Application of High-Performance Liquid-Chromatography Mass-Spectrometry Platform to Study Metabolism and Epigenetic Control of Metabolism

Naturally occurring small molecules (metabolites, signaling intermediates) are a critical component of the information flow in biology, along with DNA, RNA, and proteins. Metabolomics is an analytical approach that seeks to comprehensively analyze naturally occurring small molecules and quantify their dynamic changes in biological systems. In recent years metabolomics has begun to provide understanding of the metabolic basis of different diseases, such as heart disease, cancer, and diabetes. Our lab built a High-Performance Liquid-Chromatography Mass-Spectrometry (HPLC-MS) based metabolomics platform to analyze metabolites from mammalian cells, spent cellular media, and model organisms such as C. elegans. We used C. elegans to elucidate the metabolic changes seen after treatment with Metformin, which is a known activator of the AMPK pathway. Cancer cells exhibit high levels of glycolysis producing large amounts of lactate; circumventing the mitochondrial pathway. This phenomenon is known as the Warburg effect. We hypothesize that cancer metabolism is epigenetically regulated. Epigenetics refers to inheritable traits that are not due to alterations in the primary DNA sequence. DNA methylation is an important epigenetic modification. DNA methylation mainly occurs in the CpG islands of the promoter region of genes. It is believed that during cancer development de novo DNA methyltransferases methylate tumor-suppressing genes allowing cancer cells to proliferate uninhibited. There are two de novo DNMTs, DNMT3A, and DNMT3B. These DNMTs establish the pattern of methylation. We examined de novo DNMT-mediated control of cellular metabolism, identifying global changes in metabolism, as well as differential sensitivity towards glycolytic and mitochondrial inhibitors

Glycosylation of human cyclooxygenase-2 (COX-2) decreases the efficacy of certain COX-2 inhibitors.

Prostanoids play an important role in a variety of physiological and pathophysiological processes including inflammation and cancer. The rate-limiting step in the prostanoid biosynthesis pathway is catalyzed by cyclooxygenase-2 (COX-2). COX-2 exists as two glycoforms, 72 and 74 kDa, the latter resulting from an additional glycosylation at Asn(580). In this study, Asn(580) was mutated, and the mutant and wild-type COX-2 genes were expressed in COS-1 cells to determine how glycosylation affects the inhibition of COX-2 activity by aspirin, flurbiprofen, ibuprofen, celecoxib, and etoricoxib. Results indicate that certain inhibitors were 2-5 times more effective at inhibiting COX-2 activity when the glycosylation site was eliminated, indicating that glycosylation of COX-2 at Asn(580) decreases the efficacy of some inhibitors

Using the Systemic Immune-Inflammation Index (SII) as a mid-treatment marker for survival among patients with stage-III locally advanced non-small cell lung cancer (NSCLC)

The Systemic Immune-Inflammation Index (SII) is an important marker of immune function, defined as the product of neutrophil-to-lymphocyte ratio (NLR) and platelet count (P). Higher baseline SII levels have been associated with improved survival in various types of cancers, including lung cancer. Data were obtained from PROCLAIM, a randomized phase III trial comparing two different chemotherapy regimens pemetrexed + cisplatin (PEM) vs. etoposide + cisplatin (ETO), in combination with radiotherapy (RT) for the treatment of stage III non-squamous non-small cell lung cancer (NSCLC). We aimed to determine if SII measured at the mid-treatment window for RT (weeks 3-4) is a significant predictor of survival, and if the effect of PEM vs. ETO differs by quartile (Q) level of SII. Hazard-ratios (HR) for survival were estimated using a proportional hazards model, accounting for the underlying correlated structure of the data. A total of 548 patients were included in our analysis. The median age at baseline was 59 years. Patients were followed for a median of 24 months. Adjusting for age, body mass index, sex, race, and chemotherapy regimen, SII was a significant mid-treatment predictor of both overall (adjusted HR (aHR) = 1.6

“I lied a little bit.” A qualitative study exploring the perspectives of elite Australian athletes on self-reported data

Objectives: Explore the perceptions and experiences of elite Australian athletes’ engagement with reporting data in surveillance systems. Design: Qualitative Descriptive. Setting: Semi-structured interviews conducted using Zoom. Participants: We recruited 13 elite Australian athletes competing at a national or international level for semi-structured interviews. Main outcome measures: Audio recordings were transcribed using DeScript, checked for errors and imported into QSR NVIVO. Thematic analysis using QSR NVIVO was used to determine key themes from transcripts. Results: Thematic analysis uncovered four key themes: ‘the paradox of reporting’, ‘data for data\u27s sake’, ‘eyes on reporting’ and ‘athlete friendly reporting’. Conclusion: Athletes perceived reporting as a burden and the athlete management system presented numerous technological difficulties which led to athletes to backfill data entries and compromise data accuracy. Athletes had little knowledge on how their data was used and managed and often received minimal feedback from staff accessing the data. Athletes were unaware of who has access to their data, which is of concern as sensitive information may be collected and athletes may be underage. As a result, many athletes chose to report dishonest data to avoid their performance being questioned

A novel AhR ligand, 2AI, protects the retina from environmental stress.

