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1.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells 
Science (New York, N.Y.)  2013;343(6166):84-87.
The simplicity of programming the CRISPR-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic that inhibits mutant protein kinase BRAF. Our highest-ranking candidates include previously validated genes NF1 and MED12 as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
PMCID: PMC4089965  PMID: 24336571
2.  Drug-induced hypothermia in stroke models: does it always protect? 
Ischemic stroke is a common neurological disorder lacking a cure. Recent studies show that therapeutic hypothermia is a promising neuroprotective strategy against ischemic brain injury. Several methods to induce therapeutic hypothermia have been established; however, most of them are not clinically feasible for stroke patients. Therefore, pharmacological cooling is drawing increasing attention as a neuroprotective alternative worthy of further clinical development. We begin this review with a brief introduction to the commonly used methods for inducing hypothermia; we then focus on the hypothermic effects of eight classes of hypothermia-inducing drugs: the cannabinoids, opioid receptor activators, transient receptor potential vanilloid, neurotensins, thyroxine derivatives, dopamine receptor activators, hypothermia-inducing gases, adenosine, and adenine nucleotides. Their neuroprotective effects as well as the complications associated with their use are both considered. This article provides guidance for future clinical trials and animal studies on pharmacological cooling in the setting of acute stroke.
PMCID: PMC4081530  PMID: 23469851
Brain ischemia; Hypothermic; Neuroprotection; Pharmacological cooling
3.  Increased genome instability in human DNA segments with self-chains: homology-induced structural variations via replicative mechanisms 
Human Molecular Genetics  2013;22(13):2642-2651.
Environmental factors including ionizing radiation and chemical agents have been known to be able to induce DNA rearrangements and cause genomic structural variations (SVs); however, the roles of intrinsic characteristics of the human genome, such as regional genome architecture, in SV formation and the potential mechanisms underlying genomic instability remain to be further elucidated. Recently, locus-specific observations showed that ‘self-chain’ (SC), a group of short low-copy repeats (LCRs) in the human genome, can induce autism-associated SV mutations of the MECP2 and NRXN1 genes. In this study, we conducted a genome-wide analysis to investigate SCs and their potential roles in genomic SV formation. Utilizing a vast amount of human SV data, we observed a significant biased distribution of human germline SV breakpoints to SC regions. Notably, the breakpoint distribution pattern is different between SV types across deletion, duplication, inversion and insertion. Our observations were coincident with a mechanism of SC-induced DNA replicative errors, whereas SC may sporadically be used as substrates of nonallelic homologous recombination (NAHR). This contention was further supported by our consistent findings in somatic SV mutations of cancer genomes, suggesting a general mechanism of SC-induced genome instability in human germ and somatic cells.
PMCID: PMC3674805  PMID: 23474816
4.  Temporal Production of the Signaling Lipid Phosphatidic Acid by Phospholipase D2 Determines the Output of Extracellular Signal-Regulated Kinase Signaling in Cancer Cells 
Molecular and Cellular Biology  2014;34(1):84-95.
The Ras-extracellular signal-regulated kinase (ERK) cascade is an important signaling module in cells. One regulator of the Ras-ERK cascade is phosphatidic acid (PA) generated by phospholipase D (PLD) and diacylglycerol kinase (DGK). Using a newly developed PA biosensor, PASS (phosphatidic acid biosensor with superior sensitivity), we found that PA was generated sequentially by PLD and DGK in epidermal growth factor (EGF)-stimulated HCC1806 breast cancer cells. Inhibition of PLD2, one of the two PLD members, was sufficient to eliminate most of the PA production, whereas inhibition of DGK decreased PA production only at the later stages of EGF stimulation, suggesting that PLD2 precedes DGK activation. The temporal production of PA by PLD2 is important for the nuclear activation of ERK. While inhibition of both PLD and DGK had no effect on the overall ERK activity, inhibition of PLD2 but not PLD1 or DGK blocked the nuclear ERK activity in several cancer cell lines. The decrease of active ERK in the nucleus inhibited the activation of Elk1, c-fos, and Fra1, the ERK nuclear targets, leading to decreased proliferation of HCC1806 cells. Together, these findings reveal that PA production by PLD2 determines the output of ERK in cancer cell growth factor signaling.
