Publications by authors named "Junli Zhao"

80 Publications

Emerging Role of PD-1 in the Central Nervous System and Brain Diseases.

Neurosci Bull 2021 Apr 20. Epub 2021 Apr 20.

Department of Anesthesiology, Duke University Medical Center, Durham, 27710, USA.

Programmed cell death protein 1 (PD-1) is an immune checkpoint modulator and a major target of immunotherapy as anti-PD-1 monoclonal antibodies have demonstrated remarkable efficacy in cancer treatment. Accumulating evidence suggests an important role of PD-1 in the central nervous system (CNS). PD-1 has been implicated in CNS disorders such as brain tumors, Alzheimer's disease, ischemic stroke, spinal cord injury, multiple sclerosis, cognitive function, and pain. PD-1 signaling suppresses the CNS immune response via resident microglia and infiltrating peripheral immune cells. Notably, PD-1 is also widely expressed in neurons and suppresses neuronal activity via downstream Src homology 2 domain-containing protein tyrosine phosphatase 1 and modulation of ion channel function. An improved understanding of PD-1 signaling in the cross-talk between glial cells, neurons, and peripheral immune cells in the CNS will shed light on immunomodulation, neuromodulation, and novel strategies for treating brain diseases.
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http://dx.doi.org/10.1007/s12264-021-00683-yDOI Listing
April 2021

Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying IL-24/endostatin enhances glioma therapy.

Cancer Lett 2021 Jul 2;509:26-38. Epub 2021 Apr 2.

Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, 710062, PR China. Electronic address:

Oncolytic adenovirus-mediated gene therapy shows promise for cancer treatment; however, the systemic delivery of oncolytic adenovirus to tumors remains challenging. Recently, mesenchymal stem cells (MSCs) have emerged as potential vehicles for improving delivery. Yet, because the oncolytic adenovirus replicates in MSCs, balancing MSC viability with viral load is key to achieving optimal therapeutic effect. We thus developed an all-in-one Tet-on system that can regulate replication of oncolytic adenovirus. Then, we loaded the novel oncolytic adenovirus carrying interleukin (IL)-24 and/or Endostatin in human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) for glioma therapy. In vitro assays demonstrated that this novel oncolytic adenovirus could efficiently replicate and kill glioma cells while sparing normal cells. Moreover, doxycycline effectively regulated oncolytic adenovirus replication in the hUCB-MSCs. The doxycycline induction group with dual expression of IL-24 and Endostatin exhibited significantly greater antitumor effects than other groups in a xenograft model of glioma. Thus, this strategy for systemic delivery of oncolytic adenovirus with its oncolytic activity controlled by a Tet-on system is a promising method for achieving antitumor efficacy in glioma, especially for metastatic tumors.
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http://dx.doi.org/10.1016/j.canlet.2021.03.027DOI Listing
July 2021

CRISPR/Cas9-mediated grna gene knockout leads to neurodevelopmental defects and motor behavior changes in zebrafish.

J Neurochem 2021 May 15;157(3):520-531. Epub 2021 Feb 15.

Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi, P.R. China.

Progranulin (PGRN) is a secreted glycoprotein with multiple biological functions in early embryogenesis, anti-inflammation, and neurodegeneration. A good model for the functional study of PGRN is the zebrafish with knockdown or knockout of grn, the gene encoding PGRN. Morpholino oligonucleotides (MOs) and zinc finger nucleases have been used to generate zebrafish grn models, yet they have shown inconsistent phenotypes due to either the neurotoxicity of the MOs or possible genetic compensation responses during gene editing. In this study, we generated stable grna (one of the major grn homologues of zebrafish) knockout zebrafish by using CRISPR/Cas9-mediated genome editing. A grna sgRNA was designed to target the similar repeated sequence shared by exon 13, exon 15, and exon 19 in zebrafish. The F1 generation with the frameshift mutation of + 4 bp (the addition of 4 bp to exon15), which causes a premature termination, was obtained and subjected to morphological and behavioral evaluation. The grna knockout zebrafish showed neurodevelopmental defects, including spinal motor neurons with shorter axons, decreased sensory hair cells, thinning of the outer nuclear layer and thickening of the inner nuclear layer of the retina, decreased expression of rhodopsin in the cone cells, and motor behavior changes. Moreover, the phenotypes of grna knockout zebrafish could be rescued with the Tol2 system carrying the grna gene. The grna knockout zebrafish model generated in this study provides a useful tool to study PGRN function and has potential for high-throughput drug screening for disease therapy.
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http://dx.doi.org/10.1111/jnc.15307DOI Listing
May 2021

STING controls nociception via type I interferon signalling in sensory neurons.

Nature 2021 03 13;591(7849):275-280. Epub 2021 Jan 13.

Center for Translational Pain Medicine, Department of Anesthesiology, Duke University Medical Center, Durham, NC, USA.

The innate immune regulator STING is a critical sensor of self- and pathogen-derived DNA. DNA sensing by STING leads to the induction of type-I interferons (IFN-I) and other cytokines, which promote immune-cell-mediated eradication of pathogens and neoplastic cells. STING is also a robust driver of antitumour immunity, which has led to the development of STING activators and small-molecule agonists as adjuvants for cancer immunotherapy. Pain, transmitted by peripheral nociceptive sensory neurons (nociceptors), also aids in host defence by alerting organisms to the presence of potentially damaging stimuli, including pathogens and cancer cells. Here we demonstrate that STING is a critical regulator of nociception through IFN-I signalling in peripheral nociceptors. We show that mice lacking STING or IFN-I signalling exhibit hypersensitivity to nociceptive stimuli and heightened nociceptor excitability. Conversely, intrathecal activation of STING produces robust antinociception in mice and non-human primates. STING-mediated antinociception is governed by IFN-Is, which rapidly suppress excitability of mouse, monkey and human nociceptors. Our findings establish the STING-IFN-I signalling axis as a critical regulator of physiological nociception and a promising new target for treating chronic pain.
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http://dx.doi.org/10.1038/s41586-020-03151-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7977781PMC
March 2021

Central opioid receptors mediate morphine-induced itch and chronic itch via disinhibition.

Brain 2021 03;144(2):665-681

Center for Translational Pain Medicine, Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA.

