Temporal and Tissue Specific Regulation of RP-Associated Splicing Factor Genes PRPF3, PRPF31 and PRPC8—Implications in the Pathogenesis of RP

Genetic mutations in several ubiquitously expressed RNA splicing genes such as PRPF3, PRP31 and PRPC8, have been found to cause retina-specific diseases in humans. To understand this intriguing phenomenon, most studies have been focused on testing two major hypotheses. One hypothesis assumes that these mutations interrupt retina-specific interactions that are important for RNA splicing, implying that there are specific components in the retina interacting with these splicing factors. The second hypothesis suggests that these mutations have only a mild effect on the protein function and thus affect only the metabolically highly active cells such as retinal photoreceptors.We examined the second hypothesis using the PRPF3 gene as an example. We analyzed the spatial and temporal expression of the PRPF3 gene in mice and found that it is highly expressed in retinal cells relative to other tissues and its expression is developmentally regulated. In addition, we also found that PRP31 and PRPC8 as well as snRNAs are highly expressed in retinal cells.Our data suggest that the retina requires a relatively high level of RNA splicing activity for optimal tissue-specific physiological function. Because the RP18 mutation has neither a debilitating nor acute effect on protein function, we suggest that retinal degeneration is the accumulative effect of decades of suboptimal RNA splicing due to the mildly impaired protein

Overcoming Immunological Challenges to Helper-Dependent Adenoviral Vector-Mediated Long-Term CFTR Expression in Mouse Airways

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and CF patients require life-long treatment. Although CFTR modulators show a great potential for treating most CF patients, some individuals may not tolerate the treatment. In addition, there is no effective therapy for patients with some rare CFTR mutations, such as class I CF mutations, which lead to a lack of CFTR protein production. Therefore, other therapeutic strategies, such as gene therapy, have to be investigated. Currently, immune responses to gene therapy vectors and transgene products are a major obstacle to applying CF gene therapy to clinical applications. In this study, we examined the effects of cyclophosphamide on the modulation of host immune responses and for the improvement of the CFTR transgene expression in the repeated delivery of helper-dependent adenoviral (HD-Ad) vectors to mouse lungs. We have found that cyclophosphamide significantly decreased the expression of T cell genes, such as CD3 (cluster of differentiation 3) and CD4, and reduced their infiltration into mouse lung tissues. We have also found that the levels of the anti-adenoviral antibody and neutralizing activity as well as B-cell infiltration into the mouse lung tissues were significantly reduced with this treatment. Correspondingly, the expression of the human CFTR transgene has been significantly improved with cyclophosphamide administration compared to the group with no treatment. These data suggest that the sustained expression of the human CFTR transgene in mouse lungs through repeated vector delivery can be achieved by transient immunosuppression

Overcoming Immunological Challenges to Helper-Dependent Adenoviral Vector-Mediated Long-Term CFTR Expression in Mouse Airways

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and CF patients require life-long treatment. Although CFTR modulators show a great potential for treating most CF patients, some individuals may not tolerate the treatment. In addition, there is no effective therapy for patients with some rare CFTR mutations, such as class I CF mutations, which lead to a lack of CFTR protein production. Therefore, other therapeutic strategies, such as gene therapy, have to be investigated. Currently, immune responses to gene therapy vectors and transgene products are a major obstacle to applying CF gene therapy to clinical applications. In this study, we examined the effects of cyclophosphamide on the modulation of host immune responses and for the improvement of the CFTR transgene expression in the repeated delivery of helper-dependent adenoviral (HD-Ad) vectors to mouse lungs. We have found that cyclophosphamide significantly decreased the expression of T cell genes, such as CD3 (cluster of differentiation 3) and CD4, and reduced their infiltration into mouse lung tissues. We have also found that the levels of the anti-adenoviral antibody and neutralizing activity as well as B-cell infiltration into the mouse lung tissues were significantly reduced with this treatment. Correspondingly, the expression of the human CFTR transgene has been significantly improved with cyclophosphamide administration compared to the group with no treatment. These data suggest that the sustained expression of the human CFTR transgene in mouse lungs through repeated vector delivery can be achieved by transient immunosuppression

A Vibration Control Method Using MRASSA for 1/4 Semi-Active Suspension Systems

The multi-subpopulation refracted adaptive salp swarm algorithm (MRASSA) was proposed for vibration control in 1/4 semi-active suspension systems. The MRASSA algorithm was applied to optimize suspension damping performance by addressing the local optimal and slow convergence speed challenge of the standard salp swarm algorithm for two-degrees-of-freedom 1/4 semi-active suspension systems. The developed MRASSA contains three key improvements: (1) partitioning multi-subpopulation; (2) applying refracted opposition-based learning; (3) adopting adaptive factors. In order to verify the performance of the MRASSA approach, a 1/4 suspension Simulink model was developed for simulation experiments. To further validate the results, a physical platform was built to test the applicability of the simulation model. The optimized suspension performance of MRASSA was also compared with three optimized models, namely, standard SSA, Single-Objective Firefly (SOFA) and Whale-optimized Fuzzy-fractional Order (WOAFFO). The experimental results showed that MRASSA outperformed the other models, achieving better suspension performance in complex environments such as a random road with a speed of 60 km/h. Compared to passive suspension, MRASSA led to a 41.15% reduction in sprung mass acceleration and a 15–25% reduction compared to other models. Additionally, MRASSA had a maximum 20% reduction in suspension dynamic deflection and dynamic load. MRASSA also demonstrated a faster convergence speed, finding the optimal solution faster than the other algorithms. These results indicate that MRASSA is superior to other models and has potential as a valuable tool for suspension performance optimization

