bluuebunny/crossref_metadata_2025_split huggingface dataset:
High‐throughput Targeted Lipidomics Analysis of Dihydroceramide Desaturase‐1 (DES1) Knockout Mice
Authors: Pearson Mackenzie Jo , Kapil Santosh J. , Ubhi Baljit J. , Summers Scott 
Published: April 2020

Retention time scheduling for MRM transitions during targeted assays enables more compounds to be quantified with higher quality results. In this assay, MRMs to quantitate over 1150 lipid molecular species were combined into a single assay. The selected amide column chemistry provides reproducible isomer separation based on lipid class. This Liquid Chromatography separation strategy, is then coupled with the time scheduled targeted assay. To validate this method, we selected DES1 knockout mice to measure lipid changes in liver and adipose tissues. The MRM™ Pro Builder tool (SCIEX) was then used to optimize window width and dwell weight to enhance sensitivity and coverage of method. This method provides extensive, reproducible lipid coverage in complex biological samples like tissue, cells, and plasma.Amide column chemistry was chosen for lipid class separation and minimize isomeric interference. The target list of lipids is comprehensive, covering most major lipid classes and categories, and MRMs were selected to cover lipids containing fatty acids with 14 to 22 carbons and 0 to 6 double bonds. The method is customizable, so new lipid categories, classes and molecular species can be added to the MRMs list. This method provides quantitative measurement of over 1150 different lipid molecular species in a rapid, highly reproducible manner. The sMRM Pro Builder template which was developed to streamline the method optimization process, enables assigning the retention time, optimize dwell weight and set window size per MRM to enhanced coverage and sensitivity of the method. This optimization improved results quality especially on low abundant lipids.Liver and eWAT tissues were harvested from dihydroceramide desaturase‐1 (DES1) knockout mice. DES1 is the enzyme responsible for inserting the 4,5‐trans‐double bond into the sphingolipid backbone causing the dihydroceramide conversion to ceramide. While both of these classes of lipids are lower in abundance in the chosen tissues, this method shows significant changes in these lipid classes in a quantitative manner. However, lipids from another 17 classes were not changing.Lipid standards from 19 different classes, which are either heavy isotopic labeled lipids or odd chain lipids, served as internal standard. This method provided extensive lipid class coverages including, CE, CER, DCER, HCER, LCER, TAG, DAG, MAG, LPC, PC, LPE, PE, LPG, PG, LPI, PI, LPS and PS.
GONADAL HORMONES MODULATE HIPPOCAMPAL NUCLEAR DEK EXPRESSION IN ADULT MICE AND RATS
Authors: Greene Allie , Balmer Nikolaus , Estrada Christina , Vinnedge Lisa Privette , Solomon Matia 
Published: April 2020

Alzheimer’s Disease (AD) is currently the 6th leading cause of death in the United States, and is the most common cause of dementia. Women are more likely than men to be diagnosed with AD. The cause of this discrepancy is not known; however, the loss of circulating estrogen after menopause is hypothesized to be a leading factor for women’s increased risk of AD. Estrogen is considered neuroprotective; it can ameliorate the effects of brain injury, contribute to DNA repair in mitochondria, and maintain synapse number and spine density in the hippocampus. Our laboratory is investigating the role of an estrogen receptor alpha (ERα) target gene, DEK, as a novel player in Alzheimer’s disease and age‐related dementias. DEK is a nuclear chromatin phosphoprotein that is expressed throughout the brain, and is prominently expressed in the hippocampus, a brain region important for learning and memory. Preliminary data indicate that DEK loss in vitro and in vivo results in AD and dementia‐like phenotypes, such as cell death, increased total tau and phosphorylated tau expression, DNA damage, and decreased hippocampal microtubule associated protein (MAP2) expression. These findings suggest a neuroprotective role of DEK. Given that DEK is an ERα target gene, we postulated that DEK expression in the brain would be modulated by circulating levels of estrogen. We specifically hypothesized that loss of estrogens would decrease DEK expression in the hippocampus. To study this, we used a surgical rodent model of menopause, ovariectomy (OVX), to remove the main source of circulating estrogen in female rats. OVX was performed in 3‐month‐old Sprague Dawley rats. Cell counts and optical density analysis were done using NIH Image J. We found that following two months ovariectomy, DEK expression was decreased in the hippocampus of adult female rats. Besides estrogen, the gonadal hormones testosterone and progesterone can exhibit neuroprotective effects as well. Therefore, we included males in our next experiment in order to determine if gonadal hormone depletion in males would also affect DEK expression. Similar to rats, gonadectomy decreased hippocampal DEK expression in adult male and female mice. Interestingly, OVX significantly decreased DEK expression relative to castration in the CA1 and CA3 regions of the hippocampus. These findings suggest that the impact of gonadal hormone loss on DEK expression is evident across species. We do not yet know the effect of specific gonadal hormonal administration (e.g., E2 or testosterone) on central DEK expression in castrated males and OVX females; therefore, future studies will explore this. Our results suggest that biological factors such as circulating hormones can affect the expression of DEK, whose deficiency may be linked with neurodegeneration and brain aging in dementia and AD.Support or Funding InformationL.I.F.E. Foundation
Accelerating SWATH® Acquisition for Protein Quantitation – Up to 100 Samples per Day
Authors: Arnold Randy J. , Hunter Christie , Morrice Nick , Demianova Zuzana 
Published: April 2020