Opioids such as morphine are mainstay treatments for clinical pain conditions. Itch is a common side effect of opioids, particularly as a result of epidural or intrathecal administration. Recent progress has advanced our understanding of itch circuits in the spinal cord. However, the mechanisms underlying opioid-induced itch are not fully understood, although an interaction between µ-opioid receptor (MOR) and gastrin-releasing peptide receptor (GRPR) in spinal GRPR-expressing neurons has been implicated. In this study we investigated the cellular mechanisms of intrathecal opioid-induced itch by conditional deletion of MOR-encoding Oprm1 in distinct populations of interneurons and sensory neurons. We found that intrathecal injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching as well as licking and biting, but this pruritus was totally abolished in mice with a specific Oprm1 deletion in Vgat+ neurons [Oprm1-Vgat (Slc32a1)]. Loss of MOR in somatostatin+ interneurons and TRPV1+ sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception. In situ hybridization revealed Oprm1 expression in 30% of inhibitory and 20% of excitatory interneurons in the spinal dorsal horn. Whole-cell recordings from spinal cord slices showed that DAMGO induced outward currents in 9 of 19 Vgat+ interneurons examined. Morphine also inhibited action potentials in Vgat+ interneurons. Furthermore, morphine suppressed evoked inhibitory postsynaptic currents in postsynaptic Vgat- excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by intrathecal administration of NPY and abolished by spinal ablation of GRPR+ neurons with intrathecal injection of bombesin-saporin, whereas intrathecal GRP-induced itch response remained intact in mice lacking Oprm1-Vgat. Intrathecal bombesin-saporin treatment reduced the number of GRPR+ neurons by 97% in the lumber spinal cord and 91% in the cervical spinal cord, without changing the number of Oprm1+ neurons. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased by Oprm1-Vgat deletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the CTCL model, indicating a contribution of central MOR signalling to chronic itch. Our findings demonstrate that intrathecal morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data also provide mechanistic insights into the current treatment of chronic itch with opioid receptor antagonist such as naloxone.
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http://dx.doi.org/10.1093/brain/awaa430DOI Listing
March 2021

[Identification of a novel c.1A>G variant of GDAP1 gene in a pedigree affected with autosomal recessive fibula atrophy].

Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2020 Nov;37(11):1244-1246

Center for Reproductive Medicine, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, China.

Objective: To explore the genetic basis for a pedigree affected with Charcot-Marie-Tooth (CMT) disease through high-throughput sequencing.

Methods: Potential variants of the genes associated with CMT were screened by next-generation sequencing (NGS) of the members of the pedigree.

Results: NGS has revealed that the two affected sisters both harbored homozygous c.1A>G variant of the GDAP1 gene, which caused replacement of the first amino acid Methionine by Valine (p.Met1Val). Their parents were both carriers of the heterozygous c.1A>G variant. The variant was unreported previously and has an extremely low frequency in the population. Meanwhile, one of the sisters and the mother also carried heterozygous c.710A>T variant of the BAG3 gene.

Conclusion: The homozygous c.1A>G variant of the GDAP1 gene probably underlay the CMT in both children. Above result has enabled clinical diagnosis and genetic counseling for this pedigree.
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http://dx.doi.org/10.3760/cma.j.cn511374-20191210-00630DOI Listing
November 2020

Lysophospholipids Contribute to Oxaliplatin-Induced Acute Peripheral Pain.

J Neurosci 2020 12 6;40(49):9519-9532. Epub 2020 Nov 6.

Institute of Clinical Pharmacology, Pharmazentrum Frankfurt/ZAFES, University Hospital, Goethe-University, D-60590 Frankfurt am Main, Germany

Oxaliplatin, a platinum-based chemotherapeutic drug, which is used as first-line treatment for some types of colorectal carcinoma, causes peripheral neuropathic pain in patients. In addition, an acute peripheral pain syndrome develop in almost 90% of patients immediately after oxaliplatin treatment, which is poorly understood mechanistically but correlates with incidence and severity of the later-occurring neuropathy. Here we investigated the effects of acute oxaliplatin treatment in a murine model, showing that male and female mice develop mechanical hypersensitivity 24 h after oxaliplatin treatment. Interestingly, we found that the levels of several lipids were significantly altered in nervous tissue during oxaliplatin-induced acute pain. Specifically, the linoleic acid metabolite 9,10-EpOME (epoxide of linoleic acid) as well as the lysophospholipids lysophosphatidylcholine (LPC) 18:1 and LPC 16:0 were significantly increased 24 h after oxaliplatin treatment in sciatic nerve, DRGs, or spinal cord tissue as revealed by untargeted and targeted lipidomics. In contrast, inflammatory markers including cytokines and chemokines, ROS markers, and growth factors are unchanged in the respective nervous system tissues. Importantly, LPC 18:1 and LPC 16:0 can induce Ca transients in primary sensory neurons, and we identify LPC 18:1 as a previously unknown endogenous activator of the ligand-gated calcium channels transient receptor potential V1 and M8 (transient receptor potential vanilloid 1 and transient receptor potential melastatin 8) in primary sensory neurons using both pharmacological inhibition and genetic knockout. Additionally, a peripheral LPC 18:1 injection was sufficient to induce mechanical hypersensitivity in naive mice. Hence, targeting signaling lipid pathways may ameliorate oxaliplatin-induced acute peripheral pain and the subsequent long-lasting neuropathy. The first-line cytostatic drug oxaliplatin can cause acute peripheral pain and chronic neuropathic pain. The former is causally connected with the chronic neuropathic pain, but its mechanisms are poorly understood. Here, we performed a broad unbiased analysis of cytokines, chemokines, growth factors, and ∼200 lipids in nervous system tissues 24 h after oxaliplatin treatment, which revealed a crucial role of lysophospholipids lysophosphatidylcholine (LPC) 18:1, LPC 16:0, and 9,10-EpOME in oxaliplatin-induced acute pain. We demonstrate for the first time that LPC 18:1 contributes to the activation of the ion channels transient receptor potential vanilloid 1 and transient receptor potential melastatin 8 in sensory neurons and causes mechanical hypersensitivity after peripheral injection These findings suggest that the LPC-mediated lipid signaling is involved in oxaliplatin-induced acute peripheral pain.
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http://dx.doi.org/10.1523/JNEUROSCI.1223-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724144PMC
December 2020

PD-1 Regulates GABAergic Neurotransmission and GABA-Mediated Analgesia and Anesthesia.

iScience 2020 Oct 16;23(10):101570. Epub 2020 Sep 16.

