bluuebunny/crossref_metadata_2025_split huggingface dataset:
CREB and ERK Activation by Leptin and Angiotensin in the GT1‐7 Cell Model by Capillary Electrophoresis‐Based Western Blotting
Authors: Balapattabi Kirthikaa , Patil Chetan N. , Deng Guorui , Sigmund Curt D. , Grobe Justin L. 
Published: April 2020

Leptin (LEP) increases resting energy expenditure in part by decreasing Agouti‐related peptide (Agrp) within the arcuate nucleus (ARC) of the hypothalamus. Prolonged obesity is associated with hyperleptinemia and development of selective LEP resistance (SLR), referring to the loss of ‘metabolic’ but maintenance of ‘cardiovascular’ sympathetic nervous activity responses to LEP. Previously, we discovered that angiotensin II (ANG) type 1a (Agtr1a) receptors located on Agrp neurons of the ARC are dispensable for blood pressure control but critically required for metabolic responses to LEP. Therefore, we generally hypothesize a role for Agtr1a in Agrp neurons of the ARC in the development of SLR. We used single‐nucleus RNA‐seq followed by Ingenuity Pathway Analysis (IPA) to interrogate the transcriptomes of individual cell types of the ARC from mice after 10 weeks of 45% high fat diet (HFD) or chow. We determined that canonical LEP signaling was strongly under‐represented in Agrp neurons, but not other cell types. Similarly, CREB & ERK pathways, which are implicated in the control of Agrp expression, were significantly under‐represented within Agrp neurons from HFD‐fed mice. To explore changes in the function of these second‐messenger cascades in Agrp neurons, immortalized mouse hypothalamic GT1‐7 cells were examined for expression of relevant genes and activation of signaling cascades in response to various stimuli. First, GT1‐7 cells were confirmed to express Agrp, Lepr, and Agtr1a, but not proopiomelanocortin by qPCR. Second, GT1‐7 cells were treated with LEP (1uM), ANG (200nM), forskolin (30uM), or vehicle for 15 min and lysates were isolated for Western blotting. Capillary electrophoresis methodology (Protein Simple WES; standard matrix (12–230 kDa)/anti‐rabbit detection module) was specifically chosen to enable future studies of ARC Agrp neurons captured after dietary interventions by laser capture microdissection (LCM). Phosphorylated (p) CREB (1:50; CST # 9198), total (t) CREB (1:50; CST #9197), p‐ERK (10ug/ml; R&D Systems # AF1018‐SP), t‐ERK (10ug/ml; R&D Systems # AF1576‐SP) and β‐actin (1:100; Abcam ab8227) protein levels were assessed for all four treatments. Peak signal‐to‐noise (S/N) ratios were well above the threshold value of 10 for all protein samples: p‐CREB (ranging from 560–1500), t‐CREB (25–40), p‐ERK (115–1245) t‐ERK (150–270) and β‐actin (1400–1600). LEP, ANG, and forskolin treatment significantly (p<0.05; n=5–6 each) increased p‐CREB and p‐ERK with no change in t‐CREB or t‐ERK, compared to vehicle. Finally, the viability of protein collection from the ARC was confirmed and optimized by measuring β‐actin from cells isolated by LCM. Ongoing studies are focused on assessing ARC Agrp neuron CREB & ERK responses to acute leptin treatment after prolonged exposure to HFD.Support or Funding InformationHL134850, HL084207
Through regulating Ubiquitin E3 ligase in achieving degradation of aggregated protein and suppression of cell death in polyglutamine degeneration
Authors: Chan Edwin HY 
Published: April 2020

Polyglutamine (polyQ) diseases, including Huntington’s disease and several types of spinocerebellar ataxias, are caused by the genomic expansion of coding CAG trinucleotide sequences. This expansion leads to the production and accumulation of misfolded polyQ domain‐containing disease proteins, which cause cellular dysfunction and neuronal death. As one of the principal cellular protein clearance pathways, the activity of the ubiquitin proteasome system (UPS) is tightly regulated to ensure efficient clearance of damaged and toxic proteins. Emerging evidence demonstrates that the UPS plays a crucial role in the pathogenesis of polyQ diseases. Ubiquitin (Ub) E3 ligases catalyze the transfer of a Ub tag to label proteins destined for proteasomal clearance. We recently identified an E3 ligase Y that modulates expanded polyQ‐induced neurodegeneration in both mammalian and Drosophila disease models. We further showed that Y promotes poly‐ubiquitination and degradation of expanded polyQ protein. Our data further demonstrated the nuclear localization of Y is essential for it to function as a polyQ E3 ligase. Intriguingly, we found that an exocyst complex component X, a Y interacting partner, modulates expanded polyQ protein levels and toxicity in an opposite manner to Y. Our data suggest that X exerts, at least in part, its polyQ‐modifying effect through regulating the E3 ligase function of Y. In summary, this study allows us to better define the role of protein ubiquitination in polyQ pathogenesis and understand how the E3 ligase activity can be regulated in a pathogenic condition.Support or Funding InformationThis work was supported by General Research Fund (14122815) of The Research Grants Council of Hong Kong and CUHK Gerald Choa Neuroscience Centre Grant (7105306).
Immunogenicity of an Adenoviral Vector Vaccine (rAd‐MOMP‐CpG) Against Chlamydia Trachomatis
Authors: Ababneh Mustafa , Ghazal Ehab , khalifeh Mohammad 
Published: April 2020

