Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus

A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an in vivo side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted in vivo using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids

Modification of m5C regulators in sarcoma can guide different immune infiltrations as well as immunotherapy

BackgroundRecent studies have found that 5-methylcytosine (m5C) modulators are associated with the prognosis and treatment of cancer. However, the relevance of m5C modulators in sarcoma prognosis and the tumour microenvironment is unclear.MethodsWe selected 15 m5C regulators and performed unsupervised clustering to identify m5C modification patterns and differentially expressed genes associated with the m5C phenotype in The Cancer Genome Atlas (TCGA) sarcomas. The extent of immune cell infiltration in different clustering groups was explored using single-sample gene set enrichment analysis and estimation algorithms. A principal component analysis algorithm-based m5C scoring protocol was performed to assess the m5C modification patterns of individual tumors.ResultsWe identified two distinct m5C modification patterns in the TCGA sarcoma cohort, which possess different clinical outcomes and biological processes. Tumour microenvironment analysis revealed two groups of immune infiltration patterns highly consistent with m5C modification patterns, classified as immune inflammatory and immune desert types. We constructed m5C scores and found that high m5C scores were closely associated with leiomyosarcoma and other subtypes, and were associated with poorer prognosis, lower PD-L1 expression, and poorer immunotherapy outcomes. The best application was validated against the m5C database.ConclusionWe constructed an m5C score for sarcoma based on the TCGA database and identified a poorer prognosis in the high m5c score group. The stability and good prognostic predictive power of the m5C score was verified by an external database. We found that sarcomas in the low m5C score group may have a better response to immunotherapy

Paternal genetic affinity between western Austronesians and Daic populations

<p>Abstract</p> <p>Background</p> <p>Austronesian is a linguistic family spread in most areas of the Southeast Asia, the Pacific Ocean, and the Indian Ocean. Based on their linguistic similarity, this linguistic family included Malayo-Polynesians and Taiwan aborigines. The linguistic similarity also led to the controversial hypothesis that Taiwan is the homeland of all the Malayo-Polynesians, a hypothesis that has been debated by ethnologists, linguists, archaeologists, and geneticists. It is well accepted that the Eastern Austronesians (Micronesians and Polynesians) derived from the Western Austronesians (Island Southeast Asians and Taiwanese), and that the Daic populations on the mainland are supposed to be the headstream of all the Austronesian populations.</p> <p>Results</p> <p>In this report, we studied 20 SNPs and 7 STRs in the non-recombining region of the 1,509 Y chromosomes from 30 China Daic populations, 23 Indonesian and Vietnam Malayo-Polynesian populations, and 11 Taiwan aboriginal populations. These three groups show many resemblances in paternal lineages. Admixture analyses demonstrated that the Daic populations are hardly influenced by Han Chinese genetically, and that they make up the largest proportion of Indonesians. Most of the population samples contain a high frequency of haplogroup O1a-M119, which is nearly absent in other ethnic families. The STR network of haplogroup O1a* illustrated that Indonesian lineages did not derive from Taiwan aborigines as linguistic studies suggest, but from Daic populations.</p> <p>Conclusion</p> <p>We show that, in contrast to the Taiwan homeland hypothesis, the Island Southeast Asians do not have a Taiwan origin based on their paternal lineages. Furthermore, we show that both Taiwan aborigines and Indonesians likely derived from the Daic populations based on their paternal lineages. These two populations seem to have evolved independently of each other. Our results indicate that a super-phylum, which includes Taiwan aborigines, Daic, and Malayo-Polynesians, is genetically educible.</p

Microstructure and mechanical properties of refill friction stir spot welded joints: Effects of tool size and welding parameters

A novel refill friction stir spot welding (RFSSW) technique employing large-sized tools is proposed. The microstructure and mechanical properties of joints produced with a large-sized welding tool and a conventional tool are compared. The results show that the exit line resulting from the sleeve becomes longer with increasing the plunge depth, and the diameter of nugget increases with higher rotational speed for the joints produced by both conventional and novel tools. The plunge depth increases from 2.0 mm to 2.2 mm, then to 2.4 mm, which affects the hook defect to bend upwards, almost parallel to the lap interface, to bend downwards, respectively. The joints produced with the novel tool have a flat hook compared to the conventional tool. The microstructure evolution of the conventional and novel joints is similar. The tensile-shear and tearing forces measured of the novel joints are higher than those of the conventional joints for the same welding parameters. For conventional joints, the maximum tensile-shear and tearing forces are 8.6 ± 0.1 kN and 4.4 ± 0.2 kN, respectively. The maximum tensile-shear and tearing forces for novel joints are 10.9 ± 0.1 kN and 5.6 ± 0.1 kN, respectively. After the tensile-shear test, there are three modes of fracture, the upper-mixed, the lower-mixed, and the shear fracture one. The plunge depth has a pronounced effect on the fracture mode of joints

Table1_Modification of m5C regulators in sarcoma can guide different immune infiltrations as well as immunotherapy.xls

