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1.  LMO4 Functions As a Negative Regulator of Sensory Organ Formation in the Mammalian Cochlea 
The Journal of Neuroscience  2014;34(30):10072-10077.
In mammals, formation of the auditory sensory organ (the organ of Corti) is restricted to a specialized area of the cochlea. However, the molecular mechanisms limiting sensory formation to this discrete region in the ventral cochlear duct are not well understood, nor is it known whether other regions of the cochlea have the competence to form the organ of Corti. Here we identify LMO4, a LIM-domain-only nuclear protein, as a negative regulator of sensory organ formation in the cochlea. Inactivation of Lmo4 in mice leads to an ectopic organ of Corti (eOC) located in the lateral cochlea. The eOC retains the features of the native organ, including inner and outer hair cells, supporting cells, and other nonsensory specialized cell types. However, the eOC shows an orientation opposite to the native organ, such that the eOC appears as a mirror-image duplication to the native organ of Corti. These data demonstrate a novel sensory competent region in the lateral cochlear duct that is regulated by LMO4 and may be amenable to therapeutic manipulation.
doi:10.1523/JNEUROSCI.0352-14.2014
PMCID: PMC4107398  PMID: 25057208
inner ear development; LIM-domain; LMO4; organ of Corti; cochlea; transcriptional regulator
2.  Expression of LIM-homeodomain transcription factors in the developing and mature mouse retina 
LIM-homeodomain (LIM-HD) transcription factors have been extensively studied for their role in the development of the central nervous system. Their function is key to several developmental events like cell proliferation, differentiation and subtype specification. However, their roles in retinal neurogenesis remain largely unknown. Here we report a detailed expression study of LIM-HD transcription factors LHX9 and LHX2, LHX3 and LHX4, and LHX6 in the developing and mature mouse retina using immunohistochemistry and in situ hybridization techniques. We show that LHX9 is expressed during the early stages of development in the retinal ganglion cell layer and the inner nuclear layer. We also show that LHX9 is expressed in a subset of amacrine cells in the adult retina. LHX2 is known to be expressed in retinal progenitor cells during development and in Müller glial cells and a subset of amacrine cells in the adult retina. We found that the LHX2 subset of amacrine cells is not cholinergic and that a very few of LHX2 amacrine cells express calretinin. LHX3 and LHX4 are expressed in a subset of bipolar cells in the adult retina. LHX6 is expressed in cells in the ganglion cell layer and the neuroblast layer starting at embryonic stage 13.5 (E13.5) and continues to be expressed in cells in the ganglion cell layer and inner nuclear layer, postnatally, suggesting its likely expression in amacrine cells or a subset thereof. Taken together, our comprehensive assay of expression patterns of LIM-HD transcription factors during mouse retinal development will help further studies elucidating their biological functions in the differentiation of retinal cell subtypes.
doi:10.1016/j.gep.2013.12.001
PMCID: PMC3921069  PMID: 24333658
LIM-homeodomain; Lhx genes; retinogenesis; retinal development; transcription factors
3.  Protein-Protein Interaction and Pathway Analyses of Top Schizophrenia Genes Reveal Schizophrenia Susceptibility Genes Converge on Common Molecular Networks and Enrichment of Nucleosome (Chromatin) Assembly Genes in Schizophrenia Susceptibility Loci 
Schizophrenia Bulletin  2013;40(1):39-49.
Recent genome-wide association studies have identified many promising schizophrenia candidate genes and demonstrated that common polygenic variation contributes to schizophrenia risk. However, whether these genes represent perturbations to a common but limited set of underlying molecular processes (pathways) that modulate risk to schizophrenia remains elusive, and it is not known whether these genes converge on common biological pathways (networks) or represent different pathways. In addition, the theoretical and genetic mechanisms underlying the strong genetic heterogeneity of schizophrenia remain largely unknown. Using 4 well-defined data sets that contain top schizophrenia susceptibility genes and applying protein-protein interaction (PPI) network analysis, we investigated the interactions among proteins encoded by top schizophrenia susceptibility genes. We found proteins encoded by top schizophrenia susceptibility genes formed a highly significant interconnected network, and, compared with random networks, these PPI networks are statistically highly significant for both direct connectivity and indirect connectivity. We further validated these results using empirical functional data (transcriptome data from a clinical sample). These highly significant findings indicate that top schizophrenia susceptibility genes encode proteins that significantly directly interacted and formed a densely interconnected network, suggesting perturbations of common underlying molecular processes or pathways that modulate risk to schizophrenia. Our findings that schizophrenia susceptibility genes encode a highly interconnected protein network may also provide a novel explanation for the observed genetic heterogeneity of schizophrenia, ie, mutation in any member of this molecular network will lead to same functional consequences that eventually contribute to risk of schizophrenia.
doi:10.1093/schbul/sbt066
PMCID: PMC3885298  PMID: 23671194
genome-wide association study; schizophrenia susceptibility genes; protein-protein interaction; common molecular networks; genetic heterogeneity; enrichment
4.  Treatment of hepatic carcinoma by low-frequency ultrasound and microbubbles: A case report 
Oncology Letters  2014;9(3):1249-1253.
