Vector Integration Research Articles

In Vitro and In Vivo Specificity and Biodistribution of a Novel CD8-Targeted Fusosome

Autologouschimeric antigen receptor (CAR) T cells represent a groundbreaking therapy for treating B-cell malignancies. However, the complexity, cost, time and toxicity associated with manufacturing and administering current CAR T cell therapies hinder their broader applications. To overcome CAR T limitations, Sana has developed a novel fusosome platform that can deliver a T cell targeted CAR gene therapy via systemic administration, without ablative pre-conditioning. The fusosome is a lentiviral vector pseudotyped with a targeted fusogen that specifically directs gene transfer into CD8+ cells. Our CD8-targeted CD19-directed CAR fusosome, SG299, transduces CD8+ cells in lymphoid tissues, showing preclinical safety and high selectivity, laying the groundwork for clinical evaluation of an in vivo CAR gene therapy. In vitro tissue cross reactivity (TCR) study was assessed to evaluate binding of the CD8 fusogen binder, a CD8-binding scFv, on tissues from Nemestrina macaques and compared these with tissues from human donors. SG299 specificity was also evaluated in vitro using panel of cell lines representing a breadth of receptor types. Cells were exposed to 8 IU/cell SG299 and vector integration was evaluated using ddPCR. Reverse transcriptase inhibitor nevirapine was used as control for the in vitro studies. To evaluate in vivo specificity and biodistribution, healthy nemestrina macaques were treated with a single dose of SG299 at 1 or 4 x 10 9 IU/kg (n=2 or 4) or saline (n=1 or 2) via intravenous infusion and evaluated for up to 35 days for vector biodistribution in CD8 enriched PBMCs and at terminal timepoint in panel of 17 tissues using ddPCR. In addition, we also demonstrated SG299 specificity with help of flow cytometry, in NALM6 tumor model in NSG mice. Briefly, NSG mice engrafted with human PBMCs were challenged with NALM6 tumors and treated with either SG299 or saline. The TCR study demonstrated that the CD8 fusogen binder is specific for the target tissues containing T cells and similar binding across multiple human and Nemestrina tissues. SG299 fusosome demonstrated CD8-specificity evaluated by transduction of cell types with CD8 receptor (SupT1 and PanT cells). There was no detectable off-target transduction in sets of primary as well as immortalized cell lines evaluated including pulmonary cells, renal epithelial cells, lymphoblast, endothelial cells and CD8KO cells. VCN analysis of nemestrina macaques PBMC and tissue samples following treatment with SG299 demonstrated dose dependent integrations per diploid genome through Day 35. All animals dosed 4 x 10 9 IU/kg showed quantifiable VCN signal through Day 34 in PBMC population enriched for CD8+ cells (0.0032 - 0.6544 vc/dg, vector copy per diploid genome). One out of 4 animals dosed at 1 x 10 9 IU/kg showed quantifiable VCN signal at Day 34 (0.0276 vc/dg). Tissues collected at necropsy on Day 35 demonstrated quantifiable VCN signal in spleen, lymph node, CD8+ splenocyte and lymphocyte, draining lymph node, heart ventricle, and injection site, for animals dosed at 4 x 10 9 IU/kg. One of 4 animals dosed 1 x 10 9 IU/kg showed quantifiable VCN in spleen, CD8+ splenocyte, and injection site. Flow cytometric analysis of peripheral blood of mice treated with SG299 showed a dose dependent increase in CAR T cells, and the CAR T cells were CD8+ T cells specific with no CAR T cell detection amongst the CD4+ T cells. Collectively, these studies demonstrate high on target specificity of SG299 fusosome to transduce CD8+ T cells in vitro and in vivo. Therefore, SG299 represents a novel therapeutic opportunity to generate CAR T cells in vivo and potentially overcome challenges associated with ex vivo CAR T therapies.

