A microenvironment-inspired synthetic three-dimensional model for pancreatic ductal adenocarcinoma organoids.

Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α3/α6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro.

Widespread severe cerebral elevations of haptoglobin and haemopexin in sporadic Alzheimer's disease: Evidence for a pervasive microvasculopathy.

Sporadic Alzheimer's disease (sAD) is the commonest cause of age-related neurodegeneration but there are no available treatments with demonstrated disease-modifying actions. It is therefore relevant to study hitherto-unknown aspects of brain structure and function to seek new disease-related mechanisms that might be targeted by novel disease-modifying interventions. During hypothesis-generating proteomic investigations in a case-control study of sAD, we observed widespread elevations of haptoglobin and haemopexin in all six brain-regions studied, which together represent much of the brain. Measured perturbations were significant, with the posterior probability of upregulation generally >95% and haptoglobin doubling in expression levels on average across deep brain structures (hippocampus, entorhinal cortex and cingulate gyrus) as well as sensory and motor cortices, and cerebellum. Haptoglobin and haemopexin are often regarded as circulating proteins whose main functions are to bind, respectively, the strongly pro-inflammatory extracellular haemoglobin and haeme molecules that form following haemolysis, thereby promoting their clearance and suppressing damage they might otherwise cause, for example, acute kidney injury. To our knowledge, elevations in neither cerebral haptoglobin nor haemopexin have previously been linked to the pathogenesis of sAD. Post-mortem examination of these cases showed no signs of macroscopic cerebral haemorrhage. These findings demonstrate pervasive cerebral elevation of haptoglobin and haemopexin, consistent with low-level intracerebral leakage of haemoglobin and consequent haeme formation throughout sAD brain. They point to a widespread underlying microvasculopathy that facilitates erythrocyte leakage, thereby triggering elevated tissue-free haemoglobin and driving the measured elevations in haptoglobin and haemopexin.

Cerebral deficiency of vitamin B5 (d-pantothenic acid; pantothenate) as a potentially-reversible cause of neurodegeneration and dementia in sporadic Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of age-related neurodegeneration and dementia, and there are no available treatments with proven disease-modifying actions. It is therefore appropriate to study hitherto-unknown aspects of brain structure/function in AD to seek alternative disease-related mechanisms that might be targeted by new therapeutic interventions with disease-modifying actions. During hypothesis-generating metabolomic studies of brain, we identified apparent differences in levels of vitamin B5 between AD cases and controls. We therefore developed a method based on gas chromatography-mass spectrometry by which we quantitated vitamin B5 concentrations in seven brain regions from nine AD cases and nine controls. We found that widespread, severe cerebral deficiency of vitamin B5 occurs in AD. This deficiency was worse in those regions known to undergo severe damage, including the hippocampus, entorhinal cortex, and middle temporal gyrus. Vitamin B5 is the obligate precursor of CoA/acetyl-CoA (acetyl-coenzyme A), which plays myriad key roles in the metabolism of all organs, including the brain. In brain, acetyl-CoA is the obligate precursor of the neurotransmitter acetylcholine, and the complex fatty-acyl groups that mediate the essential insulator role of myelin, both processes being defective in AD; moreover, the large cerebral vitamin B5 concentrations co-localize almost entirely to white matter. Vitamin B5 is well tolerated when administered orally to humans and other mammals. We conclude that cerebral vitamin B5 deficiency may well cause neurodegeneration and dementia in AD, which might be preventable or even reversible in its early stages, by treatment with suitable oral doses of vitamin B5.

Vitamin B5 (d-pantothenic acid) localizes in myelinated structures of the rat brain: Potential role for cerebral vitamin B5 stores in local myelin homeostasis.

