Publications by authors named "Zi-Wei Chen"

35 Publications

Machine Learning for the Prediction of Red Blood Cell Transfusion in Patients During or After Liver Transplantation Surgery.

Front Med (Lausanne) 2021 22;8:632210. Epub 2021 Feb 22.

Department of Blood Transfusion, The Second Xiangya Hospital of Central South University, Changsha, China.

This study aimed to use machine learning algorithms to identify critical preoperative variables and predict the red blood cell (RBC) transfusion during or after liver transplantation surgery. A total of 1,193 patients undergoing liver transplantation in three large tertiary hospitals in China were examined. Twenty-four preoperative variables were collected, including essential population characteristics, diagnosis, symptoms, and laboratory parameters. The cohort was randomly split into a train set (70%) and a validation set (30%). The Recursive Feature Elimination and eXtreme Gradient Boosting algorithms (XGBOOST) were used to select variables and build machine learning prediction models, respectively. Besides, seven other machine learning models and logistic regression were developed. The area under the receiver operating characteristic (AUROC) was used to compare the prediction performance of different models. The SHapley Additive exPlanations package was applied to interpret the XGBOOST model. Data from 31 patients at one of the hospitals were prospectively collected for model validation. In this study, 72.1% of patients in the training set and 73.2% in the validation set underwent RBC transfusion during or after the surgery. Nine vital preoperative variables were finally selected, including the presence of portal hypertension, age, hemoglobin, diagnosis, direct bilirubin, activated partial thromboplastin time, globulin, aspartate aminotransferase, and alanine aminotransferase. The XGBOOST model presented significantly better predictive performance (AUROC: 0.813) than other models and also performed well in the prospective dataset (accuracy: 76.9%). A model for predicting RBC transfusion during or after liver transplantation was successfully developed using a machine learning algorithm based on nine preoperative variables, which could guide high-risk patients to take appropriate preventive measures.
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http://dx.doi.org/10.3389/fmed.2021.632210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937729PMC
February 2021

Surgical Performance Is Not Negatively Impacted by Wearing a Commercial Full-Face Mask with Ad Hoc 3D-Printed Filter Connection as a Substitute for Personal Protective Equipment during the COVID-19 Pandemic: A Randomized Controlled Cross-Over Trial.

J Clin Med 2021 Feb 2;10(3). Epub 2021 Feb 2.

Department of General, Visceral and Transplant Surgery, Heidelberg University Hospital, 69120 Heidelberg, Germany.

(1) Background: During the COVID-19 pandemic, shortages in the supply of personal protective equipment (PPE) have become apparent. The idea of using commonly available full-face diving (FFD) masks as a temporary solution was quickly spread across social media. However, it was unknown whether an FFD mask would considerably impair complex surgical tasks. Thus, we aimed to assess laparoscopic surgical performance while wearing an FFD mask as PPE. (2) Methods: In a randomized-controlled cross-over trial, 40 laparoscopically naive medical students performed laparoscopic procedures while wearing an FFD mask with ad hoc 3D-printed connections to heat and moisture exchange (HME) filters vs. wearing a common surgical face mask. The performance was evaluated using global and specific Objective Structured Assessment of Technical Skills (OSATS) checklists for suturing and cholecystectomy. (3) Results: For the laparoscopic cholecystectomy, both global OSATS scores and specific OSATS scores for the quality of procedure were similar (Group 1: 25 ± 4.3 and 45.7 ± 12.9, = 0.485, vs. Group 2: 24.1 ± 3.7 and 43.3 ± 7.6, = 0.485). For the laparoscopic suturing task, the FFD mask group needed similar times to the surgical mask group (3009 ± 1694 s vs. 2443 ± 949 s; = 0.200). Some participants reported impaired verbal communication while wearing the FFD mask, as it muffled the sound of speech, as well as discomfort in breathing. (4) Conclusions: FFD masks do not affect the quality of laparoscopic surgical performance, despite being uncomfortable, and may therefore be used as a substitute for conventional PPE in times of shortage-i.e., the global COVID-19 pandemic.
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http://dx.doi.org/10.3390/jcm10030550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867352PMC
February 2021

Risks associated with cryopreserved semen in a human sperm bank during and after the COVID-19 pandemic.

Reprod Biomed Online 2021 03 3;42(3):589-594. Epub 2020 Dec 3.

Institute of Reproductive and Stem Cell Engineering, Basic Medicine College, Central South University, Changsha Hunan 410005, China; Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, ChangshaHunan 410005, China. Electronic address:

Research Question: What are the risks associated with cryopreserved semen collected during and after the coronavirus disease 2019 (COVID-19) pandemic wave in Wuhan, China?

Design: Retrospective cohort study involving young adult men who were qualified sperm donors at the Hunan Province Human Sperm Bank (China) during the pandemic wave (1 January 2020 to 30 January 2020) and after the wave and return to work (7 April 2020 to 30 May 30 2020). One hundred paired semen and blood specimens from 100 donors were included. One-step single-tube nested quantitative real-time polymerase chain reaction (OSN-qRT-PCR) was used to detect SARS-CoV-2. Moreover, to control the unacceptable risk of false-negative results, a second round of screening was performed with pooled RNA from negative semen samples using crystal digital PCR (cd-PCR).

