Production Cross Research Articles

Production of retroviral vectors in continuous high cell density culture

Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting.Key points• A continuous high cell density process for MLV vector production was established• The tubular cell retention membrane allowed for continuous vector harvesting• The established process had a 18-fold higher space time yield compared to a batch

Counting pairs of saddle connections

We show that for almost every translation surface the number of pairs of saddle connections with bounded magnitude of the cross product has asymptotic growth like cR2 where the constant c depends only on the area and the connected component of the stratum. The proof techniques combine classical results for counting saddle connections with the crucial result that the Siegel–Veech transform is in L2. In order to capture information about pairs of saddle connections, we consider pairs with bounded magnitude of the cross product since the set of such pairs can be approximated by a fibered set which is equivariant under geodesic flow. In the case of lattice surfaces, small bounded magnitude of the cross product is equivalent to counting parallel pairs of saddle connections, which also have a quadratic growth of cR2 where c depends in this case on the given lattice surface.

Diagonalization of the cross-product matrix

The paper considers diagonalization of the cross-product matrices, i.e., skew-symmetric matrices of order three. A procedure to determine a nonsingular matrix, which yields the diagonalization is indicated. Furthermore, a method to derive the inverse of a diagonalizing matrix is proposed by means of a formula for the Moore–Penrose inverse of any matrix, which is columnwise partitioned into two matrices having disjoint ranges. This rather nonstandard method to obtain the inverse of a nonsingular matrix is appealing, as it can be applied to any diagonalizing matrix, and not only of those originating from diagonalization of the cross-product matrices. The paper provides also comments and examples demonstrating applicability of the diagonalization procedure to calculate roots of a cross-product matrix.

Science and engineering students’ difficulties in understanding vector concepts

The study assesses science and engineering students’ difficulties in understanding vector concepts. Test of understanding of vectors was administered to 101 university students who were completing their second introductory physics course. Rasch measurement software (Winsteps) was used to analyze the raw data. The concepts that the students found most difficult were identified; these included the graphical representation of a unit vector, the graphical addition and subtraction of vectors, and the interpretation of the dot and cross products, among others. On the other hand, some concepts were easier for students, such as the magnitude of a vector presented in unit-vector notation, and determination of the components of vectors. No significant differences in understanding of vector concepts could be attributed to gender; however, engineering students outperformed science students in understanding vector concepts. This study offers recommendations that physics instructors and researchers can use to improve the teaching of vector concepts.

Focusing a vortex beam with circular polarization: angular momentum

Based on the Richards-Wolf formalism, we obtain two different exact expressions for the angular momentum (AM) density in the focus of a vortex beam with the topological charge n and with right circular polarization. One expression for the AM density is derived as the cross product of the position vector and the Poynting vector and has a nonzero value at the focus for an arbitrary integer number n. The other expression for the AM density is deduced as a sum of the orbital angular momentum (OAM) and the spin angular momentum (SAM). We reveal that at the focus of the light field under analysis, the latter turns zero at n = –1. While both these expressions are not equal to each other at each point of space, 3D integrals thereof are equal. Thus, exact expressions are obtained for densities of AM, SAM and OAM at the focus of a vortex beam with right-hand circular polarization and the identity for the densities AM = SAM + OAM is shown to be violated. Besides, it is shown that the expressions for the strength vectors of the electric and magnetic fields near the sharp focus, obtained by adopting the Richards-Wolf formalism, are exact solutions of the Maxwell's equations. Thus, Richards–Wolf theory exactly describes the behavior of light near the sharp focus in free space.

Experimental and numerical study of liquid film by jet impingement: Based on contact angle model

Liquid film by jet impingement is widely applied in aerospace, steel quenching, ink-jet printing, and cleaning. In this paper, simulations with the modified contact angle model were used to describe the characteristics of the liquid film. Based on the Hoffman's law and the Tanner's correlation, the contact angle model was constructed and further nested within the Coupled Level Set and Volume of Fluid model. The model determined the movement direction of contact line according to the inner product of the normal vector at the phase interface and the lateral velocity in the cell and then outputted the value of the contact angle. Results showed that the advancing and the receding contact angles of droplet were inappropriate for the simulation of the liquid film. The size of the liquid film obtained by the static contact angle largely depended on the selection of contact angle values. Instead, the modified contact angle model provided an accurate prediction on the morphology and the size of the liquid film.