In vitro and in vivo studies have identified that low-frequency ultrasound (US) and microbubbles (MBs) mediate tumor inhibitory effects. However, the application of US in the clinical setting remains unclear. The aim of the present study was to investigate the clinically therapeutic effect of 20 kHz US in combination with MBs for the treatment of hepatic carcinoma. A 71-year-old male with a hepatic malignant tumor was admitted to Nantong University Affiliated Nantong Tumor Hospital (Nantong, China). The patient was subsequently sonicated with 20 kHz US and MBs over a period of five days. The low-frequency US parameters were set at 20 kHz, 2 W/cm2, duty cycle 40% (on 2 sec, off 3 sec) for a duration of 5 min each day for a total of five days. Computed tomography (CT), contrast-enhanced US (CEUS) and carbohydrate antigen 19-9 (CA19-9) tests were performed to evaluate the therapeutic effects. Although the tumor size increased marginally on CT from 5.4 to 5.6 cm after US treatment, the intensity and enhanced-areas on the CT scans and CEUS decreased. The abdominal lymph node decreased in size, from 2.2 to 1.9 cm, and CA19-9 levels decreased from the pretreatment value of 2,007 to 734 U/ml. Therapy with low-frequency US combined with MBs may exhibit an antivasculature effect and may be used as a palliative treatment for patients with unresectable hepatic malignant tumors.
doi:10.3892/ol.2014.2812
PMCID: PMC4314986  PMID: 25663892
hepatic tumor; low-frequency ultrasound; therapy; microbubbles; cavitation
5.  Restorative effect and mechanism of mecobalamin on sciatic nerve crush injury in mice 
Neural Regeneration Research  2014;9(22):1979-1984.
Mecobalamin, a form of vitamin B12 containing a central metal element (cobalt), is one of the most important mediators of nervous system function. In the clinic, it is often used to accelerate recovery of peripheral nerves, but its molecular mechanism remains unclear. In the present study, we performed sciatic nerve crush injury in mice, followed by daily intraperitoneal administration of mecobalamin (65 μg/kg or 130 μg/kg) or saline (negative control). Walking track analysis, histomorphological examination, and quantitative real-time PCR showed that mecobalamin significantly improved functional recovery of the sciatic nerve, thickened the myelin sheath in myelinated nerve fibers, and increased the cross-sectional area of target muscle cells. Furthermore, mecobalamin upregulated mRNA expression of growth associated protein 43 in nerve tissue ipsilateral to the injury, and of neurotrophic factors (nerve growth factor, brain-derived nerve growth factor and ciliary neurotrophic factor) in the L4–6 dorsal root ganglia. Our findings indicate that the molecular mechanism underlying the therapeutic effect of mecobalamin after sciatic nerve injury involves the upregulation of multiple neurotrophic factor genes.
doi:10.4103/1673-5374.145379
PMCID: PMC4283280  PMID: 25598780
nerve regeneration; peripheral nerve injury; mecobalamin; sciatic nerve; nerve repair; neurotrophic factor; neuroprotective effect; vitamin B12; molecular mechanism; gene expression; neural regeneration
6.  Genetic analysis of Chinese families reveals a novel truncation allele of the retinitis pigmentosa GTPase regulator gene 
AIM
To make comprehensive molecular diagnosis for retinitis pigmentosa (RP) patients in a consanguineous Han Chinese family using next generation sequencing based Capture-NGS screen technology.
METHODS
A five-generation Han Chinese family diagnosed as non-syndromic X-linked recessive RP (XLRP) was recruited, including four affected males, four obligate female carriers and eleven unaffected family members. Capture-NGS was performed using a custom designed capture panel covers 163 known retinal disease genes including 47 RP genes, followed by the validation of detected mutation using Sanger sequencing in all recruited family members.
RESULTS
Capture-NGS in one affected 47-year-old male reveals a novel mutation, c.2417_2418insG:p.E806fs, in exon ORF15 of RP GTPase regulator (RPGR) gene results in a frameshift change that results in a premature stop codon and a truncated protein product. The mutation was further validated in three of four affected males and two of four female carriers but not in the other unaffected family members.