Center for Translational Pain Medicine, Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA.

The immune checkpoint inhibitor programmed cell death protein 1 (PD-1) plays a critical role in immune regulation. Recent studies have demonstrated functional PD-1 expression in peripheral sensory neurons, which contributes to neuronal excitability, pain, and opioid analgesia. Here we report neuronal expression and function of PD-1 in the central nervous system (CNS), including the spinal cord, thalamus, and cerebral cortex. Notably, GABA-induced currents in spinal dorsal horn neurons, thalamic neurons, and cortical neurons are suppressed by the PD-1-neutralizing immunotherapeutic Nivolumab in spinal cord slices, brain slices, and dissociated cortical neurons. Reductions in GABA-mediated currents in CNS neurons were also observed in P mice without changes in GABA receptor expression. Mechanistically, Nivolumab binds spinal cord neurons and elicits ERK phosphorylation to suppress GABA currents. Finally, both GABA-mediated analgesia and anesthesia are impaired by Pd1 deficiency. Our findings reveal PD-1 as a CNS-neuronal inhibitor that regulates GABAergic signaling and GABA-mediated behaviors.
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http://dx.doi.org/10.1016/j.isci.2020.101570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530307PMC
October 2020

The Znt7-null mutation has sex dependent effects on the gut microbiota and goblet cell population in the mouse colon.

PLoS One 2020 29;15(9):e0239681. Epub 2020 Sep 29.

Department of Nutrition, University of California Davis, Davis, California, United States of America.

Cellular homeostasis of zinc, an essential element for living organisms, is tightly regulated by a family of zinc transporters. The zinc transporter 7, ZnT7, is highly expressed on the membrane of the Golgi complex of intestinal epithelial cells and goblet cells. It has previously been shown that Znt7 knockout leads to zinc deficiency and decreased weight gain in C57BL/6 mice on a defined diet. However, effects within the colon are unknown. Given the expression profile of Znt7, we set out to analyze the changes in mucin density and gut microbial composition in the mouse large intestine induced by Znt7 knockout. We fed a semi-purified diet containing 30 mg Zn/kg to Znt7-/- mice with their heterozygous and wild type littermates and found a sex specific effect on colonic mucin density, goblet cell number, and microbiome composition. In male mice Znt7 knockout led to increased goblet cell number and mucin density but had little effect on gut microbiome composition. However, in female mice Znt7 knockout was associated with decreased goblet cell number and mucin density, with increased proportions of the microbial taxa, Allobaculum, relative to wild type. The gut microbial composition was correlated with mucin density in both sexes. These findings suggest that a sex-specific relationship exists between zinc homeostasis, mucin production and the microbial community composition within the colon.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239681PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523961PMC
December 2020

Irregular Bone Defect Repair Using Tissue-Engineered Periosteum in a Rabbit Model.

Tissue Eng Regen Med 2020 10 10;17(5):717-727. Epub 2020 Sep 10.

Orthopaedic Department of the 2nd Hospital of Lanzhou University, 80 Cui Ying Men, Cheng Guan District, Lanzhou City, 730030, People's Republic of China.

Background: In previous studies, we succeeded in repairing a long bone defect with tissue-engineered periosteum (TEP), fabricated by incorporating rabbit mesenchymal stem cells with small intestinal submucosa. In this study, we investigated the feasibility of allogeneic irregular bone defect repair using TEP.

Methods: We performed a subtotal resection of the scapula in 36 rabbits to establish a large irregular bone defect model. The rabbits were then randomly divided into three groups (n = 12 per group) and the defects were treated with TEP (Group 1), allogeneic deproteinized bone (DPB) (Group 2) or a hybrid of TEP and DPB (Group 3). At 4, 8, and 12 weeks after surgery, the rabbits were sacrificed, and the implants were harvested. X-ray radiographic and histological examinations were performed to detect bone healing. Ink-formaldehyde perfusion was introduced to qualitatively analyze vascularization in TEP engineered new bone.

Results: The repair of scapular defects was diverse in all groups, shown by radiographic and histological tests. The radiographic scores in Group 1 and Group 3 were significantly higher than Group 2 at 8 and 12 weeks (p < 0.05). Histological scores further proved that Group 1 had significantly greater new bone formation compared to Group 3 (p < 0.05), while Group 2 had the lowest osteogenesis at all time-points (p < 0.001). Ink-formaldehyde perfusion revealed aboundant microvessels in TEP engineered new bone.

Conclusion: We conclude that TEP is promising for the repair of large irregular bone defects. As a 3D scaffold, DPB could provide mechanical support and a shaping guide when combined with TEP. TEP engineered new bone has aboundant microvessels.
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http://dx.doi.org/10.1007/s13770-020-00282-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524931PMC
October 2020

Enhanced anticancer efficacy of cantharidin by mPEG-PLGA micellar encapsulation: An effective strategy for application of a poisonous traditional Chinese medicine.

Colloids Surf B Biointerfaces 2020 Dec 30;196:111285. Epub 2020 Jul 30.

Institute of Pharmacy, College of Pharmacy, Henan University, Kaifeng, 475004, China. Electronic address:

Cantharidin (CTD), the main active component of a poisonous traditional Chinese medicine (PTCM) Mylabris, exhibits highly effective therapy of hepatocellular carcinoma (HCC); however, the severe toxicity of CTD on the digestive and urinary systems prevents its clinical application. Here, CTD-loaded micelles (mPEG-PLGA-CTD) were prepared for enhancement of the antitumor efficacy and reduction of the toxicity of CTD. mPEG-PLGA-CTD comprised uniform spherical particles with particle size of 25.32 ± 1.25 nm and zeta potential of -5.70 ± 0.76 mV, exhibiting good stability and biocompatibility. mPEG-PLGA-CTD showed high toxicity on HepG2 cells by improving apoptosis and inhibiting protein phosphatases 2A (PP2A) compared to the low toxicity on l-02 hepatocytes. Intravenous injection of mPEG-PLGA-CTD led to a long circulation half-life of drugs, enhanced drug accumulation in the tumor tissues, and reduced drug accumulation in the other organs (e.g., the kidney) due to the enhanced permeability and retention effect compared to injection of free CTD; more importantly, the highly efficient antitumor effect and low systemic toxicity were achieved. A micellar formulation is very useful for enhancement of therapeutic efficacy and reduction of systemic toxicity of PTCMs.
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http://dx.doi.org/10.1016/j.colsurfb.2020.111285DOI Listing
December 2020

Berberine Mediated Positive Inotropic Effects on Rat Hearts a Ca-Dependent Mechanism.