Chlamydia trachomatis (C. trachomatis) infection is categorized as an important causes of sexually transmitted diseases in humans. Currently, there is no approved vaccine against C. trachomatis. This study was conducted to develop and evaluate the immunogenicity of a recombinant adenovirus (rAd‐MOMP‐CpG) vaccine against C. trachomatis in mice. The vaccine was constructed using adenovirus type 5 based on the major outer membrane protein (MOMP) antigen of C. trachomatis. Sixty mice were divided into 4 groups. In group 1, mice received rAd‐MOMP‐CpG (5×108 PFU) intranasal, 3 times at 2‐weeks intervals. Mice in groups 2, 3 and 4 received equivalent volume of adenovirus vector, CpG 1826, and PBS, respectively. Splenic cytokine levels and mRNA expression levels of IFNg and IL‐4 were determined by ELISA and qPCR, respectively. Furthermore, serum specific murine anti‐MOMP IgG levels were determined by quantitative ELISA. The constructed rAd‐MOMP‐CpG vaccine provoked a Chlamydia‐specific T helper type 1 response. There was a significant (P = 0.047) splenic IFNg stimulation in vaccinated group in comparison to the vector‐control group. The splenic IL‐4 expression remained at minimal stimulation levels across time points. In addition, the vaccine induced significant increase (P = 0.006) of delayed humoral anti‐MOMP IgG response compared to control groups. A recombinant adenovirus vaccine against C. trachomatis could provide important clues toward a vaccine development for C. trachomatis serovar protection in humans. However, further in vitro and in vivo studies are warranted to determine the most effective dosage, vaccine efficacy and safety.Support or Funding InformationJordan University of Science and Technology, Deanship of Research (grant number: 20140275).
Introduction of Mini‐peer Teaching Sessions (MPTs) During Dental Gross Anatomy Laboratory Sessions
Authors: Nonabur Venkatesh , Rarey Kyle E. 
Published: April 2020

Dental Gross Anatomy is an integrated course during the first semester of dental education at the University Of Florida College Of Dentistry. The gross anatomy of the human body is still primarily learned in the anatomy laboratory. The purpose of this study was to assess the effectives of creating mini‐peer teaching sessions within assigned anatomy laboratory times.Multiple mini‐peer teaching sessions (MPTs) were created and held during the assigned anatomy sessions for first year dental students. During each MPT session, students at assigned tables were clustered into two stations (groups A and B). Each station consisted of students assigned to six tables; there were six to seven students presented per table. One student at each table was assigned as a teacher who was responsible for demonstrating dissected structures that their group had dissected.Other members at the assigned tables rotated every six to seven minutes intervals to learn about the anatomy at the other tables. After first round was concluded in groups A and B, the students were switched between A and B stations and a new teacher was designated to demonstrate the assigned anatomy. The MPTs allowed each student to observe the assigned cadaveric anatomy at all 12 tables during a given MPT.Students’ perspective of the value of the dental MPT, sessions were extremely positive. An online survey taken during 2018 Fall semester from the First‐Year Dental students identified that the MPTs were extremely effective. Ninety eight percent of the students strongly agreed that their class mates, serving as peer teachers, were beneficial to their learning. Ninety five percent believed rotating to different stations, with different peer teachers, facilitated their learning of the assigned anatomy. Qualitative data further confirmed the positive impact students placed on the MPTs. Although setting up individual MPTs is time‐intensive, the teaching faculty view the MPTs experiences as a means to further enrich student’s knowledge‐base of anatomy.Support or Funding InformationUniversity of Florida, College of Medicine
Factors Affecting Histone Deacetylase 1 Activity and Selectivity
Authors: Diffley Kelsey , Fierke Carol 
Published: April 2020

