The Zinc-Finger Protein ZCCHC3 Binds RNA and Facilitates Viral RNA Sensing and Activation of the RIG-I-like Receptors.

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiates innate antiviral immune response. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified ZCCHC3 as a positive regulator of the RLRs including RIG-I and MDA5. ZCCHC3 deficiency markedly inhibited RNA virus-triggered induction of downstream antiviral genes, and ZCCHC3-deficient mice were more susceptible to RNA virus infection. ZCCHC3 was associated with RIG-I and MDA5 and functions in two distinct processes for regulation of RIG-I and MDA5 activities. ZCCHC3 bound to dsRNA and enhanced the binding of RIG-I and MDA5 to dsRNA. ZCCHC3 also recruited the E3 ubiquitin ligase TRIM25 to the RIG-I and MDA5 complexes to facilitate its K63-linked polyubiquitination and activation. Thus, ZCCHC3 is a co-receptor for RIG-I and MDA5, which is critical for RLR-mediated innate immune response to RNA virus.

ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response.

The retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated gene 5 (MDA5), sense cytoplasmic viral RNA and initiate innate antiviral responses. How RIG-I and MDA5 are differentially regulated remains enigmatic. In this study, we identified the guanylate-binding protein (GBP) and zinc-finger FYVE domain-containing protein ZFYVE1 as a negative regulator of MDA5- but not RIG-I-mediated innate antiviral responses. ZFYVE1-deficiency promoted MDA5- but not RIG-I-mediated transcription of downstream antiviral genes. Comparing to wild-type mice, Zfyve1-/- mice were significantly protected from lethality induced by encephalomyocarditis virus (EMCV) that is sensed by MDA5, whereas Zfyve1-/- and Zfyve1+/+ mice were comparable to death induced by vesicular stomatitis virus (VSV) that is sensed by RIG-I. Mechanistically, ZFYVE1 interacted with MDA5 but not RIG-I. ZFYVE1 bound to viral RNA and decreased the ligand binding and oligomerization of MDA5. These findings suggest that ZFYVE1 acts as a specific negative regulator of MDA5-mediated innate immune responses by inhibiting its ligand binding and oligomerization.

SNX8 modulates innate immune response to DNA virus by mediating trafficking and activation of MITA.

MITA (also called STING) is a central adaptor protein in innate immune response to cytosolic DNA. Cellular trafficking of MITA from the ER to perinuclear microsomes after DNA virus infection is critical for MITA activation and onset of innate antiviral response. Here we found that SNX8 is a component of DNA-triggered induction of downstream effector genes and innate immune response. Snx8-/- mice infected with the DNA virus HSV-1 exhibited lower serum cytokine levels and higher viral titers in the brains, resulting in higher lethality. Mechanistically, SNX8 recruited the class III phosphatylinositol 3-kinase VPS34 to MITA, which is required for trafficking of MITA from the ER to perinuclear microsomes. Our findings suggest that SNX8 is a critical component in innate immune response to cytosolic DNA and DNA virus.

An engineered mouse embryonic stem cell model with survivin as a molecular marker and EGFP as the reporter for high throughput screening of embryotoxic chemicals in vitro.

Embryonic stem cell test (EST) is the only generally accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of EST for regulatory embryotoxicity screening are impeded by its technical complexity, long testing period, and limited endpoint data. In this study, a high throughput embryotoxicity screening based on mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by a human survivin promoter and a human cytomegalovirus promoter, respectively, was developed. These EGFP expressing mESCs were cultured in three-dimensional (3D) fibrous scaffolds in microbioreactors on a multiwell plate with EGFP fluorescence signals as cell responses to chemicals monitored noninvasively in a high throughput manner. Nine chemicals with known developmental toxicity were used to validate the survivin-based embryotoxicity assay, which showed that strongly embryotoxic compounds such as 5-fluorouracil, retinoic acid, and methotrexate downregulated survivin expression by more than 50% in 3 days, while weakly embryotoxic compounds such as boric acid, methoxyacetic acid, and tetracyclin showed modest downregulation effect and nonembryotoxic saccharin, penicillin G, and acrylamide had negligible downregulation effect on survivin expression, confirming that survivin can be used as a molecular endpoint for high throughput screening of embryotoxicants. The potential developmental toxicity of three Chinese herbal medicines were also evaluated using this assay, demonstrating its application in in vitro developmental toxicity test for drug safety assessment.

ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3.