CONCLUSION
We have identified a novel mutation, c.2417_2418insG:p.E806fs, in a Han Chinese family with XLRP. Our findings expand the mutation spectrum of RPGR and the phenotypic spectrum of XLRP in Han Chinese families, and confirms Capture-NGS could be an effective and economic approach for the comprehensive molecular diagnosis of RP.
doi:10.3980/j.issn.2222-3959.2014.05.02
PMCID: PMC4206875  PMID: 25349787
retinitis pigmentosa GTPase regulator; retinitis pigmentosa; next-generation sequencing; genetic diagnosis
7.  Activated Notch Causes Deafness by Promoting a Supporting Cell Phenotype in Developing Auditory Hair Cells 
PLoS ONE  2014;9(9):e108160.
Purpose
To determine whether activated Notch can promote a supporting cell fate during sensory cell differentiation in the inner ear.
Methods
An activated form of the Notch1 receptor (NICD) was expressed in early differentiating hair cells using a Gfi1-Cre mouse allele. To determine the effects of activated Notch on developing hair cells, Gfi1-NICD animals and their littermate controls were assessed at 5 weeks for hearing by measuring auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). The differentiation of NICD-expressing hair cells was assessed at postnatal day (P) 6, 11 and 20, using histological and molecular markers for hair cells, as well as supporting cells/progenitor cells. We also examined whether the effects of Notch were mediated by SOX2, a gene expressed in supporting cells and a likely downstream target of Notch, by crossing an inducible form of SOX2 to the Gfi1-Cre.
Results
Activation of Notch1 in developing auditory hair cells causes profound deafness. The NICD-expressing hair cells switch off a number of hair cell markers and lose their characteristic morphology. Instead, NICD-expressing hair cells adopt a morphology resembling supporting cells and upregulate a number of supporting cell markers. These effects do not appear to be mediated by SOX2, because although expression of SOX2 caused some hearing impairment, the SOX2-expressing hair cells did not downregulate hair cell markers nor exhibit a supporting cell-like phenotype.
Conclusions
Our data show that Notch signaling inhibits hair cell differentiation and promotes a supporting cell-like phenotype, and that these effects are unlikely to be mediated by SOX2.
doi:10.1371/journal.pone.0108160
PMCID: PMC4180070  PMID: 25264928
8.  GATA3 controls the specification of prosensory domain and neuronal survival in the mouse cochlea 
Human Molecular Genetics  2013;22(18):3609-3623.
HDR syndrome (also known as Barakat syndrome) is a developmental disorder characterized by hypoparathyroidism, sensorineural deafness and renal disease. Although genetic mapping and subsequent functional studies indicate that GATA3 haplo-insufficiency causes human HDR syndrome, the role of Gata3 in sensorineural deafness and auditory system development is largely unknown. In this study, we show that Gata3 is continuously expressed in the developing mouse inner ear. Conditional knockout of Gata3 in the developing inner ear disrupts the morphogenesis of mouse inner ear, resulting in a disorganized and shortened cochlear duct with significant fewer hair cells and supporting cells. Loss of Gata3 function leads to the failure in the specification of prosensory domain and subsequently, to increased cell death in the cochlear duct. Moreover, though the initial generation of cochleovestibular ganglion (CVG) cells is not affected in Gata3-null mice, spiral ganglion neurons (SGNs) are nearly depleted due to apoptosis. Our results demonstrate the essential role of Gata3 in specifying the prosensory domain in the cochlea and in regulating the survival of SGNs, thus identifying a molecular mechanism underlying human HDR syndrome.
doi:10.1093/hmg/ddt212
PMCID: PMC3749857  PMID: 23666531
9.  Expression of Islet1 Marks the Sensory and Neuronal Lineages in the Mammalian Inner Ear 
Several basic helix-loop-helix (bHLH) genes have been shown to be essential for the generation of the auditory sensory hair cells or the spiral ganglion (SG) neurons that innervate the hair cells in the cochlea, as well as a variety of cell types in the other nervous systems. However, it remains elusive what cellular context-dependent mechanisms confer the inner ear–specific neuronal or sensory competency/identities. We explored the possibility that one of the mechanisms responsible for generating cellular diversity in the nervous system through cooperative action of bHLH and LIM-homeodomain (LIM-HD) transcriptional factors might also contribute to the inner ear–specific sensory and/or neuronal competency. Here, we show that Islet1 (Isl1), a LIM-HD protein, is expressed early in the otocyst in the region that gives rise to both the auditory sensory organ, the organ of Corti, and SG neurons. Subsequently, the expression of Isl1 is maintained in SG neurons but is transitory in the sensory lineage. At embryonic day 12 (E12) in mice, the expression of Isl1 marks distinctively the ventral portion of the nascent cochlear epithelium encompassing the primordial organ of Corti. At E13, Isl1 is maintained at relatively high levels in the sensory primordium while down-regulated in the other regions of the cochlear duct. As the sensory epithelium starts to differentiate, it is down-regulated in the entire cochlear epithelium. The expression of Isl1 in the developing inner ear reveals an early and likely a common step in the development of both sensory and neuronal lineages of the inner ear, and suggests its potential role in the inner ear-specific sensory and neuronal cell development.