Results: For individual blood and semen samples, the target genes, namely the nucleocapsid protein (N) and open reading frame (ORF-1ab) genes, tested negative in all of the 100 paired samples. Further, as per cd-PCR results, there were >20,000 droplets per well in the RNA for each combined sample and no positive droplets were present for either of the aforementioned target genes. A total of 100 paired semen and blood samples from these two groups tested negative for SARS-CoV-2.

Conclusions: Cryopreserved semen at the Hunan Province Human Sperm Bank during and after the COVID-19 pandemic wave was free of SARS-CoV-2 and was judged safe for external use in the future.
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http://dx.doi.org/10.1016/j.rbmo.2020.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713547PMC
March 2021

Site-specific effects of neurosteroids on GABA receptor activation and desensitization.

Elife 2020 09 21;9. Epub 2020 Sep 21.

Department of Anesthesiology, Washington University in St. Louis, St. Louis, United States.

This study examines how site-specific binding to three identified neurosteroid-binding sites in the αβ GABA receptor (GABAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β(+)-α(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β subunit, promoting receptor desensitization and the α subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid-binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABARs.
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http://dx.doi.org/10.7554/eLife.55331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532004PMC
September 2020

[Analysis of the clinical effect on post-stroke shoulder hand syndrome stage Ⅰ treated with the along-meridian trochar acupuncture therapy].

Zhen Ci Yan Jiu 2020 Aug;45(8):657-61

School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029.

Objective: To compare the differences in the clinical effect on post-stroke shoulder hand syndrome (SHS) stage Ⅰ between the along-meridian trochar acupuncture therapy and the routine acupuncture therapy with filiform needles.

Methods: A total of 80 patients with post-stroke SHS stage I were divided into a treatment group (41 cases) and a control group (39 cases) according to the random number table. In the control group, the common filiform needles were used to stimulate Jianyu (LI15), Jianliao (TE14), Jianzhen (SI9), Jianzhongshu (SI15), Jianwaishu (SI14), 5 times a week, 3 weeks as 1 course. In the treatment group, along-meridian trochar acupuncture therapy was applied, 3 times a week, 3 weeks as 1 course. The patients in both groups were all treated with basic medications and routine rehabilitation training. Pain degree, edema degree, upper limb motor function and activity of daily living were observed in the two groups before the treatment, at the end of the treatment and in follow-up. At the end of treatment and in follow-up, the therapeutic effect was evaluated respectively in the patients of the two groups.

Results: Compared with the values before treatment, the VAS score of the upper limb was reduced obviously (< 0.001), the score of the upper limb motor function and Barthel index were increased obviously (<0.001, <0.05) in the patients of the two groups, the score of edema degree of the affected limb was reduced after treatment in the patients of the treatment group (<0.001). Compared with the control group, VAS score of the upper limb and the score of edema degree of the affected limb were obviously lower (<0.001), and the score of the upper limb motor function and Barthel index were obviously higher in the treatment group (<0.001). The total effective rate was 66.7% (26/39) after treatment and was 74.4% (29/39) in follow-up in the treatment group and they were 20.5% (8/39) and 28.2% (11/39) respectively in the control group. The total effective rates after treatment and in follow-up in the treatment group were all obviously higher than those in the control group respectively (<0.001).

Conclusion: The along-meridian trochar acupuncture therapy remarkably relieves pain and edema and improves the upper limb motor function and the activity of daily living in the patients with post-stroke shoulder hand syndrome and its clinical therapeutic effect is definite.
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http://dx.doi.org/10.13702/j.1000-0607.200164DOI Listing
August 2020

A highly sensitive one-tube nested quantitative real-time PCR assay for specific detection of Bordetella pertussis using the LNA technique.

Int J Infect Dis 2020 Apr 8;93:224-230. Epub 2020 Feb 8.

NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China. Electronic address:

Objectives: Bordetella pertussis is a highly contagious respiratory agent and is the causative pathogen of pertussis, which primarily affects children. Current diagnostic techniques for this pathogen have a variety of limitations including a long culture time, low bacterial load, and lack of specificity.

Methods: This article reports the development of a one-tube nested quantitative real-time PCR assay using the locked nucleic acid (LNA) technique (LNA-OTN-q-PCR), targeting the BP485 gene and using a simple inexpensive extraction method. A total of 130 clinical samples from patients with clinically suspected pertussis, collected from the Children's Hospital of Hebei, China, were tested by LNA-OTN-q-PCR assay. RT-PCR and two-step semi-nested PCR assays were performed in parallel for comparison.

Results: Only strains of B. pertussis were identified as positive, whereas all of the remaining strains were appropriately identified as negative by the LNA-OTN-q-PCR assay. A single copy per reaction can be detected by the LNA-OTN-q-PCR assay. Additionally, the sensitivity of this method was 100 times that of the RT-PCR assay (100 copies per reaction). Sixty-three of the 130 clinical samples were detected positive by LNA-OTN-q-PCR assay; in contrast, RT-PCR was able to detect only 41 positive samples. Following this, all 63 samples were positively identified by two-step semi-nested PCR. Compared with the two-step semi-nested PCR assay, both the specificity and sensitivity of the LNA-OTN-q-PCR assay using purified DNA and crude extract were 100%.

Conclusions: This assay was able to detect B. pertussis infection with high sensitivity and specificity. This test shows great potential as a promising technique to detect B. pertussis in both clinical laboratories and public health settings.
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http://dx.doi.org/10.1016/j.ijid.2020.01.053DOI Listing
April 2020

[Effect of Signal Molecule Combined with on Simultaneous Removal of Nitrogen and Sulfur].