Vitamin B5 (d-pantothenic acid; pantothenate) is an essential trace nutrient that functions as the obligate precursor of coenzyme A (CoA), through which it plays key roles in myriad biological processes, including many that regulate carbohydrate, lipid, protein, and nucleic acid metabolism. In the brain, acetyl-CoA is necessary for synthesis of the complex fatty-acyl chains of myelin, and of the neurotransmitter acetylcholine. We recently found that cerebral pantothenate is markedly lowered, averaging ∼55% of control values in cases of Huntington's disease (HD) including those who are pre-symptomatic, and that regions where pantothenate is lowered correspond to those which are more severely damaged. Here we sought to determine the previously unknown distribution of pantothenate in the normal-rat brain, and whether the diabetic rat might be useful as a model for altered cerebral pantothenate metabolism. We employed histological staining (Nissl) to identify brain structures; immunohistochemistry with anti-pantothenate antibodies to determine the distribution of pantothenate in caudate putamen and cerebellum; and gas-chromatography/mass-spectrometry to quantitate levels of pantothenate and other metabolites in normal- and diabetic-rat brain. Remarkably, cerebral pantothenate was almost entirely localized to myelin-containing structures in both experimental groups. Diabetes did not modify levels or disposition of cerebral pantothenate. These findings are consistent with physiological localization of pantothenate in myelinated white-matter structures, where it could serve to support myelin synthesis. Further investigation of cerebral pantothenate is warranted in neurodegenerative diseases such as HD and Alzheimer's disease, where myelin loss is a known characteristic of pathogenesis.

Physiological and metabolomics analyses of young and old leaves from wild and cultivated soybean seedlings under low-nitrogen conditions.

BACKGROUND: It is critical to study the low nitrogen tolerance in wild soybean with extensive genetic diversity for improving cultivated soybean nitrogen use efficiency. Focusing on plant young and old leaves could provide new insights to low nitrogen tolerance research. This study compared the low nitrogen group with the control group on physiological and metabolomics changes in young and old leaves, respectively, then analyzed and compared the differences of these changes between cultivated and wild soybean. This study aimed to provide a theoretical basis for the molecular mechanism of soybean low nitrogen stress tolerance. RESULTS: Wild soybean was less affected by low-nitrogen stress than cultivated soybean as assessed by plant biomass paraments, total carbon content and total nitrogen content. Gas-exchange coefficients and chlorophylls contents maintained relatively stable in wild soybean young leaves, but opposite in cultivated soybean. Wild soybean young leaves also increased the transport of beneficial ions, such as B3+, Fe3+, Mn2+, H2PO4- and C2O42-. In wild soybean old leaves, the nitrogen metabolism pathway was significant enhanced, especially the aspartic acid and GABA metabolisms. While in cultivated soybean, the nitrogen metabolism decreased obviously in young leaves but had no significant change in old leaves. The phenylpropanoid metabolism pathway was also activated in wild soybean. Contrary to cultivated soybeans, wild soybean tricarboxylic acid cycle and carbon metabolism including polyols and organic acids consolidated in old leaves and maintained a relative normal state in young leaves. These strategies could improve the antioxidant and N-fixation capacity in wild soybean. CONCLUSION: The survival and growth of wild soybean under low nitrogen stress conditions relied on physiological adjustments and metabolic changes that occurred at the cellular level. Compared with cultivated soybean, wild soybean young leaves could maintain a relatively normal growth mainly owing to a significant enhancement of key amino acids and nonprotein nitrogen metabolism in old leaves, especially aspartic acid, proline metabolism which provided basis for nitrogen reutilization from old leaves to young leaves. Consolidating the tricarboxylic acid cycle, intensifying phenylpropanoid metabolism, and accumulating more polyols and organic acids also had positive effect on it.

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes.

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy.

Plasma metals as potential biomarkers in dementia: a case-control study in patients with sporadic Alzheimer's disease.