doi:10.1002/cne.20257
PMCID: PMC4158841  PMID: 15329890
organ of Corti; spiral ganglion neurons; sensory hair cells; LIM-HD; bHLH; Math1
10.  High gene delivery efficiency of alkylated low-molecular-weight polyethylenimine through gemini surfactant-like effect 
To our knowledge, the mechanism underlying the high transfection efficiency of alkylated low-molecular-weight polyethylenimine (PEI) is not yet well understood. In this work, we grafted branched PEI (molecular weight of 1,800 Da; bPEI1800) with lauryl chains (C12), and found that bPEI1800-C12 was structurally similar to gemini surfactant and could similarly assemble into micelle-like particles. Stability, cellular uptake, and lysosome escape ability of bPEI1800-C12/DNA polyplexes were all greatly enhanced after C12 grafting. bPEI1800-C12/DNA polyplexes exhibited significantly higher transfection efficiency than Lipofectamine™ 2000 in the presence of serum. Bioluminescence imaging showed that systemic injection of bPEI1800-C12/DNA polyplexes resulted in intensive luciferase expression in vivo and bioluminescence signals that could be detected even in the head. Altogether, the high transfection efficacy of bPEI1800-C12 was because bPEI1800-C12, being an analog of gemini surfactant, facilitated lysosome escape and induced the coil–globule transition of DNA to assemble into a highly organized micelle-like structure that showed high stability.
doi:10.2147/IJN.S64554
PMCID: PMC4122513  PMID: 25114526
self-organization; alkylation; luciferase; bioluminescence imaging
11.  Towards Understanding the Roles of Heparan Sulfate Proteoglycans in Alzheimer's Disease 
BioMed Research International  2014;2014:516028.
Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of memory and cognitive dysfunctions. A central pathological event of AD is accumulation and deposition of cytotoxic amyloid-β peptide (Aβ) in the brain parenchyma. Heparan sulfate proteoglycans (HSPGs) and the side chains heparan sulfate (HS) are found associated with Aβ deposits in the brains of AD patients and transgenic animal models of AD. A growing body of evidence from in vitro and in vivo studies suggests functional roles of HSPG/HS in Aβ pathogenesis. Although the question of “how and why HSPG/HS is codeposited with Aβ?” still remains, it is within reach to understand the mechanisms of the events. Recent progress by immunohistochemical examination with advanced antibodies shed light on molecular structures of HS codeposited with Aβ. Several recent reports have provided important new insights into the roles of HSPG in Aβ pathogenesis. Particularly, experiments on mouse models revealed indispensible functions of HSPG in modulating Aβ-associated neuroinflammation and clearance of Aβ from the brain. Application of molecules to interfere with the interaction between HS and Aβ peptides has demonstrated beneficial effects on AD mouse models. Elucidating the functions of HSPG/HS in Aβ deposition and toxicity is leading to further understanding of the complex pathology of AD. The progress is encouraging development of new treatments for AD by targeting HS-Aβ interactions.
doi:10.1155/2014/516028
PMCID: PMC4135094  PMID: 25157361
12.  Bhlhb5 is Required for the Subtype Development of Retinal Amacrine and Bipolar Cells in Mice 
Background
BHLHB5, an OLIG-related basic helix-loop-helix transcription factor, is required for the development of a subset of gamma-amino butyric acid–releasing (GABAergic) amacrine cells and OFF-cone bipolar (CB) cells in mouse retinas. In order to determine BHLHB5’s functional mechanism in retinogenesis, we used the Cre-loxP recombination system to genetically trace the lineage of BHLHB5+ cells in normal and Bhlhb5-null retinas. The Bhlhb5-Cre knock-in allele was used to activate the constitutive expression of a GFP reporter in the Bhlhb5-expressing cells, and the cell fates of Bhlhb5-lineage cells were identified by using specific cell markers and were compared between normal and Bhlhb5-null retinas.
Results
In addition to GABAergic amacrine and OFF-CB cells, Bhlhb5 lineage cells give rise to ganglion, glycinergic amacrine, rod bipolar, ON-bipolar, and rod photoreceptor cells during normal retinal development. Targeted deletion of Bhlhb5 resulted in the loss of GABAergic amacrine, glycinergic amacrine, dopaminergic amacrine, and Type 2 OFF-CB cells. Furthermore, in the absence of BHLHB5, a portion of Bhlhb5 lineage cells switch their fate and differentiate into cholinergic amacrine cells.
