to promote viral infection and disease.

Rerouting the traffic from a virus perspective.

Viruses are important human and animal pathogens causing disease that affect global health and the economy. One outcome of many virus infections is the regulation of cellular trafficking machinery. Viral proteins recruit and interact with cellular trafficking proteins to divert the normal trafficking of key proteins or to induce the formation of novel membrane structures in the host cell. These alterations often increase replication efficiency by mislocalizing immune regulators or restriction factors ot by creating platforms for replication and assembly of new virus particles. Our knowledge of how viruses interact with the cellular trafficking machinery is still limited and furthering this understanding will be important for the future development of prophylactic and therapeutic treatments. This review provides a glimpse of the types of interplay between viral and cellular factors that result in a disruption of cellular trafficking or modifications to cellular membranes.

The Identification of Key Genes and Pathways in Glioma by Bioinformatics Analysis.

Glioma is the most common malignant tumor in the central nervous system. This study aims to explore the potential mechanism and identify gene signatures of glioma. The glioma gene expression profile GSE4290 was analyzed for differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were applied for the enriched pathways. A protein-protein interaction (PPI) network was constructed to find the hub genes. Survival analysis was conducted to screen and validate critical genes. In this study, 775 downregulated DEGs were identified. GO analysis demonstrated that the DEGs were enriched in cellular protein modification, regulation of cell communication, and regulation of signaling. KEGG analysis indicated that the DEGs were enriched in the MAPK signaling pathway, endocytosis, oxytocin signaling, and calcium signaling. PPI network and module analysis found 12 hub genes, which were enriched in synaptic vesicle cycling rheumatoid arthritis and collecting duct acid secretion. The four key genes CDK17, GNA13, PHF21A, and MTHFD2 were identified in both generation (GSE4412) and validation (GSE4271) dataset, respectively. Regression analysis showed that CDK13, PHF21A, and MTHFD2 were independent predictors. The results suggested that CDK17, GNA13, PHF21A, and MTHFD2 might play important roles and potentially be valuable in the prognosis and treatment of glioma.

Minimum hyaluronic acid (HA) modified magnetic nanocrystals with less facilitated cancer migration and drug resistance for targeting CD44 abundant cancer cells by MR imaging.

We report minimal amount of hyaluronic acid (HA) conjugated magnetic nanocrystals (mHMs) for targeted imaging of CD44 abundant breast cancer cells via MRI. These mHMs lead to less induced cancer migration and drug resistance, which is distinct from conventional approaches using nanoplatforms (imaging or therapeutic systems) that are completely covered with HA. To synthesize mHMs, magnetic nanocrystals (MNCs), as MRI contrast agents, were encapsulated mostly with polysorbate 80 (P80, non-reactive to HA) and partially with aminated P80 (reactive to HA). This system enabled conjugation of an immensely diminished amount of HA onto MNCs. While these nanoparticles maintained good CD44 targeted imaging efficacy, they also showed no cytotoxicity and colloidal stability. We varied the HA ratios on an equal amount of MNCs and identified that when more HA was attached on nanoparticles, there was more facilitated cancer migration and drug resistant potentials. We chose the lowest amount of HA conjugated mHMs (mHM1) and demonstrated that mHM1 selectively diagnosed tumor regions in vivo. We believe that the technique described herein can be applied to various applications using HA to detect CD44 abundant cancer cell lines and offer a basis to understand the interaction between the cellular response and surface modification of nanoparticles.

Cellular Redox State Modifications Induced by Bioactive Fe(III)-Cyclophane Complexes Approaching to Selective Therapy Drug Design

Cellular Redox State Modifications Induced by Bioactive Fe(III)-Cyclophane Complexes Approaching to Selective Therapy Drug Design

Novel β-carboline-quinazolinone hybrids disrupt Leishmania donovani redox homeostasis and show promising antileishmanial activity

