bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bbs1 gene deletion from the leptin receptor‐containing neurons causes obesity (1126.10)
Authors: Guo Deng Fu , Sheffield Val , Rahmouni Kamal 
Published: June 2021

Bardet‐Biedl syndrome (BBS) is a pleiotropic autosomal recessive disorder with several features including obesity. Systemic knock‐out mice lacking expression of Bbs2, Bbs4 and Bbs6, or Bbs1M390R knock‐in, recapitulated many BBS phenotypes including obesity. However, the role and contribution of Bbs genes in different tissues to obesity remains unclear. To address this, we generated a new conditional knockout mouse where exon 3 of the Bbs1 gene is floxed. Cre‐mediated recombination causes a frame shift resulting in a premature stop. We assessed whether deletion of the Bbs1 gene in the leptin receptor neurons (LRb) affects body weight. Importantly, Bbs1flox/LRbCre mice display an obesity phenotype as indicated by the increased body weight and fat mass measured by MRI. We found that the obesity phenotype in Bbs1flox/LRbCre mice is due to both an increase in food intake and reduction in energy expenditure as indicated by the decreased O2 consumption and heat production. These results indicate that hyperphagia and low metabolic rate explain the development of obesity in Bbs1flox/LRbCre mice. Finally, in HEK 293 cells, silencing Bbs1 or Bbs2 genes significantly decreased LRb, but not LRa or transferrin receptor, trafficking to the plasma membrane highlighting the important role of Bbs gene in the regulation of LRb signaling. These findings demonstrate that Bbs genes are critical for energy homeostasis.Grant Funding Source: NIH and AHA
Hydrogen sulfide decreases a slow rectifying potassium conductance in nucleus tractus solitarius neurons (1129.4)
Authors: Malik Rishi , Ferguson Alastair 
Published: June 2021

Hydrogen Sulfide (H2S) is an endogenously produced gasotransmitter which has been shown to play a role within the central nervous system. Prior studies have shown that H2S has presynaptic effects on neurons of the nucleus tractus solitarius (NTS) through modulation of glutamate transmission. We have also recently demonstrated postsynaptic effects of H2S on the membrane potential of NTS neurons. The current study was undertaken to investigate the ion channels in NTS neurons modulated by H2S. Using whole cell voltage‐clamp recordings we found that H2S causes a decrease in current evoked in the depolarizing range of a slow voltage ramp indicative of modulation of the voltage activated potassium conductance IK. This conductance was found to decrease by a mean of 20.2 ± 2.5% (n=11) at the end of the ramp (10 mV) following application of 10 mM NaHS, a H2S donor, with all 11 neurons influenced in a similar manner. All cells demonstrated a partial to full recovery of this conductance after wash off of NaHS. The present work suggests that H2S controls the excitability of NTS neurons, at least in part through the modulation of the delayed rectifier potassium conductance, IK.Grant Funding Source: This work was supported by the Canadian Institutes for Health Research.
Quantitative proteomics of collecting duct cells in bilateral ureteral obstruction‐induced nephrogenic diabetes insipidus (1137.4)
Authors: Khositseth Sookkasem , Somparn Poorichaya , Charngkaew Komgrid , Uawithya Panapat , Chen Shu‐hui , Pisitkun Trairak 
Published: June 2021