PMCID: PMC3911278  PMID: 24164897
5.  Association between Copy Number Variations HLA-DQA1 and Ankylosing Spondylitis in Chinese Han population 
Genes and immunity  2013;14(8):10.1038/gene.2013.46.
Ankylosing spondylitis (AS) is a chronic inflammatory disease with complex genetic traits. Multiple sequence variations have been associated with AS, but explained only a proportion of heritability. The studies herein aimed to explore potential associations between genomic copy number variation (CNV) and AS of Han Chinese. Five AS patients were examined with the high-density comparative genomic hybridization (CGH) microarrays in the first screen test for AS associated CNVs. A total of 533 AS patients and 792 unrelated controls were examined in confirmation studies with the AccuCopy assays. A significant association was observed between the CNV of the HLA-DQA1 and AS. Comparing with controls, AS patients showed an aberrant copy number (CN), and significantly increased number of patients had more than 2 copies of the HLA-DQA1. Therefore, CNV of the HLA-DQA1 may play an important role in susceptibility to AS in Han Chinese population.
PMCID: PMC3855587  PMID: 24048351
6.  The Influence of “wuqinxi” exercises on the Lumbosacral Multifidus 
[Purpose] To investigate the effect of the five animals (wuqinxi) exercises on the lumbosacral multifidus. [Subjects and Methods] This study enrolled two groups of volunteers, 15 volunteers who did the five animals exercises, the experimental group, and 15 volunteers who did aerobic exercise (walking), the control group. Both before and after the 1 year exercise intervention, the average surface electromyography (ASEMG) of the two groups in the process of flexion and extension was recorded and analyzed using DASYLab10.0 software, and the flexion extension ratio (FER) was calculated. [Results] The ASEMG in the process of flexion was lower than the ASEMG in the process of extension both before and after the 1 year exercise intervention on both sides of all volunteers. There was no significant difference in FER between the experimental group and control group before the 1 year exercise intervention; however, the FER of experimental group was lower than that of the control group after the 1 year exercise intervention. There was no significant difference between the two sides in any individual both before and after the 1 year exercise intervention in both groups. [Conclusion] The “wuqinxi” exercises improved the function of the lumbosacral multifidus, and might be an alternative method of reducing low back pain.
PMCID: PMC4085213  PMID: 25013288
Wuqinxi exercises; Flexion extension ratio (FER); Low back pain
7.  The Continual Reassessment Method for Multiple Toxicity Grades: A Bayesian Model Selection Approach 
PLoS ONE  2014;9(5):e98147.
Grade information has been considered in Yuan et al. (2007) wherein they proposed a Quasi-CRM method to incorporate the grade toxicity information in phase I trials. A potential problem with the Quasi-CRM model is that the choice of skeleton may dramatically vary the performance of the CRM model, which results in similar consequences for the Quasi-CRM model. In this paper, we propose a new model by utilizing bayesian model selection approach – Robust Quasi-CRM model – to tackle the above-mentioned pitfall with the Quasi-CRM model. The Robust Quasi-CRM model literally inherits the BMA-CRM model proposed by Yin and Yuan (2009) to consider a parallel of skeletons for Quasi-CRM. The superior performance of Robust Quasi-CRM model was demonstrated by extensive simulation studies. We conclude that the proposed method can be freely used in real practice.
PMCID: PMC4038518  PMID: 24875783
8.  Whole-Genome Sequencing in a Patient with Charcot–Marie–Tooth Neuropathy 
The New England journal of medicine  2010;362(13):1181-1191.
Whole-genome sequencing may revolutionize medical diagnostics through rapid identification of alleles that cause disease. However, even in cases with simple patterns of inheritance and unambiguous diagnoses, the relationship between disease phenotypes and their corresponding genetic changes can be complicated. Comprehensive diagnostic assays must therefore identify all possible DNA changes in each haplotype and determine which are responsible for the underlying disorder. The high number of rare, heterogeneous mutations present in all humans and the paucity of known functional variants in more than 90% of annotated genes make this challenge particularly difficult. Thus, the identification of the molecular basis of a genetic disease by means of whole-genome sequencing has remained elusive. We therefore aimed to assess the usefulness of human whole-genome sequencing for genetic diagnosis in a patient with Charcot–Marie–Tooth disease.