Visceral Leishmaniasis is a deadly parasitic disease caused by Leishmania donovani. Paucity exists in the discovery of novel chemotherapeutics against Leishmaniasis. In this study, we synthesized a natural product inspired Diversity Oriented Synthesis library of L. donovani Trypanothione reductase (LdTR) inhibitor β-carboline-quinazolinone hybrids, which are different in stereochemical architecture and diverse in the bioactive chemical space. It is noteworthy that chirality affects drug-to-protein binding affinity since proteins in any living system are present only in one of the chiral forms. Upon evaluation of the hybrids, one of the chiral forms i.e. Compound 1 showed profound cytotoxic effect in micromolar range as compared to its other chiral form i.e. Compound 2. In-silico docking studies confirmed high binding efficiency of Compound 1 with the catalytic pocket of LdTR. Treatment of L. donovani parasites with Compound 1 inhibits LdTR activity, induces imbalance in redox homeostasis by enhancing ROS, disrupts the mitochondrial membrane potential, modifies actin polymerization and alters the surface topology and architecture. All these cellular modifications eventually led to apoptosis-like death of promastigotes. Furthermore, we synthesized the analogues of Compound 1 and found that these compounds show profound antileishmanial activity in the nanomolar range both in promastigotes and intracellular amastigotes. The enhanced inhibitory potential of these compounds was further supported by in-silico analysis of protein-ligand interactions which revealed high binding efficiency towards the catalytic pocket of LdTR. Taken together, this study reports the serendipitous discovery of β-carboline-quinazolinone hybrids with enhanced antileishmanial activity along with the in-depth structure-activity relationships and mechanism of action of these analogues.

Protective effects of resveratrol against mancozeb induced apoptosis damage in mouse oocytes.

Mancozeb, a mixture of ethylene-bis-dithiocarbamate manganese and zinc salts, is one of the most widely used fungicides in agriculture. Mancozeb could lead to mitochondria dysfunction, cellular anti-oxidation enzymes depletion and apoptotic pathways activation. Previous studies indicated the exposure of mancozeb through mother would lead to irregular estrous cycles, decreased progesterone levels, reduced litter sizes, and more frequent delivery of dead fetuses. In this study, we investigated mancozeb inducing reproductive toxicity, especially focusing on its apoptotic effect and epigenetic modifications. We also showed that resveratrol, a kind of phytoalexin found in peanuts and grapes, can alleviate mancozeb's adverse effects, such as declined fertility, decreased ovary weight and primary follicles. Besides, mancozeb treated oocytes displayed suboptimal developmental competence and this can also be improved by treatment of resveratrol. More detailed investigation of these processes revealed that mancozeb increased reactive oxygen species, causing cell apoptosis and abnormal epigenetic modifications, and resveratrol can block these cytotoxic changes. Collectively, our results showed that resveratrol can alleviate mancozeb induced infertility and this was mainly through the correction of apoptotic tendency and the abnormity of cellular epigenetic modification.

Invited commentary

Ever since the first subcutaneous veins were used as arterial substitutes in 1949, surgeons have been searching for new and better ways to procure venous conduits. Technical and biochemical methods have included “no touch” harvesting, gentle inflation with heparinized saline or lactate, infusion with smooth muscle cell dilators or cellular preservatives, endoscopic excision, and/or genetic modification through vector-based gene transfer. To date, no method had conclusively been found to be superior to the combination of an olive-tipped catheter, room temperature heparinized saline, and a good surgeon. Even with superlative technique, however, vein graft narrowing still occurs at an alarming rate, as 30% to 50% of all veins will respond to arterialization by pathologic thickening resulting in hemodynamically significant stenosis. Certainly, a major impetus for the development of vein graft neointimal hyperplasia is the abrupt increase in diameter and wall tension that accompanies exposure of thin, systemic veins to the intra-arterial environment. In their in vivo animal study of vein grafting presented herein, Salinas et al sought to limit vein graft dilatation by pre-emptively cross-linking venous collagen through the process of photochemical tissue passivation (PTP). Treated vein grafts were less dilated by exposure to intra-arterial pressure at the time of grafting and, after 4 weeks, had thickened significantly less than their untreated counterparts. Although the actual flow lumens of treated and untreated grafts appeared similar, PTP produced a thinner, more pliable graft, with profoundly attenuated cellular activation and proliferation. It remains to be seen whether PTP would be similarly effective in more muscular saphenous veins and whether limitation of their “natural” expansion might lead to increased rates of thrombosis and failure. Nonetheless, the authors provide another convincing experimental example of how vein grafts pathologically respond to arterialization and how modulation of that response might prove therapeutically useful. Prevention of vein graft intimal hyperplasia with photochemical tissue passivationJournal of Vascular SurgeryVol. 65Issue 1PreviewSaphenous vein is the conduit of choice for bypass grafting. Saphenous vein grafts have poor long-term patency rates because of intimal hyperplasia (IH) and subsequent accelerated atherosclerosis. One of the primary triggers of IH is endothelial injury resulting from excessive dilation of the vein after exposure to arterial pressures. Photochemical tissue passivation (PTP) is a technology that cross-links adventitial collagen by a light-activated process, which limits dilation by improving vessel compliance. Full-Text PDF Open Archive