The present study investigated the effects of early bilateral ureteral obstruction (BUO)‐induced nephrogenic diabetes insipidus (NDI) on rat inner medullary collecting duct (IMCD). Immunoblotting of IMCD isolated from rats after 4 hours of BUO showed a 42% decrease in total Aqp2 (p <0.05), 65.5% decrease in pS256‐AQP2 (p <0.001), 34.4 % decrease in pS261‐AQP2 (p <0.01), but no significant change in total UT‐A1 and pS486‐UT‐A1 abundances. Large‐scale identification and quantification of BUO‐affected proteins in IMCD was performedby label‐free LC‐MS/MS technique. Of 1172 proteins identified, 52 proteins were found to have significant changes in abundance in early BUO rats versus sham controls, with 40 proteins increased and 12 proteins decreased. Gene Ontology terms and pathway analysis revealed that BUO‐affected proteins were associated with focal adhesion (Flnc, Actn4, Lamb2, Lama5, Lamc1, Col4a2, Ctnnb1), cytoskeleton (Krt7, Actn4, Myh10, Tubb4a, Cryab, Ctnnb1), and generation of precursor metabolites and energy (Atp6v1e1, Atp5h, Atp5a1, Atp5b, Aco2, Mdh1, Uqcrc2). Immunohistochemistry studies demonstrated aggregation of filamentous actin in IMCD of BUO rats. These results provide potential clues for understanding the mechanisms responsible for BUO‐induced NDI.Grant Funding Source: Supprted by Thailand Research Fund
Aquaporin‐2 interactome in rat inner medullary collecting duct (1137.5)
Authors: Chou Chung‐Lin , Hageman Daniel , Knepper Mark 
Published: June 2021

Vasopressin regulates membrane trafficking of the water channel aquaporin‐2 (AQP2). We ran LC‐MS/MS analysis to identify AQP2 interacting proteins in native IMCD cells. Freshly isolated rat IMCD suspensions were treated with a vasopressin analog (dDAVP) or vehicle followed by in‐cell crosslinking using a homobifunctional crosslinker BSOCOES, detergent solubilization, AQP2 immunoprecipitation, SDS‐PAGE separation, and quantitative mass spectrometry of trypsin digests of gel pieces. The peptides passing stringent spectral quality thresholds were quantified (label‐free) to identify those with high signal:noise (AQP2 antibody/preimmune IgG > 4). The overall analysis identified a total of 89 replicable AQP2‐interacting proteins distributed throughout several cellular compartments (based on Gene Ontology analysis). 76 were found in dDAVP‐treated samples and 66 in vehicle‐treated samples (overlap 53). 16 have previously been found to undergo changes in phosphorylation in response to vasopressin including proteins involved in membrane trafficking (Lrba, Ndrg1, Sec61b), signaling (β‐catenin, inositol 3‐phosphate synthase, wolframin), cell‐cell adhesion (α‐catenin, β‐catenin, periplakin) and transport (Slc14a2, Atp1a1). Additionally, four distinct Rab proteins were identified (5C,7A,14,17). These findings provide extensive data for modeling vasopressin‐regulated AQP2 trafficking.Grant Funding Source: Supported by NHLBI
Deciphering translational and transcriptional impairment in Roberts syndrome (1138.6)
Authors: Xu Baoshan , Bose Tania , Zueckert‐Gaudenz Karin , Gogol Madelaine , Gerton Jennifer 
Published: June 2021

Roberts Syndrome (RBS) is an autosomal recessive disease characterized by prenatal growth retardation caused by mutations in the cohesin acetyltransferase ESCO2 gene. Previously we revealed that mutations in human ESCO2 impaired ribosomal RNA production and protein synthesis. We recently observed mTOR (mammalian target of rapamycin) signaling was strongly downregulated in RBS cells. Stimulation of the mTOR pathway with L‐leucine helped to partially rescue both protein synthesis and cell proliferation. To further address the transcriptional and translational deficits in RBS and their response to L‐leucine, we are performing ribosome profiling to monitor translation in vivo by deep sequencing of ribosome‐protected mRNA fragments. Western blot analysis shows that a number of 40S and 60S ribosome subunits are reduced in the RBS cells. Remarkably, global 5’TOP (5′terminal oligopyrimidine tract) mRNA translation, including 79 ribosome proteins and a panel of eukaryotic translation initiation factors, and most PRTE (Pyrimidine Rich Translational Element) genes show improved translation in RBS cells treated with the mTOR stimulant L‐leucine. The different expression of hundreds of genes is rescued with L‐leucine, suggesting the differential gene expression could be translation‐driven. In conclusion, our results uncover transcriptional and translational mechanisms that contribute to the etiology of RBS.Grant Funding Source: Supported by Stowers Institute and Cornelia de Lange Syndrome Foundation Research Grants
Effects of di‐n‐butyl phthalate on neuron development (1139.1)
Authors: Feng Josh , Acey Roger 
Published: June 2021