Conclusions
Our data reveal a broad expression pattern of Bhlhb5 throughout retinogenesis and demonstrate the cell-autonomous as well as non-cell-autonomous role of Bhlhb5 in the specification of amacrine and bipolar subtypes.
doi:10.1002/dvdy.24067
PMCID: PMC4100943  PMID: 24123365
Bhlhb5; amacrine cell; bipolar cell; neurogenesis; transcription factor; retina
13.  Pou4f1 and Pou4f2 Are Dispensable for the Long-Term Survival of Adult Retinal Ganglion Cells in Mice 
PLoS ONE  2014;9(4):e94173.
Purpose
To investigate the role of Pou4f1 and Pou4f2 in the survival of adult retinal ganglion cells (RGCs).
Methods
Conditional alleles of Pou4f1 and Pou4f2 were generated (Pou4f1loxP and Pou4f2loxP respectively) for the removal of Pou4f1 and Pou4f2 in adult retinas. A tamoxifen-inducible Cre was used to delete Pou4f1 and Pou4f2 in adult mice and retinal sections and flat mounts were subjected to immunohistochemistry to confirm the deletion of both alleles and to quantify the changes in the number of RGCs and other retinal neurons. To determine the effect of loss of Pou4f1 and Pou4f2 on RGC survival after axonal injury, controlled optic nerve crush (CONC) was performed and RGC death was assessed.
Results
Pou4f1 and Pou4f2 were ablated two weeks after tamoxifen treatment. Retinal interneurons and Müller glial cells are not affected by the ablation of Pou4f1 or Pou4f2 or both. Although the deletion of both Pou4f1 and Pou4f2 slightly delays the death of RGCs at 3 days post-CONC in adult mice, it does not affect the cell death progress afterwards. Moreoever, deletion of Pou4f1 or Pou4f2 or both has no impact on the long-term viability of RGCs at up to 6 months post-tamoxifen treatment.
Conclusion
Pou4f1 and Pou4f2 are involved in the acute response to damage to RGCs but are dispensable for the long-term survival of adult RGC in mice.
doi:10.1371/journal.pone.0094173
PMCID: PMC3988073  PMID: 24736625
14.  An underlying prognosis predictor of hepatocellular carcinoma: Oncoprotein 18 
Biomedical Reports  2013;2(1):85-88.
Recent studies have reported the association between the expression of oncoprotein 18 (op18) and hepatocellular carcinoma (HCC). However, any underlying mechanistic connection between op18 expression and hepatocarcinogenesis is poorly understood. In the present study, Flag-pcDNA3.1 vector and Flag-pcDNA3.1-op18 plasmid were stably transfected in SMMC7721 cells, respectively. Stable SMMC7721 control and op18 overexpression SMMC7721 cell lines were constructed and identified by western blot analysis. Using a cell counting kit-8 (CCK8), it was shown that cell proliferation was significantly increased in the op18 overexpression SMMC7721 cell group (0.60±0.05), compared with the control group (0.29±0.03) at an absorbance of 450 nm (P<0.01). Flow cytometry was used to analyze cell apoptosis by FITC-Annexin V and propidium iodide (PI) apoptosis assay kit. The results demonstrated that the percentage of apoptotic cells was inhibited to 5.80±0.33% in the op18 overexpression group, compared with 11.79±1.09% in the control group. Using FACS, single cell analysis data showed that op18 overexpression induced cell cycle arrest by inhibiting progression from G2 to M phase. The results suggest that op18 expression is closely associated with SMMC7721 cell proliferation and apoptosis, which appears to be a potential predictor of prognosis in HCC.
doi:10.3892/br.2013.197
PMCID: PMC3916978  PMID: 24649074
oncoprotein 18; hepatocellular carcinoma; proliferation; apoptosis
15.  Transitioning Streaming to Trapping in DC Insulator-based Dielectrophoresis for Biomolecules 
Exploiting dielectrophoresis (DEP) to concentrate and separate biomolecules has recently shown large potential as a microscale bioanalytical tool. Such efforts however require tailored devices and knowledge of all interplaying transport mechanisms competing with dielectrophoresis (DEP). Specifically, a strong DEP contribution to the overall transport mechanism is necessary to exploit DEP of biomolecules for analytical applications such as separation and fractionation. Here, we present improved microfluidic devices combining optical lithography and focused ion beam milling (FIBM) for the manipulation of DNA and proteins using insulator-based dielectrophoresis (iDEP) and direct current (DC) electric fields. Experiments were performed on an elastomer platform forming the iDEP microfluidic device with integrated nanoposts and nanopost arrays. Microscale and nanoscale iDEP was studied for λ-DNA (48.5 kbp) and the protein bovine serum albumin (BSA). Numerical simulations were adapted to the various tested geometries revealing excellent qualitative agreement with experimental observations for streaming and trapping DEP. Both the experimental and simulation results indicate that DC iDEP trapping for λ-DNA occurs with tailored nanoposts fabricated via FIBM. Moreover, streaming iDEP concentration of BSA is improved with integrated nanopost arrays by a factor of 45 compared to microfabricated arrays.