We identified a family with a recessive form of Charcot–Marie–Tooth disease for which the genetic basis had not been identified. We sequenced the whole genome of the proband, identified all potential functional variants in genes likely to be related to the disease, and genotyped these variants in the affected family members.
We identified and validated compound, heterozygous, causative alleles in SH3TC2 (the SH3 domain and tetratricopeptide repeats 2 gene), involving two mutations, in the proband and in family members affected by Charcot–Marie–Tooth disease. Separate subclinical phenotypes segregated independently with each of the two mutations; heterozygous mutations confer susceptibility to neuropathy, including the carpal tunnel syndrome.
As shown in this study of a family with Charcot–Marie–Tooth disease, whole-genome sequencing can identify clinically relevant variants and provide diagnostic information to inform the care of patients.
PMCID: PMC4036802  PMID: 20220177
9.  Addition of visual noise boosts evoked potential-based brain-computer interface 
Scientific Reports  2014;4:4953.
Although noise has a proven beneficial role in brain functions, there have not been any attempts on the dedication of stochastic resonance effect in neural engineering applications, especially in researches of brain-computer interfaces (BCIs). In our study, a steady-state motion visual evoked potential (SSMVEP)-based BCI with periodic visual stimulation plus moderate spatiotemporal noise can achieve better offline and online performance due to enhancement of periodic components in brain responses, which was accompanied by suppression of high harmonics. Offline results behaved with a bell-shaped resonance-like functionality and 7–36% online performance improvements can be achieved when identical visual noise was adopted for different stimulation frequencies. Using neural encoding modeling, these phenomena can be explained as noise-induced input-output synchronization in human sensory systems which commonly possess a low-pass property. Our work demonstrated that noise could boost BCIs in addressing human needs.
PMCID: PMC4021798  PMID: 24828128
10.  A peculiar cave species of Tomocerus (Collembola, Tomoceridae, Tomocerinae) from Vietnam, with a discussion of the postantennal organ and prelabral chaetae in Tomocerinae 
ZooKeys  2014;61-70.
The first Tomocerus species with a postantennal organ (PAO) in the adult stage is described from Vietnam. Tomocerus postantennalis sp. n. differs from the other PAO-possessing tomocerid, Tomolonus reductus Mills, 1948, mainly in the morphology of PAO, the number of ocelli, the number of chaetae in trochantero-femoral organ and several features of the furca. The new species is placed in Tomocerus because of the presence of a toothlet on the outer basal mucronal tooth and the absence of the diagnostic character states of Plutomurus Yosii, 1956 and Aphaenomurus Yosii, 1956. Besides the presence of PAO, the new species is peculiar in having six prelabral chaetae, instead of four as in other Tomocerus species. The new species is similar to Tomocerus folsomi Denis, 1929 and Tomocerus ocreatus Denis, 1948 in the type of dental spines but different from them in the body colour, the relative length of antennae to body, the number of unguis inner teeth and the number of mucronal intermediate teeth.
PMCID: PMC4042826  PMID: 24899837
New species; Southeast Asia; taxonomy; Tomocerus folsomi; Tomocerus ocreatus; Tomolonus
11.  A Transduced Living Hyaline Cartilage Graft Releasing Transgenic Stromal Cell-Derived Factor-1 Inducing Endogenous Stem Cell Homing In Vivo 
Tissue Engineering. Part A  2013;19(9-10):1091-1099.
Stromal cell-derived factor-1 (SDF-1), also known as a homing factor, is a potent chemokine that activates and directs mobilization, migration, and retention of certain cell species via systemic circulation. The responding homing cells largely consist of activated stem cells, so that, in case of tissue lesions, such SDF-1-induced cell migration may execute recruitment of endogenous stem cells to perform autoreparation and compensatory regeneration in situ. In this study, a recombinant adenoviral vector carrying SDF-1 transgene was constructed and applied to transduce a novel scaffold-free living hyaline cartilage graft (SDF-t-LhCG). As an engineered transgenic living tissue, SDF-t-LhCG is capable of continuously producing and releasing SDF-1 in vitro and in vivo. The in vitro trials were examined with ELISA, while the in vivo trials were subsequently performed via a subcutaneous implantation of SDF-t-LhCG in a nude mouse model, followed by series of biochemical and biological analyses. The results indicate that transgenic SDF-1 enhanced the presence of this chemokine in mouse's circulation system; in consequence, SDF-1-induced activation and recruitment of endogenous stem cells were also augmented in both peripheral blood and SDF-t-LhCG implant per se. These results were obtained via flow cytometry analyses on mouse blood samples and implanted SDF-t-LhCG samples, indicating an upregulation of the CXCR4+(SDF-1 receptor) cell population, accompanied by upregulation of the CD34+, CD44+, and Sca-1+ cell populations as well as a downregulation of the CD11b+ cell population. With the supply of SDF-1-recruited endogenous stem cells, enhanced chondrogenesis was observed in SDF-t-LhCG implants in situ.
