Chemical and enzymatic modifications of 5-methylcytosine at the intersection of DNA damage, repair, and epigenetic reprogramming.

The DNA of all living organisms is persistently damaged by endogenous reactions including deamination and oxidation. Such damage, if not repaired correctly, can result in mutations that drive tumor development. In addition to chemical damage, recent studies have established that DNA bases can be enzymatically modified, generating many of the same modified bases. Irrespective of the mechanism of formation, modified bases can alter DNA-protein interactions and therefore modulate epigenetic control of gene transcription. The simultaneous presence of both chemically and enzymatically modified bases in DNA suggests a potential intersection, or collision, between DNA repair and epigenetic reprogramming. In this paper, we have prepared defined sequence oligonucleotides containing the complete set of oxidized and deaminated bases that could arise from 5-methylcytosine. We have probed these substrates with human glycosylases implicated in DNA repair and epigenetic reprogramming. New observations reported here include: SMUG1 excises 5-carboxyuracil (5caU) when paired with A or G. Both TDG and MBD4 cleave 5-formyluracil and 5caU when mispaired with G. Further, TDG not only removes 5-formylcytosine and 5-carboxycytosine when paired with G, but also when mispaired with A. Surprisingly, 5caU is one of the best substrates for human TDG, SMUG1 and MBD4, and a much better substrate than T. The data presented here introduces some unexpected findings that pose new questions on the interactions between endogenous DNA damage, repair, and epigenetic reprogramming pathways.

A combinatorial system to examine the enzymatic repair of multiply damaged DNA substrates.

DNA damage drives genetic mutations that underlie the development of cancer in humans. Multiple pathways have been described in mammalian cells which can repair this damage. However, most work to date has focused upon single lesions in DNA. We present here a combinatorial system which allows assembly of duplexes containing single or multiple types of damage by ligating together six oligonucleotides containing damaged or modified bases. The combinatorial system has dual fluorescent labels allowing examination of both strands simultaneously, in order to study interactions or competition between different DNA repair pathways. Using this system, we demonstrate how repair of oxidative damage in one DNA strand can convert a mispaired T:G deamination intermediate into a T:A mutation. We also demonstrate that slow repair of a T:G mispair, relative to a U:G mispair, by the human methyl-binding domain 4 DNA glycosylase provides a competitive advantage to competing repair pathways, and could explain why CpG dinucleotides are hotspots for C to T mutations in human tumors. Data is also presented that suggests repair of closely spaced lesions in opposing strands can be repaired by a combination of short and long-patch base excision repair and simultaneous repair of multiply damage sites can potentially lead to lethal double strand breaks.

Two-stage screened selection designs for randomized phase II trials with time-to-event endpoints.

Phase II exploratory multiarm studies that randomize among new treatments are found to be broadly useful and appear to be of value both scientifically and logistically, especially in the areas of unmet needs, for example, pediatric cancer. This multiarm design also has a faster recruitment rate because it provides patients with more treatment choices than traditional two-arm randomized controlled trials do. In contrast to direct formal comparisons in multiarm multistage designs, for example, umbrella or platform designs, the screened selection design (SSD) recommends using a promising treatment arm by ranking according to the effect size, which often needs lesser sample sizes than the former. In this paper, the usefulness of the phase II SSD design is exemplified by three real trials. However, the existing SSD methods can only deal with binary endpoints. Motivated by the real trials in the authors' respective institutions, we propose using the two-stage SSD and its variant for randomized phase II trials with the time-to-event endpoint. The proposed methods not only provide a high probability of selecting a superior treatment arm but also control the type I error rate for testing the efficacy of each treatment arm versus a common external control. Sample size calculations have been derived and simulation studies demonstrate desirable operating characteristics. The proposed design has been used for designing three real trials. An R package frequentistSSD has been developed and is freely accessible for practitioners.

A semi-mechanistic dose-finding design in oncology using pharmacokinetic/pharmacodynamic modeling.

