Metabolite Profiling Reveals Predictive Biomarkers and the Absence of ß-Methyl Amino-l-alanine in Plasma from Individuals Diagnosed with Amyotrophic Lateral Sclerosis.

By employing chip-based capillary zone electrophoresis coupled to high-resolution mass spectrometry, we profiled the plasma metabolome of 134 patients diagnosed with sporadic amyotrophic lateral sclerosis (ALS) (81 males and 53 females) and 118 individuals deemed healthy (49 males and 69 females). The most significant markers (p < 0.01) were creatine, which was 49% elevated, and creatinine and methylhistidine, which were decreased by 20 and 24%, respectively, in ALS patients. The ratio of creatine versus creatinine increased 370 and 200% for male and female ALS patients, respectively. In addition, male ALS patients on an average had 5-13% lower amounts of seven essential amino acids, whereas females did not significantly differ from healthy controls. We developed two models using the metabolite abundances: (1) a classification model for the separation of ALS and healthy samples and (2) a classification model for the prediction of disease progression based on the ALS functional rating score. Utilizing a Monte Carlo cross-validation approach, a linear discriminant analysis model achieved a mean area under the receiver operating characteristic curve (AUC) of 0.85 (0.06) with a mean sensitivity of 80% (9%) and specificity of 78% (10%) for the separation of ALS and controls, respectively. A support vector machine classifier predicted progression categories with an AUC of 0.90 (0.06) with a mean sensitivity of 73% (10%) and a specificity of 86% (5%). Lastly, using a previously reported assay with a stable isotope-labeled (13C315N2) spike-in standard, we were unable to detect the exogenous neurotoxic metabolite, ß-methylamino-l-alanine, in the free or protein-bound fraction of any of the 252 plasma samples.

Differential kynurenine pathway metabolism in highly metastatic aggressive breast cancer subtypes: beyond IDO1-induced immunosuppression.

BACKGROUND: Immunotherapy has recently been proposed as a promising treatment to stop breast cancer (BrCa) progression and metastasis. However, there has been limited success in the treatment of BrCa with immune checkpoint inhibitors. This implies that BrCa tumors have other mechanisms to escape immune surveillance. While the kynurenine pathway (KP) is known to be a key player mediating tumor immune evasion and while there are several studies on the roles of the KP in cancer, little is known about KP involvement in BrCa. METHODS: To understand how KP is regulated in BrCa, we examined the KP profile in BrCa cell lines and clinical samples (n = 1997) that represent major subtypes of BrCa (luminal, HER2-enriched, and triple-negative (TN)). We carried out qPCR, western blot/immunohistochemistry, and ultra-high pressure liquid chromatography on these samples to quantify the KP enzyme gene, protein, and activity, respectively. RESULTS: We revealed that the KP is highly dysregulated in the HER2-enriched and TN BrCa subtype. Gene, protein expression, and KP metabolomic profiling have shown that the downstream KP enzymes KMO and KYNU are highly upregulated in the HER2-enriched and TN BrCa subtypes, leading to increased production of the potent immunosuppressive metabolites anthranilic acid (AA) and 3-hydroxylanthranilic acid (3HAA). CONCLUSIONS: Our findings suggest that KMO and KYNU inhibitors may represent new promising therapeutic targets for BrCa. We also showed that KP metabolite profiling can be used as an accurate biomarker for BrCa subtyping, as we successfully discriminated TN BrCa from other BrCa subtypes.

Antenatal antioxidant treatment with melatonin to decrease newborn neurodevelopmental deficits and brain injury caused by fetal growth restriction.