Incremental association rules update algorithm based on the sort compression matrix

Association rule algorithm has always been a research hotspot in the field of data mining, in the context of today’s big data era, in order to efficiently obtain association rules and effectively update them, based on the original fast update pruning (FUP) algorithm, an association rule incremental update algorithm (FBSCM) based on sorting compression matrix is proposed to solve the shortcomings of frequent scanning of transaction datasets. Firstly, The algorithm maps the transaction dataset as a Boolean matrix, and changes the storage mode of the matrix(that is, adding two columns and a row vector); Secondly, the matrix is compressed many times during the generation of frequent k-itemset; After that, the items in the matrix are sorted incrementally according to the support degree of the itemset; Finally, the original string comparison operation is replaced by the vector product of each column of the matrix. Experimental results and analysis show that the FBSCM algorithm has higher temporal performance than the traditional FUP algorithm in different incremental dataset sizes, different minimum support thresholds and different feature datasets, especially when the incremental transaction volume is large or the minimum support degree is small.

The Derivation and Some Explanations of the Uncertainty Principle

This paper is a small discussion about the uncertainty principle. With the great discoveries and researches made by predecessors, I try to answer some questions within the uncertainty principle. Using the definition of momentum, the momentum in de Broglie wave, the property of cosine function, the Euler’s formula, the basic property of plane wave, the law of derivative, the energy of quantum discovered by Max Planck, the photon energy discovered by Albert Einstein, the energy equation of a system and the Hamiltonian operator, I figure out the Schrodinger equation successfully. With the help of Schrodinger equation, the possibility density of a wave function, the concept of normalization, the average value of position and momentum, the Fourier transform, the integration by parts and the limit of normalized wave functions, I find the position operator and the momentum operator. Then using some knowledge of the standard deviation and the variance, the concept of inner product of vectors, some property of Dirac symbol, the law of calculation of complex number and the law of operator calculation, I obtain the general uncertainty relationship of abstract mathematical quantities. According to the general relationship, I get the inequality of uncertainty principle, and discuss the specific relationship between the position and the momentum. After getting the inequality, I use Heisenberg’s experiment of measuring an electron by using the γ ray to hit the electron to explain why we cannot measure the position and the momentum simultaneously. Based on Heisenberg’s experiment, I give my own explanation by discussing the earth and a spacecraft. Transforming the perspective, we can easily see how the accuracies of position and momentum change. In addition, I attempt to figure out why the uncertainty exists. However, I can only explain it by discussing the time and frequency in mathematical method and have no idea to work it out in a pure physics approach.

Modeling Height–Diameter Relationship Using Artificial Neural Networks for Durango Pine (Pinus durangensis Martínez) Species in Mexico

The total tree height (h) and diameter at breast height (dbh) relationship is an essential tool in forest management and planning. Nonlinear mixed effect modeling (NLMEM) has been extensively used, and lately the artificial neural network (ANN) and the resilient backpropagation artificial neural network (RBPANN) approach has been a trending topic for modeling this relationship. The objective of this study was to evaluate and contrast the NLMEN and RBPANN approaches for modeling the h-dbh relationship for the Durango pine species (Pinus durangensis Martínez) for both training and testing datasets in a mixed-species forest in Mexico. The knowledge of this relationship is important for forest management and planning in Mexican Forestry. The total dataset considered 1000 plots (each plot 0.10 ha) (11,472 measured trees) randomly selected from 14,390 temporary forest inventory plots and the dataset was randomly divided into two parts: 50% for training and 50% for testing. An unsupervised clustering analysis was used to group the dataset into 10 cluster-groups based on the k-means clustering method. An RBPANN was performed for tangent hyperbolicus (RBPANN-tanh), softplus (RBPANN-softplus), and logistic (RBPANN-logistic) activation functions in the cross product of the covariate or neurons and the weights for the ANN analysis. Also, a different vector of hidden layers was used for training of ANNs. For both training and testing, 10 classical statistics (e.g., RMSE, AIC, BIC, and logLik) were computed for the residual values and to assess the approaches for the h-dbh relationship. For training and testing, the ANN approach outperformed the NLMEM approach, and the RBPANN-tanh had the best performance in both the training and testing of ANNs.