Huan Jing Ke Xue 2019 Sep;40(9):4177-4184

School of Environmental and Municipal Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China.

The effects of combined with signal molecules on the removal of sulfide and nitrate was investigated. By adding signal molecules and at the same, the total number of microorganisms increased, the removal of sulfide and nitrate was accelerated, and an increase in nitrogen gas and more stable accumulation of elemental sulfur was observed. The total number of microorganisms after the reaction was detected using fluorescence hybridization (FISH) technique. In this experiment, the optimal concentration for the stable accumulation of elemental sulfur from six concentrations of signal molecules was revealed. Further, the effects of adding signal molecules, , and their combination were analyzed at this concentration. The results showed that it was easier to accumulate elemental sulfur after the addition of 1.0 μmol·L signal molecule. After adding both and 1.0 μmol·L signal molecules at a sulfide concentration of 200 mg·L, the removal of sulfide and nitrate increased to 99.8% and 96.9% at 72 h, respectively, and increases in nitrogen gas and sulfur were observed. The amounts of elemental sulfur and nitrogen gas reached to 59.0 mg and 80.0 mL, respectively, after adding 2.5 μmol·L signal molecules at 72 h when the sulfide concentration was 300 mg·L. Under those conditions, the removal efficiency of sulfide and nitrate reached 99.0% and 93.9%, and the production of elemental sulfur and nitrogen reached 63.1 mg and 79.5 mL, respectively.
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http://dx.doi.org/10.13227/j.hjkx.201903012DOI Listing
September 2019

Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7.

Virol J 2019 07 1;16(1):86. Epub 2019 Jul 1.

NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing, 102206, China.

Background: Human adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control.

Methods: We developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays incorporating competitive internal controls for detection of HAdV 3 and HAdV 7, respectively. The assays were performed in a one-step in a single tube reaction at 39° for 20 min.

Results: The analytical sensitivities of the duplex RAA assays for HAdV 3 and HAdV 7 were 5.0 and 14.8 copies per reaction, respectively (at 95% probability by probit regression analysis). No cross-reaction was observed with other types of HAdV or other common respiratory viruses. The duplex RAA assays were used to detect 152 previously-defined HAdV-positive samples. These results agreed with those obtained using a published triplex quantitative real-time PCR protocol.

Conclusions: We provide the first report of internally-controlled duplex RAA assays for the detection of HAdV 3 and HAdV 7. These assays effectively reduce the rate of false negative results and may be valuable for detection of HAdV 3 and HAdV 7 in clinical laboratories, especially in resource-poor settings.
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http://dx.doi.org/10.1186/s12985-019-1178-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604330PMC
July 2019

Multiple neurosteroid and cholesterol binding sites in voltage-dependent anion channel-1 determined by photo-affinity labeling.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 10 5;1864(10):1269-1279. Epub 2019 Jun 5.

Department of Anesthesiology, Washington University in St. Louis, MO 63110, USA; Department of Developmental Biology, Washington University in St. Louis, MO 63110, USA; Department of Psychiatry, Washington University in St. Louis, MO 63110, USA; Taylor Family Institute for Innovative Psychiatric Research, Washington University in St. Louis, MO 63110, USA. Electronic address:

Voltage-dependent anion channel-1 (VDAC1) is a mitochondrial porin that is implicated in cellular metabolism and apoptosis, and modulated by numerous small molecules including lipids. VDAC1 binds sterols, including cholesterol and neurosteroids such as allopregnanolone. Biochemical and computational studies suggest that VDAC1 binds multiple cholesterol molecules, but photolabeling studies have identified only a single cholesterol and neurosteroid binding site at E73. To identify all the binding sites of neurosteroids in VDAC1, we apply photo-affinity labeling using two sterol-based photolabeling reagents with complementary photochemistry: 5α-6-AziP which contains an aliphatic diazirine, and KK200 which contains a trifluoromethyl-phenyldiazirine (TPD) group. 5α-6-AziP and KK200 photolabel multiple residues within an E73 pocket confirming the presence of this site and mapping sterol orientation within this pocket. In addition, KK200 photolabels four other sites consistent with the finding that VDAC1 co-purifies with five cholesterol molecules. Both allopregnanolone and cholesterol competitively prevent photolabeling at E73 and three other sites indicating that these are common sterol binding sites shared by both neurosteroids and cholesterol. Binding at the functionally important residue E73 suggests a possible role for sterols in regulating VDAC1 signaling and interaction with partner proteins.
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http://dx.doi.org/10.1016/j.bbalip.2019.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681461PMC
October 2019

Multiple functional neurosteroid binding sites on GABAA receptors.

PLoS Biol 2019 03 7;17(3):e3000157. Epub 2019 Mar 7.

Department of Anesthesiology, Washington University in St Louis, St Louis, Missouri, United States of America.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action.
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http://dx.doi.org/10.1371/journal.pbio.3000157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424464PMC
March 2019

Common binding sites for cholesterol and neurosteroids on a pentameric ligand-gated ion channel.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 02 22;1864(2):128-136. Epub 2018 Nov 22.