Fetal intrauterine growth restriction (IUGR) is a serious pregnancy complication associated with increased rates of perinatal morbidity and mortality, and ultimately with long-term neurodevelopmental impairments. No intervention currently exists that can improve the structure and function of the IUGR brain before birth. Here, we investigated whether maternal antenatal melatonin administration reduced brain injury in ovine IUGR. IUGR was induced in pregnant sheep at 0.7 gestation and a subset of ewes received melatonin via intravenous infusion until term. IUGR, IUGR + melatonin (IUGR + MLT) and control lambs were born naturally, neonatal behavioral assessment was used to examine neurological function and at 24 hr after birth the brain was collected for the examination of neuropathology. Compared to control lambs, IUGR lambs took significantly longer to achieve normal neonatal lamb behaviors, such as standing and suckling. IUGR brains showed widespread cellular and axonal lipid peroxidation, and white matter hypomyelination and axonal damage. Maternal melatonin administration ameliorated oxidative stress, normalized myelination and rescued axonopathy within IUGR lamb brains, and IUGR + MLT lambs demonstrated significant functional improvements including a reduced time taken to attach to and suckle at the udder after birth. Based on these observations, we began a pilot clinical trial of oral melatonin administration to women with an IUGR fetus. Maternal melatonin was not associated with adverse maternal or fetal effects and it significantly reduced oxidative stress, as evidenced by reduced malondialdehyde levels, in the IUGR + MLT placenta compared to IUGR alone. Melatonin should be considered for antenatal neuroprotective therapy in human IUGR.

Nitric oxide stimulates myoglobin gene and protein expression in vascular smooth muscle.

Mb (myoglobin) is a haemoprotein present in cardiac, skeletal and smooth muscle and is primarily responsible for the storage and 'facilitated transfer' of molecular oxygen from the cell membrane to mitochondria. Also, Mb plays a role in regulating *NO (nitric oxide) homoeostasis through (i) binding *NO (Mb-NO complex); (ii) oxidation of *NO to nitrate; and (iii) formation of vasoactive S-nitroso-Mb [Rayner, B.S., Wu, B.-J., Raftery, M., Stocker, R. and Witting, P.K. (2005) J. Biol. Chem. 280, 9985-9993]. Pathological *NO concentrations affect mitochondrial function and decrease cell viability through inducing apoptosis. Treatment of cultured rat VSMCs (vascular smooth muscle cells) with cumulative doses (0.1, 1 or 10 microM) of *NO from the donors diethylamineNONOate or spermineNONOate (N-[2-aminoethyl]-N-[2-hydroxy-3-nitrosohydrazine]-1,2-ethelenediamine) yielded a time-dependent increase in Mb gene expression. Concomitant transcriptional activation increased the concentration of Mb within cultured rat or primary human VSMCs as judged by Western blot analysis and indirect immunofluorescence microscopy. Cell viability did not decrease in these cells at the *NO doses tested. Importantly, sub-culturing isolated rat aortic segments for 7 days in the presence of L-arginine at 37 degrees C stimulated *NO production with a parallel increase in Mb in the underlying VSMCs. Overall, exposure of VSMCs (either in cell culture or intact vessels) to pathological *NO promotes an up-regulation of the Mb gene and protein, suggesting a feedback relationship between *NO and Mb that regulates the concentration of the potent cell signalling molecule in the vessel wall, similar to the role haemoglobin plays in the vessel lumen.

Application of N-methyl-D-aspartate receptor nanopore in screening ligand molecules.

N-methyl-D-aspartate receptors (NMDARs) are crucial for excitatory synaptic transmission in the central nervous system. To study NMDARs more accurately and conveniently, we developed a stable NMDAR nanopore in a planar lipid bilayer. Pharmacological properties were validated using the allosteric modulator Ro 25-6981 and antagonist D-2-amino-5-phosphonopentanoic acid (D-APV). The cyanotoxin ß-N-methylamino-L-alanine (BMAA) found in fresh water systems is suspected to be associated with the development of neurodegenerative diseases. Therefore, BMAA and its two isomers L-2, 4-Diaminobutyric acid dihydrochloride (DAB) and N-(2-aminoethyl) glycine (AEG) and an endogenous excitotoxin, quinolinic acid (QA), were studied using the NMDAR nanopores to assess their effects on NMDAR modulation. We demonstrated that the NMDAR nanopore could reliably detect its ligand molecules at the single-channel level. The study also demonstrated the practicability of NMDAR nanopores, and results were validated using two-electrode voltage-clamp (TEVC) recording. Compared with TEVC recording, the NMDAR nanopores conducted ion channel gating at the single-channel level without being affected by other proteins on the cell membrane. The highly sensitive and accurate NMDAR nanopore technique thus has a unique advantage in screening NMDAR ligand molecules that could be associated with neurodegenerative disease.

