bluuebunny/crossref_metadata_2025_split huggingface dataset:
Movements of N‐terminus of LeuT during the Substrate Transport Cycle
Authors: Khan Jawad Akbar , Sohail Azmat , Sandtner Walter , Sitte Harald H. , Stockner Thomas 
Published: June 2021

LeuT from Aquifex aeolicus aeolicus is a bacterial homologue to mammalian solute carrier family 6 (SLC6) of neurotransmitter transporters. SLC6 transporters are of great pharmacological interests because of their crucial role in neurotransmitter clearance. These proteins are also targets of many clinically relevant drugs, including antidepressant drugs and drugs of abuse such as amphetamines. The amino terminus of SLC6 transporters plays an important regulatory role for function.High resolution X‐ray crystal structures of LeuT are available in different conformations. However, these structures are not able to fully explain the dynamic nature of LeuT function during the substrate transport cycle. The aim of this project is to look at the movements of the N terminus of LeuT in the proteoliposomes using spectroscopic based technique called lanthanide based resonance energy transfer (LRET).We developed a LeuT variant in which we inserted a Lanthanide Binding Tag (LBT) at position 335, positioned at the end of extracellular loop 4. Three LeuT mutants (K4C, H7C and A9C) were created in the background of the LeuT‐LBT construct. Using LRET based distance measurements we determined the distance between the LBT bound lanthanide and the fluorophore TMR attached to the cysteines in the N‐terminal of LeuT. The results did not show the expected increase in distance towards the N‐terminus, as the distance to H7C was shorter than to A9C. In contrast, iodate quenching experiments of TMR fluorescence showed no difference in accessibility to KI for all three residues.Intramolecular distances determined by LRET suggest that the fluorophore attached to H7 could be oriented towards the inner vestibule, while the KI based accessibility experiments displays a contrasting picture. In order to elucidate the dynamics of the N‐terminus and support one of the two findings, I am further planning to stabilize the transporter in an outward open conformation using tryptophan, and as an alternative strategy, stabilize LeuT‐LBT in the inward‐facing and an outward‐facing conformation using the R5A or the R30A mutants.Support or Funding InformationThis work is jointly supported by the Austrian Science Fund (FWF) Austria and Higher Education Commission (HEC) PakistanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Co‐expression of Cdr1as and miR‐7a is associated with hypoxia‐affected fetal brain development
Authors: Lv Yehui , Li Shiying 
Published: June 2021

Hypoxia influences different cellular biological processes and hypoxia during pregnancy can adversely affect brain development. MicroRNA (miRNA) and emerging circular RNA (circRNA) are critical regulators of multiple cell processes including hypoxia. Recently, one human circRNA, antisense to the cerebellar degeneration‐related protein 1 transcript (Cdr1as) has been identified to share expression domains with miR‐7a. This study aimed to investigate the expression pattern of Cdr1as/miR‐7a and further to understand the impact of prenatal hypoxia on brain development. For this purpose, pregnant SD rats were exposed to either hypoxia or normoxia and fetal brains were further collected at day 20. Fetal cortex weight and ratio of brain‐body weight were reduced. Subsequently, real‐time PCR was performed to quantify the expression of Cdr1as and miR‐7a in both normoxic and hypoxic neonatal cortex samples. The expression levels of both Cdr1as and miR‐7a were both upregulated in the cortex tissues of the offspring exposed to hypoxia. Moreover, miR‐7a's target SP1 (Specificity Protein 1), which had been confirmed in present study (Geng Hai‐Hua, 2016), were found to be down‐regulated in both mRNA and protein levels following prenatal hypoxia. These results indicate that the co‐expression of Cdr1as and miR‐7a play roles in the hypoxia response and these observed molecular alterations may provide insights into prenatal‐hypoxia‐impaired brain development with further study.Support or Funding InformationEthical permission was granted through the Science and Ethics Committee of Shanghai University of Medicine & Health Sciences. This study was funded by the Seed Fund of Shanghai University of Medicine & Health Sciences and Open Foundation of Shanghai Key Laboratory of Forensic Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Red Blood Cells Specifically Bind a C‐peptide/Albumin Complex
Authors: Geiger Morgan , Kauffman Nathan , Zinn Kurt , Spence Dana 
Published: June 2021