Various retinal degenerative diseases including dry and neovascular age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy are associated with the degeneration of the retinal pigmented epithelial (RPE) layer of the retina. This consequently results in the death of rod and cone photoreceptors that they support, structurally and functionally leading to legal or complete blindness. Therefore, developing therapeutic strategies to preserve cellular homeostasis in the RPE would be a favorable asset in the clinic. The aryl hydrocarbon receptor (AhR) is a conserved, environmental ligand-dependent, per ARNT-sim (PAS) domain containing bHLH transcription factor that mediates adaptive response to stress via its downstream transcriptional targets. Using in silico, in vitro and in vivo assays, we identified 2,2'-aminophenyl indole (2AI) as a potent synthetic ligand of AhR that protects RPE cells in vitro from lipid peroxidation cytotoxicity mediated by 4-hydroxynonenal (4HNE) as well as the retina in vivo from light-damage. Additionally, metabolic characterization of this molecule by LC-MS suggests that 2AI alters the lipid metabolism of RPE cells, enhancing the intracellular levels of palmitoleic acid. Finally, we show that, as a downstream effector of 2AI-mediated AhR activation, palmitoleic acid protects RPE cells from 4HNE-mediated stress, and light mediated retinal degeneration in mice

A pilot study examining the prognostic utility of tumor shrinkage on cone-beam computed tomography (CBCT) for stage III locally advanced non-small cell lung cancer patients treated with definitive chemoradiation

There has been growing interest in utilizing information from cone-beam computed tomography (CBCT) to help guide both treatment delivery and prognosis. In this assessment of locally advanced unresectable stage III non-small cell lung cancer (NSCLC) treated with definitive chemoradiation, we aimed to determine the survival advantage associated with using CBCT to measure tumor regression. Patient, tumor, and treatment characteristics were collected. The serial tumor shrinkage for each patient was determined from tumor volume contours on weekly CBCTs. Survival analysis was performed using the Kaplan-Meier technique and a Cox proportional hazards model. At least two-thirds of patients had a tumor volume reduction of at least 5% after each week of chemoradiation. A weekly reduction in tumor volume of 5% or greater seen on the CBCT images during radiation therapy was significantly associated with improved overall survival, which remained significant when adjusted for age, histology, grade, and T- and N-stages

Pretreatment neutrophil-to-lymphocyte ratio as an important prognostic marker in stage III locally advanced non-small cell lung cancer: Confirmatory results from the PROCLAIM phase III clinical trial

Background: Neutrophil-to-lymphocyte ratio (NLR) is an important pretreatment marker of systemic inflammation and tumor aggressiveness. Increased levels of this ratio have been associated with reduced survival in several observational studies of lung cancer. However, supporting analyses from large clinical trial data are lacking. Methods: To validate the prognostic role of NLR, the current study evaluated data from a randomized phase III study (PROCLAIM; clinicaltrial.gov ID: NCT00686959) of patients with stage IIIA/B, unresectable, non-squamous, non-small cell lung cancer (NSCLC), originally comparing combination pemetrexed-cisplatin chemoradiotherapy with etoposide-cisplatin chemoradiotherapy. Adjusted hazard ratios (aHR) and 95% confidence intervals (CI) for survival were estimated using a Cox proportional hazards model. Models were adjusted for age, race, sex, stage, treatment, and body mass index (BMI). Patients were followed for a median of 24 months. Results: Increased NLR levels at baseline were associated with reduced overall ( Conclusions: These findings provide substantiating evidence that NLR, which is routinely available from standard blood testing of patients diagnosed with NSCLC, is an important inflammation-based prognostic marker for survival among patients with locally advanced disease undergoing chemoradiation. Future research will benefit by assessing the prognostic potential of NLR in the context of genetic mutations and molecular markers

The Koala (Phascolarctos cinereus) faecal microbiome differs with diet in a wild population