While a number of phase I dose-finding designs in oncology exist, the commonly used ones are either algorithmic or empirical model-based. We propose a new framework for modeling the dose-response relationship, by systematically incorporating the pharmacokinetic (PK) data collected in the trial and the hypothesized mechanisms of the drug effects, via dynamic PK/PD modeling, as well as modeling of the relationship between a latent cumulative pharmacologic effect and a binary toxicity outcome. This modeling framework naturally incorporates the information on the impact of dose, schedule and method of administration (e.g., drug formulation and route of administration) on toxicity. The resulting design is an extension of existing designs that make use of pre-specified summary PK information (such as the area under the concentration-time curve [AUC] or maximum serum concentration [Cmax ]). Our simulation studies show, with moderate departure from the hypothesized mechanisms of the drug action, that the performance of the proposed design on average improves upon those of the common designs, including the continual reassessment method (CRM), Bayesian optimal interval (BOIN) design, modified toxicity probability interval (mTPI) method, and a design called PKLOGIT that models the effect of the AUC on toxicity. In case of considerable departure from the underlying drug effect mechanism, the performance of the design is shown to be comparable with that of the other designs. We illustrate the proposed design by applying it to the setting of a phase I trial of a γ-secretase inhibitor in metastatic or locally advanced solid tumors. We also provide R code to implement the proposed design.

Blood DNA methylation signatures are associated with social determinants of health among survivors of childhood cancer.

Social epigenomics is an emerging field in which social scientist collaborate with computational biologists, especially epigeneticists, to address the underlying pathway for biological embedding of life experiences. This social epigenomics study included long-term childhood cancer survivors enrolled in the St. Jude Lifetime Cohort. DNA methylation (DNAm) data were generated using the Illumina EPIC BeadChip, and three social determinants of health (SDOH) factors were assessed: self-reported educational attainment, personal income, and an area deprivation index based on census track data. An epigenome-wide association study (EWAS) was performed to evaluate the relation between DNAm at each 5'-cytosine-phosphate-guanine-3' (CpG) site and each SDOH factor based on multivariable linear regression models stratified by ancestry (European ancestry, n = 1,618; African ancestry, n = 258). EWAS among survivors of European ancestry identified 130 epigenome-wide significant SDOH-CpG associations (P < 9 × 10-8), 25 of which were validated in survivors of African ancestry (P < 0.05). Thirteen CpGs were associated with all three SDOH factors and resided at pleiotropic loci in cigarette smoking-related genes (e.g., CLDND1 and CPOX). After accounting for smoking and body mass index, these associations remained significant with attenuated effect sizes. Seven of 13 CpGs were associated with gene expression level based on 57 subsamples with blood RNA sequencing data available. In conclusion, DNAm signatures, many resembling the effect of tobacco use, were associated with SDOH factors among survivors of childhood cancer, thereby suggesting that biologically distal SDOH factors influence health behaviours or related factors, the epigenome, and subsequently survivors' health.

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase.

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.

Improved Outcome in Children With Newly Diagnosed High-Risk Neuroblastoma Treated With Chemoimmunotherapy: Updated Results of a Phase II Study Using hu14.18K322A.

PURPOSE: We evaluated whether combining a humanized antidisialoganglioside monoclonal antibody (hu14.18K322A) throughout therapy improves early response and outcomes in children with newly diagnosed high-risk neuroblastoma. PATIENTS AND METHODS: We conducted a prospective, single-arm, three-stage, phase II clinical trial. Six cycles of induction chemotherapy were coadministered with hu14.18K322A, granulocyte-macrophage colony-stimulating factor (GM-CSF), and low-dose interleukin-2 (IL-2). The consolidation regimen included busulfan and melphalan. When available, an additional cycle of parent-derived natural killer cells with hu14.18K322A was administered during consolidation (n = 31). Radiation therapy was administered at the end of consolidation. Postconsolidation treatment included hu14.18K322A, GM-CSF, IL-2, and isotretinoin. Early response was assessed after the first two cycles of induction therapy. End-of-induction response, event-free survival (EFS), and overall survival (OS) were evaluated. RESULTS: Sixty-four patients received hu14.18K322A with induction chemotherapy. This regimen was well tolerated, with continuous infusion narcotics. Partial responses (PRs) or better after the first two chemoimmunotherapy cycles occurred in 42 of 63 evaluable patients (66.7%; 95% CI, 55.0 to 78.3). Primary tumor volume decreased by a median of 75% (range, 100% [complete disappearance]-5% growth). Median peak hu14.18K322A serum levels in cycle one correlated with early response to therapy (P = .0154, one-sided t-test). Sixty of 62 patients (97%) had an end-of-induction partial response or better. No patients experienced progressive disease during induction. The 3-year EFS was 73.7% (95% CI, 60.0 to 83.4), and the OS was 86.0% (95% CI, 73.8 to 92.8), respectively. CONCLUSION: Adding hu14.18K322A to induction chemotherapy improved early objective responses, significantly reduced tumor volumes in most patients, improved end-of-induction response rates, and yielded an encouraging 3-year EFS. These results, if validated in a larger study, may be practice changing.