Department of Anesthesiology, Washington University in St Louis, 660 S Euclid Ave, St Louis, MO 63110, USA; Taylor Family Institute for Innovative Psychiatric Research, Washington University in St Louis, 660 S Euclid Ave, St Louis, MO 63110, USA; Department of Developmental Biology, Washington University in St Louis, 660 S Euclid Ave, St Louis, MO 63110, USA. Electronic address:

Cholesterol is an essential component of cell membranes, and is required for mammalian pentameric ligand-gated ion channel (pLGIC) function. Computational studies suggest direct interactions between cholesterol and pLGICs but experimental evidence identifying specific binding sites is limited. In this study, we mapped cholesterol binding to Gloeobacter ligand-gated ion channel (GLIC), a model pLGIC chosen for its high level of expression, existing crystal structure, and previous use as a prototypic pLGIC. Using two cholesterol analogue photolabeling reagents with the photoreactive moiety on opposite ends of the sterol, we identified two cholesterol binding sites: an intersubunit site between TM3 and TM1 of adjacent subunits and an intrasubunit site between TM1 and TM4. In both the inter- and intrasubunit sites, cholesterol is oriented such that the 3‑OH group points toward the center of the transmembrane domains rather than toward either the cytosolic or extracellular surfaces. We then compared this binding to that of the cholesterol metabolite, allopregnanolone, a neurosteroid that allosterically modulates pLGICs. The same binding pockets were identified for allopregnanolone and cholesterol, but the binding orientation of the two ligands was markedly different, with the 3‑OH group of allopregnanolone pointing to the intra- and extracellular termini of the transmembrane domains rather than to their centers. We also found that cholesterol increases, whereas allopregnanolone decreases the thermal stability of GLIC. These data indicate that cholesterol and neurosteroids bind to common hydrophobic pockets in the model pLGIC, GLIC, but that their effects depend on the orientation and specific molecular interactions unique to each sterol.
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http://dx.doi.org/10.1016/j.bbalip.2018.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311151PMC
February 2019

Mapping two neurosteroid-modulatory sites in the prototypic pentameric ligand-gated ion channel GLIC.

J Biol Chem 2018 02 4;293(8):3013-3027. Epub 2018 Jan 4.

Department of Anesthesiology; Taylor Family Institute for Innovative Psychiatric Research, Washington University, St. Louis, Missouri 63110; Department of Developmental Biology. Electronic address:

Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand-gated ion channels (pLGICs) and can be effective anesthetics, analgesics, or anti-epileptic drugs. The complex effects of neurosteroids on pLGICs suggest the presence of multiple binding sites in these receptors. Here, using a series of novel neurosteroid-photolabeling reagents combined with top-down and middle-down mass spectrometry, we have determined the stoichiometry, sites, and orientation of binding for 3α,5α-pregnane neurosteroids in the ligand-gated ion channel (GLIC), a prototypic pLGIC. The neurosteroid-based reagents photolabeled two sites per GLIC subunit, both within the transmembrane domain; one site was an intrasubunit site, and the other was located in the interface between subunits. By using reagents with photoreactive groups positioned throughout the neurosteroid backbone, we precisely map the orientation of neurosteroid binding within each site. Amino acid substitutions introduced at either site altered neurosteroid modulation of GLIC channel activity, demonstrating the functional role of both sites. These results provide a detailed molecular model of multisite neurosteroid modulation of GLIC, which may be applicable to other mammalian pLGICs.
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http://dx.doi.org/10.1074/jbc.RA117.000359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827446PMC
February 2018

Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1.

J Biol Chem 2017 06 10;292(22):9294-9304. Epub 2017 Apr 10.

From the Departments of Anesthesiology,

Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, -tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr and Glu, respectively. When Glu was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu residue.
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http://dx.doi.org/10.1074/jbc.M116.773069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454110PMC
June 2017

Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins.

Anal Chem 2017 02 9;89(4):2636-2644. Epub 2017 Feb 9.

Department of Anesthesiology, Washington University in St. Louis , St. Louis, Missouri 63110, United States.

Identifying sites of protein-ligand interaction is important for structure-based drug discovery and understanding protein structure-function relationships. Mass spectrometry (MS) has emerged as a useful tool for identifying residues covalently modified by ligands. Current methods use database searches that are dependent on acquiring interpretable fragmentation spectra (MS2) of peptide-ligand adducts. This is problematic for identifying sites of hydrophobic ligand incorporation in integral membrane proteins (IMPs), where poor aqueous solubility and ionization of peptide-ligand adducts and collision-induced adduct loss hinder the acquisition of quality MS2 spectra. To address these issues, we developed a fast ligand identification (FLI) tag that can be attached to any alkyne-containing ligand via Cu(I)-catalyzed cycloaddition. The FLI tag adds charge to increase solubility and ionization, and utilizes stable isotope labeling for MS1 level identification of hydrophobic peptide-ligand adducts. The FLI tag was coupled to an alkyne-containing neurosteroid photolabeling reagent and used to identify peptide-steroid adducts in MS1 spectra via the stable heavy isotope pair. Peptide-steroid adducts were not identified in MS2-based database searches because collision-induced adduct loss was the dominant feature of collision-induced dissociation (CID) fragmentation, but targeted analysis of MS1 pairs using electron transfer dissociation (ETD) markedly reduced adduct loss. Using the FLI tag and ETD, we identified Glu73 as the site of photoincorporation of our neurosteroid ligand in the IMP, mouse voltage-dependent anion channel-1 (mVDAC1), and top-down MS confirmed a single site of photolabeling.
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http://dx.doi.org/10.1021/acs.analchem.6b05003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328583PMC
February 2017

Synthesis and evaluation of multifunctional ferulic and caffeic acid dimers for Alzheimer's disease.