People with Type 1 diabetes (T1D) require exogenous insulin due to the destruction of insulin‐secreting pancreatic β‐cells. However, even with insulin treatments, people with T1D develop complications such as retinopathy, neuropathy, and nephropathy that are often associated with poor microvascular blood flow. C‐peptide, a 31‐amino acid length peptide co‐secreted with insulin from the pancreatic β‐cells in a 1:1 mole ratio has been shown to improve microvascular blood flow in T1D rats and small‐scale human studies. C‐peptide indirectly enhances endothelium‐derived nitric oxide (NO) by stimulating the release of red blood cell (RBC) derived ATP, a recognized stimulus of endothelial NO synthase. Unfortunately, a receptor for C‐peptide has never been reported. Previously, using ELISA, we reported C‐peptide uptake by RBCs was approximately 1800 C‐peptide molecules/cell, but only when in the presence of albumin. In the absence of albumin, there was no measurable C‐peptide binding to the RBCs. We also report C‐peptide binding to albumin with a Kd of 5.7 ± (2.1) × 10−6 M; therefore, we hypothesize that C‐peptide binds to RBCs via a receptor that is specific for an albumin/Cpeptide complex. The binding of 99mTc labeled bovine serum albumin (BSA) to isolated RBCs, in the presence and absence of C‐peptide, was quantitatively determined by automatic gamma counting after repeated washing with a physiological salt solution. Sample counts per minute (CPM) were compared to a standard curve to determine the 99mTc‐BSA molecules per RBC. Results suggest BSA total binding to the RBCs saturates at approximately 1600 BSA molecules/RBC, whether or not C‐peptide is present, but that the BSA‐99mTc is specifically binding to RBCs in the presence of C‐peptide. Collectively, our data strongly suggest that the Cpeptide receptor has remained elusive because the receptor specifically binds an albumin/Cpeptide complex, as opposed to C‐peptide alone.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tubastatin A attenuates chronic angiotensin‐II‐induced autophagy
Authors: Rajasingh Sheeja , Czirok Andras , Isai Dona Greta , Rajasingh Johnson 
Published: June 2021

IntroductionIt has been shown that autophagy is playing one of the crucial cellular functions, which maintain the cellular homeostasis as well as cardiac hypertrophy, but the mechanism is poorly understood. Studies have shown that over activation of autophagy is not always protective but may cause autophagic cell death. Recent reports have demonstrated that STAT3 participates in the process of autophagy. Bcl2 is an anti‐apoptotic and anti‐autophagic protein that inhibits autophagy through direct interaction with the BH3 domain of pro‐autophagic protein beclin1. Here we have examined the protective role of HDAC6 inhibitor Tubastatin A (TBSA) during chronic angiotensin II‐induced autophagy in human iPSC‐derived cardiomyocytes (hiCMCs).HypothesisWe hypothesize that TBSA treatment mediated activation of STAT3‐Bcl2 is a critical regulator of angiotensin II‐induced hiCMCs autophagy and the over expression of HDAC6 in hiCMCs leads to exaggerate angiotensin II‐induced autophagy.Methods and ResultsWe have evaluated the TBSA treatment mediated effects in the contractility of hiCMCs, cell size and mitochondrial membrane potential in response to angiotensin II‐induced autophagy. Our data shows that the functional effect of hiCMCs stimulated with angiotensin II has significantly reduced the beating frequency, whereas treatment with TBSA rescued the beating frequency (Fig. A, B). Furthermore, the angiotensin II‐induced hypertrophic effect on hiCMCs is significantly reduced by TBSA treatment (Fig. C). Moreover, the immunostaining with JC1 (marker for Autophagy) shows that the mitochondrial membrane potential significantly reduced in angiotensin II‐induced hiCMCs, whereas TBSA treatment maintains the mitochondrial membrane potential (Fig D). Moreover, our electron microscopic analysis revealed a dramatic difference was observed between angiotensin II induced hiCMCs and Angiotensin II‐induced hiCMCs treated with TBSA in actin cytoskeletal protein and disorganized sarcomeres.ConclusionsWe report for the first time that the role of HDAC6 in regulation of pathological autophagy, thus can acts as a potential therapeutic target for treatment of chronic heart diseases.Support or Funding InformationAmerican Heart Association Grant‐in‐Aid Grant 16GRNT30950010This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Myosin Chaperon, UNC‐45A, Is a Key Regulator of Intestinal Epithelial Apical Junctions
Authors: Lechuga Susana , Ivanov Andrei I 
Published: June 2021