Acetylation is an important post‐translational modification (PTM). Aberrant acetylation, or deacetylation, has been linked to cancer, diabetes, neurodegeneration and auto‐immune disorders. Lysine acetylation is a reversible PTM, where deacetylation is catalyzed by histone deacetylases. Histone deacetylase function is crucial for a properly functioning cell, yet information about the specific biological pathways regulated by each isozyme is limited. HDAC1 is especially intriguing due to its known involvement in multiple stable nuclear complexes. However, the role of these complexes in regulating the deacetylase activity of HDAC1 is unclear. We have measured changes in activity and selectivity of HDAC1 upon addition of the following known protein binding partners; retinoblastoma protein 1 (Rb1), DNA methyltransferase 1 (DNMT1), rest corepressor 1 (CoREST), and lysine demethylase 1 (LSD1). We have reconstituted the HDAC1‐containing complexes with these proteins in vitro, as indicated by immunoprecipitation and size‐exclusion chromatography. Using substrate peptide analogs, we have measured the effect of binding partners on HDAC1 activity, demonstrating that these binding partners activate and increase HDAC1 activity to varying extents. Measurement of the reactivity of HDAC1 and HDAC1‐containing complexes with acetylated peptide substrates and full‐length protein substrates (i.e. histones) will provide insight into the role of binding partners in determining HDAC1 substrate selectivity.Support or Funding InformationNational Institutes of Health R01‐GM‐040602
Clinical Implications of Sexual Dimorphism in Gonadal Vein Valves
Authors: Hasan Shaina , Thomas Olivia , Kriegshauser J Scott , Oklu Rahmi , Langley Natalie R 
Published: April 2020

The clinical significance of valves within gonadal veins is controversial. In males, the absence of valves in the left testicular vein (compared to the right) has been cited as a potential mechanism for varicocele formation in the pampiniform plexus in the scrotum. In females, ovarian vein valve absence or incompetence may lead to pelvic congestion syndrome, a disputed explanation for pelvic, abdominal, and/or flank pain of unknown etiology. One study suggests that females have incompetent valves in dilated veins more frequently than males due to past pregnancies. Furthermore, inconsistencies exist in the reported incidence of gonadal vein valves, though pelvic insufficiency is more thoroughly documented and diagnosed in males than in females.This study assesses the presence, number, and location of gonadal vein valves in male and females. The right and left gonadal veins were evaluated in 13 embalmed cadavers (7 males, 6 females; mean age = 86.3 +/− 6.2 years). Twenty‐four veins were available for dissection; two veins were lost to dissection and therefore omitted. The veins were dissected from the ovary (females) or deep inguinal ring (males) to the point of drainage into the inferior vena cava (right gonadal vein) or left renal vein (left gonadal vein). The veins were incised along the entire length of this course, and the number and location of valves was noted and photographed. Anatomical variation and pathological dilation in the vasculature was also documented. A total of 8 valves were found among the 24 dissections. Valves were present in four of the six females (67%) compared to one out of seven males (14%). The valve in the male was located in the left gonadal vein; the bilateral distribution in females was more randomly patterned. The majority of the valves (87.5%) in females were located distally, adjacent to the ovary, while a single valve was located adjacent to the left renal vein confluence. No dilation was noted in any of the specimens.The male population in this study had a lower incidence of gonadal vein valves than in previous studies. However, the number of valves in female gonadal veins was considerably greater than in males. This finding has not been reported previously, perhaps due to the emphasis on male pelvic congestion as a risk for infertility versus attributing the condition in females to a psychosomatic etiology. The primary limitation of this research is the small sample size. Future work will explore the clinical implications of these preliminary findings in a patient population. Clinical correlation with patients who are suspected or known to suffer from pelvic congestion syndrome may shed further light on the role of gonadal vein valves in vein sufficiency and inform more effective treatment for this poorly understood condition in the female patient population.
7‐Dimethylamino‐4‐trifluormethylcoumarin (Coumarin‐152) as a fluorogenic substrate of CYP3A4 and a tool for studying functional interrelationships among human cytochromes P450
Authors: DANGI BIKASH , Davydova Nadezhda Y. , Davydov Dimitri R. 
Published: April 2020