Front Pharmacol 2020 5;11:821. Epub 2020 Jun 5.

College of Pharmaceutical Sciences, Southwest University, Chongqing, China.

Previous studies showed that berberine, an alkaloid from Franch, might exert a positive inotropic effect on the heart. However, the underlying mechanisms were unclear. Here, we reported that berberine at 10-20 µM increased the left ventricular (LV) developed pressure and the maximal rate of the pressure rising, and it increased the maximal rate of the pressure descending at 20 µM in Langendorff-perfused isolated rat hearts. These effects diminished with the concentration of berberine increasing to 50 µM. In the concentration range of 50-300 µM, berberine increased the isometric tension of isolated left ventricular muscle (LVM) strips with or without electrical stimulations, and it (30-300 µM) also increased the intracellular Ca level in the isolated LV myocytes. The removal of extracellular Ca hindered the berberine-induced increases in the tension of LVM strips and the intracellular Ca level of LV myocytes. These suggested that berberine might exert its positive inotropic effects enhancing Ca influx. The blockade of L-type Ca channels (LTCCs) with nifedipine significantly attenuated 300 μM berberine-induced tension increase in LVM strips but not the increase in the intracellular Ca level. Berberine (300 μM) further increased the LVM tension following the treatment with the LTCC opener FPL-64716 (10 μM), indicating an LTCC-independent effect of berberine. Lowering extracellular Na attenuated the berberine-induced increases in both the tension of LVM strips and the intracellular Ca level of LV myocytes. In conclusion, berberine might exert a positive inotropic effect on the isolated rat heart by enhancing the Ca influx in LV myocytes; these were extracellular Na-dependent.
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http://dx.doi.org/10.3389/fphar.2020.00821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289965PMC
June 2020

A disinhibitory nigra-parafascicular pathway amplifies seizure in temporal lobe epilepsy.

Nat Commun 2020 02 17;11(1):923. Epub 2020 Feb 17.

Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Department of Pharmacology, School of Basic Medical Sciences, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.

The precise circuit of the substantia nigra pars reticulata (SNr) involved in temporal lobe epilepsy (TLE) is still unclear. Here we found that optogenetic or chemogenetic activation of SNr parvalbumin (PV) GABAergic neurons amplifies seizure activities in kindling- and kainic acid-induced TLE models, whereas selective inhibition of these neurons alleviates seizure activities. The severity of seizures is bidirectionally regulated by optogenetic manipulation of SNr PV fibers projecting to the parafascicular nucleus (PF). Electrophysiology combined with rabies virus-assisted circuit mapping shows that SNr PV neurons directly project to and functionally inhibit posterior PF GABAergic neurons. Activity of these neurons also regulates seizure activity. Collectively, our results reveal that a long-range SNr-PF disinhibitory circuit participates in regulating seizure in TLE and inactivation of this circuit can alleviate severity of epileptic seizures. These findings provide a better understanding of pathological changes from a circuit perspective and suggest a possibility to precisely control epilepsy.
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http://dx.doi.org/10.1038/s41467-020-14648-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026152PMC
February 2020

Direct Septum-Hippocampus Cholinergic Circuit Attenuates Seizure Through Driving Somatostatin Inhibition.

Biol Psychiatry 2020 05 26;87(9):843-856. Epub 2019 Nov 26.

Institute of Pharmacology and Toxicology, NHC and CAMS Key Laboratory of Medical Neurobiology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China; Institute of Neuroscience, Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou, China; Epilepsy Center, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Electronic address:

Background: Previous studies indicated the involvement of cholinergic neurons in seizure; however, the specific role of the medial septum (MS)-hippocampus cholinergic circuit in temporal lobe epilepsy (TLE) has not yet been completely elucidated.

Methods: In the current study, we used magnetic resonance imaging and diffusion tensor imaging to characterize the pathological change of the MS-hippocampus circuit in 42 patients with TLE compared with 22 healthy volunteers. Using optogenetics and chemogenetics, combined with in vivo or in vitro electrophysiology and retrograde rabies virus tracing, we revealed a direct MS-hippocampus cholinergic circuit that potently attenuates seizure through driving somatostatin inhibition in animal TLE models.

Results: We found that patients with TLE with hippocampal sclerosis showed a decrease of neuronal fiber connectivity of the MS-hippocampus compared with healthy people. In the mouse TLE model, MS cholinergic neurons ceased firing during hippocampal seizures. Optogenetic and chemogenetic activation of MS cholinergic neurons (but not glutamatergic or GABAergic [gamma-aminobutyric acidergic] neurons) significantly attenuated hippocampal seizures, while specific inhibition promoted hippocampal seizures. Electrophysiology combined with modified rabies virus tracing studies showed that direct (but not indirect) MS-hippocampal cholinergic projections mediated the antiseizure effect by preferentially targeting hippocampal GABAergic neurons. Furthermore, chemogenetic inhibition of hippocampal somatostatin-positive (rather than parvalbumin-positive) subtype of GABAergic neurons reversed the antiseizure effect of the MS-hippocampus cholinergic circuit, which was mimicked by activating somatostatin-positive neurons.

Conclusions: These findings underscore the notable antiseizure role of the direct cholinergic MS-hippocampus circuit in TLE through driving the downstream somatostatin effector. This may provide a better understanding of the changes of the seizure circuit and the precise spatiotemporal control of epilepsy.
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http://dx.doi.org/10.1016/j.biopsych.2019.11.014DOI Listing
May 2020

A prospective study of hepatic safety of statins used in very elderly patients.

BMC Geriatr 2019 12 16;19(1):352. Epub 2019 Dec 16.

Department of Gereology, Shanghai University of Medicine & Health Sciences Affiliated Zhoupu Hospital, Zhou Yuan Road 1500, Pudong New Area, Shanghai, 201318, China.

Background: Statins play an important role in the care of patients with cardiovascular disease and have a good safety record in clinical practice. Hepatotoxicity is a barrier that limits the ability of primary care physicians to prescribe statins for patients with elevated liver transaminase values and/or underlying liver disease. However, limited population-based data are available on the use of statin therapy and on the hepatotoxicity of statins in very elderly patients. This prospective study evaluated the liver enzyme elevation during statin therapy in very elderly patients (≥80 years old).