The combination of microflow LC with SWATH Acquisition for large scale quantitative proteomics studies is becoming increasingly more widespread, due to the improved robustness and throughput obtained relative to the traditional nanoflow LC approach. High quality quantitative datasets have been generated using a standard 1 hour gradient, demonstrating large numbers of proteins quantified routinely.The impact of gradient length on protein identification results using data dependent acquisition (DDA) was also explored previously and summarized here. Next, an exploration into the impact of gradient length on proteins quantified using data independent acquisition (DIA) was undertaken to provide researchers expanded workflow options with microflow SWATH acquisition. Using microflow liquid chromatography gradients as short as five minutes (total run time <15 mins), SWATH acquisition parameters were first optimized then used on multiple TripleTOF® 6600 systems to study impact of shortened separations on number of proteins quantified.Testing on multiple instruments with different complex matrices, it was observed that the number of proteins quantified using SWATH acquisition for the fastest gradients was over 1300 proteins for multiple complex matrices from 1 μg protein load. When combined with larger ion libraries, over 2100 proteins were identified and quantified in a 5 min gradient from a complex HEK digest (1 μg). The combination of microflow LC with the high MS/MS acquisition rates of the TripleTOF® 6600 system enables high‐throughput yet comprehensive analysis for proteomics samples, at rates approaching 100 samples per day.
Evaluation of Adherence to Dietary Guideline for Patients with Type 2 Diabetes Mellitus According to Age
Authors: Jang Haeun , Park Kyong 
Published: April 2020

BackgroundThis study aimed to compare adherence to dietary guidelines between elderly and non‐elderly patients with type 2 diabetes mellitus (T2DM) in Korea.MethodsData of 4577 patients with T2DM were collected from the 1998–2015 Korea National Health and Nutrition Examination Surveys. The patients were classified into two groups based on age: non‐elderly T2DM group comprising patients aged 30–64 years and elderly T2DM group comprising patients aged ≥65 years. Adherence to dietary guidelines was assessed using the Korean Diabetes Association‐Korean Ministry of Health and Welfare (KDA‐KMHW) index, comprising six components of dietary guidelines for T2DM patients. Multivariate generalized linear regression analysis was performed to analyze the KDA‐KMHW index scores.ResultsThe adherence levels to the individual components of the KDA‐KMHW index were mostly lower in non‐elderly T2DM patients (P < 0.001) than elderly group, except for moderate carbohydrate consumption. The total KDA‐KMHW index score was significantly lower in non‐elderly T2DM group than in the elderly T2DM group (P < 0.001).ConclusionsThe study results suggest the need for developing patient‐specific education programs that focus on adherence to dietary guidelines, particularly for non‐elderly patients, to adequately intervene with the difficulties experienced in T2DM dietary management.Support or Funding InformationThis research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (grant number: NRF‐2017R1A1A3A04069759).
Differential Tissue‐Specific Effects of Fructokinase Blockade in Sugar‐Induced Metabolic Syndrome
Authors: Lanaspa Miguel , Andres-Hernando Ana , Johnson Richard J. 
Published: April 2020