In this study we probe the use of 7‐Dimethylamino‐4‐trifluromethylcoumarin (Coumarin 152, C152) as a fluorogenic substrate for in vitro studies of drug metabolism in human liver microsomes (HLM). Examining spectral properties of the formed fluorescent products and using LC‐MS/MS for monitoring C152 metabolism we demonstrate sequential formation of two N‐demethylated products – 7‐Methylamino‐4‐trifluoromethylcoumarin (desmethyl‐Coumarin 152, DC152) and amino‐4‐trifluoromethylcoumarin (Coumarin 151, C151). The predominant product formed by HLM is DC152, which accounts for >70% of C152 metabolized. Using insect cell microsomes containing recombinant P450 enzymes (Supersomes™) we found that C152 is metabolized by CYP2B6, CYP3A4, CYP2C19, CYP1A2 and CYP2C9 listed in the order of decreasing rates of turnover. The KM values exhibited by CYP2B6, CYP3A4, CYP1A2 and CYP2C19 were of the same order of magnitude (1.8 – 4.8 μM). Importantly, the major metabolizers of C152 – CYP2B6, CYP3A4, CYP2C19 and CYP1A2 – demonstrated different ability to catalyze the second demethylation step, with CYP1A2 being the most potent catalyst in this reaction. While over half of the substrate metabolized by CYP1A2 underwent the second demethylation, the fraction of C151 formed by other enzymes did not exceed 30%, similar to what is observed in HLM. Although the rate of turnover of C152 with CYP2B6 is higher than that observed with CYP3A4, the latter appears to be the major metabolizer of C152 due to its high abundance in HLM. This is confirmed by ketoconazole (KCZ) inhibition experiments. We demonstrate that in both CYP3A4 Supersomes™ and HLM over 80% of C152 turnover is suppressed by KCZ, whereas the activities of CYP1A2, CYP2B4 and CYP2C19 with C152 remain largely unaffected. Our results demonstrate that C152 can be used for probing functional interrelationships between multiple cytochrome P450 species in HLM as an example of a substrate that is almost exclusively metabolized by CYP3A4 though having similar affinities to most major human drug‐metabolizing cytochromes P450. In addition, we establish an easy and reliable fluorimetric assay for monitoring and analyzing the metabolism of C152 that allows discriminating between the two sequential steps of its N‐demethylation.Support or Funding InformationThis research was supported by the National Institute On Alcohol Abuse And Alcoholism of NlH under Award Number R21AA024548
Re‐expression of REST in ventricular vagal neurons aggravates myocardial infarctionevoked ventricular arrhythmia in type 2 diabetes mellitus
Authors: HU WENFENG , ZHANG DONGZE , TU HUIYIN , WADMAN MICHAEL C. , LI YULONG 
Published: April 2020

Diabetes mellitus, especially type 2 diabetes mellitus (T2DM, 90 to 95% of diabetic population), is a major public health problem worldwide and has become a leading cause of mortality. Myocardial infarction (MI) is the most common cause of mortality in T2DM. Comparing with non‐diabetic patients, patients with T2DM are twice to die from MI. Withdrawal of cardiac vagal activity is considered to be an important trigger for MI‐induced ventricular arrhythmias in T2DM. Our previous study has demonstrated that N‐type Ca2+ currents and cell excitability of atrioventricular ganglion (AVG) neurons were reduced in T2DM rats. We have recently demonstrated that reduced expression of Cav2.2‐α in AVG neurons contributes to cardiac vagal dysfunction and ventricular arrhythmogenesis. The repressor element 1‐silencing transcription factor (REST) is a key factor to block Cav2.2‐α expression. Normally REST is not expressed in neuronal cells. Here, we test the hypothesis that re‐expression of REST inhibits N‐type Ca2+ channel function in AVG neurons and further reduces cardiac vagal activity and promotes MI‐evoked fatal ventricular arrhythmias in T2DM rats. Using reverse‐phase protein array, we found that there was no expression of REST in the AVG from sham rats, while REST expression was significantly increased in the AVG from T2DM rats. In vivo infection of lentiviral REST shRNA (2 μl, 1×107 TU/ml) to the AVG markedly reduced REST expression in the AVG from T2DM rats. REST shRNA also increased Cav2.2‐α expression in AVG neurons from T2DM rats. The results from whole‐cell patch clamp recording showed that REST shRNA markedly restored T2DM‐reduced N‐type Ca2+ currents and cell excitability in AVG neurons. Additionally, infection of REST shRNA to AVG neurons markedly restored cardiac vagal activity, as evidenced by blunted response of LVSP to left efferent vagal stimulation. Moreover, REST shRNA also decreased the inducibility of ventricular arrhythmia in anesthetized T2DM rats (0.67±0.6 in T2DM+REST group vs. 3.6±1.03 in T2DM group). The ECG data obtained from 24‐hour radiotelemetry recording in conscious rat demonstrated that REST shRNA significantly reduced the duration of MI‐evoked ventricular arrhythmia (39.39±13.03 sec/hour in T2DM+REST group vs. 64.26±13.17 sec/hour in T2DM group). Based on above data, we conclude that re‐expression of REST decreases Cav2.2‐α expression and N‐type Ca2+ currents in AVG neurons, and subsequently reduces cardiac vagal function and increases susceptibility to ventricular arrhythmias in the T2DM.Support or Funding InformationDHHS/NIH/NHLBI, 34‐5370‐2003‐001
Impact of Phosphorylation on MDH2 Structure, Function and Protein
Authors: Chang Nathan M. , Rementeria Gabriel J. , Berquist Blaine E. , Provost Joseph John 
Published: April 2020