Toll-like receptor 3 (TLR3)-mediated signaling is important for host defense against RNA virus. Upon viral RNA stimulation, toll and interleukin-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) is recruited to TLR3 and then undergoes oligomerization, which is required for the recruitment of downstream molecules to transmit signals. Here, we identified zinc finger CCHC-type containing 3 (ZCCHC3) as a positive regulator of TLR3-mediated signaling. Overexpression of ZCCHC3 promoted transcription of downstream antiviral genes stimulated by the synthetic TLR3 ligand poly(I:C). ZCCHC3-deficiency markedly inhibited TLR3- but not TLR4-mediated induction of type I interferons (IFNs) and proinflammatory cytokines. Zcchc3-/- mice were more resistant to poly(I:C)- but not lipopolysaccharide-induced inflammatory death. Mechanistically, ZCCHC3 promoted recruitment of TRIF to TLR3 after poly(I:C) stimulation. Our findings reveal that ZCCHC3 plays an important role in TLR3-mediated innate immune response by promoting the recruitment of TRIF to TLR3 after ligand stimulation.

SNX8 modulates the innate immune response to RNA viruses by regulating the aggregation of VISA.

The mitochondrial virus-induced signaling adaptor (VISA, also called mitochondrial antiviral signaling, MAVS) protein is a central adaptor in the innate immune response to cytosolic viral RNA. Viral infection causes the aggregation of VISA, which is important for its recruitment of downstream signaling components. How VISA aggregation is regulated remains unknown. Here, we found that sorting nexin 8 (SNX8) is a positive regulator of the RNA virus-triggered induction of downstream effector genes and innate immune response. The brains and lungs of Snx8-/- mice infected with RNA viruses exhibited lower serum cytokine levels and higher viral titers than those of wild-type mice, resulting in higher lethality. Mechanistically, viral infection induced the translocation of SNX8 from the cytosol to mitochondria and its increased association with VISA, leading to VISA aggregation, its recruitment of downstream signaling components and the induction of downstream antiviral genes. Our findings suggest that SNX8 is a critical component of the RIG-I-like receptor (RLR)-mediated innate immune response by modulating VISA aggregation and activation.

A fluorescent 3D cell culture assay for high throughput screening of cancer drugs down-regulating survivin.

Survivin, a member of inhibitor of apoptosis family, is currently undergoing intensive investigations as a promising cancer marker due to its overexpression in multiple tumor tissues and close relationship with chemotherapy resistance. In this study, a novel 3D survivin promoter assay was developed, using enhanced green fluorescent protein (EGFP) as the reporter to assess survivin promoter activity for cancer drug screening. Breast cancer MCF-7 cells were engineered to express EGFP controlled by a human survivin promoter and a CMV promoter, respectively. These cells were cultured in three-dimensional (3D) polymer-based scaffolds on a 40-microbioreactor platform (40-MBR) with real-time monitoring of EGFP signals. The EGFP production driven by the survivin promoter was strongly correlated with survivin transcriptional level in MCF-7 cells treated with YM155, a small-molecule survivin promoter suppressant. Moreover, the potential inhibition effects of doxorubicin and cisplatin on survivin and their cytotoxicity were also evaluated in this system. This study demonstrated the potential application of the novel 3D survivin promoter-EGFP reporter assay for high-throughput screening of chemicals down-regulating survivin as a molecular target for cancer therapy.

ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response.

Cyclic GMP-AMP synthase (cGAS) senses double-strand (ds) DNA in the cytosol and then catalyzes synthesis of the second messenger cGAMP, which activates the adaptor MITA/STING to initiate innate antiviral response. How cGAS activity is regulated remains enigmatic. Here, we identify ZCCHC3, a CCHC-type zinc-finger protein, as a positive regulator of cytosolic dsDNA- and DNA virus-triggered signaling. We show that ZCCHC3-deficiency inhibits dsDNA- and DNA virus-triggered induction of downstream effector genes, and that ZCCHC3-deficient mice are more susceptible to lethal herpes simplex virus type 1 or vaccinia virus infection. ZCCHC3 directly binds to dsDNA, enhances the binding of cGAS to dsDNA, and is important for cGAS activation following viral infection. Our results suggest that ZCCHC3 is a co-sensor for recognition of dsDNA by cGAS, which is important for efficient innate immune response to cytosolic dsDNA and DNA virus.

Efficacy and safety of late-course hypofractionated radiation therapy for muscle-invasive bladder carcinoma after bladder-conserving surgery.

AIM: To evaluate the efficacy and safety of late-course hypofractionated radiation treatment of muscle-invasive bladder carcinoma after bladder-conserving surgery. METHODS: Seventy-six patients with transitional cell bladder carcinoma, stage II (T2-4N0M0), after transurethral resection, were enrolled. Pirarubicin was given at 30 mg/m2 and 100 mL physiological saline once weekly (QW) for 12 weeks through and after intravesical instillation postoperatively. Radiation schedule delivered 46 Gy in 20 fractions for planning target volume, with an additional 20 Gy in five fractions for gross tumor volume as late-course radiation. Chemotherapy was stopped if Radiation Therapy Oncology Group grade 3 or higher bladder or bowel toxicity occurred. The primary end points were acute toxicity, local control and patients' survival. RESULTS: One-, three- and five-year overall survival rates were 98, 78 and 69.5%, respectively. Mean survival time was 58.4 months (95% CI: 52.6, 64.2). In addition, 1-, 3- and 5-year local control rates were 100, 80.5 and 76.1%, respectively. Mean local control time was 60.7 months (95% CI: 55.1, 66.3). The cumulative incidence of local/regional failure and distant failure was 28.9%. The rate of single local/regional failure was 13.2%, but distant failure rate was 21.1%. CONCLUSIONS: Concurrent pirarubicin-based late-course hypofractionated radiation therapy showed desirable local control rate and acceptable toxicity. It could be used after bladder-conserving surgery to allow patients to preserve their bladder.