Secretion of apolipoprotein E from macrophages occurs via a protein kinase A and calcium-dependent pathway along the microtubule network.

Macrophage-specific expression of apolipoprotein (apo)E protects against atherosclerosis; however, the signaling and trafficking pathways regulating secretion of apoE are unknown. We investigated the roles of the actin skeleton, microtubules, protein kinase A (PKA) and calcium (Ca2+) in regulating apoE secretion from macrophages. Disrupting microtubules with vinblastine or colchicine inhibited basal secretion of apoE substantially, whereas disruption of the actin skeleton had no effect. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI(14-22)) all decreased basal secretion of apoE by between 50% to 80% (P<0.01). Pulse-chase experiments demonstrated that inhibition of PKA reduced the rate of apoE secretion without affecting its degradation. Confocal microscopy and live cell imaging of apoE-green fluorescent protein-transfected RAW macrophages identified apoE-green fluorescent protein in vesicles colocalized with the microtubular network, and inhibition of PKA markedly inhibited vesicular movement. Chelation of intracellular calcium ([Ca2+]i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM) inhibited apoE secretion by 77.2% (P<0.01). Injection of c57Bl6 apoE+/+ bone marrow-derived macrophages into the peritoneum of apoE-/- C57Bl6 mice resulted in time-dependent secretion of apoE into plasma, which was significantly inhibited by transient exposure of macrophages to BAPTA-AM and colchicine and less effectively inhibited by H89. We conclude that macrophage secretion of apoE occurs via a PKA- and calcium-dependent pathway along the microtubule network.

Effects of hydrogen peroxide and apolipoprotein E isoforms on apolipoprotein E trafficking in HepG2 cells.

1. The major source of apolipoprotein E (apoE) is the liver. In the present study, the effects of oxidative stress and apoE isoforms on apoE distribution and trafficking were established using the HepG2 liver tumour cell line. 2. Hydrogen peroxide (0, 25, 250 and 1000 micromol/L) was associated with rapid and concentration-dependent redistribution of apoE into the early endosomal compartment. This redistribution was achieved with a much lower concentration (25 micromol/L) than that needed to induce changes in intracellular apoE mRNA expression, apoE protein levels and markers of oxidative stress (250-1000 micromol/L). 3. Live cell imaging of apoE3-green fluorescent protein revealed a significant decrease in traffic velocity in response to oxidative stress. 4. The E4 isoform was associated with reduced trafficking velocity compared with the E3 isoform under basal conditions. 5. The results indicate that oxidative stress and apoE isoforms influence apoE trafficking and distribution within HepG2 cells. Altered apoE hepatocyte trafficking may provide a mechanistic link between oxidative stress, ageing and some diseases in older people.

Human Amnion Epithelial Cells Protect Against White Matter Brain Injury After Repeated Endotoxin Exposure in the Preterm Ovine Fetus.

Intrauterine inflammation is a significant cause of injury to the developing fetal brain. Using a preterm fetal sheep model of in utero infection, we asked whether human amnion epithelial cells (hAECs) were able to reduce inflammation-induced fetal brain injury. Surgery was undertaken on pregnant sheep at ∼105 days gestation (term is 147 days) for implantation of vascular catheters. Lipopolysaccharide (LPS; 150 ng/kg bolus) or saline was administered IV at 109, 110, and 111 days. Sixty million fluorescent-labeled hAECs were administered at 110, 111, and 112 days gestation via the brachial artery catheter. Brains were collected at 114 days for histological assessment. hAECs were observed within the cortex, white matter, and hippocampus. Compared to control lambs, LPS administration was associated with significant and widespread fetal brain inflammation and injury as evidenced by increased number of activated microglia in the periventricular white matter (p = 0.02), increased pyknosis, cell degeneration (p = 0.01), and a nonsignificant trend of fewer oligodendrocytes in the subcortical and periventricular white matter. Administration of hAECs to LPS-treated animals was associated with a significant mitigation in both inflammation and injury as evidenced by fewer activated microglia (p = 0.03) and pyknotic cells (p = 0.03), significantly more oligodendrocytes in the subcortical and periventricular white matter (p = 0.01 and 0.02, respectively), and more myelin basic protein-positive cells within the periventricular white matter (p = 0.02). hAEC administration to fetal sheep exposed to multiple doses of LPS dampens the resultant fetal inflammatory response and mitigates associated brain injury.