PMCID: PMC3609611  PMID: 23167782
12.  Caveolin-1 Expression in Different Types of Psoriatic Lesions: Analysis of 66 Cases 
Indian Journal of Dermatology  2014;59(3):225-229.
Caveolin-1 is a key structural and functional protein. Caveolin-1 is known to modulate multiple signal-transducing pathways involved in cell differentiation and proliferation. Psoriasis is viewed as a multifactorial pathology characterized by keratinocyte hyperproliferation and abnormal cell maturation.
To examine the expression of caveolin-1 in skin biopsies from normal subjects, patients, and subjects with the three respective isoforms of psoriasis (psoriasis vulgaris, localized pustular psoriasis, and erythrodermic psoriasis). The expression level of caveolin-1 was compared among psoriasis vulgaris, localized pustular psoriasis, erythrodermic psoriasis, and normal subjects.
Materials and Methods:
Using immunohistochemical methods, caveolin-1 protein expression was assayed in four groups. An analysis was conducted on skin samples obtained from 22 normal subjects and 28 patients with psoriasis vulgaris, 22 patients with localized pustular psoriasis, and 16 patients with erythrodermic psoriasis. The statistical analysis of the scoring criteria reflecting the level of Caveolin-1 immunostaining between different groups was determined using the Mann–Whitney U-test.
In the normal skin, intense and consistent caveolin-1 staining was present in 22 cases. The Caveolin-1 protein was significantly reduced and showed very weak or absent staining within the tissues of psoriasis vulgaris, localized pustular psoriasis, and erythrodermic psoriasis (respective P < 0.001). Caveolin-1 protein expression in psoriasis vulgaris was higher than that in localized pustular psoriasis and erythrodermic psoriasis (respective P < 0.05). Caveolin-1 protein expression was no different in localized pustular psoriasis and erythrodermic psoriasis (P > 0.05).
The finding of this study was consistent with a downregulation of Caveolin-1, which might serve as an etiological factor in the development of psoriasis vulgaris, localized pustular psoriasis, and erythrodermic psoriasis. Further mechanistic investigations are required to prove that Caveolin-1 protein has the potential and may be a novel target for therapy of psoriasis vulgaris, localized pustular psoriasis, and erythrodermic psoriasis.
PMCID: PMC4037939  PMID: 24891649
Caveolin-1; erythrodermic psoriasis; localized pustular psoriasis; psoriasis vulgaris
13.  Chinese SLE Treatment and Research Group Registry: III. Association of Autoantibodies with Clinical Manifestations in Chinese Patients with Systemic Lupus Erythematosus 
Journal of Immunology Research  2014;2014:809389.
We investigated the characteristics of Chinese SLE patients by analyzing the association between specific autoantibodies and clinical manifestations of 2104 SLE patients from registry data of CSTAR cohort. Significant (P < 0.05) associations were found between anti-Sm antibody, anti-rRNP antibody, and malar rash; between anti-RNP antibody, anti-SSA antibody, and pulmonary arterial hypertension (PAH); between anti-SSB antibody and hematologic involvement; and between anti-dsDNA antibody and nephropathy. APL antibody was associated with hematologic involvement, interstitial lung disease, and a lower prevalence of oral ulcerations (P < 0.05). Associations were also found between anti-dsDNA antibody and a lower prevalence of photosensitivity, and between anti-SSA antibody and a lower prevalence of nephropathy (P < 0.05). Most of these findings were consistent with other studies in the literature but this study is the first report on the association between anti-SSA and a lower prevalence of nephropathy. The correlations of specific autoantibodies and clinical manifestations could provide clues for physicians to predict organ damages in SLE patients. We suggest that a thorough screening of autoantibodies should be carried out when the diagnosis of SLE is established, and repeated echocardiography annually in SLE patients with anti-RNP or anti-SSA antibody should be performed.