Engineer design process assisted by explainable deep learning network.

Engineering simulation accelerates the development of reliable and repeatable design processes in various domains. However, the computing resource consumption is dramatically raised in the whole development processes. Making the most of these simulation data becomes more and more important in modern industrial product design. In the present study, we proposed a workflow comprised of a series of machine learning algorithms (mainly deep neuron networks) to be an alternative to the numerical simulation. We have applied the workflow to the field of dental implant design process. The process is based on a complex, time-dependent, multi-physical biomechanical theory, known as mechano-regulatory method. It has been used to evaluate the performance of dental implants and to assess the tissue recovery after the oral surgery procedures. We provided a deep learning network (DLN) with calibrated simulation data that came from different simulation conditions with experimental verification. The DLN achieves nearly exact result of simulated bone healing history around implants. The correlation of the predicted essential physical properties of surrounding bones (e.g. strain and fluid velocity) and performance indexes of implants (e.g. bone area and bone-implant contact) were greater than 0.980 and 0.947, respectively. The testing AUC values for the classification of each tissue phenotype were ranging from 0.90 to 0.99. The DLN reduced hours of simulation time to seconds. Moreover, our DLN is explainable via Deep Taylor decomposition, suggesting that the transverse fluid velocity, upper and lower parts of dental implants are the keys that influence bone healing and the distribution of tissue phenotypes the most. Many examples of commercial dental implants with designs which follow these design strategies can be found. This work demonstrates that DLN with proper network design is capable to replace complex, time-dependent, multi-physical models/theories, as well as to reveal the underlying features without prior professional knowledge.

Reducing Lung ATP Levels and Alleviating Asthmatic Airway Inflammation through Adeno-Associated Viral Vector-Mediated CD39 Expression.

Asthma is a chronic respiratory inflammatory disease. Patients usually suffer long-term symptoms and high medical expenses. Extracellular ATP (eATP) has been identified as a danger signal in innate immunity and serves as a potent inflammatory mediator for asthma. Hydrolyzing eATP in lungs might be a potential approach to alleviate asthmatic inflammation. Recombinant adeno-associated virus (rAAV) vectors that contain tissue-specific cap protein have been demonstrated to efficiently transfer exogenous genes into the lung tissues. To test anti-inflammation efficacy of rAAV-mediated CD39 gene transfer, rAAV-CD39 was generated and applied to OVA-mediated asthmatic mice. BALB/c mice were sensitized intraperitoneally and challenged intratracheally with OVA and treated with rAAV-CD39. At the end of procedure, some inflammatory features were examined. rAAV-CD39 treatment downregulated the levels of pulmonary eATP by the rescued expression of CD39. Several asthmatic features, such as airway hyperresponsiveness, eosinophilia, mucin deposition, and IL-5/IL-13 production in the lungs were decreased in the rAAV-CD39-treated mice. Reduced IL-5/IL-13 production and increased frequency of CD4+FoxP3+ regulatory T cells were detected in draining lymph nodes of rAAV-CD39 treated mice. This evidence suggested that rAAV-mediated CD39 gene transfer attenuated the asthmatic airway inflammation locally. The results suggest that rAAV-CD39 might have therapeutic potential for asthma.

Bayesian single-arm phase II trial designs with time-to-event endpoints.