Plasticizers such as di‐n‐butyl phthalate (DBP) and bisphenol A (BPA) are well‐documented, ubiquitously distributed environmental contaminants. The effect of these compounds on human health has been controversial but research has strongly suggested they function as estrogen mimics or “endocrine disruptors”. We have developed a human umbilical cord mesenchymal stem cell (HUC‐SC) system as a model to predict relative toxicities of phthalate esters. The default pathway of these differentiating cells is the formation of neural tissue. Differentiating cells are exposed to the potential toxin for increasing periods of time, collected, and homogenized. Cell lysates are then analyzed by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) for changes in protein expression. Our first series of experiments looked at the effect of 10 nM DBP on undifferentiated HUC‐SCs. At this concentration of phthalate, there was no effect on cell proliferation, membrane integrity, or induction of apoptosis. Next, the HUC‐SCs were induced to differentiate in the presence or absence of 10 nM DBP. Cells were collected after 20, 24, 28, 32, and 48 hours of culture and analyzed by 2D‐PAGE. Comparison of control against phthalate‐treated cell extracts showed dramatic differences in protein expression. Preliminary analysis by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectroscopy (MALDI‐TOF MS) reveals two significant differentially‐expressed proteins: enolase, which suggests that DBP may be altering the neuron differentiation pathway, and heat shock protein β1, indicating that the cells are under stress. Future experiments will be to identify other differentially expressed proteins and determine their potential as biomarkers for evaluating the extent of DBP exposure.Grant Funding Source: Supported by the Doris A. Howell‐CSUPERB Research Scholars Award Program
Effect of Ang II on angiotensinogen expression levels in hypertensive rat astrocytes (1140.1)
Authors: Vudhya Gowrisankar Yugandhar , Clark Michelle 
Published: June 2021

Objective: To investigate the effects of Angiotensin II (Ang II) on Angiotensinogen (AGT) mRNA and protein levels in spontaneous hypertensive rats (SHR) astrocytes. Background: AGT is the precursor molecule for the synthesis of Ang II produced by the renin angiotensin system (RAS). Astrocytes are the major source of AGT in the brain, and regulation of brain AGT levels alters blood pressure and electrolyte homeostasis. In this study, SHR were used as a genetic hypertension model and the results were compared to normotensive Wistar rats. Methods: Astrocytes isolated from neonatal rat’s cerebellum (CB) and brainstem (BS) regions were treated with 100nM Ang II, and the time‐dependent effect of Ang II on AGT protein and mRNA levels were measured using Western blotting and quantitative polymerase chain reaction techniques, respectively. Results: Ang II significantly increased AGT protein levels but, decreased AGT mRNA levels over most time points examined in BS and CB Wistar and SHR astrocytes. There was no significant difference between Ang II’s ability to affect AGT protein and mRNA levels in SHR as compared to Wistar astrocytes. Conclusions: These findings showed that Ang II regulates it’s synthesis by inducing basal AGT protein and mRNA levels. Further, the ability of astrocytes to produce AGT may not be contributing to the hypertensive state in the SHR model. Grant Funding Source: Supported by Nova Southeastern University Chancellor's Faculty Research & Development Grant # 335872
The role of FXR in the regulation of phase‐I drug metabolizing gene expression during mouse liver maturation (1141.1)
Authors: Peng Lai , Cui Julia , Yoo Byunggil , Tien Yun‐chen , Lu Hong , Klaassen Curtis , Zhong Xiao‐bo 
Published: June 2021