The intake of fructose and the fructose‐containing sugars, sucrose and high fructose corn syrup, are strongly associated with the development of obesity and diabetes. Here we tested the hypothesis that the metabolism of fructose is the key feature whereby these added sweeteners cause metabolic syndrome. To this end, metabolic syndrome was induced by administering fructose‐glucose (10 or 30%) solutions in the drinking water. Of interest, we found that glucose enhances fructose intake and transport and that the blockade of fructose metabolism in fructokinase deficient (KHK‐A/C KO) mice is sufficient to fully prevent sugar‐induced metabolic syndrome. Our data indicate that this protection is not only due to reduced fructose metabolism but also to decreased preference and intake of sugar. Furthermore, and by using tissue specific KHK‐A/C KO mice, we determined that the reduction in sugar preference was mainly driven by the deficiency of KHK in the jejunum. However, gut‐specific KHK‐A/C KO mice showed enhanced risk for metabolic syndrome when equal intake of fructose was consumed due to increased fructose delivery to the liver. In contrast, liver specific KHK‐A/C KO mice continued to show a preference for sugar despite being protected from metabolic syndrome. In conclusion, these studies document a pivotal role for fructose metabolism in the mechanism by which added sweeteners cause metabolic syndrome. Intestinal fructose metabolism plays a key role in sugar intake whereas liver fructose metabolism drives fatty liver, obesity and diabetes indicating that metabolic syndrome is a hepatocyte‐centered condition.Support or Funding InformationThis work has been supported by NIH Grants 1RO1DK105364‐01A1 (to M.A.L. and R.J.J.) and 1R01DK108859 (to M.A.L.)
A Quantitative Proteomic Approach for Elucidating the Effects of Antidiabetic Drugs on Coronary Artery Endothelial Cells
Authors: Rodríguez Cindy Manríquez , Ulloa Aldo Moreno 
Published: April 2020

Individuals with type 2 Diabetes Mellitus (DMT2) possess an elevated risk of mortality due to cardiovascular disease (CVD). Although there is available a repertoire of antidiabetic drugs (ADD) to treat the disease, mortality risk reduction remains an elusive goal despite improvement in glucose metabolism by drug therapy. Moreover, some drugs further increase the CVD risk in subjects with T2DM, while others appear to partially reduce the risk. Hence, new approaches and thinking are needed to explore this phenomenon beyond the classical drug’s antihyperglycemic effects and principal organs involved in glucose metabolism. In this study, we explored the effects of selected ADD (with epidemiological CVD evidence in the clinic) on Human Coronary Artery Endothelial Cells (HCAEC) —involved in the development of CVD— using a quantitative SWATH‐based proteomic and comprehensive bioinformatic approach. To create a template or network of pathways linked to cell damage in HCAEC, we generated a list of proteins modulated (Up and down‐regulated) by the treatment with the pro‐inflammatory cytokine TNF‐a and performed a Functional Enrichment Analysis against REACTOME and KEEG Databases using ClueGO software. We further expanded our network by utilizing reported data of endothelial cells treated with cytokine IL‐1B. ADD treatment consisted of cells incubated with physiological concentrations of either metformin (100 μM), rosiglitazone (1 μM), empagliflozin (1 μM), saxagliptin (1 μM), and vehicle (DMSO) for 48 h. The list of proteins modulated by the ADD was analyzed by ClueGO using as a template the pathway network of cytokines. Our results hint at anti‐inflammatory actions of all ADD tested, but at different levels within the immune system network. Rosiglitazone, followed by saxagliptin, modulated more proteins linked to anti‐inflammatory actions compared to the rest of the drugs. Neutrophil degranulation, signaling by interleukins, and interferon signaling were modulated by rosiglitazone and saxagliptin, while the rest of the drugs modulated less or none of these specific pathways. Also, ADD down‐modulated pathways associated with the metabolism of proteins and angiogenesis. In conclusion, our study provides insight into the pathways on HCAEC modulated by drugs used to treat subjects with T2DM. Further, our results contribute to the understanding of molecular mechanisms, that could explain the clinical effects noted with some ADD for which there is no clear mechanistic data beyond their classical antihyperglycemic effects.Support or Funding InformationResearch funded by an Internal Project to AMU and a Graduate Scholarship from the Mexican National Council for Science and Technology (CONACyT) to CMM
Transcutaneous Electrical Stimulation of the Vague Nerve X Depressive Symptoms in Patients with Metabolic Syndrome
Authors: Fernandes Daniella Marques , de Moraes Tércio Lemos , Costa Fernando Oliveira , Cabral Dannielly Gomes , de Albuquerque Natália Cristina Mesquita , Coelho Fernando Morgadinho Santos , Consolim-Colombo Fernanda Marciano 
Published: April 2020