A compact Heart iteration for low-rank approximations of large matrices

In this paper we present a compact version of the Heart iteration. One that computes low-rank approximations of large sparse matrices. The new iteration is a restarted Krylov method that is based on explicit restarts and Gram–Schmidt orthogonalizations. It is a simple algorithm that requires a minimal amount of computer storage as well as a minimal number of matrix–vector products per iteration. Yet it enjoys a fast rate of convergence. Numerical experiments illustrate the usefulness of the proposed approach.

Implications of AAV affinity column reuse and vector stability on product quality attributes.

In this work, the implications of AAV9 capsid design and column reuse on AAV9 vector product quality were assessed with POROS CaptureSelect (PCS) AAVX and AAV9 resins using sf9 insect cell-derived model AAV9 vectors with varying viral protein (VP) ratios. Chromatographic experiments with purified drug substance AAV9 model feeds indicated consistent vector elution profiles, independent of adeno-associated virus (AAV) VP ratio, or cycle number. In contrast, the presence of process impurities in the clarified lysate feeds resulted in clear changes in the elution patterns. This included increased aggregate content in the vector eluates over multiple cycles as well as clear differences in the performance of these affinity resin systems. The AAV9-serotype specific PCS AAV9 column, with lower vector elution pH, resulted in higher aggregate content over multiple cycles as compared to the serotype-independent PCS AAVX column. Further, the results with vectors of varying VP ratio indicated that while one vector type eluate displayed higher aggregation in both affinity columns over column reuse, the eluate with the other vector type did not exhibit changes in the aggregation profile. Interestingly, vector aggregates in the affinity eluates also contained double-stranded DNA impurities and histone proteins, with similar trends to the aggregate levels. This behavior upon column reuse indicates that these host cell impurities are likely carried over to subsequent runs due to incomplete clean-in-place (CIP). These results indicate that feed impurities, affinity resin characteristics, elution pH, column CIP, and vector stability can impact the reusability of AAV affinity columns and product quality.

Enhanced Integrator with Drift Elimination for Accurate Flux Estimation in Sensorless Controlled Interior PMSM for High-Performance Full Speed Range Hybrid Electric Vehicles Applications

Interior Permanent Magnet Synchronous Motor (IPMSM) motion-sensorless speed control necessitates precise knowledge of rotor flux, speed, and position. Due to numerous non-ideal aspects, such as converter nonlinearities, detection errors, integral initial value, and parameter mismatches, the conventional first-order integrator’s estimated rotor flux experiences a DC offset (Doff). Low-pass filters (LPF) with a constant cut-off frequency yield accurate estimates only in the medium- and high-speed range; however, at the low-speed area, both magnitude and phase estimates are inaccurate. The presented technique resolves the aforementioned issue for a broad speed range. In order to achieve precise flux estimation, this article presents an improved technique of flux estimator with two distinct drift mitigation strategies for the motion-sensorless field-oriented control (FOC) system of IPMSM. Using the orthogonality of the α- and β-axes, the proposed drift elimination system can estimate drift in different situations while maintaining a high level of dynamic performance. The stator flux linkage (SFL) computation in the synchronous coordinate is established from the estimation of the rotating shaft’s permanent magnetic flux linkage orientation and the statistical equations model of the SFL. By comparing the calculated SFL vector to the SFL vector derived from the stator winding voltage and currents integral model with a drift PI compensation loop, a feedback loop is formed to neutralize integral drift, and the rotational speed and position of an IPMSM is estimated utilizing the vector product of the two flux linkages in a phase-locked loop. Theoretical interpretation is presented, and Matlab Simulink simulations, as well as experimental outcomes, consistently demonstrate that the suggested estimation techniques can eliminate the phenomenon of flux drift.