The functions of most Phase‐I drug metabolizing enzymes are not mature at birth, and their expression changes during liver maturation. Farnesoid X receptor (FXR) is critical for bile acid and lipid homeostasis in the liver. However, the role of FXR in regulating Phase‐I drug metabolizing gene expression during liver maturation is not clear. Hence, we applied RNA sequencing to provide a “true quantification” of mRNA expression of Phase‐I genes in both FXR‐null and its control (C57BL/6) mouse livers during development. Liver samples of male C57BL/6 and FXR‐null mice at 6 different ages from prenatal to adult were used to define the ontogenic profiles of 185 Phase‐I genes from 12 different families. Compared to control livers, the numbers of Phase‐I genes with significant changes in mRNA expressions in FXR‐null livers were 75, 82, 78, 89, 103, and 78 at the ages of day‐2, 1, 5, 20, 25 and day60, respectively. Differential expression during development occurred in genes from all 12 Phase‐I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), epoxide hydrolase (Ephx); reduction: aldoketo reductase (Akr), quinone oxidoreductase (Nqo), dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). The expression levels of some genes (like Cyp7a1and genes from the Ces family) were affected at all ages, whereas others (like genes from Nqo and Fmo families) were altered only at specific ages during liver maturation. These results indicate an important role of FXR in developmental regulation of Phase‐I genes in liver.Grant Funding Source: Supported by the NIEHS [Grant R01ES‐019487] and NIGMS [Grant R01GM‐087376]
Silencing of ClC‐3 protects against preadipocyte apoptosis in type 2 diabetes (1142.6)
Authors: Wang Guan‐Lei , Huang Yun‐Ying , Zhang Feng , Zhu Xiao‐Nan , Guan Yong‐Yuan 
Published: June 2021

OBJECTIVE: ClC‐3 chloride channel, plays an important role in the regulation of cell volume,proliferation and apoptosis. Preadipocytes of type 2 diabetes show a tendency to enhanced apoptosis. We hypothesized that there is a relation between ClC‐3 and insulin resistance, and one underlying mechanism may be associated with the modulation of free fatty acid (FFA) ‐induced preadipocyte apoptosis by ClC‐3.METHODS: Type 2 diabetes mellitus (T2DM) models in ClC‐3 KO mice and rats were induced by fed with high‐sucrose‐high‐fat diet combined with a low dose of streptozocin. The cellular apoptosis was detected by Annexin V‐PI staining.RESULTS: Compared with the wild‐type T2DM models, ClC‐3 knockout T2DM mice displayed an improvement in insulin resistance, a decrease in serum plasma free fatty acid (FFA) levels and preadipocyte apoptosis in epididymal fat pad, as evidenced by significant decrease in homeostatic model assessment of insulin resistance (HOMA‐IR) and flow cytometry. In T2DM rat models, we further found that ClC‐3 protein in adipose tissue is increased, and was positively correlated to HOMA‐IR, but was not the case in liver and skeletal muscle. We further found that palmitate‐induced preadipocyte apoptosis in vitro was parallel to a significant increase in the endogenous expression of ClC‐3 protein. Transfection with ClC‐3 siRNA decreased apoptosis and activation of caspase‐3 and 9, whereas increased ratio of Bcl‐2/Bax in palmitate‐treated preadipocytes. ClC‐3 affected apoptosis mainly through regulating ER stress signaling proteins such as Grp78, CHOP and ATF4.CONCLUSION: We concluded that chloride channel ClC‐3 modulates palmitate‐induced apoptosis in preadipocytes via the ER stress pathway. ClC‐3 in adipocyte tissue may be involved in the development of type 2 diabetes.Grant Funding Source: Supported by National Natural Science Foundation of China (No. 81370897,No. 813111115 and No. 812300
Manganese accumulates in leaves of radishes grown in manganese supplemented soils (1143.10)
Authors: Clarke‐Lambert Shellyann , Stapleton Melissa , Ruddock Karl , Skeete Dereck 
Published: June 2021