Sporadic Alzheimer's disease (AD) is a neurodegenerative disorder that causes the most prevalent form of age-related dementia but its pathogenesis remains obscure. Altered regulation of metals, particularly pan-cerebral copper deficiency, and more regionally-localized perturbation of other metals, are prominent in AD brain although data on how these CNS perturbations are reflected in the peripheral bloodstream are inconsistent to date. To assess the potential use of metal dysregulation to generate biomarkers in AD, we performed a case-control study of seven essential metals and selenium, measured by inductively coupled plasma mass-spectrometry, in samples from AD and matched control cases. Metals were sodium, potassium, calcium, magnesium, iron, zinc, and copper. In the whole study-group and in female participants, plasma metal levels did not differ between cases and controls. In males by contrast, there was moderate evidence that zinc levels trended towards increase in AD [10.8 (10.2-11.5)] µmol/L, mean (± 95% CI; P = 0.021) compared with controls [10.2 (9.6-10.4)]. Thus alterations in plasma zinc levels differed between genders in AD. In correlational analysis, there was evidence for an increased number of 'strong' metal co-regulations in AD cases and differential co-modulations of metal pairs: copper-sodium (Rcontrol = - 0.03, RAD = 0.65; P = 0.009), and copper-calcium (Rcontrol = - 0.01, RAD = 0.65; P = 0.01) were significant in AD males, potentially consistent with reported evidence for dysregulation of copper in severely damaged brain regions in AD. In conclusion, our data suggest that the measurement of metals co-regulation in plasma may provide a useful representation of those metal perturbations taking place in the AD brain and therefore might be useful as plasma-based biomarkers.

Metabolite mapping reveals severe widespread perturbation of multiple metabolic processes in Huntington's disease human brain.

Huntington's disease (HD) is a genetically-mediated neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein (Htt) through lengthening of its polyglutamine tract, thus initiating a cascade that ultimately leads to premature death. However, neurodegeneration typically manifests in HD only in middle age, and mechanisms linking the causative mutation to brain disease are poorly understood. Brain metabolism is severely perturbed in HD, and some studies have indicated a potential role for mutant Htt as a driver of these metabolic aberrations. Here, our objective was to determine the effects of HD on brain metabolism by measuring levels of polar metabolites in regions known to undergo varying degrees of damage. We performed gas-chromatography/mass spectrometry-based metabolomic analyses in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine matched controls. In each patient, we measured metabolite content in representative tissue-samples from eleven brain regions that display varying degrees of damage in HD, thus identifying the presence and abundance of 63 different metabolites from several molecular classes, including carbohydrates, amino acids, nucleosides, and neurotransmitters. Robust alterations in regional brain-metabolite abundances were observed in HD patients: these included changes in levels of small molecules that play important roles as intermediates in the tricarboxylic-acid and urea cycles, and amino-acid metabolism. Our findings point to widespread disruption of brain metabolism and indicate a complex phenotype beyond the gradient of neuropathologic damage observed in HD brain.

Graded perturbations of metabolism in multiple regions of human brain in Alzheimer's disease: Snapshot of a pervasive metabolic disorder.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that displays pathological characteristics including senile plaques and neurofibrillary tangles. Metabolic defects are also present in AD-brain: for example, signs of deficient cerebral glucose uptake may occur decades before onset of cognitive dysfunction and tissue damage. There have been few systematic studies of the metabolite content of AD human brain, possibly due to scarcity of high-quality brain tissue and/or lack of reliable experimental methodologies. Here we sought to: 1) elucidate the molecular basis of metabolic defects in human AD-brain; and 2) identify endogenous metabolites that might guide new approaches for therapeutic intervention, diagnosis or monitoring of AD. Brains were obtained from nine cases with confirmed clinical/neuropathological AD and nine controls matched for age, sex and post-mortem delay. Metabolite levels were measured in post-mortem tissue from seven regions: three that undergo severe neuronal damage (hippocampus, entorhinal cortex and middle-temporal gyrus); three less severely affected (cingulate gyrus, sensory cortex and motor cortex); and one (cerebellum) that is relatively spared. We report a total of 55 metabolites that were altered in at least one AD-brain region, with different regions showing alterations in between 16 and 33 metabolites. Overall, we detected prominent global alterations in metabolites from several pathways involved in glucose clearance/utilization, the urea cycle, and amino-acid metabolism. The finding that potentially toxigenic molecular perturbations are widespread throughout all brain regions including the cerebellum is consistent with a global brain disease process rather than a localized effect of AD on regional brain metabolism.

