A Bacterial Isolate Capable of Quenching Both Diffusible Signal Factor- and N-Acylhomoserine Lactone-Family Quorum Sensing Signals Shows Much Enhanced Biocontrol Potencies.

N-Acylhomoserine lactone (AHL) and diffusible signal factor (DSF) molecules are two families of widely conserved quorum sensing (QS) signals. Quorum quenching (QQ) via enzymatic inactivation of QS signals is a promising strategy of biocontrol. In the search for biocontrol agent quenching both AHL and DSF signals, it has been recently identified that DSF-quenching biocontrol agent Pseudomonas sp. HS-18 contains at least three genes (aigA, aigB, and aigC) encoding AHL-acylases displaying strong AHL-acylase activities on various AHLs. Among them, AigA and AigC presented broad-spectrum enzyme activity against AHLs, while AigB preferred longer AHLs. Interestingly, transcriptional expression of aigC could be significantly induced by AHL signals. Heterologous expression of aigA-C in Burkholderia cenocepacia and Pseudomonas aeruginosa resulted in drastically decreased AHL accumulation, virulence factor production, biofilm formation, motility, and virulence on plants. Significantly, the two types of QQ mechanisms in HS-18 showed a strong and much desired synergistic effect for enhanced biocontrol potency against AHL- and DSF-dependent pathogens.

CRISPR/Cas9-Mediated in vivo Genetic Correction in a Mouse Model of Hemophilia A.

Hemophilia A (HA), a common bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII), has long been considered an attractive target for gene therapy studies. However, full-length F8 cDNA cannot be packaged efficiently by adeno-associated virus (AAV) vectors. As the second most prevalent mutation causing severe HA, F8 intron 1 inversion (Inv1) is caused by an intrachromosomal recombination, leaving the majority of F8 (exons 2-26) untranscribed. In theory, the truncated gene could be rescued by integrating a promoter and the coding sequence of exon 1. To test this strategy in vivo, we generated an HA mouse model by deleting the promoter region and exon 1 of F8. Donor DNA and CRISPR/SaCas9 were packaged into AAV vectors and injected into HA mice intravenously. After treatment, F8 expression was restored and activated partial thromboplastin time (aPTT) was shortened. We also compared two liver-specific promoters and two types of integrating donor vectors. When an active promoter was used, all of the treated mice survived the tail-clip challenge. This is the first report of an in vivo gene repair strategy with the potential to treat a recurrent mutation in HA patients.

Angiotensin-(1-7) Expressed From Lactobacillus Bacteria Protect Diabetic Retina in Mice.

Purpose: A multitude of animal studies substantiates the beneficial effects of Ang-(1-7), a peptide hormone in the protective axis of the renin angiotensin system, in diabetes and its associated complications including diabetic retinopathy (DR). However, the clinical application of Ang-(1-7) is limited due to unfavorable pharmacological properties. As emerging evidence implicates gut dysbiosis in pathogenesis of diabetes and supports beneficial effects of probiotics, we sought to develop probiotics-based expression and delivery system to enhance Ang-(1-7) and evaluate the efficacy of engineered probiotics expressing Ang-(1-7) in attenuation of DR in animal models. Methods: Ang-(1-7) was expressed in the Lactobacillus species as a secreted fusion protein with a trans-epithelial carrier to allow uptake into circulation. To evaluate the effects of Ang-(1-7) expressed from Lactobacillus paracasei (LP), adult diabetic eNOS-/- and Akita mice were orally gavaged with either 1 × 109 CFU of LP secreting Ang-(1-7) (LP-A), LP alone or vehicle, 3 times/week, for 8 and 12 weeks, respectively. Results: Ang-(1-7) is efficiently expressed from different Lactobacillus species and secreted into circulation in mice fed with LP-A. Oral administration of LP-A significantly reduced diabetes-induced loss of retinal vascular capillaries. LP-A treatment also prevented loss of retinal ganglion cells, and significantly decreased retinal inflammatory cytokine expression in both diabetic eNOS-/- and Akita mice. Conclusions: These results provide proof-of-concept for feasibility and efficacy of using engineered probiotic species as live vector for delivery of Ang-(1-7) with enhanced bioavailability. Translational Relevance: Probiotics-based delivery of Ang-(1-7) may hold important therapeutic potential for the treatment of DR and other diabetic complications.

