Isolation and Characterization of Paenibacillus polymyxa B7 and Inhibition of Aspergillus tubingensis A1 by Its Antifungal Substances.

Screening of Bacillus with antagonistic effects on paddy mold pathogens to provide strain resources for biological control of mold in Oryza sativa L. screening of Bacillus isolates antagonistic towards Aspergillus tubingensis from rhizosphere soil of healthy paddy; classification and identification of antagonistic strains by biological characteristics and 16S rDNA sequence analysis; transcriptome sequencing after RNA extraction from Bacillus-treated Aspergillus tubingensis; and extraction of inhibitory crude proteins of Bacillus by ammonium sulfate precipitation; inhibitory crude protein and Bacillus spp. were treated separately for A. tubingensis and observed by scanning electron microscopy (SEM). An antagonistic strain of Bacillus, named B7, was identified as Paenibacillus polymyxa by 16S rDNA identification and phylogenetic evolutionary tree comparison analysis. Analysis of the transcriptome results showed that genes related to secondary metabolite biosynthesis such as antifungal protein were significantly downregulated. SEM results showed that the mycelium of A. tubingensis underwent severe rupture after treatment with P. polymyxa and antifungal proteins, respectively. In addition, the sporocarp changed less after treatment with P. polymyxa, and the sporangium stalks had obvious folds. P. polymyxa B7 has a good antagonistic effect against A. tubingensis and has potential for biocontrol applications of paddy mold pathogens.

Mitochondrial replication, transcription, and function in obstructive sleep apnea.

We assessed mitochondrial replication, transcription, and function in the upper airways of obstructive sleep apnea (OSA) patients and the effects of uvulopalatopharyngoplasty. Twenty subjects with mild and 40 with moderate to severe OSA requiring uvulopalatopharyngoplasty were included. Mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in uvula specimens were assessed by immunohistochemical staining, and their mRNA and protein expression was examined using reverse-transcription polymerase chain reaction and western blotting, respectively. The mitochondrial to nuclear DNA (Mt/N) ratio in the blood, exhaled breath condensate (EBC), and uvula was measured using quantitative reverse-transcription polymerase chain reaction. TFAM and PGC-1α protein concentrations in the plasma and EBC were determined using enzyme-linked immunosorbent assay. All tested parameters were higher in the OSA group than in the control. Three months later, 21 uvulopalatopharyngoplasty-responsive patients with OSA showed decreased TFAM and PGC-1α concentrations and EBC Mt/N ratio while these remained high in 19 uvulopalatopharyngoplasty-unresponsive patients. The OSA group showed severe inflammation, increased mitochondrial replication and transcription-related signaling, and mitochondrial dysfunction in the uvula. Successful OSA treatment using uvulopalatopharyngoplasty restored the TFAM and PGC-1α levels and EBC Mt/N ratio.

Continuous positive airway pressure affects mitochondrial function and exhaled PGC1-α levels in obstructive sleep apnea.

Purpose: Subjects with obstructive sleep apnea (OSA) exhibit systemic and upper airway oxidative stress and inflammation, which cause mitochondrial dysfunction. The intend of this study is to estimate mitochondrial function (mitochondrial DNA/nuclear DNA [Mt/N] ratio) and protein levels of peroxisome proliferator-coactivated receptor gamma co-activator 1-alpha (PGC1-α) in the exhaled breath condensate (EBC) and plasma before and after continuous positive airway pressure (CPAP) treatment. Materials and methods: Twenty healthy individuals (control) and 40 subjects with severe or moderate OSA were recruited to undergo CPAP treatment and evaluation in a sleep study. The Mt/N ratio in the EBC and blood were assayed by quantitative real-time polymerase chain reaction. Enzyme-linked immunosorbent assay was used to measure the protein concentration of PGC1-α in the EBC and plasma. All experiments were performed after 3 months of CPAP treatment in subjects with OSA. Results: We observed no noteworthy differences between the control and treatment groups. Moreover, there were no differences in the Mt/N ratio in the blood and plasma levels of PGC1-α in subjects with OSA before and after treatment. However, the Mt/N ratio and protein levels of PGC1-α in the EBC of OSA subjects were higher than those in the control group and returned to normal levels after CPAP treatment. Conclusions: We successfully treated subjects with OSA by CPAP, which restored the Mt/N ratio and levels of PGC1-α in the EBC.