Test Study on Vortex-Induced Vibration of Deep-Sea Riser under Bidirectional Shear Flow

A model test was carried out to reveal the vortex-induced vibration characteristics of a deep-sea riser under bidirectional shear flow. Bandpass filtering and modal analysis were used to process the test strain data, and the amplitude and frequency response characteristics of the vortex-induced vibration of the riser in the bidirectional shear flow field were obtained. The results of the test data analysis show that the dominant frequency of the vortex-induced vibration of the riser model under bidirectional shear flow is locked in the natural frequency of the riser and does not increase with the increase in flow velocity, that the average resistance coefficient of the riser model has little change under different flow velocities because of the distribution characteristics of the “bidirectional shear” flow field, that there is an extreme value of the shear force in the middle of the riser model, and that the Strouhal number in the transverse direction of the vortex-induced vibration under bidirectional shear flow is less than the recommended value of the current vortex-induced vibration prediction software. The obtained results provide basic data for the prediction of vortex-induced vibration and research into the fatigue analysis method of a riser under an internal wave flow field

TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy

Cystic fibrosis (CF) is an inherited monogenic disorder, amenable to gene-based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and the lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human cystic fibrosis transmembrane conductance regulator (CFTR) minigene (cDNA) into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we were able to detect CFTR mRNA expression using qPCR and function correction using fluorometric image plate reader (FLIPR) and iodide efflux assays. Taken together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing

Overcoming the Undesirable CRISPR-Cas9 Expression in Gene Correction

The CRISPR-Cas9 system is attractive for gene therapy, as it allows for permanent genetic correction. However, as a new technology, Cas9 gene editing in clinical applications faces major challenges, such as safe delivery and gene targeting efficiency. Cas9 is a foreign protein to recipient cells; thus, its expression may prompt the immune system to eliminate gene-edited cells. To overcome these challenges, we have engineered a novel delivery system based on the helper-dependent adenoviral (HD-Ad) vector, which is capable of delivering genes to airway basal stem cells in vivo. Using this system, we demonstrate the successful co-delivery of both CRISPR-Cas9/single-guide RNA and the LacZ reporter or CFTR gene as donor DNA to cultured cells. HD-Ad vector genome integrity is compromised following donor DNA integration, and because the CRISPR-Cas9/single-guide RNA and donor DNA are carried on the same vector, CRISPR-Cas9 expression is concurrently eliminated. Thus, we show the feasibility of site-specific gene targeting with limited Cas9 expression. In addition, we achieved stable CFTR expression and functional correction in cultured cells following successful gene integration. Keywords: gene targeting, cystic fibrosis, lung disease, gene therapy, CRISP

Experimental Study on Vortex-Induced Vibration of Tension Leg and Riser for Full Depth Mooring Tension Leg Platform

According to the geometric parameters of the tension leg platform, the test model was made with a scale ratio of 1:61. The model was used to conduct the full-depth simulation test of uniform flow and wave current combination in the test pool. The model test results showed that when the reduced speed was between 5.5 and 8.5, and the lateral motion response of the platform was the most significant. In the interval of the reduced speed, the response frequency of transverse vortex-induced motion was close to the natural transverse frequency of the platform, and resonance occurred. The amplitude of surge motion increased with the increase of reduced speed. Due to the pull of the floating body, the tension of tension legs and risers increased with the flow rate but did not increase significantly in the floating body lock zone. The mooring tension had a certain limiting effect on the floating body sway. The displacement modes of tension legs and risers were greatly affected by the flow velocity. With the flow velocity increasing, the mode order increased. In addition, the increase in tension caused by the large displacement of the floating body had a certain impact on the displacement amplitude. The wave could reduce the sway of the floating body and strengthen the surge. Therefore, under the combined action of wave and current, the tension amplitude of the tension leg and riser was increased compared with that under the uniform flow. The conclusions obtained in this paper could be used for reference in the engineering design of tension legs and risers

Expression of Pinellia pedatisecta Lectin Gene in Transgenic Wheat Enhances Resistance to Wheat Aphids

Wheat aphids are major pests during the seed filling stage of wheat. Plant lectins are toxic to sap-sucking pests such as wheat aphids. In this study, Pinellia pedatisecta agglutinin (ppa), a gene encoding mannose binding lectin, was cloned, and it shared 92.69% nucleotide similarity and 94% amino acid similarity with Pinellia ternata agglutinin (pta). The ppa gene, driven by the constitutive and phloem-specific ribulose bisphosphate carboxylase small subunit gene (rbcs) promoter in pBAC-rbcs-ppa expression vector, was transferred into the wheat cultivar Baofeng104 (BF104) by particle bombardment transformation. Fifty-four T0 transgenic plants were generated. The inheritance and expression of the ppa gene were confirmed by PCR and RT-PCR analysis respectively, and seven homozygous transgenic lines were obtained. An aphid bioassay on detached leaf segments revealed that seven ppa transgenic wheat lines had lower aphid growth rates and higher inhibition rates than BF104. Furthermore, two-year aphid bioassays in isolated fields showed that aphid numbers per tiller of transgenic lines were significantly decreased, compared with wild type BF104. Therefore, ppa could be a strong biotechnological candidate to produce aphid-resistant wheat