Mass spectrometry-based metabolomics towards understanding of gene functions with a diversity of biological contexts

Currently, mass spectrometry-based metabolomics studies extend beyond conventional chemical categorization and metabolic phenotype analysis to understanding gene function in various biological contexts (e.g., mammalian, plant, and microbial). These novel utilities have led to many innovative discoveries in the following areas: disease pathogenesis, therapeutic pathway or target identification, the biochemistry of animal and plant physiological and pathological activities in response to diverse stimuli, and molecular signatures of host-pathogen interactions during microbial infection. In this review, we critically evaluate the representative applications of mass spectrometry-based metabolomics to better understand gene function in diverse biological contexts, with special emphasis on working principles, study protocols, and possible future development of this technique. Collectively, this review raises awareness within the biomedical community of the scientific value and applicability of mass spectrometry-based metabolomics strategies to better understand gene function, thus advancing this application's utility in a broad range of biological fields

Development and validation of a ultra performance LC-ESI/MS method for analysis of metabolic phenotypes of healthy men in day and night urine samples

Ultra-performance LC coupled to quadrupole TOF/MS (UPLC-QTOF/MS) in positive and negative ESI was developed and validated to analyze metabolite profiles for urine from healthy men during the day and at night. Data analysis using principal components analysis (PCA) revealed differences between metabolic phenotypes of urine in healthy men during the day and at night. Positive ions with mass-to-charge ratio (m/z) 310.24 (5.35 min), 286.24 (4.74 min) and 310.24 (5.63 min) were elevated in the urine from healthy men at night compared to that during the day. Negative ions elevated in day urine samples of healthy men included m/z 167.02 (0.66 min), 263.12 (2.55 min) and 191.03 (0.73 min), whilst ions m/z 212.01 (4.77 min) were at a lower concentration in urine of healthy men during the day compared to that at night. The ions m/z 212.01 (4.77 min), 191.03 (0.73 min) and 310.24 (5.35 min) preliminarily correspond to indoxyl sulfate, citric acid and N-acetylneuraminic acid, providing further support for an involvement of phenotypic difference in urine of healthy men in day and night samples, which may be associated with notably different activities of gut microbiota, velocity of tricarboxylic acid cycle and activity of sialic acid biosynthesis in healthy men as regulated by circadian rhythm of the mammalian bioclock.

Prospects for whole genome linkage disequilibrium mapping in domestic dog breeds

Linkage disequilibrium (LD) mapping is commonly used as a fine mapping tool in human genome mapping and has been used with some success for initial disease gene isolation in certain isolated in-bred human populations. An understanding of the population history of domestic dog breeds suggests that LD mapping could be routinely utilized in this species for initial genome-wide scans. Such an approach offers significant advantages over traditional linkage analysis. Here, we demonstrate, using canine copper toxicosis in the Bedlington terrier as the model, that LD mapping could be reasonably expected to be a useful strategy in low-resolution, genome-wide scans in pure-bred dogs. Significant LD was demonstrated over distances up to 33.3 cM. It is very unlikely, for a number of reasons discussed, that this result could be extrapolated to the rest of the genome. It is, however, consistent with the expectation given the population structure of canine breeds and, in this breed at least, with the hypothesis that it may be possible to utilize LD in a genome-wide scan. In this study, LD mapping confirmed the location of the copper toxicosis in Bedlington terrier gene (CT-BT) and was able to do so in a population that was refractory to traditional linkage analysis.

Prevalence of chronic ocular diseases in a genetic isolate : the Norfolk Island Eye Study (NIES)

Purpose: Over 40% of the permanent population of Norfolk Island possesses a unique genetic admixture dating to Pitcairn Island in the late 18 th century, with descendents having varying degrees of combined Polynesian and European ancestry. We conducted a population-based study to determine the prevalence and causes of blindness and low vision on Norfolk Island. Methods: All permanent residents of Norfolk Island aged ≥ 15 years were invited to participate. Participants completed a structured questionnaire/interview and underwent a comprehensive ophthalmic examination including slit-lamp biomicroscopy. Results: We recruited 781 people aged ≥ 15, equal to 62% of the permanent population, 44% of whom could trace their ancestry to Pitcairn Island. No one was bilaterally blind. Prevalence of unilateral blindness (visual acuity [VA] < 6/60) in those aged ≥ 40 was 1.5%. Blindness was more common in females (P=0.049) and less common in people with Pitcairn Island ancestry (P<0.001). The most common causes of unilateral blindness were age-related macular degeneration (AMD), amblyopia, and glaucoma. Five people had low vision (Best-Corrected VA < 6/18 in better eye), with 4 (80%) due to AMD. People with Pitcairn Island ancestry had a lower prevalence of AMD (P<0.001) but a similar prevalence of glaucoma to those without Pitcairn Island ancestry. Conclusions: The prevalence of blindness and visual impairment in this isolated Australian territory is low, especially amongst those with Pitcairn Island ancestry. AMD was the most common cause of unilateral blindness and low vision. The distribution of chronic ocular diseases on Norfolk Island is similar to mainland Australian estimates.