Epithelial adherens junctions (AJs) and tight junctions (TJs) play critical roles in regulating the integrity and permeability of epithelial barriers. Proper assembly and functioning of AJs and TJs depend on their associations with underlying actin filaments and the actin motor protein, nonmuscle myosin II (NM II). Our previous studies demonstrated the roles of NM II in controlling assembly and permeability of intestinal epithelial apical junctions in vitro and in vivo. However, the molecular mechanisms that regulate NM IIA functions in intestinal epithelium remain poorly understood. UNC‐45A is a highly conserved member of the UNC‐45/CRO1/She4p family proteins, which acts as a NM II chaperone and controls a proper folding and activity of myosin heavy chains. In the present study, we investigated the roles of UNC‐45A in the regulation of apical junctions in model intestinal epithelial cell monolayers.UNC‐45A protein was abundantly expressed in several commonly‐used human intestinal epithelial cell lines. Furthermore, its expression in T84 and HT‐29 cells was significantly downregulated by proinflammatory cytokines, IFN‐γ and TNF‐α, in parallel to cytokine induced TJ disassembly and barrier disruption. Consistently, UNC‐45A and NM IIA expression was downregulated in colonic epithelial cells isolated from tissue samples of ulcerative colitis patients. CRISPR/Cas9 mediated knock‐out of UNC‐45A in SK‐CO15 and HT‐29 human colonic epithelial cells resulted in multiple defects of apical junctions. These defects include the decreased transepithelial electrical resistance, increased transepithelial dextran flux, impaired steady‐state structure of AJs and TJs, and attenuated junctional reassembly during calcium switch. Together, our findings revealed a novel cytoskeleton‐dependent mechanism that regulates structure and functions of normal intestinal epithelial barrier and may contribute to barrier disruption during mucosal inflammation.Support or Funding InformationThis work was supported by NIH grant R01DK108278.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Light/Ultrasound Responsive 750 nm‐Emitted Non‐toxic Indium Phosphide Quantum Dots Hybrid Nanobubble for Brain Tumor Imaging
Authors: Chan Ming‐Hsien , Liu Ru‐Shi , Hsiao Michael 
Published: June 2021

BackgroundThe blood‐brain barrier (BBB) hardly allows all substances to pass. Except for oxygen, carbon dioxide, and blood sugar, most dyes and proteins are generally unable to pass due to their molecular structure. Therefore, bioimaging in brain tumor is very challenging. Moreover, different diagnostic methods have their own advantages and disadvantages. Composite imaging is considered to be a more efficient method of bioimaging.DesignThe study initially constructed an ultrasonic imaging material that can be used for quantum dot bonding, which allows the nanomaterial to have both supersonic and optical diagnosis. We intend to synthesize lipid‐phase nanobubbles to combine with 750 nm emitted non‐toxic indium phosphide (InP) quantum dots, providing multiple imaging functions. Choose for high biocompatibility and for in vivo and in situ diagnosis. Moreover, recent researches have demonstrated that cavitated nanobubbles can introduce a small opening in the BBB, allowing materials extravasation. It makes InP Quantum dots can enter in brain tissue to generate the near‐infrared signal for the signal tracking.ResultsNanobubble subjected to a shock wave that disrupts the BBB. The cavitation effect makes an opening space, allowing small particles extravasation. InP quantum dots‐loaded nanobubbles activated by ultrasound can temporarily permeabilize into the BBB, causing quantum dots accumulation in the brain tumor tissue, as evidenced by ultrasound and fluorescence imaging.ConclusionsNanobubbles cavitation bioimaging displays the mean intensity of acoustic cavitation over time and was correlated with areas of acoustic cavitation‐induced BBB opening. After combining the InP quantum dots to the lipid bubbles, the nanocomposite is expected to have the functions of ultrasonic and optical imaging. We chose the best near‐infrared light to make the signal of brain tumor can be detected.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of the Effects of Toll‐like Receptor 3 (TLR3) on the Pathology of Chlamydia muridarum in the Genital Tract of Wild‐Type and TLR3 Knockout C57BL/6N Mice
Authors: Billingsley Caylin , Derbigny Wilbert , Sandusky George 
Published: June 2021