doi:10.1016/j.snb.2012.07.080
PMCID: PMC3577371  PMID: 23441049
dielectrophoresis; DNA; protein; numerical simulation; trapping condition
16.  BCL2L1 (BCL-x) promotes survival of adult and developing retinal ganglion cells 
The Bcl-2 family is responsible for regulating cell death pathways in neurons during development, after injury and in disease. The activation of the pro-death family member BAX is often the final step before cell death in neurons. Pro-survival family members such as BCL-X (BCL2L1) act to inhibit BAX activation. Overexpression studies have suggested that BCL-X could play an important physiological role in mediating neuronal viability. Loss-of-function studies performed in vivo have implicated BCL-X as a mediator of neuronal survival during the early stages of neurodevelopment. To assess whether BCL-X is needed to promote the survival of neurons in the central nervous system throughout life, Bcl-x was conditionally removed from the optic cup or throughout the adult mouse. During development BCL-X was required for the survival of differentiating retinal ganglion cells (RGCs) leading up to their normal window of developmental death. Despite its expression in adult RGCs, BCL-X was not required for maintaining RGC viability in adult retinas. However, the loss of BCL-X in adult RGCs did significantly increase the rate of death of RGCs after axonal injury. Thus, in developing and injured RGCs there appears to be an active cell survival program preventing neuronal death.
doi:10.1016/j.mcn.2012.07.006
PMCID: PMC3436941  PMID: 22836101
17.  Distinct Roles of Muscle and Motoneuron LRP4 in Neuromuscular Junction Formation 
Neuron  2012;75(1):94-107.
SUMMARY
Neuromuscular junction (NMJ) formation requires precise interaction between motoneurons and muscle fibers. LRP4 is a receptor of agrin that is thought to act incis to stimulate MuSK in muscle fibers for postsynaptic differentiation. Here we dissected the roles of LRP4 in muscle fibers and motoneurons in NMJ formation by cell-specific mutation. Studies of muscle-specific mutants suggest that LRP4 is involved in deciding where to form AChR clusters in muscle fibers, postsynaptic differentiation, and axon terminal development. LRP4 in HEK293 cells increased synapsin or SV2 puncta in contacting axons of co-cultured neurons, suggesting a synaptogenic function. Analysis of LRP4 muscle and motoneuron double mutants and mechanistic studies suggest that NMJ formation may also be regulated by LRP4 in motoneurons, which could serve as agrin’s receptor in trans to induce AChR clusters. These observations uncovered distinct roles of LRP4 in motoneurons and muscles in NMJ development.
doi:10.1016/j.neuron.2012.04.033
PMCID: PMC3422364  PMID: 22794264
18.  A Mouse Model of Interstitial Pneumonitis Induced by Murine Cytomegalovirus Infection after Allogeneic Skin Transplantation 
BioMed Research International  2013;2013:341387.
We investigated the effect of murine cytomegalovirus (MCMV) on interstitial pneumonia in transplant recipients in an experimental skin allograft model. Skin transplantation between C57BL/6J and BALB/c mice was performed in the presence or absence of cyclosporin A treatment. Flow cytometry showed that the number of CD4+ and CD8+ cells and the level of IFN-γ decreased significantly in the groups treated with cyclosporin A. We either mock-infected or infected the mice with MCMV by intranasal administration and monitored pathophysiological behavior and body weight. The infected mice were sacrificed at different days postinfection for histology, immunohistochemistry, and molecular biological evaluations. Interstitial pneumonitis was observed in positive control groups as well as in experimental group that received cyclosporin A, a skin transplant, and infected with the highest dose of virus (105 PFU). Transmission electronic microscopy demonstrated the presence of herpes virus particles. MCMV DNA and glycoprotein B were demonstrated in the epithelial cells of the lung tissue in those animals by in situ hybridization and immunohistochemistry, respectively. Our data demonstrated the establishment of a mouse model of interstitial pneumonitis via MCMV infection after allogeneic skin transplantation.
doi:10.1155/2013/341387
PMCID: PMC3713605  PMID: 23936793
19.  Genetic Analysis of Clinical VZV Isolates Collected in China Reveals a More Homologous Profile 
BioMed Research International  2013;2013:681234.