BackgroundRecent studies have found that 5-methylcytosine (m5C) modulators are associated with the prognosis and treatment of cancer. However, the relevance of m5C modulators in sarcoma prognosis and the tumour microenvironment is unclear.MethodsWe selected 15 m5C regulators and performed unsupervised clustering to identify m5C modification patterns and differentially expressed genes associated with the m5C phenotype in The Cancer Genome Atlas (TCGA) sarcomas. The extent of immune cell infiltration in different clustering groups was explored using single-sample gene set enrichment analysis and estimation algorithms. A principal component analysis algorithm-based m5C scoring protocol was performed to assess the m5C modification patterns of individual tumors.ResultsWe identified two distinct m5C modification patterns in the TCGA sarcoma cohort, which possess different clinical outcomes and biological processes. Tumour microenvironment analysis revealed two groups of immune infiltration patterns highly consistent with m5C modification patterns, classified as immune inflammatory and immune desert types. We constructed m5C scores and found that high m5C scores were closely associated with leiomyosarcoma and other subtypes, and were associated with poorer prognosis, lower PD-L1 expression, and poorer immunotherapy outcomes. The best application was validated against the m5C database.ConclusionWe constructed an m5C score for sarcoma based on the TCGA database and identified a poorer prognosis in the high m5c score group. The stability and good prognostic predictive power of the m5C score was verified by an external database. We found that sarcomas in the low m5C score group may have a better response to immunotherapy.</p

Evolution of crystallographic orientation, precipitation, phase transformation and mechanical properties realized by enhancing deposition current for dual-wire arc additive manufactured Ni-rich NiTi alloy

© 2020 Elsevier B.V. Ni-rich NiTi alloys were deposited using the in-situ alloying wire arc additive manufacturing (WAAM) method, with varying deposition currents from 80 A to 120 A. The effects of deposition current on the crystal orientation, precipitation, phase transformation and mechanical properties of the WAAM-deposited NiTi alloys were investigated. The results show that increasing the deposition current during the WAAM process would result in noticeable coarsening of B2 grain and an increased volume fraction of high angle grain boundaries (HAGBs). Also, the texture intensity gradually decreased with increasing deposition current. The fabricated components are dominated by the B2 phase with quantities of Ni4Ti3 precipitates in all samples. When increasing the deposition current during the WAAM process, the size of Ni4Ti3 precipitates generally increased and gradually decomposed into a stable Ni3Ti phase which could be detected in the sample produced at 120 A. Furthermore, all of the characteristic phase transformation temperatures increased with the deposition current while the ultimate tensile strength dropped from 927.9 MPa to 613.8 MPa and elongation reduced from 8.7 % to 5.6 %. The cyclic loading-unloading tests revealed that similar trends for the evolution of irreversible strain (εir), recoverable strain (εre), recovery ratio, and elastic energy storage efficiency (η) during cycling were obtained in all samples processed with different deposition currents. The highest εre of 3.2 % and the highest recovery ratio of 53.9 % were obtained in the sample processed with 80 A at an applied stress of 700 MPa for ten cycles. The change of mechanical properties with varying deposition current is due to a combination of factors including precipitation hardening effect, grain refinement effect, and crystal orientation. These results can be useful for optimizing WAAM process parameters to fabricate NiTi components with acceptable structural properties

A small sample data-driven method: User needs elicitation from online reviews in new product iteration

Eliciting user needs from mass online reviews is playing a significant role in the product iteration process. Efficient user needs elicitation does achieve considerable benefits for maintaining higher competitiveness and a speedier lifecycle. However, there is inevitably an online review scarcity about new products due to the short time on the market and low buyer recognition compared with commonly used products. This paper proposes a small sample data-driven method for user needs elicitation from online reviews in new product iteration. In the first stage, a scraped initial online review dataset is pre-processed roughly to improve the data quality. And then, reviews are classified into multiple categories according to different topics using ERNIE. In the second stage, each topic-based dataset is reprocessed in detail. Thereafter, the key user needs set is determined and facilitated by extracting key product information phrases from every single dataset using improved SIFRank. Moreover, the case study of a smart cat feeder is carried out to demonstrate the feasibility and potential of the ERNIE-ISIFRank methodology. Finally, comparative experiments are conducted to verify the advantages of the proposed method which is primarily based on the pre-trained language model to enhance the deep understanding of the semantics of online reviews. The experimental results confirm that the proposed method can assist in identifying key user needs with high efficiency.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Design Conceptualization and Communicatio

Mechanistic study of the effect of potassium ferrate and straw fiber on the enhancement of strength in cement-based solidified municipal sludge

Abstract The high content of organic matter in sludge is the primary reason for the poor solidifying effect and excessive dosage of the cement base. In this study, potassium ferrate and straw fiber are utilized to synergistically enhance the solidifying effect of the cement and elaborate the strength mechanisms. Among them, potassium ferrate was selected to oxidize and crack the structure of organic matter in sludge and consume part of organic matter; straw fiber was used as an adsorption material to absorb some of the organic material and reduce its interference with the cement hydration reaction; the skeleton function of straw fiber in solidified sludge was used to improve the final solidified sludge strength. It is shown that the presence of these two additives significantly improved the cement solidification strength and reduced the moisture content of the solidified body. Moreover, the moisture content and strength followed an obvious linear relationship (adjusted R2 = 0.92), with the strength increasing as the moisture content decreased. After pretreatment with potassium ferrate, the free water content in the dewatered sludge increased by 4.5%, which was conducive to the adequate hydration reaction with cement. The analysis using X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDS), and mercury intrusion porosimetry (MIP) revealed potassium ferrate synergizes with straw fibers to promote the production of hemihydrate gypsum and gismondine. However, hemihydrate gypsum, calcium carbonate, and gismondine resulted in structural swelling, which was confirmed by the microscopic morphology and pore structure analysis. However, the adverse effects due to swelling were offset by the increase in strength brought by the above crystalline substances