Critical roles of mRNA m6A modification and YTHDC2 expression for meiotic initiation and progression in female germ cells.

Meiotic entry and progression require dynamic regulation of germline gene expression. m6A on mRNAs and recognition by YTHDC2 has been known as post-transcriptional regulatory complex, but the roles of this regulator remain unclear for meiotic initiation and progression in female germ cells (FGCs). This study showed that m6A modification occurred mainly in FGCs rather than ovarian somatic cells (SOMAs), and m6A levels in FGCs increased significantly with meiotic initiation. m6A inhibition suppressed expression of the meiotic markers and affected the percent of FGCs at zygotene, pachytene and diplotene stage respectively. YTHDC2 expression also increased in the same pattern with m6A. Ythdc2 knockdown decreased the percent of STRA8-positive FGCs and altered the percent of FGCs at zygotene and pachytene stage respectively. Taken together, these results suggest that mRNA m6A modification and YTHDC2 expression are essential for meiotic initiation and progression in FGCs.

Erratum: Expression of Human ACE2 in Lactobacillus and Beneficial Effects in Diabetic Retinopathy in Mice.

[This corrects the article DOI: 10.1016/j.omtm.2019.06.007.].

Putrescine Is an Intraspecies and Interkingdom Cell-Cell Communication Signal Modulating the Virulence of Dickeya zeae.

The infections caused by Dickeya zeae become a severe problem in recent years, but the regulatory mechanisms that govern the bacterial virulence remain to be fragmental. Here we report the investigation of potential involvement of polyamines in regulation of D. zeae virulence. We showed that null mutation of speA encoding arginine decarboxylase dramatically decreased the bacterial swimming motility, swarming motility and biofilm formation, and exogenous addition of putrescine effectively rescues the defective phenotypes of D. zeae. HPLC and mass spectrometry analysis validated that speA was essential for production of putrescine in D. zeae. In addition, we demonstrated that D. zeae EC1 could detect and response to putrescine molecules produced by itself or from host plant through specific transporters. Among the two transporters identified, the one represented by PotF played a dominated role over the other represented by PlaP in modulation of putrescine-dependent biological functions. Furthermore, we provided evidence that putrescine signal is critical for D. zeae EC1 bacterial invasion and virulence against rice seeds. Our data depict a novel function of putrescine signal in pathogen-host communication and in modulation of the virulence of an important plant bacterial pathogen.

Expression of Human ACE2 in Lactobacillus and Beneficial Effects in Diabetic Retinopathy in Mice.

The angiotensin converting enzyme 2 (ACE2) catalyzes the degradation of Angiotensin II (Ang II) to generate Angiotensin-(1-7), which reduces inflammation and oxidative stress stimulated by Ang II. ACE2 has been shown to be protective in cardiovascular and metabolic diseases including diabetes and its complications. However, the challenge for its clinical application is large-scale production of high-quality ACE2 with sufficient target tissue bioavailability. We developed an expression and delivery system based on the use of probiotic species Lactobacillus paracasei (LP) to serve as a live vector for oral delivery of human ACE2. We show that codon-optimized ACE2 can be efficiently expressed in LP. Mice treated with the recombinant LP expressing the secreted ACE2 in fusion with the non-toxic subunit B of cholera toxin, which acts as a carrier to facilitate transmucosal transport, showed increased ACE2 activities in serum and tissues. ACE2-LP administration reduced the number of acellular capillaries, blocked retinal ganglion cell loss, and decreased retinal inflammatory cytokine expression in two mouse models of diabetic retinopathy. These results provide proof of concept for feasibility of using engineered probiotic species as live vector for delivery of human ACE2 with enhanced tissue bioavailability for treating diabetic retinopathy, as well as other diabetic complications.

Inhibition of RNA polymerase III transcription by Triptolide attenuates colorectal tumorigenesis.