Mitochondrial malate dehydrogenase (MDH2) is an enzyme utilizing the NAD/NADH cofactor system in the citric acid cycle to reversibly oxidize malate to oxaloacetate, where citrate synthase (CS) uses oxaloacetate to produce citrate. Coupling of the MDH2‐CS of the two reactions is critical as the thermodynamics of MDH2 alone would not drive the cycle forward. However, shuttling of carbon through the cycle during gluconeogenesis necessitates an uncoupling of the two proteins, allowing for the reversal of the reaction. Thus, a mechanism that favors the known interaction and disrupts substrate channeling through these two enzymes must exist. We performed an analysis of putative MDH2 phosphorylation sites, identified the potential kinases, and conducted a meta‐analysis of mass spectrometry data bases of phosphorylation. We have identified 13 predicted, and 30 identified phosphorylated sites of mammalian MDH2. Further analysis of possible interaction sites and mapping functional domains with these phosphorylation sites highlight the important yet unknown role of phosphorylation to regulate MDH structure and function. In order to better understand MDH2‐CS interactions and MDH function, we synthesized four phosphomimics (S/T‐D) using site directed mutagenesis on a codon optimized human MDH2 gene in a His‐tagged, pET28a construct for bacterial expression. Using purified protein, we show impact of each mutation structure with circular dichroism and the thermal melting profile of the phosphomimics and the wildtype hMDH2. The impact of phosphorylation on MHD function was also studied by comparing kinetic values. Lastly the impact of the mutations on MDH‐CS binding was analyzed using a micro‐protein thermal shift assay.Support or Funding InformationNSF EHR‐IUSE Project support NSF‐1726932
Investigation of isoform‐specific interactions between citrate synthase and malate dehydrogenase
Authors: Rementeria Gabriel J. , Berquist Blaine E. , Chang Nathan M. , Ghebreigziahber Daniel , Provost Joseph John 
Published: April 2020

Malate dehydrogenase (MDH) and citrate synthase (CS) catalyze subsequent metabolic reactions in the Krebs cycle. MDH and CS interact to form enzyme complexes called metabolons where substrates are directly shuttled from one active site to the other, improving efficiency of the overall metabolic cycle. Prior studies have shown that the mitochondrial isoform (MDH2) has stronger interactions with CS than the cytosolic isoform (MDH1), suggesting that isoformspecific sites of interaction are involved in the formation of the enzyme complex. This study attempts to determine key residues of MDH2 that are responsible for isoform‐specific affinities to CS, allowing for the formation of metabolon complexes. Model metabolon docking complexes between MDH2 and CS were generated computationally in Cluspro, using predicted and published interactions between the two enzymes. Potential interacting residues were identified and then mapped using clustal alignments to identify possible domains of isoformspecific interactions. Previous studies by our lab have shown that CS displays an isoformspecific affinity to MDH. Specifically, CS binds with high affinity to MDH2, moderate affinity to MDH1, and displays undetectable interaction with watermelon glyoxosomal MDH (wgMDH). We identified several regions of residues unique to MDH2 containing shown & predicted interactions with CS. Using docking analysis, we identified five regions of potential isoformspecific interactions. This combined with residue alignment of MDH2, MDH1 and wgMDH isoforms helped to further define potential isoform specific sites of interaction. Mutations exchanging mitochondrial residues for unique cytosolic counterparts or deletion mutations defined by our bioinformatic analysis were designed, generated and expressed. Enzyme kinetic assays were performed to investigate enzymatic activity of mutant constructs for comparison to native MDH1 and MDH2. Secondary structure and thermal stability were investigated using circular dichroism. Interactions between mutant MDH2 constructs and CS were quantified in fluorescence‐based thermal shift assays.Support or Funding InformationNSF EHR‐IUSE Project support NSF‐1726932