For the cancer clinical trials with immunotherapy and molecularly targeted therapy, time-to-event endpoint is often a desired endpoint. In this paper, we present an event-driven approach for Bayesian one-stage and two-stage single-arm phase II trial designs. Two versions of Bayesian one-stage designs were proposed with executable algorithms and meanwhile, we also develop theoretical relationships between the frequentist and Bayesian designs. These findings help investigators who want to design a trial using Bayesian approach have an explicit understanding of how the frequentist properties can be achieved. Moreover, the proposed Bayesian designs using the exact posterior distributions accommodate the single-arm phase II trials with small sample sizes. We also proposed an optimal two-stage approach, which can be regarded as an extension of Simon's two-stage design with the time-to-event endpoint. Comprehensive simulations were conducted to explore the frequentist properties of the proposed Bayesian designs and an R package BayesDesign can be assessed via R CRAN for convenient use of the proposed methods.

Cognitive and Adaptive Functioning in Youth With Retinoblastoma: A Longitudinal Investigation Through 10 Years of Age.

PURPOSE: To describe the trajectory of cognitive and adaptive functioning in pediatric patients with retinoblastoma from diagnosis through age 10. This is an extension of a previous report that discussed findings from diagnosis through age 5. PATIENTS AND METHODS: Ninety-eight participants with retinoblastoma completed psychological assessments as part of their enrollment on an institutional treatment protocol, with 73 completing an additional assessment at age 10. Trajectories of adaptive and cognitive functioning were determined, with data analyzed by treatment strata, and patients with 13q- analyzed separately. RESULTS: Longitudinal trajectories identified a significant change point in trends at age 5, with functioning declining from diagnosis through age 5 and then increasing from age 5 to age 10. This pattern was observed for all strata for adaptive functioning, but only for enucleation-only patients (strata C low) for cognitive functioning. Cognitive trajectories were also influenced by laterality and enucleation status. At age 10, overall functioning was generally within the average range, although estimated intelligence quotient was significantly below the normative mean for enucleation-only (C low) patients. Patients with 13q- demonstrated very low functioning, but few analyses were significant because of small sample size. CONCLUSION: The results generally indicate that previously demonstrated declines in functioning from diagnosis through age 5 improve by age 10. However, these early declines, as well as the continuous difficulties observed in patients treated with enucleation only, suggest the need for early intervention services for young patients with retinoblastoma. Continuous monitoring of the psychological functioning of patients with retinoblastoma, increased awareness of risk factors such as unilateral disease, enucleation, race, and surgery-only treatment plans, and referral to Early Intervention for all patients are indicated.

Persistent variations of blood DNA methylation associated with treatment exposures and risk for cardiometabolic outcomes in long-term survivors of childhood cancer in the St. Jude Lifetime Cohort.

BACKGROUND: It is well-established that cancer treatment substantially increases the risk of long-term adverse health outcomes among childhood cancer survivors. However, there is limited research on the underlying mechanisms. To elucidate the pathophysiology and a possible causal pathway from treatment exposures to cardiometabolic conditions, we conducted epigenome-wide association studies (EWAS) to identify the DNA methylation (DNAm) sites associated with cancer treatment exposures and examined whether treatment-associated DNAm sites mediate associations between specific treatments and cardiometabolic conditions. METHODS: We included 2052 survivors (median age 33.7 years) of European ancestry from the St. Jude Lifetime Cohort Study, a retrospective hospital-based study with prospective clinical follow-up. Cumulative doses of chemotherapy and region-specific radiation were abstracted from medical records. Seven cardiometabolic conditions were clinically assessed. DNAm profile was measured using MethylationEPIC BeadChip with blood-derived DNA. RESULTS: By performing multiple treatment-specific EWAS, we identified 935 5'-cytosine-phosphate-guanine-3' (CpG) sites mapped to 538 genes/regions associated with one or more cancer treatments at the epigenome-wide significance level (p < 9 × 10-8). Among the treatment-associated CpGs, 8 were associated with obesity, 63 with hypercholesterolemia, and 17 with hypertriglyceridemia (false discovery rate-adjusted p < 0.05). We observed substantial mediation by methylation at four independent CpGs (cg06963130, cg21922478, cg22976567, cg07403981) for the association between abdominal field radiotherapy (abdominal-RT) and risk of hypercholesterolemia (70.3%) and by methylation at three CpGs (cg19634849, cg13552692, cg09853238) for the association between abdominal-RT and hypertriglyceridemia (54.6%). In addition, three CpGs (cg26572901, cg12715065, cg21163477) partially mediated the association between brain-RT and obesity with a 32.9% mediation effect, and two CpGs mediated the association between corticosteroids and obesity (cg22351187, 14.2%) and between brain-RT and hypertriglyceridemia (cg13360224, 10.5%). Notably, several mediator CpGs reside in the proximity of well-established dyslipidemia genes: cg21922478 (ITGA1) and cg22976567 (LMNA). CONCLUSIONS: In childhood cancer survivors, cancer treatment exposures are associated with DNAm patterns present decades following the exposure. Treatment-associated DNAm sites may mediate the causal pathway from specific treatment exposures to certain cardiometabolic conditions, suggesting the utility of DNAm sites as risk predictors and potential mechanistic targets for future intervention studies.