Dual α-Globin and Truncated EPO Receptor Knockin Restores Hemoglobin Production in α-Thalassemia-Derived Hematopoietic Stem and Progenitor Cells

Introduction: α-thalassemia major (ATM) is an autosomal recessive disorder where all four copies of the α-globin gene are deleted. A traditionally lethal disease due to severe fetal hypoxia, this condition is now survivable due to the advent of in utero blood transfusions. However, patients still require life-long transfusions postnatally. While allogeneic hematopoietic stem cell transplant (HSCT) can provide a definitive cure, it is limited by the paucity of suitable donors, has a high morbidity and mortality due the prerequisite myeloablative regimens, and carries the risk of immune rejection and graft-versus-host disease. Here, we describe a CRISPR/AAV-mediated genome editing strategy to restore a full-length copy of the α-globin gene at the β-globin locus in ATM patient-derived HSCs to restore normal hemoglobin production to a patient's red blood cells (RBCs). Methods: We identified a Cas9 gRNA that efficiently introduced indels at the HBB locus as well as an AAV6 vector that mediated efficient integration into the start codon of HBB. After designing and testing integration cassettes, we developed two candidate vectors that insert a HBA1 transgene at the HBB locus, with one bicistronic vector that also expresses a truncated form of the erythropoietin receptor (t EPOR) to increase erythropoietic output from edited HSCs. We used this approach to edit CD34 + HSCs derived from the bone marrow of three patients with ATM. Edited cells were cultured for 16 days in RBC differentiation media. Editing rates were quantified by a custom ddPCR assay. To determine the transcriptional response to these editing strategies, bulk RNA sequencing was performed comparing wild-type cells and patient cells that underwent respective editing conditions. Lastly, α-globin protein production was quantified using tetramer and single-globin high protein liquid chromatography (HPLC). Results: While effective editing frequencies in ATM HSCs were achieved by the HBA integration vector (39% edited alleles), we observed that editing rates increased over the course of RBC differentiation in the combined HBA+t EPOR edited condition, reaching 76% edited alleles by day 16. We also found that total cell counts by the end of RBC differentiation more than doubled (2.2-fold) in the HBA+t EPOR condition compared to the HBA alone condition. RNA sequencing of edited ATM-derived HSCs following RBC differentiation revealed an upregulation of HBA1, HBA2, and EPOR transcripts compared to unedited patient cells. These results were supported by HPLC, which demonstrated restoration of hemoglobin tetramers, including fetal hemoglobin (α+γ subunits), adult hemoglobin (α+β subunits), and hemoglobin A2 (α+δ subunits) compared to unedited patient cells. Complementing this with globin single chain analysis found that we had achieved 11% restoration of α-globin compared to wild-type levels. In spite of integration at the HBB locus, neither RNA sequencing nor HPLC showed disruption of β-globin production. α-globin production with a concomitant decrease in the formation of toxic β-globin dimers was confirmed by Western blot, with restoration of the normal balance of α-globin to dimeric β-globin in edited cells to wild-type levels. Conclusions: To our knowledge, these results demonstrate for the first time that gene therapy or genome editing may be used to increase α-globin production in ATM patient-derived RBCs. We found that the editing strategy that paired site-specific α-globin integration with t EPOR expression achieved the highest editing frequencies, greatest production of edited cells, and the most robust production of α-globin protein compared to α-globin integration alone. By increasing erythropoietic output using t EPOR, this approach has the potential to overcome some of the clinical challenges previously seen in HSCT for the hemoglobinopathies, including low editing and engraftment rates. Moreover, as patients with severe anemia such those with ATM have increased levels of circulating erythropoietin due to severe anemia, a t EPOR-based enrichment strategy is likely to be especially potent following transplantation into ATM bone marrow. We believe this could allow clinicians to reduce or eliminate the need for myeloablation, which stand as a major barrier to HSCT safety and efficacy. These findings support development of a definitive ex vivo autologous genome editing strategy that may be curative for ATM.

Structure-preserving discretization of fractional vector calculus using discrete exterior calculus

Fractional vector calculus is the building block of the fractional partial differential equations that model non-local or long-range phenomena, e.g., anomalous diffusion, fractional electromagnetism, and fractional advection-dispersion. In this work, we reformulate a type of fractional vector calculus that uses Caputo fractional partial derivatives and discretize this reformulation using discrete exterior calculus on a cubical complex in the structure-preserving way, meaning that the continuous-level properties curlαgradα=0 and divαcurlα=0 hold exactly on the discrete level. We discuss important properties of our fractional discrete exterior derivatives and verify their second-order convergence in the root mean square error numerically. Our proposed discretization has the potential to provide accurate and stable numerical solutions to fractional partial differential equations and exactly preserve fundamental physics laws on the discrete level regardless of the mesh size.