Polyplex nanomicelle delivery of self-amplifying RNA vaccine.

Self-amplifying RNA (SaRNA) is a burgeoning platform that exploits the replication machinery of alphaviruses such as Venezuelan equine encephalitis (VEE) virus or Sindbis virus (SIN). SaRNA has been used for development of human vaccines, but has not been evaluated for porcine vaccine development. Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the worldwide pork industry, but current vaccines trigger delayed neutralizing antibody response and confer only partial protection. Here we first compared two SaRNA systems based on VEE and SIN, and demonstrated that in vitro transcribed VEE-based SaRNA conferred prolonged reporter gene expression and RNA amplification in pig cells with low cytotoxicity, but SIN-based SaRNA imparted evident cytotoxicity and limited gene expression in pig cells. Transfection of VEE-based SaRNA that encodes the major PRRSV antigen dNGP5 (SaRNA-dNGP5) conferred persistent expression for at least 28 days in pig cells. We next complexed SaRNA-dNGP5 with the polyaspartamide block copolymer PEG-PAsp(TEP) to form polyplex nanomicelle with high packaging efficiency and narrow size distribution. The polyplex nanomicelle enabled sustained dNGP5 expression and secretion in vitro. Compared with the commercial PRRS vaccine, nanomicelle delivery of SaRNA-dNGP5 into animal models accelerated the induction of potent neutralizing antibodies with minimal side effects, and elicited stronger IL-4 and IFN-γ responses against homologous and heterologous PRRSV. These properties tackle the problems of current vaccines and implicate the potential of SaRNA-dNGP5 nanomicelle as an effective PRRS vaccine.

Effects of continuous positive airway pressure on resolvin and matrix metalloproteinase-9 in patients with obstructive sleep apnea.

PURPOSE: Resolvin is a checkpoint controller in inflammation. Matrix metalloproteinase-9 (MMP-9) is an airway remodeling regulator. We evaluated the levels of resolvin and MMP-9 protein in the serum and exhaled breath condensate (EBC) before and after continuous positive airway pressure (CPAP) treatment. METHOD: We enrolled 20 non-OSA snorers and 40 patients with moderate to severe OSA scheduled for CPAP treatment. ELISA was used to assess resolvin and MMP-9 levels in the serum and EBC. All patients underwent sleep assessment at baseline and 3 months after CPAP. RESULTS: There was no between-group difference; moreover, there were no differences in the pre- and post-treatment serum levels of resolvin and MMP-9 in patients with OSA. Compared with non-OSA snorers, patients with OSA had lower resolvin and higher MMP-9 levels in the EBC. After CPAP treatment, the EBC levels of resolvin and MMP-9 in patients with OSA returned to normal. CONCLUSIONS: Successful OSA treatment by CPAP can normalize EBC levels of resolvin and MMP-9.

CRISPR-Cas13d for Gene Knockdown and Engineering of CHO Cells.

Chinese hamster ovary (CHO) cells are the predominant cell chassis for biopharmaceutical production. Engineering cellular pathways related to cell death, metabolism, and glycosylation in CHO cells is desired but challenging. Here, we present a novel approach that exploits CRISPR-Cas13d for gene silencing and CHO cell engineering. CRISPR-Cas13d is a burgeoning system that exploits Cas13d nuclease and guide RNA (gRNA) for RNA cleavage and gene knockdown. We first showed that CRISPR-Cas13d effectively knocked down exogenous genes in CHO cell lines (K1, DG44, and DUXB11) commonly used for recombinant protein production. We next demonstrated that CRISPR-Cas13d robustly suppressed the expression of exogenous genes and various endogenous genes involved in gene amplification, apoptosis, metabolism, and glycosylation (e.g., GS, BAK, BAX, PDK1, and FUT8) in CHO cells with efficiencies ranging from 60% to 80%, simply by transient transfection. By integrating the entire CRISPR-Cas13d system with the Sleeping Beauty system and optimal gRNA design, we further improved the knockdown efficiency and rapidly generated stable cells with ≈80%-90% knockdown. With this approach, we knocked down FUT8 expression for >90% and significantly attenuated the IgG fucosylation. These data altogether implicated the potentials of CRISPR-Cas13d for gene regulation, glycoengineering, and cell engineering of CHO cells.