Background The diet of the koala (Phascolarctos cinereus) is comprised almost exclusively of foliage from the genus Eucalyptus (family Myrtaceae). Eucalyptus produces a wide variety of potentially toxic plant secondary metabolites which have evolved as chemical defences against herbivory. The koala is classified as an obligate dietary specialist, and although dietary specialisation is rare in mammalian herbivores, it has been found elsewhere to promote a highly-conserved but low-diversity gut microbiome. The gut microbes of dietary specialists have been found sometimes to enhance tolerance of dietary PSMs, facilitating competition-free access to food. Although the koala and its gut microbes have evolved together to utilise a low nutrient, potentially toxic diet, their gut microbiome has not previously been assessed in conjunction with diet quality. Thus, linking the two may provide new insights in to the ability of the koala to extract nutrients and detoxify their potentially toxic diet. Method The 16S rRNA gene was used to characterise the composition and diversity of faecal bacterial communities from a wild koala population (n = 32) comprising individuals that predominately eat either one of two different food species, one the strongly preferred and relatively nutritious species Eucalyptus viminalis, the other comprising the less preferred and less digestible species Eucalyptus obliqua. Results Alpha diversity indices indicated consistently and significantly lower diversity and richness in koalas eating E. viminalis. Assessment of beta diversity using both weighted and unweighted UniFrac matrices indicated that diet was a strong driver of both microbial community structure, and of microbial presence/absence across the combined koala population and when assessed independently. Further, principal coordinates analysis based on both the weighted and unweighted UniFrac matrices for the combined and separated populations, also revealed a separation linked to diet. During our analysis of the OTU tables we also detected a strong association between microbial community composition and host diet. We found that the phyla Bacteroidetes and Firmicutes were co-dominant in all faecal microbiomes, with Cyanobacteria also co-dominant in some individuals; however, the E. viminalis diet produced communities dominated by the genera Parabacteroides and/or Bacteroides, whereas the E. obliqua-associated diets were dominated by unidentified genera from the family Ruminococcaceae. Discussion We show that diet differences, even those caused by differential consumption of the foliage of two species from the same plant genus, can profoundly affect the gut microbiome of a specialist folivorous mammal, even amongst individuals in the same population. We identify key microbiota associated with each diet type and predict functions within the microbial community based on 80 previously identified Parabacteroides and Ruminococcaceae genomes

Child and family experiences with inborn errors of metabolism: a qualitative interview study with representatives of patient groups

Background: Patient-centered health care for children with inborn errors of metabolism (IEM) and their families is important and requires an understanding of patient experiences, needs, and priorities. IEM-specific patient groups have emerged as important voices within these rare disease communities and are uniquely positioned to contribute to this understanding. We conducted qualitative interviews with IEM patient group representatives to increase understanding of patient and family experiences, needs, and priorities and inform patient-centered research and care. Methods: We developed a sampling frame of patient groups representing IEM disease communities from Canada, the United States, and United Kingdom. With consent, we interviewed participants to explore their views on experiences, needs, and outcomes that are most important to children with IEM and their families. We analyzed the data using a qualitative descriptive approach to identify key themes and sub-themes. Results: We interviewed 18 organizational representatives between February 28 and September 17, 2014, representing 16 IEMs and/or disease categories. Twelve participants voluntarily self-identified as parents and/or were themselves patients. Three key themes emerged from the coded data: managing the uncertainty associated with raising and caring for a child with a rare disease; challenges associated with the affected child’s life transitions, and; the collective struggle for improved outcomes and interventions that rare disease communities navigate. Conclusion: Health care providers can support children with IEM and their families by acknowledging and reducing uncertainty, supporting families through children’s life transitions, and contributing to rare disease communities’ progress toward improved interventions, experiences, and outcomes

Experiences of caregivers of children with inherited metabolic diseases: a qualitative study

Background: We sought to understand the experiences of parents/caregivers of children with inherited metabolic diseases (IMD) in order to inform strategies for supporting patients and their families. We investigated their experiences regarding the management of disease, its impact on child and family life, and interactions with the health care system. Methods: From four Canadian centres, we conducted semi-structured telephone interviews with parents/caregivers of children with an IMD who were born between 2006 and 2015 and who were participating in a larger cohort study. Participants were selected with the aim of achieving a diverse sample with respect to treatment centre, IMD, and age of the child. Interviews emphasized the impacts of the disease and its treatment on the child and family and explicitly queried perceptions of interactions with the health care system. We identified emergent themes from the interview data. Results: We completed interviews with 21 parents/caregivers. The 21 children were aged \u3c1 to 7 years old with IMD that included amino acid disorders, urea cycle disorders, fatty acid oxidation disorders, and organic acid disorders or \u27other\u27 IMD. Most parents reported that they and their families had adapted well to their child\u27s diagnosis. Parents used proactive coping strategies to integrate complex disease management protocols into routine family life. An important source of stress was concern about the social challenges faced by their children. Participants reported positive interactions with their most involved health care providers within the metabolic clinic. However, they reported challenges associated with the health care system outside of disease-specific metabolic care, when encountering systems and providers unfamiliar with the child\u27s disease. Conclusions: The successful use of proactive coping strategies among parents of children with IMD in this study suggests the potential value of promoting positive coping and is an important direction for future study. Parents\u27 social concerns for their children were important stressors that warrant consideration by health care providers positioned to support families. Our results with respect to experiences with care highlight the important role of specialized metabolic clinics and point to a need for better coordination of the care that takes place outside the disease-specific management of IMD