Selective inhibition of the master regulator transcription factor Egr-1 with catalytic oligonucleotides reduces myocardial injury and improves left ventricular systolic function in a preclinical model of myocardial infarction.

BACKGROUND: Egr-1 is implicated in the pathogenesis of myocardial ischemia-reperfusion injury. The aim of this study was to ascertain the effectiveness of intracoronary delivery of DNAzyme targeting the transcription factor Egr-1 at reperfusion following experimental myocardial ischemia. METHODS AND RESULTS: Functional DNAzyme targeting Egr-1 or a size-matched scrambled control were delivered via the intracoronary route immediately on reperfusion after 60 minutes' balloon occlusion of the left anterior descending coronary artery in a pig model of myocardial I/R injury (n=7 per treatment group). Heart function and extent of myocardial infarction were determined following intervention by echocardiography and cardiac magnetic resonance imaging, respectively. Hearts were removed and examined for molecular and histological markers of inflammation and apoptosis. Administration of functional DNAzyme led to an overall decrease in the expression of inflammatory markers including intracellular adhesion molecule-1, tissue factor, and complement 3, with associated decreases in the extent of neutrophil infiltration, oxidative damage, and subsequent apoptosis within the infarct border zone. Functional significance was indicated by an increase in salvaged left ventricular myocardium (P=0.012), ejection fraction (P=0.002), and fractional area change (P=0.039) in the functional DNAzyme-treated group compared with the control. CONCLUSIONS: Egr-1 silencing through intracoronary delivery of a targeting DNAzyme at the time of reperfusion following acute myocardial ischemia decreases myocardial inflammation and apoptosis leading to improved cardiac function.

The effect of old age on apolipoprotein E and its receptors in rat liver.

Apolipoprotein E (apoE) is associated with aging and some age-related diseases. The majority of apoE is produced by hepatocytes for the receptor-mediated uptake of lipoproteins. Here, the effects of age on the hepatic expression and distribution of apoE and its receptors were determined using immunofluorescence, Western blots, and quantitative PCR in rat liver tissue and isolated hepatocytes. The expression of apoE mRNA and protein was not influenced significantly by aging. Immunofluorescence studies in isolated hepatocytes showed that apoE was more likely to be co-localized with early endosomes, golgi, and microtubules in isolated old hepatocytes. The mRNA expression of the receptor involved in sequestration of apoE, heparan sulfate proteoglycan was reduced in old age, without any significant effect on the expression of either the low-density lipoprotein receptor or low density-lipoprotein receptor-related protein. Old age is associated with changes in hepatic apoE intracellular trafficking and heparan sulfate proteoglycan expression that might contribute to age-related disease.

The effect of aging on the response of isolated hepatocytes to hydrogen peroxide and tert-butyl hydroperoxide.

Aging is associated with increased susceptibility to oxidative stress. To study this in the liver and to elucidate underlying mechanisms, hepatocytes from young (4-6 months) and old (24-26 months) rats were exposed to two oxidants, hydrogen peroxide and tert-butyl hydroperoxide. ATP content and mitochondrial activity were lower in old hepatocytes and decreased further with oxidative stress. Expression of Cu/Zn superoxide dismutase, Mn superoxide dismutase and catalase was not substantially influenced by oxidative stress in young and old hepatocytes, whereas glutathione peroxidase 1 expression was markedly increased only in young hepatocytes. Oxidative stress in young hepatocytes led to increased expression of apoE and movement of apoE to the early endosomes. In old hepatocytes, oxidative stress did not increase apoE expression and apoE was co-localized with early endosomes under control conditions. The results show that old age is associated with impaired hepatocyte responses of mitochondria, ATP, glutathione peroxidase 1 and apoE to oxidative stress.