Nat Prod Res 2017 Mar 17;31(6):734-737. Epub 2016 Aug 17.

b Department of Pharmacology, School of Pharmaceutical Sciences , Sun Yat-Sen University , Guangzhou , China.

In this study, a series of novel ferulic and caffeic acid dimers was designed and synthesised, and their multifunctional properties against Alzheimer's disease (AD) were evaluated. Results showed that our multifunctional strategy was great supported by enhancing the inhibition of Aβ self-induced aggregation. Moreover, 7b also had potent protective effects against glutamate-induced cell death without significant cell toxicity in mouse hippocampal neuronal HT22 cells and 10c effectively scavenged diphenylpicrylhydrazyl free radicals. Collectively, these data strongly encourage further optimisation of 7b as a new hit to develop multifunctional agents for the treatment of AD.
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http://dx.doi.org/10.1080/14786419.2016.1219862DOI Listing
March 2017

Gartanin Protects Neurons against Glutamate-Induced Cell Death in HT22 Cells: Independence of Nrf-2 but Involvement of HO-1 and AMPK.

Neurochem Res 2016 Sep 9;41(9):2267-77. Epub 2016 May 9.

Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, 510120, China.

Oxidative stress mediates the pathogenesis of neurodegenerative disorders. Gartanin, a natural xanthone of mangosteen, possesses multipharmacological activities. Herein, the neuroprotection capacity of gartanin against glutamate-induced damage in HT22 cells and its possible mechanism(s) were investigated for the first time. Glutamate resulted in cell death in a dose-dependent manner and supplementation of 1-10 µM gartanin prevented the detrimental effects of glutamate on cell survival. Additional investigations on the underlying mechanisms suggested that gartanin could effectively reduce glutamate-induced intracellular ROS generation and mitochondrial depolarization. We further found that gartanin induced HO-1 expression independent of nuclear factor erythroid-derived 2-like 2 (Nrf2). Subsequent studies revealed that the inhibitory effects of gartanin on glutamate-induced apoptosis were partially blocked by small interfering RNA-mediated knockdown of HO-1. Finally, the protein expression of phosphorylation of AMP-activated protein kinase (AMPK) and its downstream signal molecules, Sirtuin activator (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), increased after gartanin treatment. Taken together, these findings suggest gartanin is a potential neuroprotective agent against glutamate-induced oxidative injury partially through increasing Nrf-2-independed HO-1 and AMPK/SIRT1/PGC-1α signaling pathways.
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http://dx.doi.org/10.1007/s11064-016-1941-xDOI Listing
September 2016

MEF2D Mediates the Neuroprotective Effect of Methylene Blue Against Glutamate-Induced Oxidative Damage in HT22 Hippocampal Cells.

Mol Neurobiol 2017 04 3;54(3):2209-2222. Epub 2016 Mar 3.

Department of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510080, China.

Methylene blue (MB) can ameliorate behavioral, neurochemical, and neuropathological impairments in animal models of acute and chronic neurodegenerative disorders, but the underlying mechanism remains unclear. Myocyte enhancer factor 2 (MEF2D) is known to promote neuronal survival in several models, and several survival and death signals converge on MEF2D and regulate its activity. Here, we investigated the role of MEF2D in the neuroprotective effect of MB against glutamate-induced toxicity in HT22 neuronal cells. Our results showed that MB, event at less than 100 nM, improved the viability of HT22 cells exposed to 2 mM glutamate. MB attenuated the mitochondrial impairment and quenches the reactive oxygen species (ROS) induced by glutamate. Surprisingly, MB at 50-200 nM did not affect the Nrf2/HO-1 pathway, an important endogenous anti-oxidative system. Further study showed that MB increased the transcription and translation of MEF2D. In addition, MB upregulated the expression of mitochondrial NADH dehydrogenase 6 (ND6) in a MEF2D-dependent manner. Knockdown of MEF2D abolished both MB-medicated increase of ND6 and MB-induced neuroprotection against glutamate-induced toxicity. Moreover, we showed that MB promoted Akt function activity, suppressed GSK-3β activity, and increased MEF2D level in hippocampus of mice and HT22 cells. These findings for the first time demonstrate that MB protects HT22 neuronal cells against glutamate-induced cell death partially via the regulation of MEF2D-associated survival pathway.
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http://dx.doi.org/10.1007/s12035-016-9818-1DOI Listing
April 2017

Neolignans from Aristolochia fordiana Prevent Oxidative Stress-Induced Neuronal Death through Maintaining the Nrf2/HO-1 Pathway in HT22 Cells.

J Nat Prod 2015 Aug 30;78(8):1894-903. Epub 2015 Jul 30.

School of Pharmaceutical Sciences, Sun Yat-sen University , Guangzhou 510006, People's Republic of China.