In 2017, a total 1,708,569 chlamydial infections were reported to Centers for Disease Control and Prevention; the most prevalent sexually transmitted disease (STD). Chlamydia trachomatis primarily replicates in epithelial cells lining the reproductive tract. Epithelial cells recognize chlamydia through cell surface and cytosolic receptors, and/or endosomal innate receptors such as Toll‐like receptors (TLRs). The activation of these receptors triggers immune mechanisms that are required for chlamydial clearance. It was previously shown that Chlamydia muridarum (Cm) induces IFN‐β in oviduct epithelial cells in a TLR3‐dependent manner. TLR3's regulation of inflammatory chemicals involved in host defense against Cm infection led to the hypothesis that TLR3 plays a protective role against Cm‐induced genital tract pathology in congenic C57BL/6N mice. To test this hypothesis, wild‐type and TLR3‐deficient mice were treated with vehicle and Cm by swabbing the vaginal vault and cervix. Tissues were extracted from mice sacrificed on day 7, 21, or 42 post‐infection and fixed in 4% paraformaldehyde for 48 hrs. Paraffin‐embedded, 4‐μm sections containing cervix and both uterine horns and oviducts were stained with H&E. Genital tract sections (cervix, uterine body and horns, and oviducts) were microscopically examined and graded for the presence of acute inflammation (neutrophilic infiltrates), chronic inflammation (lymphocytic infiltrates), epithelial erosion and disruption, and luminal distension of uterine horns and oviducts using a semi qualitative pathology assessment. Inflammatory cell infiltrates, epithelial erosion and disruption were scored as follows: 0, normal; 1, mild; 2, moderate; 3, severe. All scoring was performed in a double‐blinded fashion with regards to the treatment groups. The results revealed an inflammatory immune response showing minute differences in both groups at day 7 (acute inflammation) in the oviducts, cervix and uterus. By day 21 (chronic inflammation), there was more inflammation seen in the WT group compared to the knock out TLR3 group. By day 42, there was mild chronic inflammation in the uterus in both groups; however, the knockout group had more cystic endometrial change in the uterus compared to the wild type group. In conclusion, the data suggest that TLR3 may promote the clearance of Cm.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTOR Inhibition Delays Hepatoblastoma Growth in a Relevant Mouse Model
Authors: Monga Satdarshan Pal , Molina Laura Maria , Yang Hong , Adebayo Michael Adeola O. , Tao Junyan , Bell Aaron 
Published: June 2021