An exploratory study of the potential of resurfacing articular cartilage with synthetic phospholipids

This thesis is aimed at further understanding the uppermost lipid-filled membranous layer (i.e. surface amorphous layer (SAL)) of articular cartilage and to develop a scientific framework for re-introducing lipids onto the surface of lipid-depleted articular cartilage (i.e. "resurfacing"). The outcome will potentially contribute to knowledge that will facilitate the repair of the articular surface of cartilage where degradation is limited to the loss of the lipids of the SAL only. The surface amorphous layer is of utmost importance to the effective load-spreading, lubrication, and semipermeability (which controls its fluid management, nutrient transport and waste removal) of articular cartilage in the mammalian joints. However, because this uppermost layer of cartilage is often in contact during physiological function, it is prone to wear and tear, and thus, is the site for damage initiation that can lead to the early stages of joint condition like osteoarthritis, and related conditions that cause pain and discomfort leading to low quality of life in patients. It is therefore imperative to conduct a study which offers insight into remedying this problem. It is hypothesized that restoration (resurfacing) of the surface amorphous layer can be achieved by re-introducing synthetic surface-active phospholipids (SAPL) into the joint space. This hypothesis was tested in this thesis by exposing cartilage samples whose surface lipids had been depleted to individual and mixtures of synthetic saturated and unsaturated phospholipids. The surfaces of normal, delipidized, and relipidized samples of cartilage were characterized for their structural integrity and functionality using atomic force microscope (AFM), confocal microscope (COFM), Raman spectroscopy, magnetic resonance imaging (MRI) with image processing in the MATLAB® environment and mechanical loading experiments. The results from AFM imaging, confocal microscopy, and Raman spectroscopy revealed a successful deposition of new surface layer on delipidized cartilage when incubated in synthetic phospholipids. The relipidization resulted in a significant improvement in the surface nanostructure of the artificially degraded cartilage, with the complete SAPL mixture providing better outcomes in comparison to those created with the single SAPL components (palmitoyl-oleoyl-phosphatidylcholine, POPC and dipalmitoyl-phosphatidylcholine, DPPC). MRI analysis revealed that the surface created with the complete mixture of synthetic lipids was capable of providing semipermeability to the surface layer of the treated cartilage samples relative to the normal intact surface. Furthermore, deformation energy analysis revealed that the treated samples were capable of delivering the elastic properties required for load bearing and recovery of the tissue relative to the normal intact samples, with this capability closer between the normal and the samples incubated in the complete lipid mixture. In conclusion, this thesis has established that it is possible to deposit/create a potentially viable layer on the surface of cartilage following degradation/lipid loss through incubation in synthetic lipid solutions. However, further studies will be required to advance the ideas developed in this thesis, for the development of synthetic lipid-based injections/drugs for treatment of osteoarthritis and other related joint conditions.

An androgen receptor mutation in the MDA-MB-453 cell line model of molecular apocrine breast cancer compromises receptor activity