A possible S-glutathionylation of specific proteins by glyoxalase II: An in vitro and in silico study.

This article reports for the first time a possible additional role of Glo2 that, after interacting with a target protein, is able to promote S-glutathionylation using its natural substrate SLG, a glutathione derived compound. In this perspective, Glo2 can play a new important regulatory role inS-glutathionylation, acquiring further significance in cellular post-translational modifications of proteins.

Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinases inInflammation and Fibrosis of Skeletal Muscles.

In skeletal muscles, levels and activity of Matrix MetalloProteinases (MMPs) and Tissue Inhibitors of MetalloProteinases (TIMPs) have been involved in myoblast migration, fusion and various physiological and pathological remodeling situations including neuromuscular diseases. This has opened perspectives for the use of MMPs’ overexpression to improve the efficiency of cell therapy in muscular dystrophies and resolve fibrosis. Alternatively, inhibition of individual MMPs in animal models of muscular dystrophies has provided evidence of beneficial, dual or adverse effects on muscle morphology or function. We review here the role played by MMPs/TIMPs in skeletal muscle inflammation and fibrosis, two major hurdles that limit the success of cell and gene therapy. We report and analyze the consequences of genetic or pharmacological modulation of MMP levels on the inflammation of skeletal muscles and their repair in light of experimental findings. We further discuss how the interplay between MMPs/TIMPs levels, cytokines/chemokines, growth factors and permanent low-grade inflammation favor cellular and molecular modifications resulting in fibrosis.

Diet-sourced carbon-based nanoparticles induce lipid alterations in tissues of zebrafish (Danio rerio) with genomic hypermethylation changes in brain.

With rising environmental levels of carbon-based nanoparticles (CBNs), there is an urgent need to develop an understanding of their biological effects in order to generate appropriate risk assessment strategies. Herein, we exposed zebrafish via their diet to one of four different CBNs: C60 fullerene (C60), single-walled carbon nanotubes (SWCNT), short multi-walled carbon nanotubes (MWCNTs) or long MWCNTs. Lipid alterations in male and female zebrafish were explored post-exposure in three target tissues (brain, gonads and gastrointestinal tract) using ‘omic’ procedures based in liquid chromatography coupled with mass spectrometry (LC-MS) data files. These tissues were chosen as they are often target tissues following environmental exposure. Marked alterations in lipid species are noted in all three tissues. To further explore CBN-induced brain alterations, Raman microspectroscopy analysis of lipid extracts was conducted. Marked lipid alterations are observed with males responding differently to females; in addition, there also appears to be consistent elevations in global genomic methylation. This latter observation is most profound in female zebrafish brain tissues post-exposure to short MWCNTs or SWCNTs (P < 0.05). This study demonstrates that even at low levels, CBNs are capable of inducing significant cellular and genomic modifications in a range of tissues. Such alterations could result in modified susceptibility to other influences such as environmental exposures, pathology and, in the case of brain, developmental alterations.