Forty-four varicella-zoster virus (VZV) isolates from China were genotyped by using a scattered single nucleotide polymorphism (SNP) method, including open reading frames (ORFs) 1, 22, 31, 37, 60, 62, 67, and 68. Based on the analysis of the polymorphic markers in the 8 ORFs, all of the 44 isolates can be placed in genotype J defined by the SNP profiles in ORF22 or clade B defined by the SNP profiles in ORFs 31, 37, 60, 62, 67, and 68. The three consecutive nucleotide (CGG) in-frame insertions in ORF 1 were found in 8 (18.2%) isolates, which has not been described in VZV strains from any other part of the world. A novel synonymous A>G substitution in ORF60 was revealed in 4 (9.1%) of the isolates. In addition, a previously described three consecutive nucleotide (ATC) insertion in ORF 60 was found in all the Chinese isolates but not in the US isolate MLS. The results showed all the 44 strains that belong to genotype J/clade B with significantly high homogeneity, and phylogenetic analysis suggested that the 44 Chinese isolates consist of 4 clusters, but interstrain variations also exist. Overall, VZV isolates obtained in China showed significantly higher genetic homogeneity than isolates reported from other countries.
doi:10.1155/2013/681234
PMCID: PMC3678451  PMID: 23781507
20.  CVD Growth of Large Area Smooth-edged Graphene Nanomesh by Nanosphere Lithography 
Scientific Reports  2013;3:1238.
Current etching routes to process large graphene sheets into nanoscale graphene so as to open up a bandgap tend to produce structures with rough and disordered edges. This leads to detrimental electron scattering and reduces carrier mobility. In this work, we present a novel yet simple direct-growth strategy to yield graphene nanomesh (GNM) on a patterned Cu foil via nanosphere lithography. Raman spectroscopy and TEM characterizations show that the as-grown GNM has significantly smoother edges than post-growth etched GNM. More importantly, the transistors based on as-grown GNM with neck widths of 65-75 nm have a near 3-fold higher mobility than those derived from etched GNM with the similar neck widths.
doi:10.1038/srep01238
PMCID: PMC3566595  PMID: 23393620
21.  The Interleukin 3 Gene (IL3) Contributes to Human Brain Volume Variation by Regulating Proliferation and Survival of Neural Progenitors 
PLoS ONE  2012;7(11):e50375.
One of the most significant evolutionary changes underlying the highly developed cognitive abilities of humans is the greatly enlarged brain volume. In addition to being far greater than in most other species, the volume of the human brain exhibits extensive variation and distinct sexual dimorphism in the general population. However, little is known about the genetic mechanisms underlying normal variation as well as the observed sex difference in human brain volume. Here we show that interleukin-3 (IL3) is strongly associated with brain volume variation in four genetically divergent populations. We identified a sequence polymorphism (rs31480) in the IL3 promoter which alters the expression of IL3 by affecting the binding affinity of transcription factor SP1. Further analysis indicated that IL3 and its receptors are continuously expressed in the developing mouse brain, reaching highest levels at postnatal day 1–4. Furthermore, we found IL3 receptor alpha (IL3RA) was mainly expressed in neural progenitors and neurons, and IL3 could promote proliferation and survival of the neural progenitors. The expression level of IL3 thus played pivotal roles in the expansion and maintenance of the neural progenitor pool and the number of surviving neurons. Moreover, we found that IL3 activated both estrogen receptors, but estrogen didn’t directly regulate the expression of IL3. Our results demonstrate that genetic variation in the IL3 promoter regulates human brain volume and reveals novel roles of IL3 in regulating brain development.
doi:10.1371/journal.pone.0050375
PMCID: PMC3511536  PMID: 23226269
22.  Age-dependent in vivo conversion of mouse cochlear pillar and Deiters’ cells to immature hair cells by Atoh1 ectopic expression 
The Journal of Neuroscience  2012;32(19):6600-6610.
Unlike non-mammalian vertebrates, mammals cannot convert inner ear cochlear supporting cells (SCs) into sensory hair cells (HCs) after damage, thus causing permanent deafness. Here, we achieved in vivo conversion of two SC subtypes, pillar cells (PCs) and Deiters’ cells (DCs), into HCs by inducing targeted expression of Atoh1 at neonatal and juvenile ages using novel mouse models. The conversion only occurred in ~10% of PCs and DCs with ectopic Atoh1 expression and started with reactivation of endogenous Atoh1, followed by expression of 11 HC and synaptic markers; a process that took at least 3 weeks in vivo. These new HCs resided in the outer HC region, formed stereocilia, contained mechanoelectrical transduction channels, and survived for more than 2 months in vivo; however, they surprisingly lacked prestin and oncomodulin expression and mature HC morphology. In contrast, adult PCs and DCs no longer responded to ectopic Atoh1 expression, even after outer HC damage. Finally, permanent Atoh1 expression in endogenous HCs did not affect prestin expression, but caused cell loss of mature HCs. Together our results demonstrate that in vivo conversion of PCs and DCs into immature HCs by Atoh1 is age-dependent, and resembles normal HC development. Therefore combined expression of Atoh1 with additional factors holds therapeutic promise to convert PCs and DCs into functional HCs in vivo for regenerative purposes.
doi:10.1523/JNEUROSCI.0818-12.2012
PMCID: PMC3359704  PMID: 22573682
23.  Direct Optical Characterization of Graphene Growth and Domains on Growth Substrates 
Scientific Reports  2012;2:707.