Manganese (Mn) is a naturally occurring element, essential in trace amounts for living organisms, but is potentially toxic in high concentrations. Certain occupations including mining, welding and steel manufacturing can expose workers to chronically high levels of airborne Mn, leading to a clinical condition known as Manganism, which has Parkinson like symptoms. Recent studies report excess dietary Mn can impair immune and reproductive functions in birds. Previously we showed Mn is present in some commercially available fertilizers. We hypothesize plants grown in soils high in Mn or supplemented with fertilizers containing Mn will accumulate Mn in leaves and fruits. To test this we grew radishes in soils supplemented with fertilizers containing Mn, as well as in soil without added Mn. Samples (0.5 g) of each of the fertilizers as well as radish leaves were digested with HNO3 in a CEM Discovery Microwave Digester. Digested samples were analyzed for Mn levels using electrothermal vaporization with deuterium lamp background correction in a Perkin Elmer AA800 Atomic Absorption spectrophotometer with a THGA graphite furnace. We found that leaves from plants grown in soils supplemented with Mn accumulated Mn up to about 104 µg/gm tissue. The control leaves contained 25.28 µg/gm of Mn, which was significantly less than all the experimental groups. On the other hand, the soil that contained fertilizer had a significantly higher concentration of Mne than the plant tissue. The highest concentration of Mn recorded in the soil with Mn supplement fertilizer was 1709 µg/gm, while the lowest concentration was 1280 µg/gm. The study shows that plants will accumulate Mn from the soil and that use of fertilizers with high concentrations of Mn will increase the accumulations, possible creating a situation where animals and people ingesting the fruits and vegetables might be subjected to elevated Mn levels.Grant Funding Source: Supported by 0516041071 of NYSDOE and 0622197 of the DUE Program of NSF
Alleviation of neuronal oxidative stress by thioredoxin through PDE2 inhibition (1143.13)
Authors: Klabnik Jonathan , O'Donnell James 
Published: June 2021

Purpose Reactive oxygen species (ROS) are a normal byproduct of metabolism within the cell. Normally the cell is able to sequester these species by the various redox agents but in some cases, the stress can be too great for the system as ROS is prone to act in positive feedback manner. This redox imbalance then places the cell into oxidative stress where severe consequences arise, such as protein damage and even apoptosis. Recent studies suggest this can have global effects on CNS function and overall health. The working hypothesis of this study is that the use of PDE2 inhibitors can be used to upregulate redox agents and return ROS to homeostatic levels. The rationale behind this is that is has been shown thioredoxin upregulation is downstream of the mediator Protein Kinase G (PKG) leading to reduced ROS levels. One way to increase PKG levels is to promote cGMP, which can be done through inhibition of phosphodiesterases (PDE), which catalyze the degradation of cyclic nucleotides. The idea of the use of PDE2 versus other inhibitors is that reported expression levels found relative to available PKG expression compared to other cGMP‐metabolizing PDEs. Previous work done by our lab has shown that PDE2 inhibitors were able to decrease ROS activity as well as total antioxidant capacity. Presently, however, it is not known the exact mechanism of those results and this research hopes to illustrate a possible mechanism.Results Currently, the results indicate that, indeed, PDE2 inhibition by the use of Bay 60‐7550 is involved in increasing thioredoxin response in a dose‐dependent manner. This upregulation also correlates with a decrease in overall ROS and peroxynitrite levels.Conclusion The implications of this research can shed light on a novel way to treat patients suffering from diseases that are largely associated with ROS activity, such as Parkinson’s disease or autoimmune disorders.
Antidepressant potential of organic cation transporter blockade: the role of norepinephrine (1144.5)
Authors: Mitchell Nathan , Horton Rebecca , Vitela Melissa , Gould Georgianna , Koek Wounter , Daws Lynette 
Published: June 2021