Bioassay-guided fractionation of the ethanolic extract of the stems of Aristolochia fordiana led to the isolation of six new dihydrobenzofuran neolignans (1-3 and 7-9), three new 2-aryldihydrobenzofurans (4-6), a new 8-O-4' neolignan (10), and 14 known analogues (11-24). The structures of compounds 1-10 were established by spectroscopic methods, and their absolute configurations were determined by analyses of the specific rotation and electronic circular dichroism data. The neuroprotective effects of compounds 1-24 against glutamate-induced cell death were tested in hippocampal neuronal cell line HT22. Compounds 17 and 20-24 exhibited moderate neuroprotective activity by increasing the endogenous antioxidant defense system. In addition, the neolignans activated the Nrf2 (nuclear factor E2-related factor 2) pathway, resulting in the increase of the expression of endogenous antioxidant protein HO-1 (heme oxygenase-1). The active compounds also preserved the levels of antiapoptotic protein Bcl-2 (B cell lymphoma/leukemia-2), which was decreased by glutamate. Collectively, these results suggested that the active neolignans protect neurons against glutamate-induced cell death through maintaining the Nrf2/HO-1 signaling pathway as well as preserving the Bcl-2 protein and might be promising novel beneficial agents for oxidative stress-associated diseases.
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http://dx.doi.org/10.1021/acs.jnatprod.5b00220DOI Listing
August 2015

Effect of tacrine-3-caffeic acid, a novel multifunctional anti-Alzheimer's dimer, against oxidative-stress-induced cell death in HT22 hippocampal neurons: involvement of Nrf2/HO-1 pathway.

CNS Neurosci Ther 2014 Sep 12;20(9):840-50. Epub 2014 Jun 12.

Department of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.

Aims: Oxidative stress (OS) plays an important role in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). This study was designed to uncover the cellular and biochemical mechanisms underlying the neuroprotective effects of tacrine-3-caffeic acid (T3CA), a novel promising multifunctional anti-Alzheimer's dimer, against OS-induced neuronal death.

Methods And Results: T3CA protected HT22 cells against high-concentration-glutamate-induced cell death in time- and concentration-dependent manners and potently attenuated glutamate-induced intracellular reactive oxygen species (ROS) production as well as mitochondrial membrane-potential (ΔΨ) disruption. Besides, T3CA significantly induced nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and increased its transcriptional activity, which were demonstrated by Western blotting, immunofluorescence, and antioxidant response element (ARE)-luciferase reporter gene assay. Further studies showed that T3CA potently up-regulated heme oxygenase-1 (HO-1), an endogenous antioxidative enzyme and a downstream effector of Nrf2, at both mRNA and protein levels. The neuroprotective effects of T3CA were partially reversed by brusatol, which reduced protein level of Nrf2, or by inhibiting HO-1 with siRNA or ZnPP-IX, a specific inhibitor of HO-1.

Conclusions: Taken together, these results clearly demonstrate that T3CA protects neurons against OS-induced cell death partially through Nrf2/ARE/HO-1 signaling pathway, which further supports that T3CA might be a promising novel therapeutic agent for OS-associated diseases.
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http://dx.doi.org/10.1111/cns.12286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493203PMC
September 2014

11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors.

Psychopharmacology (Berl) 2014 Sep 23;231(17):3479-91. Epub 2014 Apr 23.

Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO, 63110, USA.

Rationale: While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified.

Objectives: This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors.

Results: Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C-H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1β2γ2L subunits, modulated [(35)S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [(3)H]CW12 and [(3)H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and β2 subunits or β3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific.

Conclusions: CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.
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http://dx.doi.org/10.1007/s00213-014-3568-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263769PMC
September 2014

Design and syntheses of electron-transfer photochromic metal-organic complexes using nonphotochromic ligands: a model compound and the roles of its ligands.

Inorg Chem 2014 Jan 23;53(2):847-51. Epub 2013 Dec 23.

State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences , Fuzhou, Fujian 350002, People's Republic of China.

The model compound [Zn(HCOO)2(4,4'-bipy)] (1; 4,4'-bipy = 4,4'-bipyridine) is selected in this work to demonstrate the effectiveness of our previously proposed design strategy for electron-transfer photochromic metal-organic complexes. The electron-transfer photochromic behavior of 1 has been discovered for the first time. Experimental and theoretical data illustrate that the photochromism of 1 can be attributed to the electron transfer from formato to 4,4'-bipy and the formation of a radical photoproduct. The electron transfer prefers to occur between formato and 4,4'-bipy, which are combined directly by the Zn(II) atoms. A high-contrast (up to 8.3 times) photoluminescence switch occurs during the photochromic process. The similarity of photochromic behaviors among 1 and its analogues as well as viologen compounds has also been found. Photochromic studies of this model compound indicate that new electron-transfer photochromic metal-organic complexes can be largely designed and synthesized by the rational assembly of nonphotochromic electron-donating and electron-accepting ligands.
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http://dx.doi.org/10.1021/ic402182jDOI Listing
January 2014

A propofol binding site on mammalian GABAA receptors identified by photolabeling.

Nat Chem Biol 2013 Nov 22;9(11):715-20. Epub 2013 Sep 22.

1] Department of Life Sciences, Imperial College, London, UK. [2].

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA (γ-aminobutyric acid type A) receptors, but where it binds to this receptor is not known and has been a matter of some debate. We synthesized a new propofol analog photolabeling reagent whose biological activity is very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin that have been identified using X-ray crystallography. Using a combination of protiated and deuterated versions of the reagent to label mammalian receptors in intact membranes, we identified a new binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit at the interface between the transmembrane domains and the extracellular domain and lies close to known determinants of anesthetic sensitivity in the transmembrane segments TM1 and TM2.
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http://dx.doi.org/10.1038/nchembio.1340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951778PMC
November 2013

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABA(A) receptor.