CRISPR interference-mediated noggin knockdown promotes BMP2-induced osteogenesis and calvarial bone healing.

Healing of large calvarial bone defects remains a challenging task in the clinical setting. Although BMP2 (bone morphogenetic protein 2) is a potent growth factor that can induce bone repair, BMP2 provokes the expression of antagonist Noggin that self-restricts its bioactivity. CRISPR interference (CRISPRi) is a technology for programmable gene suppression but its application in regenerative medicine is still in its infancy. We reasoned that Nog inhibition, concurrent with BMP2 overexpression, can promote the osteogenesis of adipose-derived stem cells (ASC) and improve calvarial bone healing. We designed and exploited a hybrid baculovirus (BV) system for the delivery of BMP2 gene and CRISPRi system targeting Nog. After BV-mediated co-delivery into ASC, the system conferred prolonged BMP2 expression and stimulated Nog expression while the CRISPRi system effectively repressed Nog upregulation for at least 14 days. The CRISPRi-mediated Nog knockdown, along with BMP2 overexpression, additively stimulated the osteogenic differentiation of ASC. Implantation of the CRISPRi-engineered ASC into the critical size defects at the calvaria significantly enhanced the calvarial bone healing and matrix mineralization. These data altogether implicate the potentials of CRISPRi-mediated gene knockdown for cell fate regulation and tissue regeneration.

Effects of continuous positive airway pressure on exhaled transforming growth factor-ß and vascular endothelial growth factor in patients with obstructive sleep apnea.

BACKGROUND: Both transforming growth factor ß (TGF-ß) and vascular endothelial growth factor (VEGF) are master regulators of airway remodeling; however, their pathological roles in obstructive sleep apnea (OSA) remain unclear. The aim of the present study was to evaluate the expression of TGF-ß and VEGF protein in the serum and exhaled breath condensate (EBC) before and after continuous positive airway pressure (CPAP) treatment in OSA patients. METHODS: Forty patients with moderate to severe OSA requiring CPAP and 20 healthy subjects were prospectively recruited. The concentrations of TGF-ß and VEGF protein in the serum and EBC were evaluated by enzyme-linked immunosorbent assay. All OSA patients underwent a sleep study that was repeated 3 months after receiving CPAP therapy. RESULTS: Protein concentrations of TGF-ß and VEGF in the serum did not differ between healthy controls and OSA patients before CPAP treatment. There was also no difference in the serum protein concentrations of TGF-ß and VEGF of the OSA patients before and after CPAP treatment. However, both the TGF-ß and VEGF protein concentrations in the EBC were higher in the OSA patients than those in control subjects, and recovered to normal levels after CPAP. CONCLUSIONS: Successful treatment of OSA by CPAP can restore the TGF-ß and VEGF protein concentrations in the EBC.

Coactivation of Endogenous Wnt10b and Foxc2 by CRISPR Activation Enhances BMSC Osteogenesis and Promotes Calvarial Bone Regeneration.

CRISPR activation (CRISPRa) is a burgeoning technology for programmable gene activation, but its potential for tissue regeneration has yet to be fully explored. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into osteogenic or adipogenic pathways, which are governed by the Wnt (Wingless-related integration site) signaling cascade. To promote BMSC differentiation toward osteogenesis and improve calvarial bone healing by BMSCs, we harnessed a highly efficient hybrid baculovirus vector for gene delivery and exploited a synergistic activation mediator (SAM)-based CRISPRa system to activate Wnt10b (that triggers the canonical Wnt pathway) and forkhead c2 (Foxc2) (that elicits the noncanonical Wnt pathway) in BMSCs. We constructed a Bac-CRISPRa vector to deliver the SAM-based CRISPRa system into rat BMSCs. We showed that Bac-CRISPRa enabled CRISPRa delivery and potently activated endogenous Wnt10b and Foxc2 expression in BMSCs for >14 days. Activation of Wnt10b or Foxc2 alone was sufficient to promote osteogenesis and repress adipogenesis in vitro. Furthermore, the robust and prolonged coactivation of both Wnt10b and Foxc2 additively enhanced osteogenic differentiation while inhibiting adipogenic differentiation of BMSCs. The CRISPRa-engineered BMSCs with activated Wnt10b and Foxc2 remarkably improved the calvarial bone healing after implantation into the critical-sized calvarial defects in rats. These data implicate the potentials of CRISPRa technology for bone tissue regeneration.