Phenotypic features of dystrophin gene knockout pigs harboring a human artificial chromosome containing the entire dystrophin gene

Mammalian artificial chromosomes have enabled the introduction of extremely large amounts of genetic information into animal cells in an autonomously replicating, nonintegrating format. However, the evaluation of human artificial chromosomes (HACs) as novel tools for curing intractable hereditary disorders has been hindered by the limited efficacy of the delivery system. We generated dystrophin gene knockout (DMD-KO) pigs harboring the HAC bearing the entire human DMD via asomatic cell cloning procedure (DYS-HAC-cloned pig). Restored human dystrophin expression was confirmed by immunofluorescence staining in the skeletal muscle of the DYS-HAC-cloned pigs. Viability at the first month postpartum of the DYS-HAC-cloned pigs, including motor function in the hind leg and serum creatinine kinase level, was improved significantly when compared with that in the original DMD-KO pigs. However, decrease in systemic retention of the DYS-HAC vector and limited production of the DMD protein might have caused severe respiratory impairment with general prostration by 3months postpartum. The results demonstrate that the use of transchromosomic cloned pigs permitted a straightforward estimation of the efficacy of the DYS-HAC carried in affected tissues/organs in a large-animal disease model, providing novel insights into the therapeutic application of exogenous mammalian artificial chromosomes.

Fast hierarchical low-rank view factor matrices for thermal irradiance on planetary surfaces

We present an algorithm for compressing the radiosity view factor model commonly used in radiation heat transfer and computer graphics. We use a format inspired by the hierarchical off-diagonal low rank format, where elements are recursively partitioned using a quadtree or octree and blocks are compressed using a sparse singular value decomposition—the hierarchical matrix is assembled using dynamic programming. The motivating application is time-dependent thermal modeling on vast planetary surfaces, with a focus on permanently shadowed craters which receive energy through indirect irradiance. In this setting, shape models are comprised of a large number of triangular facets which conform to a rough surface. At each time step, a quadratic number of triangle-to-triangle scattered fluxes must be summed; that is, as the sun moves through the sky, we must solve the same view factor system of equations for a potentially unlimited number of time-varying righthand sides. We first conduct numerical experiments with a synthetic spherical cap-shaped crater, where the equilibrium temperature is analytically available. We also test our implementation with triangle meshes of planetary surfaces derived from digital elevation models recovered by orbiting spacecraft. Our results indicate that the compressed view factor matrix can be assembled in quadratic time, which is comparable to the time it takes to assemble the full view matrix itself. Memory requirements during assembly are reduced by a large factor. Finally, for a range of compression tolerances, the size of the compressed view factor matrix and the speed of the resulting matrix vector product both scale linearly (as opposed to quadratically for the full matrix), resulting in orders of magnitude savings in processing time and memory space.

CRISPR library screening to develop HEK293-derived cell lines with improved lentiviral vector titers.

Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers. Briefly, LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) were used to modify HEK293 and HEK293T cells. These cell populations were then expanded, and integrated LV vector genomes were rescued by transfection. LV vectors were harvested, and the process of sequential transduction and rescue-transfection was iterated. Through this workflow, guide RNAs (gRNAs) that target genes that may suppress or enhance LV vector production were enriched and identified with Next-Generation Sequencing (NGS). Though more work is needed to test genes identified in this screen, we expect that perturbations of genes we identified here, such as TTLL12, which is an inhibitor of antiviral innate immunity may be introduced and multiplexed to yield cell lines with improved LV vector productivity.

Identification of a psychiatric risk gene NISCH at 3p21.1 GWAS locus mediating dendritic spine morphogenesis and cognitive function