Induction and Blockade of Adolescent Cocaine-Induced Habits

BackgroundCocaine use during adolescence increases vulnerability to drug dependence and decreases the likelihood that individuals will seek treatment as adults. Understanding how early-life cocaine exposure influences decision-making processes in adulthood is thus critically important. MethodsAdolescent or adult mice were exposed to subchronic cocaine, then behavioral sensitivity to changes in the predictive relationship between actions and their consequences was tested. Dendritic spines on the principal pyramidal neurons of the orbitofrontal prefrontal cortex (oPFC) were also imaged and enumerated. To determine whether cytoskeletal regulatory systems in the oPFC influenced decision-making strategies, we then inhibited the activity of Abl family and Rho kinases as well as NR2B-containing N-methyl-D-aspartate receptors. We also attempted to block the reinstatement of cocaine seeking in cocaine self-administering mice. ResultsAdult mice with a history of subchronic cocaine exposure in adolescence engaged habit-based response strategies at the expense of goal-directed decision-making strategies and had fewer dendritic spines in the oPFC. Inhibition of the cytoskeletal regulatory Abl family kinases in the oPFC recapitulated these neurobehavioral deficiencies, whereas Rho kinase inhibition corrected response strategies. Additionally, the NR2B-selective N-methyl-D-aspartate receptor antagonists ifenprodil and CP-101,606 blocked cocaine-induced habits; this was dependent on Abl family signaling in the oPFC. Ifenprodil also mitigated cue-induced reinstatement of cocaine seeking in mice self-administering cocaine. ConclusionsWe suggest that adolescent cocaine exposure confers a bias toward habit-based behavior in adulthood via long-term cellular structural modifications in the oPFC. Treatments aimed at mitigating the durable consequences of early-life cocaine use may benefit from targeting cytoskeletal regulatory systems.

Degradation of Redox-Sensitive Proteins including Peroxiredoxins and DJ-1 is Promoted by Oxidation-induced Conformational Changes and Ubiquitination.

Reactive oxygen species (ROS) are key molecules regulating various cellular processes. However, what the cellular targets of ROS are and how their functions are regulated is unclear. This study explored the cellular proteomic changes in response to oxidative stress using H2O2 in dose- and recovery time-dependent ways. We found discernible changes in 76 proteins appearing as 103 spots on 2D-PAGE. Of these, Prxs, DJ-1, UCH-L3 and Rla0 are readily oxidized in response to mild H2O2 stress, and then degraded and active proteins are newly synthesized during recovery. In studies designed to understand the degradation process, multiple cellular modifications of redox-sensitive proteins were identified by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry and the oxidative structural changes of Prx2 explored employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that hydrogen/deuterium exchange rate increased in C-terminal region of oxidized Prx2, suggesting the exposure of this region to solvent under oxidation. We also found that Lys191 residue in this exposed C-terminal region of oxidized Prx2 is polyubiquitinated and the ubiquitinated Prx2 is readily degraded in proteasome and autophagy. These findings suggest that oxidation-induced ubiquitination and degradation can be a quality control mechanism of oxidized redox-sensitive proteins including Prxs and DJ-1.

Cancer: fundamentals behind pH targeting and the double-edged approach.

The highly regulated pH of cells and the less-regulated pH of the surrounding extracellular matrix (ECM) is the result of a delicate balance between metabolic processes and proton production, proton transportation, chemical buffering, and vascular removal of waste products. Malignant cells show a pronounced increase in metabolic processes where the 10- to 15-fold rise in glucose consumption is only the tip of the iceberg. Aerobic glycolysis (Warburg effect) is one of the hallmarks of cancer metabolism that implies excessive production of protons, which if stayed inside the cells would result in fatal intracellular acidosis (maintaining a strict acid–base balance is essential for the survival of eukaryotic cells). Malignant cells solve this problem by increasing mechanisms of proton transportation which expel the excess acidity. This allows cancer cells to keep a normal intracellular pH, or even overshooting this mechanism permits a slightly alkaline intracellular tendency. The proton excess expelled from malignant cells accumulates in the ECM, where chronic hypoxia and relative lack of enough blood vessels impede adequate proton clearance, thus creating an acidic microenvironment. This microenvironment is quite heterogeneous due to the tumor’s metabolic heterogeneity and variable degrees of hypoxia inside the tumor mass. The acidic environment (plus other necessary cellular modifications) stimulates migration and invasion and finally intravasation of malignant cells which eventually may result in metastasis. Targeting tumor pH may go in two directions: 1) increasing extracellular pH which should result in less migration, invasion, and metastasis; and 2) decreasing intracellular pH which may result in acidic stress and apoptosis. Both objectives seem achievable at the present state of the art with repurposed drugs. This hypothesis analyzes the altered pH of tumors and its implications for progression and metastasis and also possible repurposed drug combinations targeting this vulnerable side of cancer development. It also analyzes the double-edged approach, which consists in pharmacologically increasing intracellular proton production and simultaneously decreasing proton extrusion creating intracellular acidity, acid stress, and eventual apoptosis.

