Production Cross Research Articles

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

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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.

Vector properties of geometric calculus

After its introduction by Grossman and Katz, the geometry part of geometric calculus has been mostly left unattended. In this paper, we give the elementary theorems to build the geometry on geometric space. We apply the well‐known theorems for inner product in geometric space. The terms geometric determinant and geometric vector product are introduced, and their properties are studied. We give the definitions of the circle, the plane, and the sphere and visualize them along with a line in geometric space. Finally, we discuss the Gram–Schmidt process in geometric space.

Interferon alpha and beta receptor 1 knockout in human embryonic kidney 293 cells enhances the production efficiency of proteins or adenoviral vectors related to type I interferons.

Human embryonic kidney (HEK) 293 cells are widely used in protein and viral vector production owing to their high transfection efficiency, rapid growth, and suspension growth capability. Given their antiviral, anticancer, and immune-enhancing effects, type I interferons (IFNs) have been used to prevent and treat human and animal diseases. However, the binding of type I IFNs to the IFN-α and-β receptor (IFNAR) stimulates the expression of IFN-stimulated genes (ISGs). This phenomenon induces an antiviral state and promotes apoptosis in cells, thereby impeding protein or viral vector production. In this study, we generated an IFNAR subtype 1 knockout (KO) HEK 293 suspension (IFNAR-KO) cell line by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology. Upon treatment with human IFN-α, the IFNAR-KO cells showed a constant expression of ISGs, including 2'-5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance 1 (Mx1), protein kinase RNA-activated (PKR), and IFN-induced transmembrane protein 1 (IFITM1), when compared with the wild-type HEK 293 (WT) cells, wherein the ISGs were significantly upregulated. As a result, the titer of recombinant adenovirus expressing porcine IFN-α was significantly higher in the IFNAR-KO cells than in the WT cells. Furthermore, the IFNAR-KO cells continuously produced higher amounts of IFN-α protein than the WT cells. Thus, the CRISPR-Cas9-mediated IFNAR1 KO cell line can improve the production efficiency of proteins or viral vectors related to IFNs. The novel cell line may be used for producing vaccines and elucidating the type I IFN signaling pathway in cells.

Optimizing and developing a scalable, chemically defined, animal component-free lentiviral vector production process in a fixed-bed bioreactor

Lentiviral vectors (LVVs) play a critical role in gene delivery for exvivo gene-modified cell therapies. However, the lack of scalable LVV production methods and the high cost associated with them may limit their use. In this work, we demonstrate the optimization and development of a scalable, chemically defined, animal component-free LVV production process using adherent human embryonic kidney 293T cells in a fixed-bed bioreactor. The initial studies focused on the optimization of the culture process in 2D static cultures. Process changes such as decreasing cell seeding density on day 0 from 2.5×104 to 5×103 cells/cm2, delaying the transient transfection from 24 to 120h post-seeding, reducing plasmid DNA to 167ng/cm2, and adding 5mM sodium butyrate 6h post-transfection improved functional LVV titers by 26.9-fold. The optimized animal component-free production process was then transferred to the iCELLis Nano bioreactor, a fixed-bed bioreactor, where titers of 1.2×106 TU/cm2 were achieved when it was operated in perfusion. In this work, comparable functional LVV titers were obtained with FreeStyle 293 Expression medium and the conventional Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum both at small and large scale.

Random Integration Analysis of Recombinant Adeno-Associated Virus 6 Packaged in Sf9 Insect Cells

Recently, there have been growing concerns over the integration of recombinant adeno-associated virus (rAAV) used in gene therapy. Wild-type adeno-associated virus (AAV) site specifically integrates into AAVS1 site of human genome, while rAAV randomly integrates into host chromosomes at low frequencies. This research aims to study the random integration events of rAAV6-EGFP packaged in Sf9 insect cells. Baculo-Sf9 manufacturing platform has the advantages of high-density suspension culture of Sf9 insect cells and large-scale production of rAAV vectors. In this study, we used different doses of Baculo-Sf9 produced rAAV6-EGFP to transduce HEK293T cells and A549-implanted tumors in vitro and in vivo. Using flow cytometry and fluorescence microscopy, we studied their EGFP gene expression efficiencies and EGFP fluorescence intensities. Using inverse nested PCR and DNA sequencing, random integration sites of rAAV6-EGFP genome into human chromosomes were identified. In vitro results showed that gene expression efficiencies became stable after 20 days and random integration frequencies were 0.2‒4.2%. Both in vitro and in vivo results indicated that random integration of Baculo-Sf9 rAAV6 was dose-dependent. Sequencing results showed two random integration sites, which were on human chromosomes 8 and 12. The findings suggest that we should use as low dose of rAAV vector as possible for safe gene therapy.

2D-DOA Estimation in Multipath Using EMVS Rectangle Array

Electromagnetic vector sensor (EMVS) arrays bring an epochal opportunity for direction finding, as they enable the estimation of two-dimensional direction of arrival (2D-DOA) and polarization characteristics. In this paper, we revisit the 2D-DOA estimation problem in an EMVS rectangle array under multipath propagation. An improved subspace estimator is proposed, which addresses the rank-deficit problem through matrix arrangement, and the 2D-DOA and polarization parameters are estimated via combining the normalized vector cross-product with the least squares method. Our proposed method is suitable for a single snapshot scenario and offers superior accuracy compared to existing methods. To validate its effectiveness, several numerical simulations have been designed and conducted.

Universal Geometric Relations of Acoustic Spin, Energy Flux, and Reactive Power

An understanding of acoustic spin, energy flux, and reactive power is crucial for manipulating and directing near-field sound. However, despite their importance, the relationship between these physical quantities has not been fully explored. In this paper, we aim to address this gap in knowledge by presenting a proof of some universal geometric relations between the acoustic spin, the cross product of the time-averaged energy flux and reactive power, and the local vorticity of the energy flux. These relations are independent of the specific wave structure and are verified for various acoustic systems. Moreover, our study demonstrates the mechanical torques and forces on small lossy particles generated by three different types of acoustic near-field sources, namely Janus, Huygens, and spin sources. These findings reveal hidden locking relations beyond the simple spin-momentum locking of near-field waves and provide valuable insights into the fundamental principles underlying the relationship between acoustic spin, radiation torque, energy flux, and reactive power in the field of acoustics.

Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture.

Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.