We detailed a facile detection technique to optically characterize graphene growth and domains directly on growth substrates through a simple thermal annealing process. It was found that thermal annealing transformed the naked Cu to Cu oxides while keeping graphene and graphene-covered Cu intact. This increases the interference color contrast between Cu oxides and Cu, thus making graphene easily visible under an optical microscope. By using this simple method, we studied the factors that affect graphene nucleation and growth and achieved graphene domains with the domain size as large as ~100 μm. The concept of chemically making graphene visible is universal, as demonstrated by the fact that a solution process based on selective H2O2 oxidation has been developed to achieve the similar results in a shorter time. These techniques should be valuable for studies towards elucidating the parameters that control the grains, boundaries, structures and properties of graphene.
doi:10.1038/srep00707
PMCID: PMC3463814  PMID: 23050091
24.  Novel optoelectronic devices based on single semiconductor nanowires (nanobelts) 
Nanoscale Research Letters  2012;7(1):218.
Semiconductor nanowires (NWs) or nanobelts (NBs) have attracted more and more attention due to their potential application in novel optoelectronic devices. In this review, we present our recent work on novel NB photodetectors, where a three-terminal metal–semiconductor field-effect transistor (MESFET) device structure was exploited. In contrast to the common two-terminal NB (NW) photodetectors, the MESFET-based photodetector can make a balance among overall performance parameters, which is desired for practical device applications. We also present our recent work on graphene nanoribbon/semiconductor NW (SNW) heterojunction light-emitting diodes (LEDs). Herein, by taking advantage of both graphene and SNWs, we have fabricated, for the first time, the graphene-based nano-LEDs. This achievement opens a new avenue for developing graphene-based nano-electroluminescence devices. Moreover, the novel graphene/SNW hybrid devices can also find use in other applications, such as high-sensitivity sensor and transparent flexible devices in the future.
doi:10.1186/1556-276X-7-218
PMCID: PMC3443073  PMID: 22501032
Schottky junction; Graphene; Nanowires; Nanobelts; Optoelectronics
25.  Biosafety of Non-Surface Modified Carbon Nanocapsules as a Potential Alternative to Carbon Nanotubes for Drug Delivery Purposes 
PLoS ONE  2012;7(3):e32893.
Background
Carbon nanotubes (CNTs) have found wide success in circuitry, photovoltaics, and other applications. In contrast, several hurdles exist in using CNTs towards applications in drug delivery. Raw, non-modified CNTs are widely known for their toxicity. As such, many have attempted to reduce CNT toxicity for intravenous drug delivery purposes by post-process surface modification. Alternatively, a novel sphere-like carbon nanocapsule (CNC) developed by the arc-discharge method holds similar electric and thermal conductivities, as well as high strength. This study investigated the systemic toxicity and biocompatibility of different non-surface modified carbon nanomaterials in mice, including multi-walled carbon nanotubes (MWCNTs), single-walled carbon nanotubes (SWCNTs), carbon nanocapsules (CNCs), and C60 fullerene (C60). The retention of the nanomaterials and systemic effects after intravenous injections were studied.
Methodology and Principal Findings
MWCNTs, SWCNTs, CNCs, and C60 were injected intravenously into FVB mice and then sacrificed for tissue section examination. Inflammatory cytokine levels were evaluated with ELISA. Mice receiving injection of MWCNTs or SWCNTs at 50 µg/g b.w. died while C60 injected group survived at a 50% rate. Surprisingly, mortality rate of mice injected with CNCs was only at 10%. Tissue sections revealed that most carbon nanomaterials retained in the lung. Furthermore, serum and lung-tissue cytokine levels did not reveal any inflammatory response compared to those in mice receiving normal saline injection.
Conclusion
Carbon nanocapsules are more biocompatible than other carbon nanomaterials and are more suitable for intravenous drug delivery. These results indicate potential biomedical use of non-surface modified carbon allotrope. Additionally, functionalization of the carbon nanocapsules could further enhance dispersion and biocompatibility for intravenous injection.
doi:10.1371/journal.pone.0032893
PMCID: PMC3310837  PMID: 22457723

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