BackgroundSchizophrenia and bipolar disorder (BD) are believed to share clinical symptoms, genetic risk, etiological factors, and pathogenic mechanisms. We previously reported that single nucleotide polymorphisms spanning chromosome 3p21.1 showed significant associations with both schizophrenia and BD, and a risk SNP rs2251219 was in linkage disequilibrium with a human specific Alu polymorphism rs71052682, which showed enhancer effects on transcriptional activities using luciferase reporter assays in U251 and U87MG cells.MethodsCRISPR/Cas9-directed genome editing, real-time quantitative PCR, and public Hi-C data were utilized to investigate the correlation between the Alu polymorphism rs71052682 and NISCH. Primary neuronal culture, immunofluorescence staining, co-immunoprecipitation, lentiviral vector production, intracranial stereotaxic injection, behavioral assessment, and drug treatment were used to examine the physiological impacts of Nischarin (encoded by NISCH).ResultsDeleting the Alu sequence in U251 and U87MG cells reduced mRNA expression of NISCH, the gene locates 180 kb from rs71052682, and Hi-C data in brain tissues confirmed the extensive chromatin contacts. These data suggested that the genetic risk of schizophrenia and BD predicted elevated NISCH expression, which was also consistent with the observed higher NISCH mRNA levels in the brain tissues from psychiatric patients compared with controls. We then found that overexpression of NISCH resulted in a significantly decreased density of mushroom dendritic spines with a simultaneously increased density of thin dendritic spines in primary cultured neurons. Intriguingly, elevated expression of this gene in mice also led to impaired spatial working memory in the Y-maze. Given that Nischarin is the target of anti-hypertensive agents clonidine and tizanidine, which have shown therapeutic effects in patients with schizophrenia and patients with BD in preliminary clinical trials, we demonstrated that treatment with those antihypertensive drugs could reduce NISCH mRNA expression and rescue the impaired working memory in mice.ConclusionsWe identify a psychiatric risk gene NISCH at 3p21.1 GWAS locus influencing dendritic spine morphogenesis and cognitive function, and Nischarin may have potentials for future therapeutic development.

Goody two plasmids: An optimized transient transfection system for AAV vector production

Goody two plasmids: An optimized transient transfection system for AAV vector production

Cubic Sublattices

A sublattice of the three-dimensional integer lattice Z3\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\mathbb {Z}}^3$$\\end{document} is called cubic sublattice if there exists a basis of the sublattice whose elements are pairwise orthogonal and of equal lengths. We show that for an integer vector v∈Z3\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ extbf{v}}\\in {\\mathbb {Z}}^3$$\\end{document} whose squared length is divisible by d2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$d^2$$\\end{document}, there exists a cubic sublattice containing v\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ extbf{v}}$$\\end{document} with edge length d. This improves one of the main result of a paper of Goswick et al. (J. Number Theory 132(1), 37–53 (2012)), where similar theorem was proved by using the decomposition theory of Hurwitz integral quaternions. We give an elementary proof heavily using cross product. This method allows us to characterize the cubic sublattices.

Classification and quantification of entanglement through wedge product and geometry

The wedge product of post-measurement vectors of a two-qubit state gives rise to a parallelogram, whose ‘area’ has been shown to be equivalent to the generalized I-concurrence measure of entanglement. In multi-qudit systems, the wedge product of post-measurement vectors naturally leads to a higher dimensional parallelepiped which yields a modified faithful entanglement measure. Our new measure fine grains the entanglement monotone, wherein different entangled classes manifest with different geometries. We present a complete analysis of the bipartite qutrit case considering all possible geometric structures where three entanglement classes of pure bipartite qutrit states can be identified with different geometries of post-measurement vectors: three planar vectors; three mutually orthogonal vectors; and three vectors that are neither planar and not all of them are mutually orthogonal. It is further demonstrated that the geometric condition of area and volume maximization naturally leads to the maximization of entanglement. The wedge product approach uncovers an inherent geometry of entanglement and is found to be very useful for the characterization and quantification of entanglement in higher dimensional systems.

SP3-based host cell protein monitoring in AAV-based gene therapy products using LC-MS/MS

Residual host cell proteins (HCPs) represent a critical quality attribute of biotherapeutic drug products. Workflows enabling reliable HCP detection in monoclonal antibodies and recombinant proteins have been developed, which facilitated process optimization to improve product stability and safety, and allowed setting of acceptance limits for HCP content. However, the detection of HCPs in gene therapy products such as adeno-associated viral (AAV) vectors has been limited. Here, the use of SP3 sample preparation followed by liquid chromatography-mass spectrometry (LC-MS) analysis for HCP profiling in various AAV samples is reported. Suitability of the workflow is demonstrated and provided data constitutes an important reference for future work aiming towards a knowledge-driven improvement of manufacturing conditions and characterization of AAV vector products.