Concept of an elementary work as introduction to the line integral in engineering studies

The paper is devoted to the line integral topic belonging to the section of vector calculus in Mathematical Analysis applied to the undergraduate Mechanical Engineering program. An efficient way of teaching line integrals is proposed and developed based on the elementary work/force principles. By this way, the mathematical concept of the line integral is supposed to be learned in harmony with the elementary mechanics to appreciate its diversity and to set up the right idea about the scientific area covered by Mechanical Engineering and related academic and technical fields. In terms of practical training, the research also offers two new techniques of analytical calculus for line integrals containing singularities and provides a new coherent engineering approach to deal with vector fields as integrands in line integrals.

Versatile allelic replacement and self-excising integrative vectors for plasmid genome mutation and complementation.

In spite of the dissemination of multidrug-resistant plasmids among Gram-negative pathogens, including those carrying virulence genes, vector tools for studying plasmid-born genes are lacking. The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens including in vivo studies without the need for antibiotic selection. Overall, our newly developed vectors can be applied for the assessment of the function of plasmid-encoded genes by specifically creating mutations, moving large operons between plasmids and to/from the chromosome, and complementing phenotypes associated with gene mutation. Furthermore, the vectors express chromophores for the detection of target gene modification or colony isolation, avoiding time-consuming screening procedures.

POMBOX: A Fission Yeast Cloning Toolkit for Molecular and Synthetic Biology.

The fission yeast Schizosaccharomyces pombe is a popular model organism in molecular biology and cell physiology. With its ease of genetic manipulation and growth, supported by in-depth functional annotations in the PomBase database and genome-wide metabolic models,S. pombe is an attractive option for synthetic biology applications. However,S. pombe currently lacks modular tools for generating genetic circuits with more than 1 transcriptional unit. We developed a toolkit to address this gap. Adapted from the MoClo-YTK plasmid kit for Saccharomyces cerevisiae and using the same modular cloning grammar, our POMBOX toolkit is designed to facilitate fast, efficient, and modular construction of genetic circuits inS. pombe. It allows for interoperability when working with DNA sequences that are functional in bothS. cerevisiae and S. pombe (e.g., protein tags, antibiotic resistance cassettes, and coding sequences). Moreover, POMBOX enables the modular assembly of multigene pathways and increases the possible pathway length from 6 to 12 transcriptional units. We also adapted the stable integration vector homology arms to Golden Gate assembly and tested the genomic integration success rates depending on different sequence sizes, from 4 to 24 kb. We included 14 S. pombe promoters that we characterized using two fluorescent proteins, in both minimally defined (EMM2─Edinburgh minimal media) and complex (YES─yeast extract with supplements) media. Then, we examined the efficacy of 6 S. cerevisiae and 6 synthetic terminators in S. pombe. Finally, we used the POMBOX kit for a synthetic biology application in metabolic engineering and expressed plant enzymes in S. pombe to produce specialized metabolite precursors, namely, methylxanthine, amorpha-4,11-diene, and cinnamic acid from the purine, mevalonate, and aromatic amino acid pathways.

Denovo production of resveratrol by engineered Saccharomyces cerevisiae W303-1a using pretreated Gracilaria corticata extracts.

Assembly and construction of resveratrol production pathway in Saccharomyces cerevisiae for denovo production of resveratrol using seaweed extract as fermentation medium. Genes involved in the production of resveratrol from tyrosine pathway, tyrosine ammonia lyase (FTAL) gene from Flavobacterium johnsoniae (FjTAL), the 4-coumarate:CoA ligase gene from Arabidopsis thaliana (4CL1) and the stilbene synthase gene from Vitis vinifera (VvSTS) were introduced into low copy, high copy and integrative vector and transformed into S. cerevisiae W303-1a. The resulting strains W303-1a/pARS-res5, W303-1a/2µ-res1 and W303-1a/IntUra-res9 produced a level of 2.39 ± 0.01, 3.33 ± 0.03 and 8.34 ± 0.03mg resveratrol l-1 respectively. CRISPR mediated integration at the δ locus resulted in 17.13 ± 1.1mg resveratrol l-1. Gracilaria corticata extract was tested as a substrate for the growth of transformant to produce resveratrol. The strain produced a comparable level, 13.6 ± 0.54mg resveratrol l-1 when grown in seaweed extract medium. The strain W303-1a/IntδC-res1 utilized Gracillaria hydrolysate and produced 13.6 ± 0.54mg resveratrol l-1 and further investigations are being carried out focusing on pathway engineering and optimization of process parameters to enhance resveratrol yield.