Recent evidence indicates that the estrogen receptor-a-negative, androgen receptor (AR)- positive molecular apocrine subtype of breast cancer is driven by AR signaling. The MDA-MB-453 cell line is the prototypical model of this breast cancer subtype; its proliferation is stimulated by androgens such as 5a-dihydrotestosterone (DHT) but inhibited by the progestin medroxyprogesterone acetate (MPA) via AR-mediated mechanisms. We report here that the AR gene in MDAMB- 453 cells contains a G-T transversion in exon 7, resulting in a receptor variant with a glutamine to histidine substitution at amino acid 865 (Q865H) in the ligand binding domain. Compared with wild-type AR, the Q865H variant exhibited reduced sensitivity to DHT and MPA in transactivation assays in MDA-MB-453 and PC-3 cells but did not respond to non-androgenic ligands or receptor antagonists. Ligand binding, molecular modeling, mammalian two-hybrid and immunoblot assays revealed effects of the Q865H mutation on ligand dissociation, AR intramolecular interactions, and receptor stability. Microarray expression profiling demonstrated that DHT and MPA regulate distinct transcriptional programs in MDA-MB-453 cells. Gene Set Enrichment Analysis revealed that DHT- but not MPA-regulated genes were associated with estrogen-responsive transcriptomes from MCF-7 cells and the Wnt signaling pathway. These findings suggest that the divergent proliferative responses of MDA-MB-453 cells to DHT and MPA result from the different genetic programs elicited by these two ligands through the AR-Q865H variant. This work highlights the necessity to characterize additional models of molecular apocrine breast cancer to determine the precise role of AR signaling in this breast cancer subtype. Endocrine-Related Cancer (2012) 19 599–613

Cone signals and activity in myopia and emmetropia

Myopia (short-sightedness) is a common ocular disorder of children and young adults. Studies primarily using animal models have shown that the retina controls eye growth and the outer retina is likely to have a key role. One theory is that the proportion of L (long-wavelength-sensitive) and M (medium-wavelength-sensitive) cones is related to myopia development; with a high L/M cone ratio predisposing individuals to myopia. However, not all dichromats (persons with red-green colour vision deficiency) with extreme L/M cone ratios have high refractive errors. We predict that the L/M cone ratio will vary in individuals with normal trichromatic colour vision but not show a systematic difference simply due to refractive error. The aim of this study was to determine if L/M cone ratios in the central 30° are different between myopic and emmetropic young, colour normal adults. Information about L/M cone ratios was determined using the multifocal visual evoked potential (mfVEP). The mfVEP can be used to measure the response of visual cortex to different visual stimuli. The visual stimuli were generated and measurements performed using the Visual Evoked Response Imaging System (VERIS 5.1). The mfVEP was measured when the L and M cone systems were separately stimulated using the method of silent substitution. The method of silent substitution alters the output of three primary lights, each with physically different spectral distributions to control the excitation of one or more photoreceptor classes without changing the excitation of the unmodulated photoreceptor classes. The stimulus was a dartboard array subtending 30° horizontally and 30° vertically on a calibrated LCD screen. The m-sequence of the stimulus was 215-1. The N1-P1 amplitude ratio of the mfVEP was used to estimate the L/M cone ratio. Data were collected for 30 young adults (22 to 33 years of age), consisting of 10 emmetropes (+0.3±0.4 D) and 20 myopes (–3.4±1.7 D). The stimulus and analysis techniques were confirmed using responses of two dichromats. For the entire participant group, the estimated central L/M cone ratios ranged from 0.56 to 1.80 in the central 3°-13° diameter ring and from 0.94 to 1.91 in the more peripheral 13°-30° diameter ring. Within 3°-13°, the mean L/M cone ratio of the emmetropic group was 1.20±0.33 and the mean was similar, 1.20±0.26, for the myopic group. For the 13°-30° ring, the mean L/M cone ratio of the emmetropic group was 1.48±0.27 and it was slightly lower in the myopic group, 1.30±0.27. Independent-samples t-test indicated no significant difference between the L/M cone ratios of the emmetropic and myopic group for either the central 3°-13° ring (p=0.986) or the more peripheral 13°-30° ring (p=0.108). The similar distributions of estimated L/M cone ratios in the sample of emmetropes and myopes indicates that there is likely to be no association between the L/M cone ratio and refractive error in humans.

Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise

Background: Ingestion of whey or casein yields divergent patterns of aminoacidemia that influence whole-body and skeletal muscle myofibrillar protein synthesis (MPS) after exercise. Direct comparisons of the effects of contrasting absorption rates exhibited by these proteins are confounded by their differing amino acid contents. Objective: Our objective was to determine the effect of divergent aminoacidemia by manipulating ingestion patterns of whey protein alone on MPS and anabolic signaling after resistance exercise. Design: In separate trials, 8 healthy men consumed whey protein either as a single bolus (BOLUS; 25-g dose) or as repeated, small, "pulsed" drinks (PULSE; ten 2.5-g drinks every 20 min) to mimic a more slowly digested protein. MPS and phosphorylation of signaling proteins involved in protein synthesis were measured at rest and after resistance exercise. Results: BOLUS increased blood essential amino acid (EAA) concentrations above those of PULSE (162% compared with 53%, P < 0.001) 60 min after exercise, whereas PULSE resulted in a smaller but sustained increase in aminoacidemia that remained elevated above BOLUS amounts later (180-220 min after exercise, P < 0.05). Despite an identical net area under the EAA curve, MPS was elevated to a greater extent after BOLUS than after PULSE early (1-3 h: 95% compared with 42%) and later (3-5 h: 193% compared with 121%) (both P < 0.05). There were greater changes in the phosphorylation of the Akt-mammalian target of rapamycin pathway after BOLUS than after PULSE. Conclusions: Rapid aminoacidemia in the postexercise period enhances MPS and anabolic signaling to a greater extent than an identical amount of protein fed in small pulses that mimic a more slowly digested protein. A pronounced peak aminoacidemia after exercise enhances protein synthesis.

mTOR function in skeletal muscle : a focal point for overnutrition and exercise

The mammalian target of rapamycin (mTOR) is a highly conserved atypical serine-threonine kinase that controls numerous functions essential for cell homeostasis and adaptation in mammalian cells via 2 distinct protein complex formations. Moreover, mTOR is a key regulatory protein in the insulin signalling cascade and has also been characterized as an insulin-independent nutrient sensor that may represent a critical mediator in obesity-related impairments of insulin action in skeletal muscle. Exercise characterizes a remedial modality that enhances mTOR activity and subsequently promotes beneficial metabolic adaptation in skeletal muscle. Thus, the metabolic effects of nutrients and exercise have the capacity to converge at the mTOR protein complexes and subsequently modify mTOR function. Accordingly, the aim of the present review is to highlight the role of mTOR in the regulation of insulin action in response to overnutrition and the capacity for exercise to enhance mTOR activity in skeletal muscle.

Hemi-field and full-field form-deprivation induce timing changes in multifocal ERG responses in chick

Purpose: In animal models hemi-field deprivation results in localised, graded vitreous chamber elongation and presumably deprivation induced localised changes in retinal processing. The aim of this research was to determine if there are variations in ERG responses across the retina in normal chick eyes and to examine the effect of hemi-field and full-field deprivation on ERG responses across the retina and at earlier times than have previously been examined electrophysiologically. Methods: Chicks were either untreated, wore monocular full-diffusers or half-diffusers (depriving nasal retina) (n = 6-8 each group) from day 8. mfERG responses were measured using the VERIS mfERG system across the central 18.2º× 16.7º (H × V) field. The stimulus consisted of 61 unscaled hexagons with each hexagon modulated between black and white according to a pseudorandom binary m-sequence. The mfERG was measured on day 12 in untreated chicks, following 4 days of hemi-field diffuser wear, and 2, 48 and 96 h after application of full-field diffusers. Results: The ERG response of untreated chick eyes did not vary across the measured field; there was no effect of retinal location on the N1-P1 amplitude (p = 0.108) or on P1 implicit time (p > 0.05). This finding is consistent with retinal ganglion cell density of the chick varying by only a factor of two across the entire retina. Half-diffusers produced a ramped retina and a graded effect of negative lens correction (p < 0.0001); changes in retinal processing were localized. The untreated retina showed increasing complexity of the ERG waveform with development; form-deprivation prevented the increasing complexity of the response at the 2, 48 and 96 h measurement times and produced alterations in response timing. Conclusions: Form-deprivation and its concomitant loss of image contrast and high spatial frequency images prevented development of the ERG responses, consistent with a disruption of development of retinal feedback systems. The characterisation of ERG responses in normal and deprived chick eyes across the retina allows the assessment of concurrent visual and retinal manipulations in this model. (Ophthalmic & Physiological Optics © 2013 The College of Optometrists.)