Currently, some techniques such as nerve stimulation have been used as a treatment to reduce inflammation levels in the organism. Inflammation is present in both metabolic syndrome and depression. In brain stimulation through the vagus nerve, the main neurotransmitter acetylcholine modulates the actions of various peptides, hypothalamic incretins and cholecystokinin, consequently modulating the actions of the Autonomic Nervous System. According to the World Health Organization, depression occurs in people of all genders and ages, affects 350 million people worldwide, is among the leading causes of disability. This study aimed to verify the impact of transcutaneous electrical stimulation of the vagus nerve on depressive symptoms in patients with metabolic syndrome. This is the analysis of patients with metabolic syndrome who underwent transcutaneous electrical stimulation in the left ear once a week for 30 minutes for a period of 8 weeks. Pre‐ and post‐Transcutaneous Electrical Stimulation analyzes were performed through interviews with the application of three psychometric depression assessment scales of Beck I (IDB), Hamilton (HAM‐D) and Montgomery‐Asberg (MADRS). Nine patients with Metabolic Syndrome from the city of São Paulo, Brazil, were interviewed. Of the 09 respondents, the study showed in the IDB Scale there are 06 (66.6%) with depression, HAM‐D 08 (88.8%) and MADRS all 08 (88.8%) with depression before Transcutaneous Electrical Stimulation. The results showed a significance level of 55.5% for depression scores after Transcutaneous Electrical Stimulation. A high prevalence of depressive symptoms was observed in the study sample. The research allowed us to reflect on the Transcutaneous Electrical Stimulation of the vagus nerve as an alternative treatment for individuals with Metabolic Syndrome and depressive symptoms aiming to prevent or mitigate the effects of these pathologies.Support or Funding InformationWithout funding.
Effect of sulfated galactans from red seaweed Gracilaria fisheri on extracellular matrix production in human dermal fibroblast
Authors: Pratoomthai Benjamart , Gangnonngiw Warachin , Songtavisin Thanitsara , Wongprasert Kanokpan 
Published: April 2020

Fibroblasts are derived from mesenchymal stem cells that play an important role in produce the extracellular matrix (ECM). ECM creates a tissue microenvironment for regulation of cell signaling. The loss of ECM plasticity is associated with several pathologies, especially those involving chronic inflammation, therefore, the ECM represents a potential therapeutic target for certain conditions. Sulfated galactans was extracted from red seaweed Gracilaria fisheri that previous studies have been reported about its activities on anti‐oxidant, anti‐tumour, anti‐melanogenesis, and anti‐bacteria. Therefore, the aim of this study was to investigate the effect of potential of sulfated galactans from red seaweed G. fisheri on enhancing the ECM production. Cytotoxicity was tested following determination of ECM‐related gene and protein expression on human dermal fibroblast (HDF) by quantitative RT‐PCR and enzyme‐linked immunosorbent assay (ELISA), respectively. The result showed that sulfated galactans not effects to survival rate of HDF. Additionally, we found that sulfated galactans was able to modulate ECM production by regulating the collagen, elastin, hyaluronic acid, MMP‐1, and TIMP production. The present results demonstrating the effects of a natural multi‐component compound on ECM composition, highlighted the possibility of pharmacologically modulating ECM molecules. The recovery and the maintenance of ECM homeostasis might be considered as a potential therapeutic goal to ameliorate pathological conditions.Support or Funding InformationThis work was financially supported by Navamindradhiraj University Research Fund.
Molecular Hydrogen Manages Mitochondrial Proteostatic Stress and Induces Cellular Response
Authors: Hasegawa Tomoya , Ito Mikako , Hasegawa Satoru , Takeda Kouki , Nishiwaki Hiroshi , Negishi Shuto , Ohno Kinji 
Published: April 2020

