High SEPT9_v1 Expression Is Associated with Poor Clinical Outcomes in Head and Neck Squamous Cell Carcinoma

The purpose of this work was to determine SEPT9_v1 expression levels in head and neck squamous cell carcinoma (HNSCC) and to analyze whether SEPT9_v1 expression is relevant to clinical outcomes. Recently, the SEPT9 isoform SEPT9_v1 has been implicated in oncogenesis, and methylation of the SEPT9 promoter region was reported in HNSCC. These findings led us to hypothesize that SEPT9_v1 could be differently expressed in HNSCC. To determine whether SEPT9_v1 is expressed in HNSCC, tissue microarray immunohistochemical analysis was performed using a SEPT9_v1-specific antibody. Tissue microarrays stained with a polyclonal SEPT9_v1-specific antibody was used to determine protein expression levels in HNSCC tissue samples, some with known clinical outcomes. This analysis showed that SEPT9_v1 is in fact highly expressed in HNSCC compared with normal epithelium, and high expression levels directly correlated with poor clinical outcomes. Specifically, a high SEPT9_v1 expression was associated with decreased disease-specific survival (P = .012), time to indication of surgery at primary site (P = .008), response to induction chemotherapy (P = .0002), and response to chemotherapy (P = .02), as well as advanced tumor stage (P = .012) and N stage (P = .0014). The expression of SEPT9_v1 was also strongly correlated with smoking status (P = .00094). SEPT9_v1 is highly expressed in HNSCC, and a high expression of SEPT9_v1 is associated with poor clinical outcomes. These data indicate that SEPT9_v1 warrants additional investigation as a potential biomarker for HNSCC.     

Alcohol dehydrogenase-coupled spectrophotometric assay of plasmalogenase

Plasmalogenase has been assayed by conversion of the fatty aldehydes, released by hydrolysis of the vinyl ether bond of plasmalogens, to long-chain alcohols by horse liver alcohol dehydrogenase. The reaction was followed spectrophotometrically by measuring the oxidation of NADH. The assay is sufficiently sensitive to enable plasmalogenase activity to be determined in isolated oligodendroglia and derived membranes and in brain microsomal membranes using 50-250 micrograms protein.     

History of safe exposure and bioinformatic assessment of phosphomannose-isomerase (PMI) for allergenic risk

Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.

     

Mechanistic Phenotypes: An Aggregative Phenotyping Strategy to Identify Disease Mechanisms Using GWAS Data

A single mutation can alter cellular and global homeostatic mechanisms and give rise to multiple clinical diseases. We hypothesized that these disease mechanisms could be identified using low minor allele frequency (MAF<0.1) non-synonymous SNPs (nsSNPs) associated with “mechanistic phenotypes”, comprised of collections of related diagnoses. We studied two mechanistic phenotypes: (1) thrombosis, evaluated in a population of 1,655 African Americans; and (2) four groupings of cancer diagnoses, evaluated in 3,009 white European Americans. We tested associations between nsSNPs represented on GWAS platforms and mechanistic phenotypes ascertained from electronic medical records (EMRs), and sought enrichment in functional ontologies across the top-ranked associations. We used a two-step analytic approach whereby nsSNPs were first sorted by the strength of their association with a phenotype. We tested associations using two reverse genetic models and standard additive and recessive models. In the second step, we employed a hypothesis-free ontological enrichment analysis using the sorted nsSNPs to identify functional mechanisms underlying the diagnoses comprising the mechanistic phenotypes. The thrombosis phenotype was solely associated with ontologies related to blood coagulation (Fisher''s p = 0.0001, FDR p = 0.03), driven by the F5, P2RY12 and F2RL2 genes. For the cancer phenotypes, the reverse genetics models were enriched in DNA repair functions (p = 2×10−5, FDR p = 0.03) (POLG/FANCI, SLX4/FANCP, XRCC1, BRCA1, FANCA, CHD1L) while the additive model showed enrichment related to chromatid segregation (p = 4×10−6, FDR p = 0.005) (KIF25, PINX1). We were able to replicate nsSNP associations for POLG/FANCI, BRCA1, FANCA and CHD1L in independent data sets. Mechanism-oriented phenotyping using collections of EMR-derived diagnoses can elucidate fundamental disease mechanisms.     

Condition-Dependent Effects of Mating on Longevity and Fecundity of Female Medflies: The Interplay between Nutrition and Age of Mating

Background

In various species mating exerts direct and indirect effects on female demographic traits ranging from life span shortening to behavioural shifts. A wealth of data regarding effects of nutrition on longevity and reproduction output also exists. Nonetheless, little is known regarding the interaction between the age of mating and nutrition on female fitness.

Methodology

We studied, the effects of protein deprivation and age of mating on female fitness traits, using a wild population of the Mediterranean fruit fly (medfly). We tested the hypotheses that (a) protein availability increases female lifespan and fecundity, (b) female longevity and egg production are independent of mating and the age of mating, and (c) female mating behaviour is independent of their age and nutritional status. Thus, we recorded the mating success and the copulation characteristics, as well as the egg production and survival of females mated at young or at old age and fed a full or a protein-deprived diet.

Results

Mating boosts egg production and reduces longevity of protein-fed females. On the contrary, mating increases the longevity of protein-deprived females. Mortality responses (negative or positive) to mating are expressed after a long lag phase. Old females are more receptive and less selective than young females regardless of the food regime.

Conclusions

Our findings suggest that condition (nutritional status and age) defines the positive or negative output of mating in female medflies. These results contribute towards understanding the effects of mating, aging, resource allocation and their interactions on survival and female reproduction.     

Hexosamine Biosynthesis Is a Possible Mechanism Underlying Hypoxia’s Effects on Lipid Metabolism in Human Adipocytes

Introduction

Hypoxia regulates adipocyte metabolism. Hexosamine biosynthesis is implicated in murine 3T3L1 adipocyte differentiation and is a possible underlying mechanism for hypoxia’s effects on adipocyte metabolism.

Methods

Lipid metabolism was studied in human visceral and subcutaneous adipocytes in in vitro hypoxic culture with adipophilic staining, glycerol release, and palmitate oxidation assays. Gene expression and hexosamine biosynthesis activation was studied with QRTPCR, immunofluorescence microscopy, and Western blotting.

Results

Hypoxia inhibits lipogenesis and induces basal lipolysis in visceral and subcutaneous human adipocytes. Hypoxia induces fatty acid oxidation in visceral adipocytes but had no effect on fatty acid oxidation in subcutaneous adipocytes. Hypoxia inhibits hexosamine biosynthesis in adipocytes. Inhibition of hexosamine biosynthesis with azaserine attenuates lipogenesis and induces lipolysis in adipocytes in normoxic conditions, while promotion of hexosamine biosynthesis with glucosamine in hypoxic conditions slightly increases lipogenesis.

Conclusions

Hypoxia’s net effect on human adipocyte lipid metabolism would be expected to impair adipocyte buffering capacity and contribute to systemic lipotoxicity. Our data suggest that hypoxia may mediate its effects on lipogenesis and lipolysis through inhibition of hexosamine biosynthesis. Hexosamine biosynthesis represents a target for manipulation of adipocyte metabolism.