PMCID: PMC4017718  PMID: 24864270
14.  The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor 
Nucleic Acids Research  2014;42(9):5765-5775.
Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption.
PMCID: PMC4027211  PMID: 24728990
15.  Mechanisms of Nerve Capping Technique in Prevention of Painful Neuroma Formation 
PLoS ONE  2014;9(4):e93973.
Nerve capping techniques have been introduced as a promising treatment modality for the treatment of painful neuroma with varied outcomes; however, its exact mechanism is still unknown. RhoA is one of the members of the RAS superfamily of GTPases that operate as molecular switches and plays an important role in peripheral nerve regeneration. Our aim was to investigate the structural and morphologic mechanisms by which the nerve capping technique prevents the formation of painful neuromas after neuroectomy. We also hoped to provide a theoretical basis for this treatment approach. An aligned nanofiber conduit was used for the capping procedure and the sciatic nerve of Sprague-Dawley rats was selected as the animal model. Behavioral analysis, extent of neuroma formation, histological assessment, expressions of pain markers of substance P and c-fos, molecular biological changes as well as ultrastructural features were investigated and compared with the findings in a no-capping control group. The formation of traumatic neuromas was significantly inhibited in the capping group with relatively “normal” structural and morphological features and no occurrence of autotomy and significantly lower expression of pain markers compared to the no-capping group. The gene expression of RhoA was consistently in a higher level in the capping group within 8 weeks after surgery. This study shows that capping technique will alter the regeneration state of transected nerves and reduce painful neuroma formation, indicating a promising approach for the treatment of painful neuroma. The initiation of the “regenerative brake” induced by structural as well as morphological improvements in the severed nerve is theorized to be most likely a key mechanism for the capping technique in the prevention of painful neuroma formation.
PMCID: PMC3976365  PMID: 24705579
16.  Rapamycin Regulates iTreg Function through CD39 and Runx1 Pathways 
Journal of Immunology Research  2014;2014:989434.
It has been shown that rapamycin is able to significantly increase the expression of FoxP3 and suppress activity in induced Treg (iTreg) cells in vivo and in vitro. CD39 is a newly determined Treg marker that relates to cell suppression. Runx1, a regulator of FoxP3, controls the expression of adenosine deaminase (ADA) gene, which is found recently in the downstream of CD39 pathway in trophoblast cells. Whether rapamycin would influence CD39 pathway and regulate the expression of Runx1 remains to be determined. The addition of rapamycin to human CD4+ naïve cells in the presence of IL-2, TGF-β promotes the expression of FoxP3. In this paper, we found that CD39 positively correlated with the FoxP3 expression in iTreg cells. Rapamycin induced iTreg cells showed a stronger CD39/Runx1 expression with the enhanced suppressive function. These data suggested that CD39 expression was involved in iTreg generation and the enhanced suppressive ability of rapamycin induced Treg was partly due to Runx1 pathway. We conclude that rapamycin favors CD39/Runx1 expression in human iTreg and provides a novel insight into the mechanisms of iTreg generation enhanced by rapamycin.
PMCID: PMC3988749  PMID: 24741640
17.  DNA targeting specificity of RNA-guided Cas9 nucleases 
Nature biotechnology  2013;31(9):827-832.
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of singleguide RNAs (sgRNAs) to enable genome editing1–10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
PMCID: PMC3969858  PMID: 23873081
18.  One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering 
Cell  2013;153(4):910-918.
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.
PMCID: PMC3969854  PMID: 23643243
19.  Parvalbumin neurons and gamma rhythms enhance cortical circuit performance 
Nature  2009;459(7247):698-702.