Effects of aminophylline on airway epithelial-mesenchymal transition in brown Norway rats after repeated allergen challenge.

Purpose: Chronic asthma is characterized by airway inflammation and remodeling. The aim of this study is to evaluate the effects of aminophylline on airway epithelial-mesenchymal transition (EMT). Materials and methods: Two experimental groups of brown Norway rats that were repeatedly challenged with aerosolized ovalbumin (OA) were given oral aminophylline (OA-aminophylline group) or saline only (OA-saline group). A third group was challenged by saline as a control. The rats were anesthetized and pulmonary function were performed. Immuno-histochemical staining of epithelial markers (zonula occludens-1 (ZO-1)) and mesenchymal markers (vimentin) in the airway were performed. The protein expressions of ZO-1, E-cadherin, vimentin, fibronectine, TGF-ß1, SMAD 2/3, JNK, and p38 MAPK were examined by western blot. Results: Aminophylline had beneficial effects on airway inflammation, and airway remodeling in the OA-aminophylline group compared to the OA-saline group. The OA-saline group had decreased ZO-1 but increased vimentin according to immuno-histochemical staining. The protein expression indicated decreases in ZO-1 and E-cadherin but increases in vimentin, fibronectine, TGF-ß1, SMAD 2/3, JNK, and p38 MAPK in comparison to the other two groups. The OA-aminophylline group had higher ZO-1 but lower vimentin in immuno-histochemical staining compared to the OA-saline group. The protein expression showed higher ZO-1 and E-cadherin but lower vimentin, fibronectine, TGF-ß1, SMAD 2/3, JNK, and p38 MAPK when compared to the OA-saline group. Conclusions: Ovalbumin increases airway remodeling and airway EMT. Aminophylline is effective in preventing airway remodeling and airway EMT in Brown Norway rats after repeated allergen challenge.

CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.

Calvarial bone healing remains difficult but may be improved by stimulating chondrogenesis of implanted stem cells. To simultaneously promote chondrogenesis and repress adipogenesis of stem cells, we built a CRISPRai system that comprised inactive Cas9 (dCas9), two fusion proteins as activation/repression complexes and two single guide RNA (sgRNA) as scaffolds for recruiting activator (sgRNAa) or inhibitor (sgRNAi). By plasmid transfection and co-expression in CHO cells, we validated that dCas9 coordinated with sgRNAa to recruit the activator for mCherry activation and also orchestrated with sgRNAi to recruit the repressor for d2EGFP inhibition, without cross interference. After changing the sgRNA sequence to target endogenous Sox9/PPAR-γ, we packaged the entire CRISPRai system into an all-in-one baculovirus for efficient delivery into rat bone marrow-derived mesenchymal stem cells (rBMSC) and verified simultaneous Sox9 activation and PPAR-γ repression. The activation/inhibition effects were further enhanced/prolonged by using the Cre/loxP-based hybrid baculovirus. The CRISPRai system delivered by the hybrid baculovirus stimulated chondrogenesis and repressed adipogenesis of rBMSC in 2D culture and promoted the formation of engineered cartilage in 3D culture. Importantly, implantation of the rBMSC engineered by the CRISPRai improved calvarial bone healing. This study paves a new avenue to translate the CRISPRai technology to regenerative medicine.

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.

Effect of oral appliance on circulating leukocyte telomere length and SIRT1 in obstructive sleep apnea.