Identification of elevated urea as a severe, ubiquitous metabolic defect in the brain of patients with Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein through the lengthening of a polyglutamine tract and initiates a cascade that ultimately leads to dementia and premature death. However, neurodegeneration typically manifests in HD only in middle age, and processes linking the causative mutation to brain disease are poorly understood. Here, our objective was to elucidate further the processes that cause neurodegeneration in HD, by measuring levels of metabolites in brain regions known to undergo varying degrees of damage. We applied gas-chromatography/mass spectrometry-based metabolomics in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine controls. Unexpectedly, a single major abnormality was evident in all eleven brain regions studied across the forebrain, midbrain and hindbrain, namely marked elevation of urea, a metabolite formed in the urea cycle by arginase-mediated cleavage of arginine. Urea cycle activity localizes primarily in the liver, where it functions to incorporate protein-derived amine-nitrogen into urea for recycling or urinary excretion. It also occurs in other cell-types, but systemic over-production of urea is not known in HD. These findings are consistent with impaired local urea regulation in brain, by up-regulation of synthesis and/or defective clearance. We hypothesize that defective brain urea metabolism could play a substantive role in the pathogenesis of neurodegeneration, perhaps via defects in osmoregulation or nitrogen metabolism. Brain urea metabolism is therefore a target for generating novel monitoring/imaging strategies and/or therapeutic interventions aimed at ameliorating the impact of HD in patients.

Diabetic cardiomyopathy is associated with defective myocellular copper regulation and both defects are rectified by divalent copper chelation.

BACKGROUND: Heart disease is the leading cause of death in diabetic patients, and defective copper metabolism may play important roles in the pathogenesis of diabetic cardiomyopathy (DCM). The present study sought to determine how myocardial copper status and key copper-proteins might become impaired by diabetes, and how they respond to treatment with the Cu (II)-selective chelator triethylenetetramine (TETA) in DCM. METHODS: Experiments were performed in Wistar rats with streptozotocin (STZ)-induced diabetes with or without TETA treatment. Cardiac function was analyzed in isolated-perfused working hearts, and myocardial total copper content measured by particle-induced x-ray emission spectroscopy (PIXE) coupled with Rutherford backscattering spectrometry (RBS). Quantitative expression (mRNA and protein) and/or activity of key proteins that mediate LV-tissue-copper binding and transport, were analyzed by combined RT-qPCR, western blotting, immunofluorescence microscopy, and enzyme activity assays. Statistical analysis was performed using Student's t-tests or ANOVA and p-values of < 0.05 have been considered significant. RESULTS: Left-ventricular (LV) copper levels and function were severely depressed in rats following 16-weeks' diabetes, but both were unexpectedly normalized 8-weeks after treatment with TETA was instituted. Localized myocardial copper deficiency was accompanied by decreased expression and increased polymerization of the copper-responsive transition-metal-binding metallothionein proteins (MT1/MT2), consistent with impaired anti-oxidant defences and elevated susceptibility to pro-oxidant stress. Levels of the high-affinity copper transporter-1 (CTR1) were depressed in diabetes, consistent with impaired membrane copper uptake, and were not modified by TETA which, contrastingly, renormalized myocardial copper and increased levels and cell-membrane localization of the low-affinity copper transporter-2 (CTR2). Diabetes also lowered indexes of intracellular (IC) copper delivery via the copper chaperone for superoxide dismutase (CCS) to its target cuproenzyme, superoxide dismutase-1 (SOD1): this pathway was rectified by TETA treatment, which normalized SOD1 activity with consequent bolstering of anti-oxidant defenses. Furthermore, diabetes depressed levels of additional intracellular copper-transporting proteins, including antioxidant-protein-1 (ATOX1) and copper-transporting-ATPase-2 (ATP7B), whereas TETA elevated copper-transporting-ATPase-1 (ATP7A). CONCLUSIONS: Myocardial copper deficiency and defective cellular copper transport/trafficking are revealed as key molecular defects underlying LV impairment in diabetes, and TETA-mediated restoration of copper regulation provides a potential new class of therapeutic molecules for DCM.

