bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of Vascular Endothelial Growth Factor Receptor 2 (VEGFR‐2) Glycosylation and its Role in Angiogenesis
Authors: Chandler Kevin B , Khatri Kshitij , Leon Deborah R , Meyer Rosana D , Rahimi Nader , Costello Catherine E 
Published: November 2023

Angiogenesis, the formation of new blood vessels from pre‐existing vessels, is required for tumor growth and metastasis. Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is one of the most important receptor tyrosine kinases (RTKs) among the VEGF receptor subfamily, and activation of VEGFR‐2 is essential for tumor angiogenesis. The extracellular domain of VEGFR‐2 contains seven immunoglobulin‐like (Ig) domains, each with multiple potential N‐glycosylation sites, and N‐glycosylation is thought to play a central role in receptor stability, ligand binding and trafficking. However, to date the N‐glycosylation sites and their putative role(s) in VEGFR‐2 function remain largely unknown. The objective of this study is to characterize VEGFR‐2 N‐glycosylation sites using tandem mass spectrometry (MS/MS) and investigate the functional importance of these modifications on VEGFR‐2 angiogenic signaling.VEGFR‐2 isolated from porcine aortic endothelial cell (PAEC) lysate was immunoprecipitated with a polyclonal anti‐VEGFR2 antibody. Evaluation of the purity and site occupancy of VEGFR‐2 were carried out by gel electrophoresis, followed by protease digestion, PNGaseF/H218O treatment, and MS/MS analysis. MS/MS data were processed using Proteome Discoverer 1.4. To obtain site‐specific glycosylation information we performed proteolysis of VEGFR‐2, glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC) and subsequent analysis of glycopeptides via an Agilent 6550 Quadrupole Time‐of‐Flight (Q‐TOF) MS using collision‐induced dissociation.MS/MS analysis of the VEGFR‐2 tryptic digest achieved 56% sequence coverage. We detected candidate N‐glycosylated peptides in the extracellular domain of VEGFR‐2 after treatment with PNGaseF/H218O, including N98, N160, N247, N297, N521, N578, N702 and N719. MS/MS spectra were used to define the locations of the glycosylation sites. These results are the first evidence for which potential VEGFR‐2 N‐glycosylation sites are occupied by N‐linked glycans. In addition, VEGFR‐2 glycopeptide MS/MS data provide information on the glycoform distributions at each occupied site. All seven VEGFR‐2 immunoglobulin domains contain at least one occupied N‐glycosylation site. Observed glycosylation on Ig domain 2, involved in VEGFR‐2 ligand binding, and on Ig domain 7, involved in receptor dimerization and activation, could have important implications for protein folding, ligand binding and receptor dimerization. These analyses are essential for determination of the role of glycosylation in VEGFR‐2 function and trafficking.Support or Funding InformationThis research is supported by NIH grants P41 GM104603, R01 CA191970, and F32 CA196157.
Functional Reconstitution of P‐glycoprotein into Nanodiscs: Biochemical and Biophysical Evaluation
Authors: Chen Gang , Oliveira Maisa , Wise John G. , Vogel Pia D. 
Published: November 2023

Multidrug resistance (MDR) proteins protect normal cells from xenobiotic attack by pumping a large range of toxins out of the cell. Unfortunately, over‐expression in cancer cells increases efflux of therapeutic drugs rendering them ineffective. In order to re‐sensitize multidrug resistance cancers to chemotherapy, we have found inhibitors of P‐gp by in silico screening methods (Brewer, F. K., Follit, C. A., Vogel, P. D., & Wise, J. G. (2014) Molecular Pharmacology, 86(6), 716–726). To further investigate the mechanism underlying the inhibition, we have employed biochemical assays and electron spin resonance spectroscopy (ESR), and have established nanodisc technologies that provide native‐like phospholipid bilayer environments to incorporate and stabilize P‐gp for our drug finding and mechanistic studies. A cysteine‐less mutant of the mouse MDR3 P‐gp (MDR3CL) has been incorporated into nanodiscs and showed several fold enhanced ATPase activities. The stability of P‐gp in nanodiscs was also dramatically increased. Biophysical studies using ESR showed that P‐gp in nanodiscs as compared to micelles retains the maximum ATP binding value of 2.2 mole/mole, while the dissociation constant Kd was decreased to 40 μM. Enzymatic activity assays showed similar inhibition of P‐gp in nanodiscs by potential inhibitors that were identified using P‐gp in micelles. These results indicate that the performance of P‐gp in nanodiscs was superior to that of micelles in terms of activity, stability and ATP binding. These nanodisc technologies are currently being established as a uniform platform to study different mutants of P‐pg including human MDR1, which was previously assumed to be too unstable to be used in these biochemical, biophysical and drug discovery studies.Support or Funding InformationThis work is supported by NIH NIGMS [Grants R15‐GM094771‐01A1 and R15‐GM094771‐02] to PDV and JGW, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzann Ruff of Dallas, Texas.
Altered Zinc Ion Levels and Transporter Expression Indicate That High Liver Copper Disrupts Zinc Metabolism in the Atp7b−/− Mouse Model of Wilson Disease
Authors: Meacham Kelsey , Burkhead Jason 
Published: November 2023