OBJECTIVES: The increased cardiovascular risk seen in patients with obstructive sleep apnea (OSA) may be due to combination of oxidative stress, systemic inflammation and damage to leukocyte telomere length (LTL) seen with aging. Another molecule, Sirtuin 1 (SIRT1), a histone/protein deacetylase, regulates endothelial nitric oxide synthase and is involved in different aspects of cardiovascular disease, aging and stress resistance. The aim of this study was to evaluate the effects of mandibular advancement device (MAD) on the circulating LTL and SIRT1 protein level in peripheral blood mononuclear cells (PBMCs) in patients with OSA. MATERIALS AND METHODS: Forty patients with moderately severe to severe OSA who desired MAD and 20 healthy controls were prospectively enrolled. The LTL was measured by quantitative polymerase chain reaction while SIRT1 protein levels in PBMC was assessed using a Sirtuin 1 ELISA Kit. All study subjects underwent baseline sleep study, with OSA patients having repeat testing at 3 months after MAD. RESULTS: Compared to healthy subjects, patients with OSA at baseline had lower LTL and SIRT1 protein levels in PBMC. After 3 months of MAD, 24 OSA patients, designated as MAD responders, median (range) LTL increased from (0.556 [0.393-0.748]) to (0.708 [0.533-0.893]) and SIRT1 protein levels in PBMC increased from 0.58 ± 0.23 pg/µg of total protein to 0.95 ± 0.26 pg/µg of total protein. For the 16 MAD unresponsive patients, LTL and SIRT1 protein levels remained low. CONCLUSIONS: Successful treatment of OSA with MAD can restore LTL and SIRT1 protein levels in PBMC. CLINICAL RELEVANCE: LTL and SIRT1 protein levels in PBMC can be improved following effective treatment of OSA using MAD.

Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation.

CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel 'hit and run' strategy for in vivo genome editing.

Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells.

Baculovirus (BV) holds promise as a vector for anticancer gene delivery to combat the most common liver cancer-hepatocellular carcinoma (HCC). However, in vivo BV administration inevitably results in BV entry into non-HCC normal cells, leaky anticancer gene expression and possible toxicity. To improve the safety, we employed synthetic biology to engineer BV for transgene expression regulation. We first uncovered that miR-196a and miR-126 are exclusively expressed in HCC and normal cells, respectively, which allowed us to engineer a sensor based on distinct miRNA expression signature. We next assembled a synthetic switch by coupling the miRNA sensor and RNA binding protein L7Ae for translational repression, and incorporated the entire device into a single BV. The recombinant BV efficiently entered HCC and normal cells and enabled cis-acting transgene expression control, by turning OFF transgene expression in normal cells while switching ON transgene expression in HCC cells. Using pro-apoptotic hBax as the transgene, the switch-based BV selectively killed HCC cells in separate culture and mixed culture of HCC and normal cells. These data demonstrate the potential of synthetic switch-based BV to distinguish HCC and non-HCC normal cells for selective transgene expression control and killing of HCC cells.

Graphene oxide sensitizes cancer cells to chemotherapeutics by inducing early autophagy events, promoting nuclear trafficking and necrosis.

Rationale: Cisplatin (CDDP) is a broad-spectrum anticancer drug but chemoresistance to CDDP impedes its wide use for cancer therapy. Autophagy is an event occurring in the cytoplasm and cytoplasmic LC3 puncta formation is a hallmark of autophagy. Graphene oxide (GO) is a nanomaterial that provokes autophagy in CT26 colon cancer cells and confers antitumor effects. Here we aimed to evaluate whether combined use of GO with CDDP (GO/CDDP) overcomes chemoresistance in different cancer cells and uncover the underlying mechanism. Methods: We treated different cancer cells with GO/CDDP and evaluated the cytotoxicity, death mechanism, autophagy induction and nuclear entry of CDDP. We further knocked down genes essential for autophagic flux and deciphered which step is critical to nuclear import and cell death. Finally, we performed immunoprecipitation, mass spectrometry and immunofluorescence labeling to evaluate the association of LC3 and CDDP. Results: We uncovered that combination of GO and CDDP (GO/CDDP) promoted the killing of not only CT26 cells, but also ovarian, cervical and prostate cancer cells. In the highly chemosensitized Skov-3 cells, GO/CDDP significantly enhanced concurrent nuclear import of CDDP and autophagy marker LC3 and elevated cell necrosis, which required autophagy initiation and progression but did not necessitate late autophagy events (e.g., autophagosome completion and autolysosome formation). The GO/CDDP-elicited nuclear trafficking and cell death also required importin α/ß, and LC3 also co-migrated with CDDP and histone H1/H4 into the nucleus. In particular, GO/CDDP triggered histone H4 acetylation in the nucleus, which could decondense the chromosome and enable CDDP to more effectively access chromosomal DNA to trigger cell death. Conclusion: These findings shed light on the mechanisms of GO/CDDP-induced chemosensitization and implicate the potential applications of GO/CDDP to treat multiple cancers.