Human dementia with Lewy bodies brain shows widespread urea elevations.

INTRODUCTION: Several recent studies have uncovered the presence of widespread urea elevations in multiple neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease dementia (PDD), vascular dementia (VaD), and Huntington's disease (HD). However, it is currently unknown whether dementia with Lewy bodies also shows these alterations in urea. This study aimed to investigate if and where urea is perturbed in the DLB brain. METHODS: Tissues from ten brain regions were obtained from 20 diagnosed cases of DLB and 19 controls. Urea concentrations were measured using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Case-control differences were assessed by nonparametric Mann-Whitney U tests, and s-values, E-values, effect sizes, and risk ratios were determined for each brain region. The results were compared to those previously obtained for AD, PDD, VaD, and HD. RESULTS: As with other previously investigated dementia diseases, DLB shows widespread urea elevations, affecting all ten regions investigated in the current study; the degree of these elevations is lower than that seen in AD or PDD, similar to that seen in HD, and higher than that observed in VaD. The highest urea fold-change was observed in the pons and the lowest in the primary visual cortex. CONCLUSION: Urea elevations appear to be a shared alterations across at least five neurodegenerative diseases, despite their many differences in clinical and neuropathological presentation. The cause and effects of this perturbation should be the focus of future studies, for its possible contributions to the pathology of these conditions.

Localized Pantothenic Acid (Vitamin B5) Reductions Present Throughout the Dementia with Lewy Bodies Brain.

Background: Localized pantothenic acid deficiencies have been observed in several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease dementia (PDD), and Huntington's disease (HD), indicating downstream energetic pathway perturbations. However, no studies have yet been performed to see whether such deficiencies occur across the dementia with Lewy bodies (DLB) brain, or what the pattern of such dysregulation may be. Objective: Firstly, this study aimed to quantify pantothenic acid levels across ten regions of the brain in order to determine the localization of any pantothenic acid dysregulation in DLB. Secondly, the localization of pantothenic acid alterations was compared to that previously in AD, PDD, and HD brains. Methods: Pantothenic acid levels were determined in 20 individuals with DLB and 19 controls by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) across ten brain regions. Case-control differences were determined by nonparametric Mann-Whitney U test, with the calculation of S-values, risk ratios, E-values, and effect sizes. The results were compared with those previously obtained in DLB, AD, and HD. Results: Pantothenic acid levels were significantly decreased in six of the ten investigated brain regions: the pons, substantia nigra, motor cortex, middle temporal gyrus, primary visual cortex, and hippocampus. This level of pantothenic acid dysregulation is most similar to that of the AD brain, in which pantothenic acid is also decreased in the motor cortex, middle temporal gyrus, primary visual cortex, and hippocampus. DLB appears to differ from other neurodegenerative diseases in being the only of the four to not show pantothenic acid dysregulation in the cerebellum. Conclusions: Pantothenic acid deficiency appears to be a shared mechanism of several neurodegenerative diseases, although differences in the localization of this dysregulation may contribute to the differing clinical pathways observed in these conditions.