Methods: Patients with hypercholesterolemia (LDL-C levels ≥3.4 and < 5.7 mmol/L), atherosclerosis, coronary heart disease (CHD), or a CHD-risk equivalent were enrolled and received once-daily statin treatment. Multivariate logistic regression models were used to study the impact of age, gender, hepatitis B infection, fatty liver disease, biliary calculus, other chronic diseases, drug kinds, alcohol abuse, statin variety, and statin dose variables.

Results: A total of 515 consecutive patients ranging from 80 to 98 years old were included in the analysis. These patients were treated with simvastatin, fluvastatin, pravastatin, rosuvastatin, or atorvastatin. Twenty-four patients (4.7, 95% CI 2.7-6.6) showed an increase in their hepatic aminotransferase levels. No significant difference of hepatic aminotransferase elevation rates was observed in different statin treatment groups. The incidence of mild, moderate, and severe elevation of aminotransferase levels was 62.5% (15/24), 29.2% (7/24), and 8.3% (2/24), respectively. None of the patients developed hepatic failure. Nine patients with moderate or severe aminotransferase elevations discontinued therapy. The time of onset of hepatic aminotransferase elevation ranged from 2 weeks to 6 months after statin treatment. The onset of hepatic aminotransferase elevation was within 1 month for 70.8% of patients. The patients took 2 weeks to 3 months to recover their liver function after statin therapy cessation. Multivariate analysis identified chronic hepatitis B infection and alcohol consumption as independent factors associated with the hepatic response to statins: OR, 12.83; 95% CI (4.36-37.759) and OR, 2.736; 95% CI (1.373-5.454), respectively.

Conclusion: The prevalence of elevated transaminases was higher than published data in very elderly patients. Overall, statin treatment is safe for patients ≥80 years old.
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http://dx.doi.org/10.1186/s12877-019-1361-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915904PMC
December 2019

HMGB1 Is a Therapeutic Target and Biomarker in Diazepam-Refractory Status Epilepticus with Wide Time Window.

Neurotherapeutics 2020 04;17(2):710-721

Institute of Pharmacology & Toxicology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.

Status epilepticus (SE), a life-threatening neurologic emergency, is often poorly controlled by the current pharmacological therapeutics, which are limited to a narrow time window. Here, we investigated the proinflammatory cytokine high mobility group box-1 (HMGB1) as a candidate therapeutic target for diazepam (DZP)-refractory SE. We found that HMGB1 was upregulated and translocated rapidly during refractory SE period. Exogenous HMGB1 was sufficient to directly induce DZP-refractory SE in nonrefractory SE. Neutralization of HMGB1 with an anti-HMGB1 monoclonal antibody decreased the incidence of SE and alleviated the severity of seizure activity in DZP-refractory SE, which was mediated by a Toll-like receptor 4 (TLR4)-dependent pathway. Importantly, anti-HMGB1 mAb reversed DZP-refractory SE with a wide time window, extending the therapeutic window from 30 to 180 min. Furthermore, we found the upregulation of plasma HMGB1 level is closely correlated with the therapeutic response of anti-HMGB1 mAb in DZP-refractory SE. All these results indicated that HMGB1 is a potential therapeutic target and a useful predictive biomarker in DZP-refractory SE.
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http://dx.doi.org/10.1007/s13311-019-00815-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283397PMC
April 2020

A simple, efficient and economical method for isolating and culturing human umbilical cord blood‑derived mesenchymal stromal cells.

Mol Med Rep 2019 Dec 23;20(6):5257-5264. Epub 2019 Oct 23.

Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi 710062, P.R. China.

Mesenchymal stromal cells (MSCs) hold broad therapeutic potential in various diseases, however, it is difficult to produce sufficient numbers of MSCs for clinical application, therefore, improved culture systems are required. The present study aimed to develop a novel method for isolating and culturing human umbilical cord blood‑derived mesenchymal stromal cells (hUCB‑MSCs). A sequential culture method was developed that uses two types of culture media to optimize the isolation and culture of hUCB‑MSCs. First, DMEM supplemented with mesenchymal stem cell growth supplement was used to improve the colony formation and primary culture success rates of hUCB‑MSCs. Then, after removing the heterogeneous cell population, ordinary DMEM was used from the fourth passage. This method obtained hUCB‑MSCs with high culture efficiency and at a greatly reduced cost. The optimal culture conditions were determined and the hUCB‑MSCs were phenotypically characterized after passaging. Taken together, this simple, efficient and economical method can produce a large number of high‑quality hUCB‑MSCs in <1 month, therefore facilitating the future clinical applications of hUCB‑MSCs.
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http://dx.doi.org/10.3892/mmr.2019.10767DOI Listing
December 2019

Suppression of Progranulin Expression Leads to Formation of Intranuclear TDP-43 Inclusions In Vitro: A Cell Model of Frontotemporal Lobar Degeneration.

J Neuropathol Exp Neurol 2019 12;78(12):1124-1129

Mesulam Center for Cognitive Neurology and Alzheimer's Disease, Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Mutations in the GRN gene coding for progranulin (PGRN) are responsible for many cases of familial frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein 43 (TDP-43)-positive inclusions (FTLD-TDP). GRN mutations create null alleles resulting in decreased progranulin protein or haploinsufficiency. FTLD-TDP with GRN mutations is characterized by lentiform neuronal intranuclear inclusions that are positive for TDP-43 in affected brain regions. In this study, by stably expressed short hairpin RNA, we established a neuroblastoma cell line with decreased PGRN level. This cell line reveals TDP-43-positive intranuclear inclusions. In addition, replacement with purified PGRN protein restores normal TDP-43 nuclear distribution. This cell model can be valuable for the study of the role of PGRN in the pathogenesis in FTLD-TDP.
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http://dx.doi.org/10.1093/jnen/nlz102DOI Listing
December 2019

Intracytoplasmic sperm injection (ICSI) versus conventional in vitro fertilisation (IVF) in couples with non-severe male infertility (NSMI-ICSI): protocol for a multicentre randomised controlled trial.

BMJ Open 2019 09 30;9(9):e030366. Epub 2019 Sep 30.