Synchronized oscillations and inhibitory interneurons have important and interconnected roles within cortical microcircuits. In particular, interneurons defined by the fast-spiking phenotype and expression of the calcium-binding protein parvalbumin1,2 have been suggested to be involved in gamma (30–80 Hz) oscillations3–7, which are hypothesized to enhance information processing8,9. However, because parvalbumin interneurons cannot be selectively controlled, definitive tests of their functional significance in gamma oscillations, and quantitative assessment of the impact of parvalbumin interneurons and gamma oscillations on cortical circuits, have been lacking despite potentially enormous significance (for example, abnormalities in parvalbumin interneurons may underlie altered gamma-frequency synchronization and cognition in schizophrenia10 and autism11). Here we use a panel of optogenetic technologies12–14 in mice to selectively modulate multiple distinct circuit elements in neocortex, alone or in combination. We find that inhibiting parvalbumin interneurons suppresses gamma oscillations in vivo, whereas driving these interneurons (even by means of non-rhythmic principal cell activity) is sufficient to generate emergent gamma-frequency rhythmicity. Moreover, gamma-frequency modulation of excitatory input in turn was found to enhance signal transmission in neocortex by reducing circuit noise and amplifying circuit signals, including inputs to parvalbumin interneurons. As demonstrated here, optogenetics opens the door to a new kind of informational analysis of brain function, permitting quantitative delineation of the functional significance of individual elements in the emergent operation and function of intact neural circuitry.
PMCID: PMC3969859  PMID: 19396159
20.  Genome engineering using the CRISPR-Cas9 system 
Nature protocols  2013;8(11):2281-2308.
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. to minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1–2 weeks, and modified clonal cell lines can be derived within 2–3 weeks.
PMCID: PMC3969860  PMID: 24157548
21.  Apoptosis of THP-1 Derived Macrophages Induced by Sonodynamic Therapy Using a New Sonosensitizer Hydroxyl Acetylated Curcumin 
PLoS ONE  2014;9(3):e93133.
Curcumin is extracted from the rhizomes of the traditional Chinese herb Curcuma longa. Our previous study indicated curcumin was able to function as a sonosensitizer. Hydroxyl acylated curcumin was synthesized from curcumin to eliminate the unstable hydroxy perssad in our group. The potential use of Hydroxyl acylated curcumin as a sonosensitizer for sonodynamic therapy (SDT) requires further exploration. This study investigated the sonodynamic effect of Hydroxyl acylated curcumin on THP-1 macrophage. THP-1 macrophages were cultured with Hydroxyl acylated curcumin at a concentration of 5.0 μg/mL for 4 hours and then exposed to pulse ultrasound irradiation (0.5 W/cm2 with 1.0 MHz ) for 5 min, 10 min and 15 min. Six hours later, cell viability decreased significantly by CCK-8 assay. After ultrasound irradiation, the ratio of apoptosis and necrosis in SDT group was higher than that in control, Hydroxyl acylated curcumin alone and ultrasound alone. Moreover, the apoptotic rate was higher than necrotic rate with the flow cytometry analysis. Furthermore, Hydroxyl acylated curcumin-SDT induced reactive oxygen species (ROS) generation in THP-1 macrophages immediately after the ultrasound treatment while ROS generation was reduced significantly with the scavenger of singlet oxygen Sodium azide (NaN3). Hydroxyl acylated curcumin-SDT led to a conspicuous loss of mitochondrial membrane potential (MMP) compared with other groups, while MMP was increased significantly with the scavenger of singlet oxygen Sodium azide (NaN3), ROS inhibitor N-acetyl cysteine (NAC) and Mitochondrial Permeability Transition Pore (MPTP) inhibitor Cyclosporin A (CsA). The cytochrome C, cleaved-Caspase-9, cleaved-Caspase-3 and cleaved-PARP upregulated after SDT through Western blotting. These findings suggested that Hydroxyl acylated curcumin under low-intensity ultrasound had sonodynamic effect on THP-1 macrophages via generation of intracellular singlet oxygen and mitochondria-caspase signaling pathway, indicating that Hydroxyl acylated curcumin could be used as a novel sonosensitizer in SDT for atherosclerosis.
PMCID: PMC3968056  PMID: 24676101
22.  Genome-wide association studies for hematological traits in Chinese Sutai pigs 
BMC Genetics  2014;15:41.
It has been shown that hematological traits are strongly associated with the metabolism and the immune system in domestic pig. However, little is known about the genetic architecture of hematological traits. To identify quantitative trait loci (QTL) controlling hematological traits, we performed single marker Genome-wide association studies (GWAS) and haplotype analysis for 15 hematological traits in 495 Chinese Sutai pigs.
We identified 161 significant SNPs including 44 genome-wide significant SNPs associated with 11 hematological traits by single marker GWAS. Most of them were located on SSC2. Meanwhile, we detected 499 significant SNPs containing 154 genome-wide significant SNPs associated with 9 hematological traits by haplotype analysis. Most of the identified loci were located on SSC7 and SSC9.