Contour integration for the exchange of radiant energy between diffuse rectangular geometries

View factor calculation is a key point in radiation heat transfer between surfaces. In thermal engineering it is required to compute the view factor between complex geometries, which include several parallel and inclined surfaces. Currently, no analytical expressions are available for this purpose, so the use of Summation Rules (SR) is required, however, when analyzing complex geometries, the handling of SR is extremely laborious. Therefore, this work aims to develop an equation for the calculation of the view factor in the radiative heat transfer of two arbitrary rectangular inclined or parallel surfaces, using the advantages provided by vector calculus. The exact solutions obtained with contour integration were compared with the values calculated via the Algebra Factor (AF) using the Hottel and Hamilton-Morgan Solution and with the results computed from numerical integration of the quadruple integral using Simpson's rule 1/3 with five intervals. The lack of similar precedents in the technical literature reinforces the practical and scientific nature of the proposed method of analysis.

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations.

Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.

Standardization of Molecular MRD Levels in AML Using an Integral Vector Bearing ABL and the Mutation of Interest.

Quantitative PCR for specific mutation is being increasingly used in Acute Myeloid Leukemia (AML) to assess Measurable Residual Disease (MRD), allowing for more tailored clinical decisions. To date, standardized molecular MRD is limited to typical NPM1 mutations and core binding factor translocations, with clear prognostic and clinical implications. The monitoring of other identified mutations lacks standardization, limiting its use and incorporation in clinical trials. To overcome this problem, we designed a plasmid bearing both the sequence of the mutation of interest and the ABL reference gene. This allows the use of commercial standards for ABL to determine the MRD response in copy number. We provide technical aspects of this approach as well as our experience with 19 patients with atypical NPM1, RUNX1 and IDH1/2 mutations. In all cases, we demonstrate a correlation between response and copy number. We further demonstrate how copy number monitoring can modulate the clinical management. Taken together, we provide proof of concept of a novel yet simple tool, which allows in-house MRD monitoring for identified mutations, with ABL-based commercial standards. This approach would facilitate large multi-center studies assessing the clinical relevance of selected MRD monitoring.

Geometric approach to solving inverse kinematics of six DOF robot with spherical joints

Inverse kinematics is a fundamental concept in robotics that plays a crucial role in a robot’s ability to perform tasks. In this contribution, we propose a novel geometric approach based on vector calculus to solve the inverse kinematics problem. The primary advantage of this approach originates from the solutions, which exhibit a linear form and uncoupled equations. To validate the effectiveness and correctness of our proposed method, we constructed a six-degrees-of-freedom robot. This robot is controlled by an Arduino Mega 2650 on which we have implemented the inverse kinematics algorithm. The validation process involved considering various desired trajectories of the end-effector, which were simulated in Matlab and then performed by the physical robot. Importantly, our findings confirm that the end-effector successfully tracks the predefined trajectories. Furthermore, we conducted a comparative analysis between Paul’s method and the results obtained from joint angles using our proposed approach. Interestingly, our study reveals a significant similarity between the two sets of results, reaffirming the accuracy and validity of the approach presented in this study.