Novel plasma metabolite markers of attention-deficit/hyperactivity disorder identified using high-performance chemical isotope labelling-based liquid chromatography-mass spectrometry.

OBJECTIVES: Metabolites are the intermediate and final products of biological processes and ultimately reflect the responses of these processes to genetic regulation and environmental perturbations, including those involved in attention deficit/hyperactivity disorder (ADHD). METHODS: We identified a quantitative profile of plasma metabolites in 58 ADHD patients (mean age 9.0 years, 77.6% males) and 38 healthy control subjects (mean age 10.2 years, 55.3% males) using the high-performance chemical isotope labelling (CIL)-based liquid chromatography-mass spectrometry (LC-MS). Using a volcano plot and orthogonal projections to latent structure-discriminant analysis (OPLS-DA), we determined nine metabolites with differentially expressed levels in ADHD plasma samples. RESULTS: Compared to the control group, the plasma levels of guanosine, O-phosphoethanolamine, phenyl-leucine, hypoxanthine, 4-aminohippuric acid, 5-hydroxylysine, and L-cystine appeared increased in the ADHD patients, whilegentisic acid and tryptophyl-phenylalanine were down-regulated in the patients with ADHD. We found that the plasma levels of all nine metabolites were able to discriminate the ADHD group from the control group. Levels of O-phosphoethanolamine, 4-aminohippuric acid, 5-hydroxylysine, L-cystine, tryptophyl-phenylalanine, and gentisic acid were significantly correlated with clinical ADHD symptoms. CONCLUSIONS: This study is the first to use the CIL-based LC-MS to profile ADHD plasma metabolites, and we identified nine novel metabolite biomarkers of ADHD.

Metabolomic profiling of parapneumonic effusion reveals a regulatory role of dipeptides in interleukin-8 production in neutrophil-like cells.

Bacterial pneumonia is a lethal condition, and approximately 40% of bacterial pneumonia patients experience parapneumonic effusion (PPE). Based on the severity of inflammation, PPEs can be categorized as early-stage uncomplicated PPE (UPPE), advanced-stage complicated PPE (CPPE) and, most seriously, thoracic empyema. Appropriate antibiotic treatment at the early stage of PPE can prevent PPE progression and reduce mortality, indicating that understanding PPE generation and components can help researchers develop corresponding treatment strategies for PPE. To this end, metabolomes of 73 PPE (38 UPPE and 35 CPPE samples) and 30 malignant pleural effusion (MPE) samples were profiled with differential 12C2-/13C2-isotope dansylation labeling-based mass spectrometry. We found that PPE is characterized by elevated levels of dipeptides, especially for PPEs at advanced stages. Furthermore, with integrated proteomic and transcriptomic analyses of PPEs, the levels of dipeptides were strongly associated with the production of interleukin-8 (IL-8), an inflammation-associated cytokine. The production of IL-8 indeed increased upon the treatment of HL-60-derived neutrophilic cells with dipeptides, Gly-Val and Gly-Tyr. Our findings help to elucidate the metabolic perturbations present in PPE and indicate for the first time that dipeptides may be involved in the immune regulation observed during PPE progression.

Activation of M1 Macrophages in Response to Recombinant TB Vaccines With Enhanced Antimycobacterial Activity.