Enhancing Protein Production Yield from Chinese Hamster Ovary Cells by CRISPR Interference.

Chinese hamster ovary (CHO) cells are an important host for biopharmaceutical production. Generation of stable CHO cells typically requires cointegration of dhfr and a foreign gene into chromosomes and subsequent methotrexate (MTX) selection for coamplification of dhfr and foreign gene. CRISPR interference (CRISPRi) is an emerging system that effectively suppresses gene transcription through the coordination of dCas9 protein and guide RNA (gRNA). However, CRISPRi has yet to be exploited in CHO cells. Here we constructed vectors expressing the functional CRISPRi system and proved effective CRISPRi-mediated suppression of dhfr transcription in CHO cells. We next generated stable CHO cell clones coexpressing DHFR, the model protein (EGFP), dCas9 and gRNA targeting dhfr. Combined with MTX selection, CRISPRi-mediated repression of dhfr imparted extra selective pressure to force CHO cells to coamplify more copies of dhfr and egfp genes. Compared with the traditional method relying on MTX selection (up to 250 nM), the CRISPRi approach increased the dhfr copy number ∼3-fold, egfp copy number ∼3.6-fold and enhanced the EGFP expression ∼3.8-fold, without impeding the cell growth. Furthermore, we exploited the CRISPRi approach to enhance the productivity of granulocyte colony stimulating factor (G-CSF) ∼2.3-fold. Our data demonstrate, for the first time, the application of CRISPRi in CHO cells to enhance recombinant protein production and may pave a new avenue to CHO cell engineering.

Antiangiogenic and antihepatocellular carcinoma activities of the Juniperus chinensis extract.

BACKGROUND: To identify a novel therapeutic agent for hepatocellular carcinoma (HCC), for which no promising therapeutic agent exists, we screened a panel of plants and found that Juniperus chinensis exhibited potential antiangiogenic and anti-HCC activities. We further investigated the antiangiogenic and anti-HCC effects of the active ingredient of J. chinensis extract, CBT-143-S-F6F7, both in vitro and in vivo. METHODS: A tube formation assay conducted using human umbilical vein endothelial cells (HUVECs) was first performed to identify the active ingredient of CBT-143-S-F6F7. A series of angiogenesis studies, including HUVEC migration, Matrigel plug, and chorioallantoic membrane (CAM) assays, were then performed to confirm the effects of CBT-143-S-F6F7 on angiogenesis. The effects of CBT-143-S-F6F7 on tumor growth were investigated using a subcutaneous and orthotopic mouse model of HCC. In vitro studies were performed to investigate the effects of CBT-143-S-F6F7 on the cell cycle and apoptosis in HCC cells. Moreover, protein arrays for angiogenesis and apoptosis were used to discover biomarkers that may be influenced by CBT-143-S-F6F7. Finally, nuclear magnetic resonance analysis was conducted to identify the compounds of CBT-143-S-F6F7. RESULTS: CBT-143-S-F6F7 showed significantly antiangiogenic activity in various assays, including HUVEC tube formation and migration, CAM, and Matrigel plug assays. In in vivo studies, gavage with CBT-143-S-F6F7 significantly repressed subcutaneous Huh7 tumor growth in severe combined immunodeficient (SCID) mice, and prolonged the survival of orthotopic Huh7 tumor-bearing SCID mice (a 40 % increase in median survival duration compared with the vehicle-treated mice). Immunohistochemical staining of subcutaneous Huh7 tumors in CBT-143-S-F6F7-treated mice showed a significantly decrease in the cell cycle regulatory protein cyclin D1, cellular proliferation marker Ki-67, and endothelial marker CD31. CBT-143-S-F6F7 caused arrest of the G2/M phase and induced Huh7 cell apoptosis, possibly contributing to the inhibition of HCC tumors. Protein array analysis revealed that several angiogenic and antiapoptotic factors were suppressed in CBT-143-S-F6F7-treated Huh7 cells. Finally, five compounds from CBT-143-S-F6F7 were identified. CONCLUSIONS: According to these results, we report for the first time the antiangiogenic and anti-HCC activities of CBT-143-S-F6F7, the active fractional extract of J. chinensis. We believe that CBT-143-S-F6F7 warrants further evaluation as a new anti-HCC drug.