Centre for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China

Introduction: Intracytoplasmic sperm injection (ICSI), originally introduced as add-on to in vitro fertilisation (IVF) for couples with severe male infertility, is in current clinical practice also used in couples with mild male or even unexplained infertility. However, ICSI has involved unresolved concerns regarding the selection and damage to gametes and the health conditions of the offspring, and it is also labour intensive and therefore more expensive than conventional IVF. High-quality well-powered randomised clinical trials (RCTs) comparing ICSI and IVF are lacking.

Methods And Analysis: We propose a multicentre, open-label RCT in 10 reproductive medical centres across China. We will study couples with non-severe male infertility (defined as a semen concentrate 5-15×10/mL or sperm with a progressive motility 10%-32%) scheduled for their first or second ICSI or IVF cycle, as low fertility rate after fertilisation are more frequent in this population, which could lead to controversy about ICSI or conventional IVF for fertilisation. On the day of oocyte retrieval, eligible participants are after informed consent be randomised to undergo either ICSI or conventional IVF in a 1:1 treatment ratio. Other standard assisted reproductive treatments are similar and parallel between two groups. Our primary outcome is ongoing pregnancy leading to live birth after the first cycle with embryo transfer. To demonstrate or refute a difference of 7% between ICSI and conventional IVF, we need to include 2346 women (1173 in each intervention arm). In addition, we will follow-up neonatal outcomes after delivery to identify the influence of ICSI on offspring.

Ethics And Dissemination: Ethical approval was obtained from Peking University Third Hospital medical science research ethics committee. The findings will be disseminated to the public through conference presentations and peer-reviewed scientific journals.

Trial Registration Number: ClinicalTrials.gov registry (NCT03298633).
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http://dx.doi.org/10.1136/bmjopen-2019-030366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773417PMC
September 2019

Occurrence, Distribution and Risk Assessment of Mercury in Multimedia of Soil-Dust-Plants in Shanghai, China.

Int J Environ Res Public Health 2019 08 21;16(17). Epub 2019 Aug 21.

Key Laboratory of Geographic Information Science (Ministry of Education), East China Normal University, Shanghai 200241, China.

The urban environment is a complex ecosystem influenced by strong human disturbances in multi-environmental media, so it is necessary to analyze urban environmental pollutants through the comprehensive analysis of different media. Soil, road dust, foliar dust, and camphor leaves from 32 sample sites in Shanghai were collected for the analysis of mercury contamination in soil-road dust-leaves-foliar dust systems. Mercury concentrations in surface soils in Shanghai were the highest, followed by road dust, foliar dust, and leaves, successively. The spatial distribution of mercury in the four environmental media presented different distribution patterns. Except for the significant correlation between mercury concentrations in road dust and mercury concentrations in leaves (r = 0.56, < 0.001), there was no significant correlation between the other groups in the four media. Besides this, there was no significant correlation between mercury concentrations and land types. The LUR (Land use regression) model was used to assess the impact of urbanization factors on mercury distribution in the environment. The results showed that soil mercury was affected by factories and residential areas. Foliar dust mercury was affected by road density and power plants. Leaf mercury was affected by power plants and road dust mercury was affected by public service areas. The highest average HI (Hazard index) value of mercury in Shanghai was found in road dust, followed by surface soil and foliar dust. The HI values for children were much higher than those for adults. However, the HI values of mercury exposure in all sampling sites were less than one, suggesting a lower health risk level. The microscopic mechanism of mercury in different environmental media was suggested to be studied further in order to learn the quantitative effects of urbanization factors on mercury concentrations.
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http://dx.doi.org/10.3390/ijerph16173028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747141PMC
August 2019

A Telemedicine-Based Registration System for the Management of Renal Anemia in Patients on Maintenance Hemodialysis: Multicenter Study.

J Med Internet Res 2019 05 8;21(5):e13168. Epub 2019 May 8.

Clinical Research Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Background: Renal anemia is one of the most important complications in patients on maintenance hemodialysis (MHD). Telehealth-based dialysis registration systems have the advantage of real-time monitoring and have gradually been applied to the management of chronic diseases.

Objective: The objective of our study was to evaluate the impact of a telehealth-based dialysis registration system on patients on MHD in terms of renal anemia control.

Methods: The Red China project aimed to develop a dialysis registration system based on the WeChat mobile platform. Demographic and baseline laboratory parameters such as age, gender, primary disease, dialysis age, and baseline creatinine levels were recorded using this system. In addition, the hemoglobin and hematocrit levels were recorded monthly. The platform then generated a hemoglobin and hematocrit statistics report for each hemodialysis center monthly, including the detection rate, target rate, and distribution of hemoglobin and released it to physicians via the WeChat mobile phone app. The physicians were then able to treat the individual's anemia appropriately by changing the doses of erythropoiesis-stimulating agents or iron use on the basis of this report. We analyzed the demographic and baseline laboratory parameters, detection rate, target rate, and average level and distribution of hemoglobin 28 months after the launch of the project.

Results: A total of 8392 patients on MHD from 28 hemodialysis centers in Shanghai were enrolled from June 2015 to October 2017. The detection rate of hemoglobin increased from 54.18% to 73.61% (P<.001), the target rate of hemoglobin increased from 47.55% to 56.07% (P<.001), and the mean level of hemoglobin increased from 10.83 (SD 1. 60) g/dL to 11.07 (SD 1.60) g/dL (P<.001). In addition, the proportion of patients with hemoglobin levels ≥11 g/dL but <13 g/dL increased from 40.40% to 47.48%.

Conclusions: This telehealth-based dialysis registration system can provide timely reporting of the anemia status in patients on MHD, which may improve the awareness of anemia and the attention to and compliance with anemia monitoring.
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http://dx.doi.org/10.2196/13168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682288PMC
May 2019

A Highly Sensitive Sandwich ELISA to Detect CSF Progranulin: A Potential Biomarker for CNS Disorders.