Pulmonary tuberculosis (TB) is a difficult-to-eliminate disease. Although the Bacille Calmette-Guérin (BCG) vaccine against Mycobacterium tuberculosis (MTB) has been available for decades, its efficacy is variable and has lessened over time. Furthermore, the BCG vaccine no longer protects against newly emerged Beijing strains which are responsible for many current infections in adults. Development of a novel vaccine is urgently needed. In this study, we first tested the efficacy of our recombinant BCG vaccines rBCG1 and rBCG2, compared to parental BCG, against MTB strain H37Ra in mice. Both the bacterial load and the level of lymphocyte infiltration decreased dramatically in the three groups treated with vaccine, especially rBCG1 and rBCG2. Furthermore, the Th1 and Th17 responses increased and macrophage numbers rose in the vaccination groups. Th1-mediated production of cytokines TNF-α, IFN-γ, and MCP-1 as well as M1-polarized cells all increased in lung tissue of the rBCG1 and rBCG2 groups. Clodronate-induced depletion of macrophages reduced the level of protection. Based on these results, we conclude that rBCG vaccines induce a significant increase in the number of M1 macrophages, which augments their potential as TB vaccine candidates.

Fiber optic nanogold-linked immunosorbent assay for rapid detection of procalcitonin at femtomolar concentration level.

A rapid and ultrasensitive biosensing method based on fiber optic nanogold-linked immunosorbent assay is reported. The method employs an immobilized capture probe on the fiber core surface of an optical fiber and a detection probe conjugated to gold nanoparticles (AuNPs) in a solution. Introduction of a sample containing an analyte and the detection probe into a biosensor chip leads to the formation of a sandwich-like complex of capture probe-analyte-detection probe on the fiber core surface, through which nanoplasmonic absorption of the fiber optic evanescent wave occurs. The performance of this method has been evaluated by its application to the detection of procalcitonin (PCT), an important biomarker for sepsis. In this study, anti-PCT capture antibody is functionalized on an unclad segment of an optical fiber to yield a fiber sensor and anti-PCT detection antibody is conjugated to AuNPs to afford nanoplasmonic probes. The method provides a wide linear response range from 1 pg/mL to 100 ng/mL (5 orders) and an extremely low limit of detection of 95 fg/mL (7.3 fM) for PCT. In addition, the method shows a good correlation in determining PCT in blood plasma with the clinically validated electrochemiluninescent immunoassay. Furthermore, the method is quick (analysis time ≤15 min), requires low-cost instrumentation and sensor chips, and is also potentially applicable to the detection of many other biomarkers.

Proteomic Profiling of Paired Interstitial Fluids Reveals Dysregulated Pathways and Salivary NID1 as a Biomarker of Oral Cavity Squamous Cell Carcinoma.

Patients with oral cavity squamous cell carcinoma (OSCC) are frequently first diagnosed at an advanced stage, leading to poor prognosis and high mortality rates. Early detection of OSCC using body fluid-accessible biomarkers may improve the prognosis and survival rate of OSCC patients. As tumor interstitial fluid is a proximal fluid enriched with cancer-related proteins, it is a useful reservoir suitable for the discovery of cancer biomarkers and dysregulated biological pathways in tumor microenvironments. Thus, paired interstitial fluids of tumor (TIF) and adjacent noncancerous (NIF) tissues from 10 OSCC patients were harvested and analyzed using one-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Using label-free spectral counting-based quantification, 113 proteins were found to be up-regulated in the TIFs compared with the NIFs. The gene set enrichment analysis (GSEA) revealed that the differentially expressed TIF proteins were highly associated with aminoacyl tRNA biosynthesis pathway. The elevated levels of 4 proteins (IARS, KARS, WARS, and YARS) involved in the aminoacyl tRNA biosynthesis were verified in the OSCC tissues with immunohistochemistry (IHC). In addition, nidogen-1 (NID1) was selected for verification as an OSCC biomarker. Salivary level of NID1 in OSCC patients (n = 48) was significantly higher than that in the healthy individuals (n = 51) and subjects with oral potentially malignant disorder (OPMD; n = 53). IHC analysis showed that NID1 level in OSCC tissues was increased compared with adjacent noncancerous epithelium (n = 222). Importantly, the elevated NID1 level was correlated with the advanced stages of OSCC, as well as the poor survival of OSCC patients. Collectively, the results suggested that TIF analysis facilitates understanding of the OSCC microenvironment and that salivary NID1 may be a useful biomarker for OSCC.