Mol Pharmacol 2012 Sep 30;82(3):408-19. Epub 2012 May 30.

Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-β3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-β3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-β3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the β3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3β,5β)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-β3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole β3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.
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http://dx.doi.org/10.1124/mol.112.078410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422701PMC
September 2012

Analysis of humoral immune responses to LM1 ganglioside in guinea pigs.

J Neuroimmunol 2012 May 30;246(1-2):58-64. Epub 2012 Mar 30.

Department of Neurology and Neurosciences, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA.

Guillain-Barré syndrome (GBS) is an autoimmune-mediated disease triggered by a preceding infection. A substantial body of evidence implicates antibodies to various gangliosides in subtypes of GBS. A significant proportion of patients with acute demyelinating subset of GBS have IgG antibodies against peripheral nervous system myelin specific neolactogangliosides such as LM1 and Hex-LM1. Although anti-neolactoganglioside antibodies in GBS were described more than two decades ago, their pathogenic role in neuropathy remains unknown due to the lack of suitable experimental models. In this study, we immunized ten guinea pigs with purified LM1 ganglioside mixed with keyhole limpet hemocyanin (KLH) and emulsified in complete Freund's adjuvant (CFA). Control guinea pigs were injected with KLH emulsified in CFA only. The animals were bled every four week intervals. The animals were boosted 3 times every four weeks. Experiments were terminated four months after initial immunization. Nine of 10 guinea pigs immunized with LM1 exhibited antibody responses to LM1. Anti-LM1 IgG titers in nine guinea pigs ranged from 1:400 to 1:12,800 at 16-weeks after initial immunization. Anti-LM1 antibodies were predominantly of IgG2 subclass. One guinea pig with the highest levels of IgG antibodies exhibited mild signs of neuropathy. There was no evidence of demyelination or inflammation in the sciatic nerves of LM1-immunized guinea pigs. Anti-LM1 antibodies bound to rat sciatic nerve myelin and to isolated rat Schwann cells. In summary, our findings suggest that relatively high levels of anti-LM1 IgG antibodies can be induced in guinea pigs and that LM1 is localized in peripheral nerve myelin and in Schwann cells. Further studies are needed to determine the pathogenic potential of anti-neolactoganglioside antibodies in neuropathy.
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http://dx.doi.org/10.1016/j.jneuroim.2012.03.001DOI Listing
May 2012

A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis.

Electrophoresis 2012 Feb;33(4):666-74

Departments of Anesthesiology, Washington University School of Medicine, St. Louis, MO, USA.

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of β-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-β-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z = 1287.77), a β-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and β-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.
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http://dx.doi.org/10.1002/elps.201100434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3690291PMC
February 2012

Deep amino acid sequencing of native brain GABAA receptors using high-resolution mass spectrometry.

Mol Cell Proteomics 2012 Jan;11(1):M111.011445

Department of Anesthesiology, Washington University in St. Louis, St. Louis, Missouri, 63110, USA.

Mass spectrometric sequencing of low abundance, integral membrane proteins, particularly the transmembrane domains, presents challenges that span the multiple phases of sample preparation including solubilization, purification, enzymatic digestion, peptide extraction, and chromatographic separation. We describe a method through which we have obtained high peptide coverage for 12 γ-aminobutyric acid type A receptor (GABAA receptor) subunits from 2 picomoles of affinity-purified GABAA receptors from rat brain neocortex. Focusing on the α₁ subunit, we identified peptides covering 96% of the protein sequence from fragmentation spectra (MS2) using a database searching algorithm and deduced 80% of the amino acid residues in the protein from de novo sequencing of Orbitrap spectra. The workflow combined microscale membrane protein solubilization, protein delipidation, in-solution multi-enzyme digestion, multiple stationary phases for peptide extraction, and acquisition of high-resolution full scan and fragmentation spectra. For de novo sequencing of peptides containing the transmembrane domains, timed digestions with chymotrypsin were utilized to generate peptides with overlapping sequences that were then recovered by sequential solid phase extraction using a C4 followed by a porous graphitic carbon stationary phase. The specificity of peptide identifications and amino acid residue sequences was increased by high mass accuracy and charge state assignment to parent and fragment ions. Analysis of three separate brain samples demonstrated that 78% of the sequence of the α₁ subunit was observed in all three replicates with an additional 13% covered in two of the three replicates, indicating a high degree of sequence coverage reproducibility. Label-free quantitative analysis was applied to the three replicates to determine the relative abundances of 11 γ-aminobutyric acid type A receptor subunits. The deep sequence MS data also revealed two N-glycosylation sites on the α₁ subunit, confirmed two splice variants of the γ₂ subunit (γ₂L and γ₂S) and resolved a database discrepancy in the sequence of the α₅ subunit.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3270104PMC
January 2012

Neurosteroid analogues. 16. A new explanation for the lack of anesthetic effects of δ(16)-alphaxalone and identification of a δ(17(20)) analogue with potent anesthetic activity.

J Med Chem 2011 Jun 6;54(11):3926-34. Epub 2011 May 6.

Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA.