Multiwalled carbon nanotube-coated polyethylene terephthalate fibrous matrices for enhanced neuronal differentiation of mouse embryonic stem cells.

The next-generation of tissue scaffolds should incorporate 3-D structures with nanofeatures. Fibrous polyethylene terephthalate (PET) matrices have been widely studied as tissue engineering scaffolds, but their performance is limited by the lack of nanotopography. Carbon nanotubes can provide nanoscale structures similar to those present in natural extracellular matrices in vivo, but are difficult to use as 3-D scaffolds for tissue engineering. In this study, multiwalled carbon nanotubes (MWCNTs) were used to coat and provide nanofeatures on the surface of PET membranes and fibers. The effects of MWCNTs on the cellular functions of mouse embryonic stem (mES) cells cultured on the nanoengineered PET surface and 3-D scaffolds were investigated. In general, MWCNTs promoted cell adhesion and proliferation of mES cells while maintaining excellent cell viability in the growth medium. Neuronal differentiation was also significantly enhanced when cells were cultured in the differentiation medium. Different cell morphologies were observed in the presence of MWCNTs. Cells were stretched and well spread out in MWCNT-coated PET scaffolds, whereas cells were sporadically distributed in non-coated PET scaffolds. Furthermore, more filopodia were formed in MWCNT-coated PET matrices, suggesting increased interactions between scaffolds and cells. Also, neuronal cells differentiated from mES cells in the 3-D nanoengineered PET scaffolds formed extensive nerve networks around each fiber and neurite bridges between fibers. These findings suggest that MWCNTs can provide nanofeatures on PET fibrous matrices and enhance their performance as 3-D nanoengineered tissue engineering scaffolds.

Cell-based screening of traditional Chinese medicines for proliferation enhancers of mouse embryonic stem cells.

A high-throughput cell-based method was developed for screening traditional Chinese herbal medicines (TCHMs) for potential stem cell growth promoters. Mouse embryonic stem (mES) cells expressing enhanced green fluorescent protein (EGFP) were cultured in growth media supplemented with various TCHM extracts. The dosage-dependent effects of TCHM extracts on cell growth, including proliferation and cytotoxicity, were assessed via EGFP fluorescence measurement. Seven TCHMs were investigated, and among them Panax notoginseng (PN), Rhizoma Atractylodis macrocephalae, Rhizoma chuanxiong, and Ganoderma lucidum spores (GLS) showed potential to improve mES cell proliferation. Eleven mixtures of these four TCHMs were then studied, and the results showed that the mixture of PN and GLS had the strongest growth promoting effect, increasing the specific growth rate of mES cells by 29.5% at a low dosage of 0.01% (wt/vol) PN/GLS (P<0.01) and 34.2% at 0.1% (wt/vol) PN/GLS (P<0.05) compared to the control. The growth promoting effect of PN/GLS was further confirmed with ES cells cultured in spinner flasks. A 29.3-fold increase in the total cell number was achieved in the medium supplemented with 0.01% PN/GLS after 5 days, while the control culture only gave a 16.8-fold increase. This cell-based screening method thus can provide an efficient and high-throughput way to explore potential stem cell growth promoters from TCHMs.

Engineering stem cell niches in bioreactors.

Stem cells, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells and amniotic fluid stem cells have the potential to be expanded and differentiated into various cell types in the body. Efficient differentiation of stem cells with the desired tissue-specific function is critical for stem cell-based cell therapy, tissue engineering, drug discovery and disease modeling. Bioreactors provide a great platform to regulate the stem cell microenvironment, known as "niches", to impact stem cell fate decision. The niche factors include the regulatory factors such as oxygen, extracellular matrix (synthetic and decellularized), paracrine/autocrine signaling and physical forces (i.e., mechanical force, electrical force and flow shear). The use of novel bioreactors with precise control and recapitulation of niche factors through modulating reactor operation parameters can enable efficient stem cell expansion and differentiation. Recently, the development of microfluidic devices and microbioreactors also provides powerful tools to manipulate the stem cell microenvironment by adjusting flow rate and cytokine gradients. In general, bioreactor engineering can be used to better modulate stem cell niches critical for stem cell expansion, differentiation and applications as novel cell-based biomedicines. This paper reviews important factors that can be more precisely controlled in bioreactors and their effects on stem cell engineering.