Identification of Salivary Biomarkers for Oral Cancer Detection with Untargeted and Targeted Quantitative Proteomics Approaches.

Oral cavity squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. In Taiwan, OSCC is the fifth leading cause of cancer-related mortality and leads to 2800 deaths per year. The poor outcome of OSCC patients is principally ascribed to the fact that this disease is often advanced at the time of diagnosis, suggesting that early detection of OSCC is urgently needed. Analysis of cancer-related body fluids is one promising approach to identify biomarker candidates of cancers. To identify OSCC biomarkers, salivary proteomes of OSCC patients, individuals with oral potentially malignant disorders (OPMDs), and healthy volunteers were comparatively profiled with isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry (MS). The salivary levels of 67 and 18 proteins in the OSCC group are elevated and decreased compared with that in the noncancerous group (OPMD and healthy groups), respectively. The candidate biomarkers were further selected using the multiple reaction monitoring (MRM)-MS and validated with the immunoassays. More importantly, the higher salivary level of three proteins, complement factor H (CFH), fibrinogen alpha chain (FGA), and alpha-1-antitrypsin (SERPINA1) was correlated with advanced stages of OSCC. Our results indicate that analysis of salivary proteome is a feasible strategy for biomarker discovery, and the three proteins are potential salivary markers for OSCC diagnosis.

Integrated analyses utilizing metabolomics and transcriptomics reveal perturbation of the polyamine pathway in oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC), the most common malignancy of the oral cavity, is associated with poor prognosis and high mortality worldwide. Moreover, knowledge of the metabolic alterations that occur in OSCC is still limited. In the present study, we used a quantitative metabolomic approach with chemical isotope labeling (CIL) to analyze alterations in the metabolite levels in paired cancerous (T) and adjacent noncancerous (AN) tissues from 31 OSCC patients. Using volcano plot and orthogonal projections to latent structure-discriminant analysis (OPLS-DA), we uncovered 99 dysregulated metabolites in OSCC and verified the identities of seven metabolites via comparison with authenticated standards. From these seven metabolites, we constructed a 3-marker panel, consisting of putrescine, glycyl-leucine, and phenylalanine, using a support vector machine (SVM) model that can discriminate T from AN with high sensitivity and specificity based on receiver operator characteristic (ROC) analysis. Furthermore, by integrating the metabolomics profiles with transcriptomics data obtained from the same sample set, we revealed the dysregulation of the polyamine pathway in OSCC. Our findings provide insights into the metabolic perturbations present in OSCC and have uncovered potential metabolic biomarkers and therapeutic targets for use in the treatment of OSCC.

Bretschneider solution-induced alterations in the urine metabolome in cardiac surgery patients.

The development of Bretschneider's histidine-tryptophan-ketoglutarate (HTK) cardioplegia solution represented a major advancement in cardiac surgery, offering significant myocardial protection. However, metabolic changes induced by this additive in the whole body have not been systematically investigated. Using an untargeted mass spectrometry-based method to deeply explore the urine metabolome, we sought to provide a holistic and systematic view of metabolic perturbations occurred in patients receiving HTK. Prospective urine samples were collected from 100 patients who had undergone cardiac surgery, and metabolomic changes were profiled using a high-performance chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method. A total of 14,642 peak pairs or metabolites were quantified using differential 13C-/12C-dansyl labeling LC-MS, which targets the amine/phenol submetabolome from urine specimens. We identified 223 metabolites that showed significant concentration change (fold change > 5) and assembled several potential metabolic pathway maps derived from these dysregulated metabolites. Our data indicated upregulated histidine metabolism with subsequently increased glutamine/glutamate metabolism, altered purine and pyrimidine metabolism, and enhanced vitamin B6 metabolism in patients receiving HTK. Our findings provide solid evidence that HTK solution causes significant perturbations in several metabolic pathways and establish a basis for further study of key mechanisms underlying its organ-protective or potential harmful effects.