Decreases in a molecule called pantothenic acid (also known as vitamin B5) have been observed in several areas of the brain in multiple dementia disease, including Alzheimer's disease, Parkinson's disease dementia, and Huntington's disease. However, it is unknown whether such changes also occur in another dementia disease, dementia with Lewy bodies, which shows many of the same symptoms and molecular changes as these conditions. As such, this study was performed in order to determine if and where changes in pantothenic acid occur throughout the dementia with Lewy bodies brain. Using a methodology called liquid chromatography­mass spectrometry, which is able to measure pantothenic acid levels in a highly precise manner in brain tissues, we found that several regions of the dementia with Lewy bodies brain show decreases in pantothenic acid, including some involved in movement such as the substantia nigra and motor cortex, as well as regions associated with cognition and memory such as the hippocampus­looking most similar to the pattern of changes already seen in Alzheimer's disease. It is possible that these changes contribute to the progression of dementia with Lewy bodies; however, further studies need to be performed to determine at what point these changes happen during the disease and how they may contribute to the development of symptoms.

Widespread selenium deficiency in the brain of cases with Huntington's disease presents a new potential therapeutic target.

BACKGROUND: Huntington or Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterised by both progressive motor and cognitive dysfunction; its pathogenic mechanisms remain poorly understood and no treatment can currently slow, stop, or reverse its progression. There is some evidence of metallomic dysfunction in limited regions of the HD brain; we hypothesised that these alterations are more widespread than the current literature suggests and may contribute to pathogenesis in HD. METHODS: We measured the concentrations of eight essential metals (sodium, potassium, magnesium, calcium, iron, zinc, copper, and manganese) and the metalloid selenium across 11 brain regions in nine genetically confirmed, clinically manifest cases of HD and nine controls using inductively-coupled plasma mass spectrometry. Case-control differences were assessed by non-parametric Mann-Whitney U test (p < 0.05), risk ratios, E-values, and effect sizes. FINDINGS: We observed striking decreases in selenium levels in 11 out of 11 investigated brain regions in HD, with risk ratios and effect sizes ranging 2.3-9.0 and 0.7-1.9, respectively. Increased sodium/potassium ratios were observed in every region (risk ratio = 2.5-8.0; effect size = 1.2-5.8) except the substantia nigra (risk ratio = 0.25; effect size = 0.1). Multiple regions showed increased calcium and/or zinc levels, and localised decreases in iron, copper, and manganese were present in the globus pallidus, cerebellum, and substantia nigra, respectively. INTERPRETATION: The observed metallomic alterations in the HD brain may contribute to several pathogenic mechanisms, including mitochondrial dysfunction, oxidative stress, and blood-brain barrier dysfunction. Selenium supplementation may represent a potential, much-needed therapeutic pathway for the treatment of HD that would not require localised delivery in the brain due to the widespread presence of selenium deficiency in regions that show both high and low levels of neurodegeneration. FUNDING: In Acknowledgments, includes the Lee Trust, the Endocore Research Trust, Cure Huntington's Disease Initiative, the Oakley Mental Health Research Foundation, the Medical Research Council (MRC), the New Zealand Neurological Foundation, and others.

Advances in the Specificity of Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy Based Structural Characterisation Methods for Synthetic Oligonucleotides.

Identity testing is a critical part in the development of a therapeutic synthetic oligonucleotide. Tandem Mass Spectrometry (MS/MS) is commonly used for the analysis of oligonucleotides to obtain structural and sequence information, however there are challenges resulting from chemical modifications introduced to improve their pharmacokinetics and stability. For these structurally complex oligonucleotides, Nuclear Magnetic Resonance (NMR) Spectroscopy has found limited use for characterisation and identity testing, as only partial NMR resonance assignment for oligonucleotides is achieved without isotopic labelling methodologies. Regardless of the choice of method used for oligonucleotide analysis, the specificity is of critical importance. In this work, in-source dissociation mass spectrometry and proton (1H) and carbon (13C) NMR at high temperature were used to analyse danvatirsen, a 16 nucleotide phosphorothioate antisense oligonucleotide, and its closely related switch sequences. Both approaches have shown specificity to distinguish danvatirsen from these similar sequences.