Political Preferences Of Residents Of Donbass Republics (In The Context Of The Elections To The State Duma Of The Russian Federation In 2021)

Very approximate information is available about the electoral preferences of the residents of Donbass within the framework of the Russian political and legal field. The article considers the issue of establishing the exact results of the will of the voters of the L-DPR, who were citizens of the Russian Federation, in the elections to the State Duma in 2021. Conclusions about the electoral preferences of residents of the L-DPR are made on the basis of the difference in the results of voting on the proportional and majority components of the electoral system of the Russian Federation, within which residents of Donbass could not vote for majority candidates. The results of remote electronic voting (REV), “exit” voting at the polling stations of the Rostov region and post-election polling are analyzed. It is established that at least 200 thousand L-DPR voters voted in these elections. The results of the REV and “exit” voting differed somewhat. The greatest support was enjoyed by: “United Russia” (the “exit” vote of the DPR — 81 %, the LPR — 83 %, the REV — 88 %) and the LDPR (up to 10 % on the “exit” vote). The electoral culture of Donbass had features of “periphery” in relation to the whole of the Russian Federation: many residents are poorly versed in the intricacies of Russian politics, vote for patriotic forces, identify support for the Russian Federation and its leadership. According to the results of surveys, more than 60 % of residents of the L-DPR ethnically identify themselves as Russians. Depending on the wording of the questions, from 65 % to 80 % of respondents called joining the Russian Federation a priority vector of integration of the region. During the period of the existence of the L-DPR, the prevailing pro-Russian sentiments in this territory have only intensified.

InCliniGene enables high-throughput andcomprehensive in vivo clonal tracking toward clinical genomics data integration.

High-throughput clonal tracking in patients under hematopoietic stem cell gene therapy with integrating vector is instrumental in assessing bio-safety and efficacy. Monitoring the fate of millions of transplanted clones and their progeny across differentiation and proliferation over time leverages the identification of the vector integration sites, used as surrogates of clonal identity. Although γ-tracking retroviral insertion sites (γ-TRIS) is the state-of-the-art algorithm for clonal identification, the computational drawbacks in the tracking algorithm, based on a combinatorial all-versus-all strategy, limit its use in clinical studies with several thousands of samples per patient. We developed the first clonal tracking graph database, InCliniGene (https://github.com/calabrialab/InCliniGene), that imports the output files of γ-TRIS and generates the graph of clones (nodes) connected by arches if two nodes share common genomic features as defined by the γ-TRIS rules. Embedding both clonal data and their connections in the graph, InCliniGene can track all clones longitudinally over samples through data queries that fully explore the graph. This approach resulted in being highly accurate and scalable. We validated InCliniGene using an in vitro dataset, specifically designed to mimic clinical cases, and tested the accuracy and precision. InCliniGene allows extensive use of γ-TRIS in large gene therapy clinical applications and naturally realizes the full data integration of molecular and genomics data, clinical and treatment measurements and genomic annotations. Further extensions of InCliniGene with data federation and with application programming interface will support data mining toward precision, personalized and predictive medicine in gene therapy. Database URL: https://github.com/calabrialab/InCliniGene.

A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes.

For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.

Chlamydia suis displays high transformation capacity with complete cloning vector integration into the chromosomal rrn-nqrF plasticity zone.

Chlamydia, comprising several human and zoonotic pathogens, is a genus of the conserved bacterial phylum Chlamydiota. Their obligate intracellular niche serves as a barrier for natural genetic exchange via horizontal gene transfer (HGT), and further limits the development and application of genetic tools. To date, the only example for recent inter-phylum HGT among the Chlamydiota is tetracycline resistance in the potentially zoonotic species Chlamydia suis, a close phylogenetic relative of human C. trachomatis, which causes bacterial sexually transmitted infections and ocular trachoma. Tetracycline resistance in porcine C. suis strains has been described worldwide and is always part of a genomic island dividing invasin (inv), located within a chromosomal region between the rRNA operon (rrn) and the nqrF reductase. Here, we aimed to expand the still modest number of available genetic manipulation systems for Chlamydia by generating allele-replacement and integration vectors for C. suis. These vectors comprised homologous C. suis sequences of the chromosomal region of interest, an E. coli origin of replication (ori) and selection markers but lacked the native chlamydial plasmids or its ori. We first recovered allele-replacement mutants using a vector that targets the tryptophan (trp) operon of C. suis. The vector was further successfully maintained as a free plasmid in C. trachomatis without allele replacement, suggesting complex plasmid dynamics in the absence of a chlamydial ori. Moreover, we showed that the hypervariable rrn-nqrF intergenic region of C. suis is highly susceptible to transformation, resulting in complete vector integration upstream of nqrF without interruption of the targeted inv gene.IMPORTANCEThe obligate intracellular Chlamydia genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of Chlamydia with the opportunistic porcine pathogen Chlamydia suis, the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer. C. suis is transmissible to humans, posing a potential public health concern. We report that C. suis can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that C. trachomatis, the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with C. suis vectors. Finally, the chromosomal region that harbors the resistance gene of C. suis is highly susceptible to complete vector integration.