Wilson disease is an autosomal recessive disorder caused by a mutation in the ATP7B gene, which encodes for a copper‐transporting P‐type ATPase. This mutation impacts regulation of copper metabolism primarily in the liver, but in the kidneys and brain as well. Impaired function of the transporter ultimately results in a hyper‐accumulation of copper in these organs with subsequent toxicity. While Wilson disease is traditionally viewed as a copper‐related disorder, significantly elevated hepatic zinc levels (p <0.01) were observed via inductively‐coupled plasma mass spectroscopy in the Atp7b−/− mouse model used to study Wilson disease. In order to determine the mechanisms of this observed hepatic hyperzincemia, relative transcription levels of hepatic specific zinc transporters belonging to the ZIP and ZnT families were observed through the use of RT‐qPCR. The results obtained showed differences in mRNA levels of zinc transporters in the Atp7b−/− mice. Specifically, in 6 week old animals there was an increase in the relative amount of mRNA for the transporters ZIP9 and ZnT10. Further, differences in relative mRNA abundance were observed between 6 week old and greater than 20 week old animals, including decreased expression of ZIP7, ZIP9, ZnT4, ZnT6, and ZnT10 in the greater than 20 week old animals. This suggests there is indeed some connection between transcriptional regulation of hepatic zinc transporters and the abnormal zinc concentrations as a result of elevated hepatic copper levels.Support or Funding InformationAlaska INBRE: NIH Grant Number P20GM103395; Alaska Heart Institute Fellowship
Hepatic Nuclear Factor 4 Alpha (HNF4α) Regulates Transcriptional Control of Amino Acid Catabolism
Authors: Tobon Sandra , Velazquez‐Villegas Laura A , Ortiz Victor , Torres Nimbe , Tovar Armando R 
Published: November 2023

The liver is the main organ involved in the metabolism of amino acids (AA), which are oxidized by amino acid catabolizing enzymes (AACE). There is evidence that peroxisome proliferator‐activated receptor α (PPARα) can modulate hepatic amino acids oxidation, and it function as a physiological switch to preserve body proteins during the shift of the type of energy fuel. PPARα activates fatty acid oxidation and suppresses amino acid oxidation. However, it has been postulated by microarray analysis, that PPARα does not perform this effect directly. Our data suggest that PPARa regulates the expression of genes associated with amino acid oxidation via HNF4α. Therefore, the aim of the study was to demonstrate that HNF4α regulates the transcription of amino acid‐catabolizing enzymes (AACE) genes using the enzyme serine dehydratase (SDH) as a model of study.Database MatInspector and Promoter database were consulted to analyze the potential HNF4α elements in the promoter sequences of the AACE including serine dehydratase. The analysis was done with a core of similarity above 0.7. The functionality of the putative response element in the promoter of serine dehydratase was tested overexpressing HNF4α and using the luciferase gene reporter assay. Also the silencing of HNF4α using shRNA HNF4α was assessed. Additionally, the binding specificity of the response element HNF4α was examined with electrophoretic mobility shift assays (EMSA) and Chromatin Inmunoprecipitation (ChIP).After in silico analysis of the promoter sequences for the enzymes of amino acid catabolism, it was found that over 70% of the analyzed potential sites for enzymes HNF4α had a core of similarity higher than 0.8. In the SDH promoter, the potential HNF4α site turned out to be functionally active as detected with the luciferase reporter assays. The presence of specific binding of HNF4α to the response element of the SDH promoter was corroborated by EMSA and ChIP indicating the HNF4a is capable to activate SDH transcription. Finally, with the silencing of HNF4a it was possible to demonstrate the importance of this nuclear receptor in the regulatory mechanism of gene transcription of SDH, since the expression of SDH only reached basal levels.The results indicated that HNF4α upregulates the transcription of SDH. Additionally, in silico analysis revealed the presence of HNF4α‐response element in promoter of enzymes involved in amino acid catabolism, suggesting that this transcription factor could function as a global regulator of the amino acid catabolism.Support or Funding InformationCONACYT (grant ART)
Inhibition of Angiotensin Converting Enzyme Induces Beige Adipocytes through Angiotensin‐(1–7) in 3T1‐L1 Adipocytes and Obese Mice
Authors: Vargas‐Castillo Ariana E , Del Valle‐Mondragon Leonardo , Torres Nimbe , Hong Enrique , Tovar Armando R 
Published: November 2023