Depression is a major health problem for which most patients are not effectively treated. This underscores a need to identify new targets for the development of antidepressants with improved efficacy. Our previous studies have shown that blockade of organic cation transporters (OCTs) and the plasma membrane monoamine transporter (PMAT) with decynium‐22 (D‐22) can produce antidepressant‐like effects, which are correlated with the ability of D‐22 to inhibit serotonin (5‐HT) clearance in brain, when the 5‐HT transporter (SERT) is pharmacologically or genetically compromised. In vitro studies show that OCT3 takes up norepinephrine (NE) more efficiently than 5‐HT, which raises the possibility that D‐22 might enhance the antidepressant‐like actions of drugs that block NE uptake via the NE transporter (NET). Using in vivo chronoamperometry, an electrochemical technique which allows second by second recording of extracellular biogenic amine concentrations, we show D‐22 increases time to clear NE from the extracellular space, and show D‐22 potentiates the effects of NET blockers venlafaxine (VEN) and desipramine (DMI) to increase clearance time of NE in the dentate gyrus and CA3 of the hippocampus. We next investigated if D‐22 can enhance the antidepressant‐like effects of VEN/DMI. Both VEN and DMI reduced immobility on the tail suspension test (TST), a test commonly used to assay antidepressant‐like activity of drugs in mice. Unlike the enhancing effect of D‐22 on increasing clearance time of NE, D‐22 did not enhance the antidepressant‐like effect of VEN/DMI in wild‐type mice. Ongoing studies are investigating the antidepressant‐like effect of D22 in SERT KO and heterozygous mice.Grant Funding Source: Supported by MH093320 (LD & WK), MH064489 (LD), NARSAD (LD) and UL1RR025767 (R25 START UP).
Functional characterization of human α7 nicotinic acetylcholine receptor genetic variants (1147.4)
Authors: Baez‐Pagan Carlos , Holder‐Viera Mileyshmi , Santana‐Rodriguez Zuleirys , Aponte‐Santiago Nicole , Aviles‐Pagan Emir , Lasalde‐Dominicci Jose 
Published: June 2021

The α7 nicotinic acetylcholine receptor (nAChR) is a ligand‐gated ion channel that is abundantly expressed throughout the central nervous system (CNS) and has been linked to Alzheimer’s disease, Parkinson’s, and Schizophrenia. In addition, the α7 nAChR has been identified in non‐neuronal cells such as macrophages, where it plays an essential role in the control of inflammation through the cholinergic anti‐inflammatory pathway; and cancer cells, where its activation promotes tumor growth, angiogenesis, metastasis, and cancer stem cell proliferation. Consistent with its protagonistic role in various diseases, the α7 nAChR is extensively studied by the pharmaceutical industry. The CHRFAM7A gene, a partial duplication of the gene encoding the α7 nAChR (CHRNA7), encodes dupα7, which is a protein that was recently demonstrated to act as negative regulator of the α7 functionality. The objective of the research presented herein was to examine whether genetic variants of the CHRNA7 gene could result in α7 receptors with significant changes in functionality, expression, and proneness to dupα7 regulation. To achieve this objective, two‐electrode voltage clamp, cell‐attached patch clamp and confocal microscopy experiments were performed using Xenopus laevis oocytes as expression system. We found that genetic variants of the CHRNA7 gene can result in significant changes in functionality and expression of the α7 nAChR in addition to significantly affecting the capacity of dupα7 to negatively regulate α7. In conclusion, our results demonstrate that genetic variants of the CHRNA7 gene can modify the functionality, expression, and regulation of the α7 nAChR, and should thus be considered when designing drugs that target this important receptor.Grant Funding Source: Supported by NIMH grant # P30MH075673‐07 and NIMHD grant # 8U54MD007587‐03.