BACKGROUND: Upregulation of RNA polymerase (Pol) III products, including tRNAs and 5S rRNA, in tumor cells leads to enhanced protein synthesis and tumor formation, making it a potential target for cancer treatment. In this study, we evaluated the inhibition of Pol III transcription by triptolide and the anti-cancer effect of this drug in colorectal tumorigenesis. METHODS: The effect of triptolide on colorectal cancer development was assessed in colorectal cancer mouse models, 3D organoids, and cultured cells. Colorectal cancer cells were treated with triptolide. Pol III transcription was measured by real-time quantitative polymerase chain reaction (PCR). The formation of TFIIIB, a multi-subunit transcription factor for Pol III, was determined by chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP), and fluorescence resonance energy transfer (FRET). RESULTS: Triptolide reduced both tumor number and tumor size in adenomatous polyposis coli (Apc) mutated (ApcMin/+) mice as well as AOM/DSS-induced mice. Moreover, triptolide effectively inhibited colorectal cancer cell proliferation, colony formation, and organoid growth in vitro, which was associated with decreased Pol III target genes. Mechanistically, triptolide treatment blocked TBP/Brf1interaction, leading to the reduced formation of TFIIIB at the promoters of tRNAs and 5S rRNA. CONCLUSIONS: Together, our data suggest that inhibition of Pol III transcription with existing drugs such as triptolide provides a new avenue for developing novel therapies for colorectal cancer.

Research on a learning rate with energy index in deep learning.

The stochastic gradient descent algorithm (SGD) is the main optimization solution in deep learning. The performance of SGD depends critically on how learning rates are tuned over time. In this paper, we propose a novel energy index based optimization method (EIOM) to automatically adjust the learning rate in the backpropagation. Since a frequently occurring feature is more important than a rarely occurring feature, we update the features to different extents according to their frequencies. We first define an energy neuron model and then design an energy index to describe the frequency of a feature. The learning rate is taken as a hyperparameter function according to the energy index. To empirically evaluate the EIOM, we investigate different optimizers with three popular machine learning models: logistic regression, multilayer perceptron, and convolutional neural network. The experiments demonstrate the promising performance of the proposed EIOM compared with that of other optimization algorithms.

Improved GGIW-PHD filter for maneuvering non-ellipsoidal extended targets or group targets tracking based on sub-random matrices.

For non-ellipsoidal extended targets and group targets tracking (NETT and NGTT), using an ellipsoid to approximate the target extension may not be accurate enough because of the lack of shape and orientation information. In consideration of this, we model a non-ellipsoidal extended target or target group as a combination of multiple ellipsoidal sub-objects, each represented by a random matrix. Based on these models, an improved gamma Gaussian inverse Wishart probability hypothesis density (GGIW-PHD) filter is proposed to estimate the measurement rates, kinematic states, and extension states of the sub-objects for each extended target or target group. For maneuvering NETT and NGTT, a multi-model (MM) approach based GGIW-PHD (MM-GGIW-PHD) filter is proposed. The common and the individual dynamics of the sub-objects belonging to the same extended target or target group are described by means of the combination between the overall maneuver model and the sub-object models. For the merging of updating components, an improved merging criterion and a new merging method are derived. A specific implementation of prediction partition with pseudo-likelihood method is presented. Two scenarios for non-maneuvering and maneuvering NETT and NGTT are simulated. The results demonstrate the effectiveness of the proposed algorithms.

Silencing the double-stranded RNA binding protein DGCR8 inhibits ovarian cancer cell proliferation, migration, and invasion.

PURPOSE: To evaluate the role of DiGeorge Critical Region 8 (DGCR8), a key component of miRNA biogenesis pathway in ovarian cancer. METHODS: The expression of DGCR8 in ovarian cancer was detected by immunostaining and DGCR8 knockdown in ovarian cancer cells was achieved using lentiviral shRNA. Differential expression of miRNAs was determined using Nanostring miRNA arrays and validated by real-time RT-PCR. RESULTS: DGCR8 was highly expressed in ovarian cancer. Knockdown of DGCR8 expression inhibits cell proliferation, migration, and invasion, as well as sensitizes cells to apoptosis induced by the chemotherapeutic drug cisplatin. Cellular survival pathways including ERK1/2 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT were attenuated in DGCR8 knockdown cells. DGCR8 knockdown results in dysregulated miRNA gene expression. miR-27b was identified as the most highly down-regulated miRNA in DGCR8 knockdown cells and promoted cell proliferation in ovarian cancer cells. CONCLUSIONS: DGCR8 functions as an oncogene in ovarian cancer, which is in part mediated by miR-27b.