Exploring the complexities of swapping the order of integration in double integrals

This article explores the conceptual challenges that engineering students encounter with double integrals in a vector calculus course. Drawing on previous literature and utilising APOS (Activity- Process- Objects- Schema) as a theoretical framework, this study investigates the difficulties that students face in understanding and applying double integrals. By analysing responses to a written test that assesses students’ conceptual understanding of double integrals and student responses in interviews, this research sheds light on specific areas where students struggle, such as curve sketching, identifying regions of integration and changing the order of integration. Swapping the order of integration in double integrals is the focus of this research study. The findings of this study have the potential to contribute to the enhancement of teaching and learning strategies for double integrals to better support engineering students’ conceptual comprehension and proficiency in this important mathematical concept.

Nonlocal half-ball vector operators on bounded domains: Poincaré inequality and its applications

This work contributes to nonlocal vector calculus as an indispensable mathematical tool for the study of nonlocal models that arises in a variety of applications. We define the nonlocal half-ball gradient, divergence and curl operators with general kernel functions (integrable or fractional type with finite or infinite supports) and study the associated nonlocal vector identities. We study the nonlocal function space on bounded domains associated with zero Dirichlet boundary conditions and the half-ball gradient operator and show it is a separable Hilbert space with smooth functions dense in it. A major result is the nonlocal Poincaré inequality, based on which a few applications are discussed, and these include applications to nonlocal convection–diffusion, nonlocal correspondence model of linear elasticity and nonlocal Helmholtz decomposition on bounded domains.

Non-expansive matrix number systems with bases similar to certain Jordan blocks

We study representations of integral vectors in a number system with a matrix base M and vector digits. We focus on the case when M is equal or similar to Jn, the Jordan block with eigenvalue 1 and dimension n. If M=J2, we classify all digit sets of size two allowing representation for all of Z2. For M=Jn with n≥3, we show that a digit set of size three suffice to represent all of Zn. For bases M similar to Jn, n≥2, we construct a digit set of size n such that all of Zn is represented. The language of words representing the zero vector with M=J2 and the digits (0,±1)T is shown not to be context-free, but to be recognizable by a Turing machine with logarithmic memory.

On the spurious effects in Lamb-vector-based force decomposition methods

Recent methods based on Lamb vector integration allow for a far-field formulation and decomposition of the whole aerodynamic force, with identification of local flow structures associated with the force generation. Different exact Lamb-vector-based force formulations were proposed in the past and the decomposition of total drag into lift-induced and parasite contributions was subject of very recent studies. However, inconsistencies were also reported, raising questions on the physical role of a non-zero induced drag found in two-dimensional flows. Moreover, the effect of the arbitrary pole (appearing in Lamb-vector-based formulas) on the numerical accuracy of such methods was never investigated. Additionally, the separation of parasite drag into viscous and wave drag contributions by Lamb vector field analysis is still questioned. In this paper, the accuracy of different force formulas and of the drag decomposition in lift-induced and parasite contributions is first analyzed, for steady flows. Two-dimensional numerical solutions around RAE2822 and NACA0012 airfoils are considered, the latter in both inviscid and viscous flows (at low and high Reynolds number), to analyze consistency between lift-induced/parasite drag definitions and the drag generated in isentropic flow conditions (reversible drag) or due to entropy production (irreversible drag). A spurious induced drag contribution (related to the streamwise momentum perturbation) is identified, resulting in negative induced drag values in two-dimensional flows. The effect of a shift in the pole location is also analysed, with reference to its impact on the total force and on drag decomposition accuracy, then a new approach to parasite drag breakdown into physical constituents (viscous and wave contributions) is proposed and results are compared against predictions obtained using consolidated thermodynamic-based methods and against available results from recent literature.

Integrome signatures of lentiviral gene therapy for SCID-X1 patients.

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.