Molecular Pap smear: HPV genotype and DNA methylation of ADCY8, CDH8, and ZNF582 as an integrated biomarker for high-grade cervical cytology.

BackgroundThe Pap smear has remained the foundation for cervical cancer screening for over 70 years. With advancements in molecular diagnostics, primary high-risk human papillomavirus (hrHPV) screening has recently become an accepted stand-alone or co-test with conventional cytology. However, both diagnostic tests have distinct limitations. The aim of this study was to determine the association between HPV genotypes and cellular epigenetic modifications in three grades of cervical cytology for screening biomarker discovery.MethodsThis prospective, cross-sectional study used residual liquid-based cytology samples for HPV genotyping and epigenetic analysis. Extracted DNA was subjected to parallel polymerase chain reactions using three primer sets (MY09/11, FAP59/64, E6-E7 F/B) for HPV DNA amplification. HPV+ samples were genotyped by DNA sequencing. Promoter methylation of four candidate tumor suppressor genes (adenylate cyclase 8 (ADCY8), cadherin 8, type 2 (CDH8), MGMT, and zinc finger protein 582 (ZNF582)) out of 48 genes screened was quantified by bisulfite-pyrosequencing of genomic DNA. Independent validation of methylation profiles was performed by analyzing data from cervical cancer cell lines and clinical samples from The Cancer Genome Atlas (TCGA).ResultsTwo hundred seventy-seven quality cytology samples were analyzed. HPV was detected in 31/100 (31 %) negative for intraepithelial lesion or malignancy (NILM), 95/100 (95 %) low-grade squamous intraepithelial lesion (LSIL), and 71/77 (92 %) high-grade squamous intraepithelial lesion (HSIL) samples. The proportion of IARC-defined carcinogenic HPV types in sequenced samples correlated with worsening grade: NILM 7/29 (24 %), LSIL 53/92 (58 %), and HSIL 65/70 (93 %). Promoter methylation of ADCY8, CDH8, and ZNF582 was measured in 170 samples: NILM (N = 33), LSIL (N = 70), and HSIL (N = 67) also correlated with worsening grade. Similar hypermethylation patterns were found in cancer cell lines and TCGA samples. The combination of four biomarkers, i.e., HPV genotype and three-gene promoter methylation, predicted HSIL (AUC 0.89) better than HPV alone (AUC 0.74) by logistic regression and probabilistic modeling.ConclusionsHPV genotype and DNA methylation of ADCY8, CDH8, and ZNF582 are correlated with cytological grade. Collectively, these biomarkers may serve as a molecular classifier of Pap smears.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0263-9) contains supplementary material, which is available to authorized users.

Accumulation of Deleterious Mutations on the Neo-Y Chromosome of Japan Sea Stickleback (Gasterosteus nipponicus).

Degeneration of Y chromosomes is a common evolutionary path of XY sex chromosome systems. Recent genomic studies in flies and plants have revealed that even young neo-sex chromosomes with the age of a few million years show signs of Y degeneration, such as the accumulation of nonsense and frameshift mutations. However, it remains unclear whether neo-Y chromosomes also show rapid degeneration in fishes, which often have homomorphic sex chromosomes. Here, we investigated whether a neo-Y chromosome of Japan Sea stickleback (Gasterosteus nipponicus), which was formed by a Y-autosome fusion within the last 2 million years, accumulates deleterious mutations. Our previous genomic analyses did not detect excess nonsense and frameshift mutations on the Japan Sea stickleback neo-Y. In the present study, we found that the nonrecombining region of the neo-Y near the fusion end has accumulated nonsynonymous mutations altering amino acids of evolutionarily highly conserved residues. Enrichment of gene ontology terms related to protein phosphorylation and cellular protein modification process was found in the genes with potentially deleterious mutations on the neo-Y. These results suggest that the neo-Y of the Japan Sea stickleback has already accumulated mutations that may impair protein functions.