GABAergic agents modify the response of chick scleral fibroblasts to myopic and hyperopic eye cup tissues

Purpose: GABA antagonists inhibit experimental myopia in chick and GABA receptors have been localized to chick sclera and the retinal pigment epithelium (RPE). The RPE and the choroid alter scleral DNA and glycosaminoglycan (GAG) content in vitro; opposite effects have been observed for tissues from myopic and hyperopic eyes. The aim was to determine the effect of GABAergic agents on the DNA and GAG content of chick scleral fibroblasts directly and in co-culture with ocular tissues from myopic and hyperopic chick eyes. Materials and Methods: Primary cultures of fibroblastic cells expressing vimentin and α-smooth muscle actin were established. GABAergic agents were added separately (i) to the culture medium of the scleral cells and (ii) to the culture medium of the scleral cells with the addition of posterior eye cup tissue (retina, RPE, retina + RPE, choroid + RPE) to cell culture inserts. Ocular tissues were obtained from chick eyes wearing + 15D (lens-induced hyperopia, LIH) or −15D lenses (lens-induced myopia, LIM) for three days (post-hatch day 5–8) (n = 12). GAG and DNA content of scleral fibroblasts were measured. Results: GABA agents had a small direct effect on scleral cell GAG and DNA content but a larger effect was measured when GABA agents were added to the culture medium with myopic and hyperopic RPE and choroid + RPE tissues. GABA agonists increased (p = 0.002) whereas antagonists decreased (p = 0.0004) DNA content of scleral cells; effects were opposite for scleral GAG content. GABA agents significantly altered the effect of both LIM and LIH tissues (p = 0.0005) compared to control; the effects were greater for LIM tissue versus LIH tissue co-culture (p = 0.0004). Conclusion: GABAergic agents affect the DNA and GAG content of scleral fibroblasts both directly and when co-cultured with ocular tissues. GABA antagonists that prevent myopia development in chick model could act via a scleral mechanism utilizing the RPE/choroid.

Identification of GABA receptors in chick retinal pigment epithelium

Purpose: The retinal pigment epithelium (RPE) is a multifunctional, monolayer of cells located between the neural retina and the choroicapillaris. γ-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and GABA receptors are known to be present in chick retina, sclera and cornea. There is a report of genes involved in GABA receptor signaling being expressed in human RPE, however, whether GABA receptors are present in chick RPE is unknown. Methods: Real time PCR and western blot were used to determine the expression of GABA receptors (alpha1 GABAA, GABABR2, and rho1 GABAC receptors) in isolated chicken RPE. Immunofluorescence using antibodies against one of the GABA receptor sub-types was used to determine receptor localization. Results: Both real-time PCR and western blot demonstrated that alpha1 GABAA, GABABR2 and rho1 GABAC receptors were expressed in isolated chick RPE. Immunofluorescence further demonstrated that GABA receptors were localized to the cell membrane and plasma of RPE cells. Conclusions: Alpha1 GABAA, GABABR2 and rho1 GABAC receptors were expressed in chick RPE. The purpose of the GABA receptors within the RPE remains to be explored.

Daxx regulates mitotic progression and prostate cancer predisposition

Mitotic progression of mammalian cells is tightly regulated by the E3 ubiquitin ligase anaphase promoting complex (APC)/C. Deregulation of APC/C is frequently observed in cancer cells and is suggested to contribute to chromosome instability and cancer predisposition. In this study, we identified Daxx as a novel APC/C inhibitor frequently overexpressed in prostate cancer. Daxx interacts with the APC/C coactivators Cdc20 and Cdh1 in vivo, with the binding of Cdc20 dependent on the consensus destruction boxes near the N-terminal of the Daxx protein. Ectopic expression of Daxx, but not the D-box deleted mutant (DaxxΔD-box), inhibited the degradation of APC/Cdc20 and APC/Cdh1 substrates, leading to a transient delay in mitotic progression. Daxx is frequently upregulated in prostate cancer tissues; the expression level positively correlated with the Gleason score and disease metastasis (P = 0.027 and 0.032, respectively). Furthermore, ectopic expression of Daxx in a non-malignant prostate epithelial cell line induced polyploidy under mitotic stress. Our data suggest that Daxx may function as a novel APC/C inhibitor, which promotes chromosome instability during prostate cancer development.

An extracellular signal-regulated kinase 2 survival pathway mediates resistance of human mesothelioma cells to asbestos-induced injury

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR) - linked survival pathways (phosphoinositol-3-kinase/AKT/ mammalian target of rapamycin and extracellular signal - regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death - related gene expression. Our results suggest that EGFR phosphorylation is causally linkedto pERK and pAKT activation by asbestos in normal and SV40 Tag - immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. ©2011 AACR.