Extensive multiregional urea elevations in a case-control study of vascular dementia point toward a novel shared mechanism of disease amongst the age-related dementias.

Introduction: Vascular dementia (VaD) is one of the most common causes of dementia among the elderly. Despite this, the molecular basis of VaD remains poorly characterized when compared to other age-related dementias. Pervasive cerebral elevations of urea have recently been reported in several dementias; however, a similar analysis was not yet available for VaD. Methods: Here, we utilized ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to measure urea levels from seven brain regions in post-mortem tissue from cases of VaD (n = 10) and controls (n = 8/9). Brain-urea measurements from our previous investigations of several dementias were also used to generate comparisons with VaD. Results: Elevated urea levels ranging from 2.2- to 2.4-fold-change in VaD cases were identified in six out of the seven regions analysed, which are similar in magnitude to those observed in uremic encephalopathy. Fold-elevation of urea was highest in the basal ganglia and hippocampus (2.4-fold-change), consistent with the observation that these regions are severely affected in VaD. Discussion: Taken together, these data not only describe a multiregional elevation of brain-urea levels in VaD but also imply the existence of a common urea-mediated disease mechanism that is now known to be present in at least four of the main age-related dementias.

Coenzyme A-Dependent Tricarboxylic Acid Cycle Enzymes Are Decreased in Alzheimer's Disease Consistent With Cerebral Pantothenate Deficiency.

Sporadic Alzheimer's disease (sAD) is the commonest cause of age-related neurodegeneration and dementia globally, and a leading cause of premature disability and death. To date, the quest for a disease-modifying therapy for sAD has failed, probably reflecting our incomplete understanding of aetiology and pathogenesis. Drugs that target aggregated Aß/tau are ineffective, and metabolic defects are now considered to play substantive roles in sAD pathobiology. We tested the hypothesis that the recently identified, pervasive cerebral deficiency of pantothenate (vitamin B5) in sAD, might undermine brain energy metabolism by impairing levels of tricarboxylic acid (TCA)-cycle enzymes and enzyme complexes, some of which require the pantothenate-derived cofactor, coenzyme A (CoA) for their normal functioning. We applied proteomics to measure levels of the multi-subunit TCA-cycle enzymes and their cytoplasmic homologues. We analysed six functionally distinct brain regions from nine sAD cases and nine controls, measuring 33 cerebral proteins that comprise the nine enzymes of the mitochondrial-TCA cycle. Remarkably, we found widespread perturbations affecting only two multi-subunit enzymes and two enzyme complexes, whose function is modulated, directly or indirectly by CoA: pyruvate dehydrogenase complex, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase complex, and succinyl-CoA synthetase. The sAD cases we studied here displayed widespread deficiency of pantothenate, the obligatory precursor of CoA. Therefore, deficient cerebral pantothenate can damage brain-energy metabolism in sAD, at least in part through impairing levels of these four mitochondrial-TCA-cycle enzymes.

Contrasting Sodium and Potassium Perturbations in the Hippocampus Indicate Potential Na+/K+-ATPase Dysfunction in Vascular Dementia.

Vascular dementia (VaD) is thought to be the second most common cause of age-related dementia amongst the elderly. However, at present, there are no available disease-modifying therapies for VaD, probably due to insufficient understanding about the molecular basis of the disease. While the notion of metal dyshomeostasis in various age-related dementias has gained considerable attention in recent years, there remains little comparable investigation in VaD. To address this evident gap, we employed inductively coupled-plasma mass spectrometry to measure the concentrations of nine essential metals in both dry- and wet-weight hippocampal post-mortem tissue from cases with VaD (n = 10) and age-/sex-matched controls (n = 10). We also applied principal component analysis to compare the metallomic pattern of VaD in the hippocampus with our previous hippocampal metal datasets for Alzheimer's disease, Huntington's disease, Parkinson's disease, and type-2 diabetes, which had been measured using the same methodology. We found substantive novel evidence for elevated hippocampal Na levels and Na/K ratios in both wet- and dry-weight analyses, whereas decreased K levels were present only in wet tissue. Multivariate analysis revealed no distinguishable hippocampal differences in metal-evoked patterns between these dementia-causing diseases in this study. Contrasting levels of Na and K in hippocampal VaD tissue may suggest dysfunction of the Na+/K+-exchanging ATPase (EC 7.2.2.13), possibly stemming from deficient metabolic energy (ATP) generation. These findings therefore highlight the potential diagnostic importance of cerebral sodium measurement in VaD patients.