We detected 4 SNPs with pleiotropic effects on SSC2 by single marker GWAS and (or) on SSC7 by haplotype analysis. Furthermore, through checking the gene functional annotations, positions and their expression variation, we finally selected 7 genes as potential candidates. Specially, we found that three genes (TRIM58, TRIM26 and TRIM21) of them originated from the same gene family and executed similar function of innate and adaptive immune. The findings will contribute to dissection the immune gene network, further identification of causative mutations underlying the identified QTLs and providing insights into the molecular basis of hematological trait in domestic pig.
PMCID: PMC3986688  PMID: 24674592
Single marker GWAS; Haplotype analysis; Hematological traits; Pig
23.  Nanoroughened Surfaces for Efficient Capture of Circulating Tumor Cells without Using Capture Antibodies 
ACS nano  2012;7(1):566-575.
Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers. Here we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilized the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.
PMCID: PMC3962680  PMID: 23194329
Cancer; circulating tumor cell; nanotopography; biomaterials; microfabrication
24.  RNF111-dependent neddylation activates DNA damage-induced ubiquitination 
Molecular cell  2013;49(5):897-907.
Ubiquitin-like proteins have been shown to be covalently conjugated to targets. However, the functions of these ubiquitin-like proteins are largely unknown. Here, we have screened most known ubiquitin-like proteins after DNA damage and found that NEDD8 is involved in the DNA damage response. Following various DNA damage stimuli, NEDD8 accumulated at DNA damage sites, and this accumulation was dependent on an E2 enzyme UBE2M and an E3 ubiquitin ligase RNF111. We further found that histone H4 was polyneddylated in response to DNA damage, and NEDD8 was conjugated to the N-terminal lysine residues of H4. Interestingly, the DNA damage-induced polyneddylation chain could be recognized by the MIU (Motif Interacting with Ubiquitin) domain of RNF168. Loss of DNA damage-induced neddylation negatively regulated DNA damage-induced foci formation of RNF168 and its downstream functional partners, such as 53BP1 and BRCA1, thus affecting the normal DNA damage repair process.
PMCID: PMC3595365  PMID: 23394999
25.  Adenosine Triphosphate (ATP) Is a Candidate Signaling Molecule in the Mitochondria-to-Nucleus Retrograde Response Pathway 
Genes  2013;4(1):86-100.
Intracellular communication from the mitochondria to the nucleus is achieved via the retrograde response. In budding yeast, the retrograde response, also known as the RTG pathway, is regulated positively by Rtg1, Rtg2, Rtg3 and Grr1 and negatively by Mks1, Lst8 and two 14-3-3 proteins, Bmh1/2. Activation of retrograde signaling leads to activation of Rtg1/3, two basic helix-loop-helix leucine zipper transcription factors. Rtg1/3 activation requires Rtg2, a cytoplasmic protein with an N-terminal adenosine triphosphate (ATP) binding domain belonging to the actin/Hsp70/sugar kinase superfamily. The critical regulatory step of the retrograde response is the interaction between Rtg2 and Mks1. Rtg2 binds to and inactivates Mks1, allowing for activation of Rtg1/3 and the RTG pathway. When the pathway is inactive, Mks1 has dissociated from Rtg2 and bound to Bmh1/2, preventing activation of Rtg1/3. What signals association or disassociation of Mks1 and Rtg2 is unknown. Here, we show that ATP at physiological concentrations dissociates Mks1 from Rtg2 in a highly cooperative fashion. We report that ATP-mediated dissociation of Mks1 from Rtg2 is conserved in two other fungal species, K. lactis and K. waltii. Activation of Rtg1/3 upregulates expression of genes encoding enzymes catalyzing the first three reactions of the Krebs cycle, which is coupled to ATP synthesis through oxidative phosphorylation. Therefore, we propose that the retrograde response is an ATP homeostasis pathway coupling ATP production with ATP-mediated repression of the retrograde response by releasing Mks1 from Rtg2.
PMCID: PMC3899953  PMID: 24605246
retrograde response; mitochondria to nucleus signaling; Rtg2; Mks1; ATP sensing; Saccharomyces cerevisiae; K. lactis; K. waltii

Results 1-25 (302)