The renin angiotensin system (RAS) has recently been recognized as an important factor in the regulation of energy balance. Adipocytes express all the components of renin angiotensin system (RAS), including Angiotensin‐(1–7) [Ang‐(1–7)] and Mas receptors. Ang‐(1–7), through Mas receptors, induces PPARy expression in 3T3‐L1 adipocytes and releases nitric oxide (NO) in diverse cells. White adipose tissue differentiates to beige adipose tissue through PPARy and NO stimulates mitochondrial biogenesis in brown adipose tissue (BAT). In recent years, beige adipocytes have been found, and they have some similar characteristics of BAT including a high thermogenic capacity due to a higher content of mitochondria and uncoupling protein 1 (UCP1). An increase of beige adipocytes has been associated with beneficial effects in health preventing obesity. The aim of this work is to determine if Ang‐(1–7) induces beige adipocytes in 3T3‐L1 cells and in high fat diet (HFD) mice. Captopril was used as a pharmacological strategy to increase Ang‐(1–7) concentrations. 3T3.L1 adipocytes were incubated with 3.16 μM of captopril during the maturation phase and then brown‐like genes and beige genes were measured by real‐time PCR. C57BL/6 mice were fed with a HFD for 24 weeks and captopril was administrated during the last four weeks in drinking water (5 mg/kg/day). Body weight, body composition, glucose tolerance test, cold resistance and energy expenditure were measured. Plasma Ang‐(1–7) was quantified with capilar electrophoresis and nitric oxide thorugh Griess reaction. Captopril increased the expression of UCP1 and beige marker genes in 3T3‐L1 adipocytes. In obese HFD‐mice, treatment with captopril reduced weight gain and decreased fat mass continuosly during the four weeks, although food intake was reduced only the first week, there was no change in remaining weeks. Moreover, HFD captopril‐treated group had improved glucose tolerance, increased energy expenditure and had an increase resistance to cold temperatures compared with the HFD group. On the other hand, HFD captopril‐treated group had increased plasma concentrations of Ang(1–7) and nitric oxide. In conclution, Ang‐(1–7) may be a new factor that induces browning process and partly explains the beneficial effects of ACE inhibitors in glucose and energy homeostasis.Support or Funding InformationCONACYT
Investigating the Regulation of Guanylate‐Binding Protein1 Expression during Adipose Tissue Inflammation
Authors: Qiu Xiaoxue , Chen Xiaoli 
Published: November 2023

Guanylate‐binding proteins (Gbps) are large interferon (IFN) γ‐inducible GTPase, mainly functioning in innate immunity to protect against pathogen infections by boosting autophagic machinery and phagocyte oxidase. Attentions have been mostly given to understanding roles of Gbps in host defense against intracellular bacteria in macrophages. Little is known about the involvement of Gbps in adipose tissue inflammation in obesity. Infiltration of IFNγ‐secreting T helper type 1 cells into adipose tissue during obesity has been known to promote macrophage recruitment and polarization, leading to a shift of macrophages from a non‐inflammatory M2 toward a pro‐inflammatory M1 phenotype. We found that Gbp1 gene expression was significantly decreased in inguinal white adipose tissue of high fat diet‐induced obese mice. Therefore, our first aim was to identify the Gbp1‐expressing and IFNγ responsive cell types in adipose tissue. To achieve this aim, we determined the regulation of Gbp1 expression in both adipocytes and macrophages by different cytokines and fatty acids. Fully differentiated 3T3‐L1 adipocytes as well as Raw264 macrophages were treated with various above‐mentioned factors for 24h. Our results showed that Gbp1 was not highly expressed in 3T3‐L1 adipocytes, while Gbp1 protein levels were markedly increased in macrophages when treated with IFNγ. However, tumor necrosis factor (TNF) α and interleukin (IL)‐1β failed to induce Gbp1 expression in macrophages; lipopolysaccharide (LPS) was able to induce the expression of Gbp1 at a much lower level compared to IFNγ treatment. Additionally, Gbp1 protein expression levels were not altered in macrophages in response to the treatment with various fatty acids, including palmitic, oleic, linoleic, eicosapentaenoic and docosahexaenoic acids. Furthermore, we found that Gbp1 proteins were detected in both cytosol and mitochondrial fractions. Our data suggest that IFNγ is a specific and potent inducer of Gbp1 expression; macrophages but not adipocytes are the primary Gbp1‐expressing and IFNγ responsive cells in adipose tissue. Due to the mitochondrial localization of Gbp1 protein, further studies will focus on the role of Gbp1 in regulating mitochondrial oxidative metabolism and M2‐M1 polarization in macrophages.Support or Funding InformationNIDDK‐funded Minnesota Obesity Center (P30DK050456)
Patterns of 16S rRNA Microbial Regions in Aggressor‐Exposed Mice with Features of Post‐Traumatic Stress Disorder
Authors: Gautam Aarti , Hoke Allison , Kumar Raina , Chakraborty Nabarun , Muhie Seid , Hammamieh Rasha , Jett Marti 
Published: November 2023