Effects of nasal CPAP on exhaled SIRT1 and tumor necrosis factor-α in patients with obstructive sleep apnea.

Exhaled breath condensate (EBC) has been used to examine airway inflammation and oxidative stress. This study aimed to evaluate if there were abnormal Sirtuin 1 (SIRT1) protein and tumor necrosis factor (TNF)-α levels in EBC and to determine if these levels could be improved after nasal continuous positive airway pressure (CPAP) treatment. Thirty-five patients with moderately severe to severe obstructive sleep apnea syndrome (OSAS) who wanted nasal CPAP treatment and 20 healthy controls were prospectively enrolled. The EBC SIRT1 protein levels and EBC TNF-α protein levels were assessed by ELISA. All patients underwent sleep studies that were repeated 3 months after nasal CPAP treatment in patients with OSAS. Results showed that in OSAS before nasal CPAP treatment, the EBC SIRT1 protein levels were lower than that in normal subjects, whereas the EBC TNF-α protein levels were higher. After nasal CPAP treatment, the EBC SIRT1 levels increased and EBC TNF-α levels decreased. In conclusion, successful treatment of OSAS by nasal CPAP can normalize the levels of EBC SIRT1 and EBC TNF-α.

Effect of Nasal CPAP on SIRT1 and Endothelial Function in Obstructive Sleep Apnea Syndrome.

BACKGROUND: Sirtuin 1 (SIRT1), a histone/protein deacetylase, has been implicated in aging, metabolism, and stress resistance. SIRT1 regulates endothelial nitric oxide (NO) synthase, restores NO availability, and is involved in different aspects of cardiovascular disease. The aim of this study was to evaluate any abnormalities with regard to SIRT1 protein level in the blood, SIRT1 activity, and impaired endothelial function in patients with obstructive sleep apnea syndrome (OSAS). We also investigated whether or not OSAS patients who received nasal continuous positive airway pressure (CPAP) treatment showed improvements in the levels of SIRT1. METHODS: Thirty-five patients with moderately severe to severe OSAS who requested nasal CPAP treatment and 20 healthy controls were prospectively enrolled. The SIRT1 protein levels in blood and its activity, and the serum levels of nitric oxide derivative (NO x ) were assessed. All subjects participated in sleep studies, which were repeated 3 months after nasal CPAP treatment in the patients with OSAS. RESULTS: In the patients with OSAS, the level of SIRT1 in the blood, its activity, and that of NO x was lower than those of normal subjects before nasal CPAP treatment. After nasal CPAP treatment, the level of SIRT1 in the blood and its activity increased from 0.55 ± 0.32 pg/µg of total protein and 3085.53 ± 1071.57 arbitrary fluorescence units (AFUs)/µg of total protein to 1.13 ± 0.43 pg/µg of total protein and 5344.65 ± 1579.71 AFUs/µg of total protein. The serum levels of NO x in the patients with OSAS increased from 16.36 ± 5.78 to 25.94 ± 5.17 µM. CONCLUSIONS: Successful treatment for OSAS with nasal CPAP can restore blood levels of the SIRT1 protein and its activity and serum levels of NO x .