Hepatoblastoma (HB) is the most common pediatric liver malignancy, affecting children in their first few years of life. Around 80% of HB demonstrate simultaneous activation of β‐catenin and Yes‐associated protein 1 (YAP1), which are transcriptional coactivators downstream of the Wnt and Hippo signaling pathways, respectively. Recently, mTORC1 activation was reported in human HB cell lines as well as HB tumors in a mouse model driven by β‐catenin and YAP1. In the current study we investigated directly the impact of mTOR inhibition on HB development in a YAP1‐β‐catenin HB mouse model. Hydrodynamic tail vein injection (HTVI) was used to co‐deliver Sleeping Beauty transposase along with plasmids coding for ΔN90‐β‐catenin and S127A‐YAP1. Five weeks post‐HTVI, when HB tumors are evident, we treated half of the mice with rapamycin via diet, and both control and treated groups were monitored with ultrasound imaging to assess tumor growth over time. All mice were sacrificed at 10 weeks post‐HTVI. Our results show that mice treated with rapamycin for 5 weeks developed significantly less HB tumor burden than control mice, with almost normal liver weight to body weight ratio and fewer, smaller tumors observed histologically. We show by immunohistochemistry that rapamycin treatment reduces activation of downstream targets of mTORC1 signaling such as phosphorylation of 4EBP1 and S6‐ribosomal protein. Importantly, we show by ultrasound imaging that control mice exhibit a dramatic rise in tumor number and volume starting at ~5 weeks post‐HTVI, while rapamycin‐treated mice begin to show a similar increase only at 8–9 weeks post‐HTVI (after 3–4 weeks of rapamycin treatment). Interestingly, while a majority of HB tumors in the control group exhibit embryonal histology, the tumors that developed in rapamycin‐treated mice exhibit more well differentiated, fetal histology. Thus we show that while rapamycin does not completely block HB tumor formation, it significantly delays the development of tumors driven by β‐catenin and YAP1. Our study also demonstrates the utility of standard and 3D ultrasound imaging for the monitoring of liver tumor formation in mice and supports the use of mTORC1 inhibitors for HB treatment.Support or Funding InformationThis work was supported by NIH grants 1R01DK62277, 1R01DK100287, 1R01CA204586, and the Endowed Chair for Experimental Pathology (S.P.M.), as well as CATER T32 grant 5T32EB001026‐14 (L.M.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isoflurane‐Induced Hypothermia Does Not Aggravate Memory Impairment in A Sporadic Alzheimer's Disease Rat Model Produced by Intracerebroventricular Streptozotocin
Authors: Vizin Robson Cristiano Lillo , Harris Grant , Kunstetter Ana Cançado , Almeida Maria Camila , Carrettiero Daniel Carneiro , Romanovsky Andrej A. 
Published: June 2021

Several studies have shown that hypothermia and anesthesia are risk factors for Alzheimer's disease. Most of them were performed on healthy elderly rodents. However, the effect of anesthesia‐induced hypothermia in memory in a sporadic Alzheimer's disease animal model has never been studied. In this study, Wistar rats (3‐month‐old) were induced to anesthesia with 3% isoflurane and maintained with 1.5–2% isoflurane in 100% oxygen for 3 hours. In the first 30 min of anesthesia, rats were subjected to a single bilateral intracerebroventricular injection of streptozotocin (STZ, 1 mg/kg) to produce the sporadic Alzheimer's disease model, or its vehicle (0.05 mol/L citrate buffer, pH 4.5, 2 μL/ventricle). The rats were either subjected to hypothermia or not, induced by isoflurane (hypothermic and normothermic groups, respectively). In this sense, during anesthesia, normothermic rats were kept on a heating pad, and their rectal temperature was maintained at 37.5 ± 0.5°C. Hypothermic rats were kept without a heating pad. Spatial working memory was evaluated using Morris Water Maze from the 3rd to 28th‐day postsurgery, and by Y‐maze test on the 30th‐day postsurgery. Recognition memory was evaluated on the 31st‐day postsurgery using the Novel Object Recognition Test. All the procedures were approved by and are in accordance with the local ethics committee from St Joseph's Hospital and Medical Center (protocol n° 485). In the Morris water maze test, both hypothermic and normothermic STZ‐injected rats shown similar impairment in spatial working memory compared to their respective controls, i.e., hypothermic and normothermic vehicle‐injected rats, respectively. In the Y‐maze test, normothermic STZ‐injected rats shown a deficit in spatial working memory compared to normothermic vehicle‐injected rats (p = 0.028). However, this effect was missed in hypothermic groups with no difference between STZ‐ and vehicleinjected groups (p = 0.073). Similarly, in the Novel Object Recognition Test, recognition memory was impaired in normothermic STZ‐injected rats compared to normothermic vehicleinjected rats (p = 0.012) and no effect was observed between hypothermic STZ‐ and vehicleinject rats (p = 0.118). In conclusion, isoflurane‐induced hypothermia does not aggravate the STZ‐deleterious effect in recognition memory and spacial work memory in rats.Support or Funding InformationThis work was supported by the Brazilian National Council for Scientific and Technological Development (grant 449102/2014‐9 to D.C.C.) and the São Paulo Research Foundation (grants 2015/02991‐0 to M.C.A., 2015/23426‐9 to D.C.C., and 2016/01836‐3 to R.C.L.V.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.