We evaluated repeated exposures of mice to a trained aggressor mouse as a model for aspects of post‐traumatic stress disorder (PTSD), which was adapted from “social stress” models of traumatic stress, and used a “cage‐within‐cage resident‐intruder” protocol where subject C57BL/6J mice were exposed to aggressor mice for 10 days. With the increased awareness of the significance of the symbiotic influence of microbiota on the nervous system and behavior, this longitudinal study focused on the effect of social stress on the microbiome. Fecal samples were collected daily during the 10 day exposure time‐course from control and aggressor‐exposed (Agg‐E) mice. DNA was isolated; followed by amplification of variable V3 and V4 region of the 16S ribosomal RNA (rRNA) amplicon using the Illumina 16S Metagenomics Library Preparation Protocol. The samples were barcoded with Nextera index primers and pooled prior to sequencing on the Illumina MiSeq platform. Our results indicate on day 4 in Agg‐E mice, the phylum Bacteriodetes shows lower relative abundance, whereas phyla Firmicutes and Verrucomicrobia exhibit higher abundance. The predicted functional enrichment of abundant bacterial composition corresponds with the PPAR and Adipocytokine signaling pathways being highly activated in Agg‐E mice in comparison to control mice.DISCLAIMERS: Research was conducted in compliance with the Animal Welfare Act and all other Federal Requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.Support or Funding InformationResearch supported by CRADA # W81XWH‐11‐0076.
Genesis of Antibiotic Resistance XIX: An Alternative to incineration of Prescription Drug collection &[uml]en masse”
Authors: Kannan Subburaj , Echeverria Leticia , Munoz Janet , Luna Jennifer , Rosales Ramiro , Rodriguez Jennifer , Ruiz Vanessa , Velez Micaela , Rodriguez Lizzeth , Chavez Dariela , Subburaj Prabavathy , Villarreal‐Moreno Raquel 
Published: November 2023

The grandeur of “Prescription Drug Take Back Program” (including Pharmaceutical, Personal Care Products (PPCP)) administered by the Drug Enforcement Administration, division of United States Department of Justice (DEA‐US‐DOJ), though it is a stellar success, frill will be flounced by adopting Bioremediation for its disposal. It is essential to point out that the medications of PPCP should be categorized according to their chemical potency and possible contaminants prior to the incineration process. At this time, it is not clear whether DEA‐US‐DOJ or commercial vendors are performing this task prior to the incineration. The DEA‐US‐DOJ's strategy of mass incinerating drugs taken by the aforesaid program, carry a heavy burden of acting as a source of known and unknown pollutants damaging the public health by contaminating ground water table, water reservoirs, land and air. By adopting an appropriate bioremediation method (aerobic or anaerobic) encompassing several specific modus operandi (Cometabolism, biostimulation biosorption, bioleaching, bioaccumulation, bioaugumentation) will definitely alleviate the concerns over contaminating the sand, water and air by the pollutants generated by the current disposal methods, however a multitude of factors such as: competition with/among microorganisms, insufficient supply with essential substrates, unfavorable external conditions (aeration, moisture, pH, temperature), and low bioavailability of the pollutant, biodegradation would potentially act as a limiting factor for choosing an appropriate method for the disposal process. Systematic analysis of merits and demerits of all available methods for disposing the PPCP shall be presented in Experimental Biology 2016.Support or Funding InformationSupported by professional development funds by SWTJC to S.Kannan for 2014–2015
Genesis of Antibiotic Resistance XI: Critical Appraisal of Disposal Method of National Prescription Drug Take‐Back Day
Authors: KANNAN SUBBURAJ , ROBLES LIZET , MOODY NADINE , Delgado Mayra , Garcia Valerie , Moore Kelly , Echeverria Leticia , Ruiz Pauline , Martinez Consuelo 
Published: November 2023