Gene expression profiling associated with angiotensin II type 2 receptor-induced apoptosis in human prostate cancer cells.

Increased expression of angiotensin II type 2 receptor (AT2R) induces apoptosis in numerous tumor cell lines, with either Angiotensin II-dependent or Angiotensin II-independent regulation, but its molecular mechanism remains poorly understood. Here, we used PCR Array analysis to determine the gene and microRNA expression profiles in human prostate cancer cell lines transduced with AT2R recombinant adenovirus. Our results demonstrated that AT2R over expression leads to up-regulation of 6 apoptosis-related genes (TRAIL-R2, BAG3, BNIPI, HRK, Gadd45a, TP53BP2), 2 cytokine genes (IL6 and IL8) and 1 microRNA, and down-regulation of 1 apoptosis-related gene TNFSF10 and 2 cytokine genes (BMP6, BMP7) in transduced DU145 cells. HRK was identified as an up-regulated gene in AT2R-transduced PC-3 cells by real-time RT-PCR. Next, we utilized siRNAs to silence the up-regulated genes to further determine their roles on AT2R overexpression mediated apoptosis. The results showed downregulation of Gadd45a reduced the apoptotic effect by ∼30% in DU145 cells, downregulation of HRK reduced AT2R-mediated apoptosis by more than 50% in PC-3 cells, while downregulation of TRAIL-R2 enhanced AT2R-mediated apoptosis more than 4 times in DU145 cells. We also found that the effects on AT2R-mediated apoptosis caused by downregulation of Gadd45a, TRAIL-R2 and HRK were independent in activation of p38 MAPK, p44/42 MAPK and p53. Taken together, our results demonstrated that TRAIL-R2, Gadd45a and HRK may be novel target genes for further study of the mechanism of AT2R-mediated apoptosis in prostate cancer cells.

Effects of angiotensin II type 2 receptor overexpression on the growth of hepatocellular carcinoma cells in vitro and in vivo.

Increasing evidence suggests that the renin-angiotensin system (RAS) plays an important role in tumorigenesis. The interaction between Angiotensin II (AngII) and angiotensin type 1 receptor (AT1R) may have a pivotal role in hepatocellular carcinoma (HCC) and therefore, AT1R blocker and angiotensin I-converting enzyme (ACE) inhibitors may have therapeutic potential in the treatment of hepatic cancer. Although the involvement of AT1R has been well explored, the role of the angiotensin II Type 2 receptor (AT2R) in HCC progression remains poorly understood. Thus, the aim of this study was to explore the effects of AT2R overexpression on HCC cells in vitro and in mouse models of human HCC. An AT2R recombinant adenoviral vector (Ad-G-AT2R-EGFP) was transduced into HCC cell lines and orthotopic tumor grafts. The results indicate that the high dose of Ad-G-AT2R-EGFP-induced overexpression of AT2R in transduced HCC cell lines produced apoptosis. AT2R overexpression in SMMC7721 cells inhibited cell proliferation with a significant reduction of S-phase cells and an enrichment of G1-phase cells through changing expression of CDK4 and cyclinD1. The data also indicate that overexpression of AT2R led to apoptosis via cell death signaling pathway that is dependent on activation of p38 MAPK, pJNK, caspase-8 and caspase-3 and inactivation of pp42/44 MAPK (Erk1/2). Finally, we demonstrated that moderately increasing AT2R expression could increase the growth of HCC tumors and the proliferation of HCC cells in vivo. Our findings suggest that AT2R overexpression regulates proliferation of hepatocellular carcinoma cells in vitro and in vivo, and the precise mechanisms of this phenomenon are yet to be fully determined.