Elevated hippocampal copper in cases of type 2 diabetes.

BACKGROUND: Type-2 diabetes (T2D) is characterized by chronic hyperglycaemia and glucose-evoked organ damage, and displays systemic copper overload, elevated risk of impaired cognitive function, and epidemiological links to sporadic Alzheimer's disease (sAD). Contrastingly, sAD exhibits impaired cerebral-glucose uptake, elevation of cerebral glucose but not blood glucose levels, and widespread cerebral-copper deficiency. We hypothesized that sAD-like brain-metal perturbations would occur in T2D. METHODS: We measured nine essential elements in an observational case-control study of T2D without dementia (6 cases and 6 controls) in four brain regions and compared the results with those from our study of brain metals in sAD (9 cases and 9 controls), which employed equivalent analytical methodology. We evaluated intergroup differences by supervised and unsupervised multivariate-statistical approaches to contrast between T2D cases and controls, and to compare them with cerebral-metal patterns in sAD. FINDINGS: Unexpectedly, we found that hippocampal-copper levels in T2D were markedly elevated compared with controls (P = 0.005 and 0.007 by Welch's t-test in two technical-replicate experiments), to levels similar to those in cases of untreated Wilson's disease (WD), wherein elevated cerebral copper causes neurodegeneration. By contrast, hippocampal-copper levels in sAD were markedly deficient. Multivariate analysis identified marked differences in patterns of essential metals between hippocampal datasets from cases of T2D and of sAD. INTERPRETATION: Elevated hippocampal copper could contribute to the pathogenesis of cerebral neurodegeneration and cognitive impairment in T2D, similar to known impacts of elevated brain copper in WD. Therapeutic approaches with copper-lowering agents similar to those currently employed in pharmacotherapy of WD, may also be applicable in patients with T2D and impaired cognitive function. Further studies will be required to replicate and extend these findings and to investigate their potential therapeutic implications. FUNDING: In Acknowledgments, includes Endocore Research Trust; Lee Trust; Oakley Mental Health Research Foundation; Ministry of Business, Innovation & Employment; The Universities of Auckland and Manchester, and others.

Pan-cerebral sodium elevations in vascular dementia: Evidence for disturbed brain-sodium homeostasis.

Vascular dementia (VaD) is the second most common cause of cognitive impairment amongst the elderly. However, there are no known disease-modifying therapies for VaD, probably due to incomplete understanding of the molecular basis of the disease. Despite the complex etiology of neurodegenerative conditions, a growing body of research now suggests the potential involvement of metal dyshomeostasis in the pathogenesis of several of the age-related dementias. However, by comparison, there remains little research investigating brain metal levels in VaD. In order to shed light on the possible involvement of metal dyshomeostasis in VaD, we employed inductively coupled plasma-mass spectrometry to quantify the levels of essential metals in post-mortem VaD brain tissue (n = 10) and age-/sex-matched controls (n = 10) from seven brain regions. We found novel evidence for elevated wet-weight cerebral sodium levels in VaD brain tissue in six out of the seven regions analyzed. Decreased cerebral-potassium levels as well as increased Na/K ratios (consistent with high tissue sodium and low potassium levels) were also observed in several brain regions. These data suggest that reduced Na+/K+-exchanging ATPase (EC 7.2.2.13) activity could contribute to the contrasting changes in sodium and potassium measured here.