J Neuropathol Exp Neurol 2019 05;78(5):406-415

Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Progranulin (PGRN) plays critical roles in inflammation, tumorigenesis, and neurodegeneration. PGRN levels in blood and cerebrospinal fluid (CSF) are being increasingly investigated as potential biomarkers for these disorders. However, the value of CSF PGRN as a biomarker has been limited because currently available commercial enzyme-linked immunosorbent assay (ELISA) kits have suboptimal sensitivity for detecting CSF PGRN. In this study, pairs of monoclonal antibodies (MAbs) were first screened from eleven monoclonal antiPGRN antibodies using indirect ELISA, then a sandwich ELISA was established using the 2 optimized MAbs. This system displayed high sensitivity, with a lower limit of detection of 60.0 pg/mL and a lower limit of quantification of 150 pg/mL. By using this ELISA system, we showed varied CSF PGRN levels in different brain disorders. For example, as compared with the normal controls, patients with Alzheimer disease or multiple sclerosis showed mildly increased CSF PGRN; those with aseptic encephalitis or neuropsychiatric systemic lupus erythematosus showed moderately increased CSF PGRN; those with bacterial leptomeningitis showed severely increased CSF PGRN. Additionally, determining CSF PGRN levels could monitor CNS metastasis and CSF seeding of carcinomas. These results indicate that this system can be valuable in studying the diagnostic and prognostic value of CSF PGRN in brain disorders.
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http://dx.doi.org/10.1093/jnen/nlz022DOI Listing
May 2019

Novel mutations in WEE2: Expanding the spectrum of mutations responsible for human fertilization failure.

Clin Genet 2019 04 20;95(4):520-524. Epub 2019 Feb 20.

State Key Laboratory of Medical Neurobiology, Institutes of Biomedical Sciences, Zhongshan Hospital, Fudan University, Shanghai, China.

Successful fertilization is fundamental for sexual reproduction. After undergoing a series of molecular and morphological changes, the haploid sperm fuses with the haploid oocyte to create a diploid zygote. Defects in this process might lead to human fertilization failure. We have previously found homozygous mutations in WEE2 that are responsible for human fertilization failure, but the genetic basis of human fertilization failure requires further investigation. In the present study, we screened for WEE2 mutations in a new cohort of patients with fertilization failure. Through Sanger sequencing of WEE2 exons, we identified seven novel mutations and two reported mutations in WEE2 from six affected individuals. Morphologically normal PB1 oocytes can be retrieved from all patients. However, most of the oocytes cannot be fertilized successfully. These findings confirmed our previous research and expanded the mutational spectrum of WEE2, making it a potential genetic diagnostic marker for those suffering from human fertilization failure.
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http://dx.doi.org/10.1111/cge.13505DOI Listing
April 2019

Combined Use of Circulating miR-133a and NT-proBNP Improves Heart Failure Diagnostic Accuracy in Elderly Patients.

Med Sci Monit 2018 Dec 7;24:8840-8848. Epub 2018 Dec 7.

Department of Gereology and Cardiology, Shanghai University of Medicine and Health Sciences Affiliated Zhoupu Hospital, Shanghai, China (mainland).

BACKGROUND Circulating microRNAs (miRNAs) are emerging as novel biomarkers for detecting cardiovascular diseases. Here, circulating miR-133a and miR-221 were investigated as potential diagnostic biomarkers for heart failure (HF) patients, particularly in elderly patients. MATERIAL AND METHODS A total of 94 elderly HF patients (mean age=77.4 years old) and 31 healthy controls (age- and sex-matched) participated in this study. Plasma NT-proBNP levels were measured using an electrochemiluminescence immunoassay, and circulating miR-133a and miR-221 levels were examined using real-time quantitative PCR, with diagnostic efficacies determined for each independently and in combination. RESULTS MiR-133a expression increased by 4.6-fold (P<0.001) and miR-221 expression increased by 2.0-fold (P<0.001) in the elderly HF patients relative to the healthy controls. ROC curves were generated and AUC values of 0.863 for miR-133a (CI95%: 0.800-0.927), 0.718 for miR-221 (CI95%: 0.622-0.813), and 0.895 for NT-proBNP (CI95%: 0.841-0.948) were obtained. Unlike NT-proBNP, miR-133a and miR-221 were found to be unaffected by age, BMI, renal function, albumin, or Hb levels. More importantly, the diagnostic value of NT-proBNP was found to be improved when combined with any of the examined miRNA biomarkers alone or in a panel. When combining miR-133a with NT-proBNP, an AUC value of 0.975 (CI95%: 0.950-0.999) was obtained, which was significantly higher than for NT-proBNP alone (z=2.395, P=0.016). CONCLUSIONS miR-133a and miR-221 can serve as potential HF diagnostic biomarkers in elderly patients. Moreover, the diagnostic accuracy of NT-proBNP can be improved by the addition of miR-133a.
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http://dx.doi.org/10.12659/MSM.911632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294044PMC
December 2018

Aptazyme-mediated direct modulation of post-transcriptional sgRNA level for conditional genome editing and gene expression.

J Biotechnol 2018 Dec 1;288:23-29. Epub 2018 Nov 1.

Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an, 710062, Shaanxi, PR China. Electronic address:

RNA-guided endonuclease Cas9 derived from microbial CRISPR-Cas adaptive immune systems is a powerful tool for genome editing, which has been widely used in eukaryotic systems, prokaryotic systems, and plants. However, the off-target effects caused by Cas9/sgRNA remain a major concern. Currently, the efforts to reduce the off-target effects mainly focus on improving the targeting specificity of sgRNA/Cas9, regulating the activity of the Cas9 protein or the sgRNA, and controlling the time window of their expression. In this study, a novel system was established to regulate the post-transcriptional sgRNA level by small molecule-controlled aptazyme. This system was shown to reduce the off-target effects caused by Cas9/sgRNA, while enabling precise temporal control over gene editing and regulatory activity. This new system could provide a potentially safer and more powerful tool for genome editing and therapeutic application.
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http://dx.doi.org/10.1016/j.jbiotec.2018.10.011DOI Listing
December 2018

Establishment of a HEK293 cell line by CRISPR/Cas9-mediated luciferase knock-in to study transcriptional regulation of the human SREBP1 gene.

Biotechnol Lett 2018 Dec 19;40(11-12):1495-1506. Epub 2018 Sep 19.

Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an, 710062, Shaanxi, People's Republic of China.

Objectives: To establish a HEK293 cell line with a luciferase knock-in reporter controlled by the endogenous SREBP1 promoter for investigating transcriptional regulation of the SREBP1 gene.

Results: PCR confirmed the site-specific integration of a single copy of the exogenous luciferase gene into one allele of the genome and a 14 bp deletion of the targeted sequence in the other. Luciferase activity was directly correlated with the promoter activity of the endogenous SREBP1 gene in the HEK293-SREBP1-T2A-luciferase-KI cell line cell line.