Molecular hydrogen (H2) has an anti‐oxidant effect on hundreds of animal models. However, the exact molecular mechanisms of molecular hydrogen still remains elusive. Mitochondrial unfolded protein response (mtUPR) is a known stress response to perturbed mitochondrial proteostasis caused by oxidative stress or mitochondrial dysfunction.We examined the effects of molecular hydrogen on mtUPR by examining mtUPR‐related protein expression in vivo and in vitro. Mice drinking hydrogen water showed higher expression of the HSP60 chaperone protein, which is an mtUPR marker, as well as other mtUPR‐related proteins including protein kinase R (PKR), phosphorylated eukaryotic translation initiation factor 2a (eIf2a), and activating transcription factor 5 (ATF5). Incubation of cultured cells in 10% hydrogen gas increased expression of HSP60 and some mtUPR‐related proteins in four human cell lines (A549, HeLa, HT1080, and PC3 cells, which are called “responders” hereafter), but not in three other human cell lines (HCT116, HepG2, and SH‐SY5Y cells, which are called “non‐responders”). Flowcytometric analysis disclosed that higher mitochondrial mass, higher superoxide production, higher mitochondrial membrane potential in “responders” compared to “non‐responders’. Similarly, measurement of the oxygen consumption rate revealed high spare capacity of the electron transport complex (ETC) activity in “responders”. Indeed, the gene set enrichment analysis (GSEA) of gene expression profiles in gene expression omnibus (GEO) of two groups of cell lines demonstrated that ETC‐related pathways were upregulated in “responders”. In addition, quantification of DNA synthesis by BrdU assay revealed that incubation of cultured cells in 10% hydrogen gas for 48 h induced cell proliferation, and rescued proliferation blockage by rotenone in “responders”.We report that hydrogen induces mtUPR and cellular proliferation in cell lines with higher mitochondrial ETC activity. Molecular hydrogen ameliorates a variety of diseases including Parkinson’s disease, myocardiac or cerebral infarction, and inflammatory diseases. Molecular hydrogen is thus likely to manage mitochondrial stress through activation of mtUPR, and exerts its effects on these oxidative stress‐related diseases.Support or Funding InformationThis research was supported by grants‐in‐aids from MEXT, MHLW, and AMED of Japan.
A Novel Interaction Reveals LC8 as a Potential Hijacking Point for a Respiratory Virus
Authors: Rodriguez Joaquin , Howe Jesse James , Donner Brianna , Barbar Elisar 
Published: April 2020

Lower respiratory tract infections (LRTIs) are a major cause of death among immunocompromised patients. Human parainfluenza viruses (hPIVs) are non‐segmented, negative‐stranded RNA viruses that frequently give rise to these infections. In order to transcribe genes or replicate their genomes, these viruses encode three major genes: a nucleocapsid (N), a phosphoprotein (P) and a large RNA‐dependent RNA polymerase (L). P is indispensable for transcription and replication as it positions L at the replication/transcriptional start site and recruits accessory proteins. Phosphoprotein structure has been characterized in related viruses such as measles and mumps, where P has been shown to be an oligomer, featuring an intrinsically disordered region as well as a flexible multimerization domain. However, P has not been well studied in the hPIVs and its oligomerization state and dynamics remain unclear. In this study, we show that the phosphoprotein of hPIV type 3 (hPIV3‐P) interacts with host ‘hub’ protein LC8 resulting in structural changes. LC8 is known to bind over a hundred different proteins in eukaryotic cells, facilitating a functional structure. LC8 preferentially binds to intrinsically disordered regions within binding partners which ultimately results in self association, support, oligomerization or other structural rearrangements required for proper function.We have identified two conserved potential LC8 binding sites within hPIV3‐P using the LC8Pred algorithm. We recombinantly expressed and purified a construct featuring the coiled coil domain of the protein and the LC8 binding site in closest proximity to it. LC8 binding was confirmed via pulldown assays, isothermal titration calorimetry (ITC) and size exclusion chromatography (SEC). Further, we characterized the stability of the coiled coil domain using circular dichroism spectroscopy (CD) and the oligomerization state of the protein bound and unbound to LC8 using analytical ultracentrifugation (AUC). Our results show a well‐defined novel interaction that might be indispensable for phosphoprotein activity and overall viral viability. This adds to the evidence that LC8 is a common target for viral hijacking due to its structural properties.Support or Funding InformationThis work is funded by NSF MCB 1617019