A systematic analysis of the data pertaining to the disposal method of the drugs collected by National Prescription Drug Take‐Back Day has been performed in association with United States Drug Enforcement Administration's (US‐DEA's) – Department of Justice (DOJ), DEA Public Affairs, Washington D.C. Based on the data collected from the National Prescription Drug Take‐Back Day all across United States by US DEA–DOJ, as of September 5, 2014, an estimated 4.1 million pounds (2,123 tons) of prescription medications have been removed from the circulation. As per the US DEA b– DOJ public affairs, the drugs that are collected by this program were disposed by incineration facility monitored and regulated by the United States – Environmental Protection Agency (US‐EPA). The entire disposal of the drugs that are collected by National Prescription Drug Take ‐ Back day is completed by a vendor destined by US DEA ‐DOJ. Further analysis of the disposal process reveal following pitfalls of this process: i. There are no data on file regarding pollutants generated from the incineration of Drugs from the take back programs by US DEA ‐ DOJ, 2. The practice of incineration at the rate of en mass is a potential resource for reaction intermediates, such as dioxins, fly ash, and other unknown pollutants, 3. The practice of not sorting the drugs according to the chemical toxicity prior to incineration potential harm to the environment and biota. Specific information on the class of the medication as per the chemical potencies for generating combustion based pollutants are investigated. Data analysis on the Impact of the disposal practices of drugs from take back day will be presented in Experimental Biology 2016.Support or Funding InformationProfessional Development Funds to S.Kannan at SWTJC for 2014–2015
Genesis of Antibiotic Resistance (AR) XIV: Inter agency anomaly in data sharing debilitate the mitigation of AR Pandemic
Authors: Kannan Subburaj , Villarreal Ana , Valdez Edith , Villarreal‐Moreno Raquel , Sanchez Jema , Serna Gysel , Salazar Ketsia , Rodriguez Victor , Padilla Maria , Navejas Crystal 
Published: November 2023

Multitude of agencies within the United States – Department of Health and Human Services, Center for Disease Control and Prevention (US‐DHH‐CDC&P), as identified in the Frieden's annual report on AR, shoulder the responsibility of data collection, analysis, and implementing new strategies for prevention of AR pandemic. We have identified that Frieden's report falls short to incorporate the specific details on the categories of drugs including the antibiotics collected through the National drug Take back day organized by the Drug Enforcement Administration sections of the US Department of Justice (DEA‐US‐DOJ). Regarding the antibiotic prescribing/stewardship, the role of prescription drug monitoring program (PDMP) which is an integral component of National Alliance for Model State Drug Laws (NAMSDL), falls short of the annual progress report. PDMP is a statewide electronic database which collects designated data on substances dispensed including antibiotics / antimicrobials in each of 50 states and also United States Territories. Whereas Prescription Monitoring Information Exchange (PMIX), on the other hand allows the stakeholders to freely exchange the information pertaining to the prescription data at a patent level, including antibiotics and anti‐microbials. Neither the physicians in private practice, local pharmacies, nor the Maverick County Health District officials are aware of PDMP, and PMIX, by CDC&P. Further inquiry confirms that there is a communication gap between CDC & P with District 8 of Texas DHH on PDMP‐PMIX. Frieden's report on AR falls short of details on agencies and current status of data (Quantitative details/refills/duration of being on the specific antibiotics/ antibiotic‐ Drug interaction [blood pressure / diuretics / weight loss medication / anti‐psychotic / illicit drugs ] subsequent emergency room visits / Mortality ratio) sharing on annual rate of prescription of antibiotics in each state and also in the United States Territories. It is of pivotal concern that the integration of data on multinational effort in mitigating the AR along side of CDC&P's annual report on AR is of global significance. Prospects of an enhanced prediction and analysis of morbidity and mortality rate due to the misuse or unaccounted use of antibiotics, in AR pandemic shall be presented in EB2016. (http://www.cdc.gov/drugresistance/threat‐report‐2013/pdf/ar‐threats‐2013‐508.pdf)Support or Funding InformationProfessional development funds provided by the SWTJC to S.Kannan for 2014–2015