Conclusions: We successfully generated a novel luciferase knock-in reporter system, which will be very useful for studying transcriptional regulation of the SREBP1 gene and for screening drugs or chemical molecules that regulate SREBP1 gene expression.
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http://dx.doi.org/10.1007/s10529-018-2608-2DOI Listing
December 2018

Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system.

Cytotechnology 2018 Dec 1;70(6):1595-1605. Epub 2018 Sep 1.

Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an, 710062, Shaanxi, People's Republic of China.

The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.
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http://dx.doi.org/10.1007/s10616-018-0252-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269370PMC
December 2018

Pharmaco-genetic therapeutics targeting parvalbumin neurons attenuate temporal lobe epilepsy.

Neurobiol Dis 2018 09 9;117:149-160. Epub 2018 Jun 9.

Institute of Pharmacology & Toxicology, Department of Pharmacology, Key Laboratory of Medical Neurobiology, Ministry of Health of China, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China; Epilepsy Center, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Electronic address:

Temporal lobe epilepsy (TLE) is the most common type of epilepsy and is often medically refractory. Previous studies suggest that selective pharmaco-genetic inhibition of pyramidal neurons has therapeutic value for the treatment of epilepsy, however there is a risk of disrupting normal physical functions. Here, we test whether pharmaco-genetic activation of parvalbumin neurons, which are transgenetically transduced with the modified muscarinic receptor hM3Dq can attenuate TLE. We found that pharmaco-genetic activation of hippocampal parvalbumin neurons in epileptogenic zone not only significantly extends the latency to different seizure stages and attenuates seizure activities in acute seizure model, but also greatly alleviates the severity of seizure onsets in two chronic epilepsy models. This manipulation did not affect the normal physical function evaluated in various cognitive tasks. Further, the activation of parvalbumin neurons produced an inhibition on parts of surrounding pyramidal neurons, and the direct inactivation of pyramidal neurons via the viral expression of a modified muscarinic receptor hM4Di produced a similar anti-ictogenic effect. Interestingly, pharmaco-genetic inactivation of pyramidal neurons was more sensitive to impair cognitive function. Those data demonstrated that pharmaco-genetic seizure attenuation through targeting parvalbumin neurons rather than pyramidal neurons may be a novel and relatively safe approach for treating refractory TLE.
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http://dx.doi.org/10.1016/j.nbd.2018.06.006DOI Listing
September 2018

Establishment of a novel hepatic steatosis cell model by Cas9/sgRNA-mediated DGKθ gene knockout.

Mol Med Rep 2018 Feb 22;17(2):2169-2176. Epub 2017 Nov 22.

Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an, Shaanxi 710062, P.R. China.

To investigate the role of diacylglycerol kinase θ (DGKθ) in lipid metabolism and insulin resistance, the present study generated an in vitro hepatic steatosis cell model by knockout of the DGKθ gene in liver cancer cell line HepG2 using CRISPR/Cas9 technology. The cell line was characterized by Oil Red O staining and shown to exhibit increased intracellular lipid accumulation, compared with that in wild‑type liver cancer cell line HepG2. The gene expression levels of signaling proteins in pathways involved in lipid metabolism, insulin resistance and gluconeogenesis were also examined. The DGKθ‑knockout HepG2 cells showed increased mRNA and protein expression levels of lipid synthesis‑related genes, fatty acid synthase, peroxisome proliferator‑activated receptor‑γ and sterol regulatory element‑binding protein‑1c, and decreased expression levels of the lipolysis‑related gene, carnitine palmitoyltransferase1A. These changes may account for the increased intracellular lipid content of this cell line. The DGKθ‑knockout HepG2 cells also exhibited an increased phosphorylation level of protein kinase Cε and decreased phosphorylation levels of insulin receptor substrate 1, mechanistic target of rapamycin and protein kinase B (also known as Akt). These changes have been reported to mediate insulin resistance. Taken together, an in vitro hepatic steatosis cell model was established in the present study, providing a valuable tool for understanding the pathogenesis of nonalcoholic fatty liver disease and associated insulin resistance, and for developing treatment strategies for this disease.
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http://dx.doi.org/10.3892/mmr.2017.8140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5783457PMC
February 2018

mPEG-PLA Micelle for Delivery of Effective Parts of Andrographis Paniculata.

Curr Drug Deliv 2018 ;15(4):532-540

Institute of Pharmacy, Pharmacy College of Henan University, Kaifeng, China.

Background: Many studies have shown that Andrographis paniculata (Burm. f.) Nees has a good anti-tumor effect, but poor solubility in water and poor bioavailability hinder the modernization of it.

Method: To formulate the effective parts (mainly diterpene lactones) of Andrographis paniculata (AEP) into targeting drug delivery system, a series of poly(ethylene glycol)-poly(D.L-lactic acid)(mPEG-PLA) with different ratio of hydrophilic and hydrophobic segment was synthetized to encapsulate AEP. AEP micelles were prepared by a simple solvent-evaporation method. According to the loading capacity, the best polymer was chosen. mPEG-PLA micelles were characterized in terms of drug entrapping efficiency, loading capacity, size, the crystalline state of AEP, stability and release profile. Meanwhile, the cytotoxicity of micelles on mouse breast cancer 4T-1 was investigated.

Results: These micelle (mPEG-PLA-AEP) particles had a size of (92.84±5.63) nm and a high entrapping efficiency and loading capacity of (91.00±11.53)% and (32.14±3.02)%(w/w), respectively. The powder DSC showed that drugs were well encapsulated in the core of micelles. mPEG-PLA-AEP had a good stability against salt dissociation, protein adsorption and anion substitution and the solubility of andrographolide (AG) and 14-deoxy-11,12-didehydroandrographolide(DDAG) in AEP increased 4.51 times and 2.12 times in water, and the solubility of DAG showed no difference. mPEG-PLA-AEP had the same release profile in different dissolution medium. Cytotoxicity testing in vitro demonstrated that mPEG-PLA-AEP exhibited higher cell viability inhibition in mouse breast cancer 4T-1 than free AEP.

Conclusion: mPEG-PLA micelles offer a promising alternative for TCM therapy with higher solubility and improved antitumor effect.
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http://dx.doi.org/10.2174/1567201814666171120113521DOI Listing
October 2018