This study addresses the hypothesis that the lack of anesthetic activity for (3α,5α)-3-hydroxypregn-16-ene-11,20-dione (Δ(16)-alphaxalone) is explained by the steroid Δ(16) double bond constraining the steroid 20-carbonyl group to a position that prevents it from favorably interacting with γ-aminobutyric acid type A (GABA(A)) receptors. A series of Δ(16) and Δ(17(20)) analogues of Δ(16)-alphaxalone was prepared to evaluate this hypothesis in binding, electrophysiological, and tadpole anesthesia experiments. The results obtained failed to support the hypothesis. Instead, the results indicate that it is the presence of the C-21 methyl group in Δ(16)-alphaxalone, not the location of the constrained C-20 carbonyl group, that prevents Δ(16)-alphaxalone from interacting strongly with the GABA(A) receptor and having anesthetic activity. Consistent with this conclusion, a Δ(17(20)) analogue of Δ(16)-alphaxalone without a C-21 methyl group was found to be very similar to the anesthetic steroid (3α,5α)-3-hydroxypregnane-11,20-dione (alphaxalone) with regard to time of onset and rate of recovery from anesthesia when administered to mice by tail vein injection.
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http://dx.doi.org/10.1021/jm2002487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794474PMC
June 2011

Arabidopsis kinesin KP1 specifically interacts with VDAC3, a mitochondrial protein, and regulates respiration during seed germination at low temperature.

Plant Cell 2011 Mar 15;23(3):1093-106. Epub 2011 Mar 15.

State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.

The involvement of cytoskeleton-related proteins in regulating mitochondrial respiration has been revealed in mammalian cells. However, it is unclear if there is a relationship between the microtubule-based motor protein kinesin and mitochondrial respiration. In this research, we demonstrate that a plant-specific kinesin, Kinesin-like protein 1 (KP1; At KIN14 h), is involved in respiratory regulation during seed germination at a low temperature. Using in vitro biochemical methods and in vivo transgenic cell observations, we demonstrate that KP1 is able to localize to mitochondria via its tail domain (C terminus) and specifically interacts with a mitochondrial outer membrane protein, voltage-dependent anion channel 3 (VDAC3). Targeting of the KP1-tail to mitochondria is dependent on the presence of VDAC3. When grown at 4° C, KP1 dominant-negative mutants (TAILOEs) and vdac3 mutants exhibited a higher seed germination frequency. All germinating seeds of the kp1 and vdac3 mutants had increased oxygen consumption; the respiration balance between the cytochrome pathway and the alternative oxidase pathway was disrupted, and the ATP level was reduced. We conclude that the plant-specific kinesin, KP1, specifically interacts with VDAC3 on the mitochondrial outer membrane and that both KP1 and VDAC3 regulate aerobic respiration during seed germination at low temperature.
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http://dx.doi.org/10.1105/tpc.110.082420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082256PMC
March 2011

A synthetic 18-norsteroid distinguishes between two neuroactive steroid binding sites on GABAA receptors.

J Pharmacol Exp Ther 2010 May 2;333(2):404-13. Epub 2010 Feb 2.

Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [35S]t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3alpha5beta-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABAA receptors. 3alpha5beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3alpha,5alpha,17beta)-3-hydroxy-17-carbonitrile (3alpha5alphaACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABAA receptors show that 3alpha5beta-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABAA receptors. 3alpha5beta-18-norACN also inhibits direct activation of GABAA receptors by 3alpha5alphaACN. 3alpha5beta-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABAA receptor.
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http://dx.doi.org/10.1124/jpet.109.164079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872955PMC
May 2010

C-terminal modification is required for GABARAP-mediated GABA(A) receptor trafficking.

J Neurosci 2007 Jun;27(25):6655-63

Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1735, USA.

We investigated the ubiquitin-like modification of GABA(A) receptor-associated protein (GABARAP) and its function. A fusion protein of GABARAP with v5 in the N terminus and myc in the C terminus was expressed in rat cultured hippocampal neurons and PC12 cells. Western blotting with antibodies to v5 and myc revealed that the C terminus of GABARAP was cleaved off. Cleavage was blocked by mutating the C-terminal Gly116 to Ala, suggesting that G116 is required for the processing. Unlike ubiquitin, GABARAP was not incorporated covalently into higher-molecular-weight protein complexes. Nor was GABARAP degraded by ubiquitinylation through the proteasome, although GABARAP formed noncovalent dimers. Immunofluorescent confocal microscopy demonstrated that recombinantly expressed GABARAP was diffusely localized in PC12 cells. However, prevention of C-terminal processing in the mutant GABARAP(G116A) resulted in redistribution to the Golgi. In neurons, punctate cytoplasmic staining of GABARAP was seen in soma and processes, whereas GABARAP(G116A) was limited to soma. Compared with wild-type GABARAP, the colocalization and interaction of GABARAP(G116A) with GABA(A) receptors were significantly reduced, resulting in a reduction in expression of receptors in the plasma membrane. When alpha1beta2gamma2S-containing GABA(A) receptors were expressed in oocytes, the increased surface expression of GABA(A) receptors, as shown by increased GABA currents and surface-accessible GABA(A) receptor subunit polypeptides resulting from GABARAP coexpression, was prevented by mutation G116A. In addition, the distribution of NSF (N-ethylmaleimide-sensitive factor) was affected in GABARAP(G116A)-expressing neurons. These results suggest that glycine 116 is required for C-terminal processing of GABARAP and that processing is essential for the localization of GABARAP and its functions as a trafficking protein.
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http://dx.doi.org/10.1523/JNEUROSCI.0919-07.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6672693PMC
June 2007