Lentiviral Vector Production at High Cell Density by Transient Transfection of Suspended Culture HEK Cells

Viral vectors are gene carriers that efficiently deliver therapeutic gene constructs to target cells. Viral vectors are frequently produced in suspension-growing cells of mammalian origin, referred to as packaging cells. Lentiviral vectors have become widely used as gene vectors since they were initially developed two and a half decades ago, particularly in research settings. The advantages of lentiviral vectors for treating diseases of genetic origin have driven research into their large-scale manufacture for clinical settings. Currently, three main challenges exist that limit mass production of lentiviral vectors. First, plasmid DNA used in the production process is very expensive. Second, high vector titers have historically only been achievable at low packaging cell densities, limiting the scalability of suspended-culture technologies. And third, vector production frequently suffers from batch-to-batch variability in vector yield. In conjunction, these challenges diminish the economic viability and practical implementation of industrially-scaled processes for lentiviral vector production. Recent advances in commercial reagents can help mitigate these stated challenges. There is currently a knowledge gap in the utility of cationic lipid-based transfection reagents for producing lentivirus in suspended cultures at high cell density. In the present work, we investigate the production of lentiviral vectors by transient transfection of high density Human Embryonic Kidney packaging cells with a commercial cationic lipid transfection reagent. Several parameters in the production process were monitored to identify sources of variability in vector titers. We found that as packaging cells were cultured to increasing densities pre-transfection, there was a corresponding increase in lentiviral vector yield. Further, we found that aside from pre-culture density, consistency in culture routine leading up to transfection had the most significant influence on lentiviral vector yields

Application of Safe Harbor Provisions to Early Termination of Swap Agreement

(Excerpt) Credit default swap agreements (“swaps”) are contracts between two entities in which the counterparties are effectively taking opposing positions on the credit worthiness of a debt instrument that acts as the collateral underlying the swap. For example, counterparty A makes payments to counterparty B in the hopes that the debtor defaults or a counterparty commits an event of default specified in the agreement, such as filing for bankruptcy. Swaps are generally executory contracts and thus may generally be rejected by a trustee or a debtor in bankruptcy pursuant to section 365 of the United States Bankruptcy Code (“the Code”). However, pursuant to section 365 of the Code, a trustee may not reject an executory contract solely because of a provision in the agreement conditioned on a party filing for bankruptcy (“Anti-Ipso Facto Provision”). Swaps are also subject to section 560 of the Code (the “Safe Harbor Provision”), one of the Code’s safe harbor provisions. Section 560 allows a swap participant to “cause the liquidation, termination, or acceleration” of a swap agreement even if a counterparty files for bankruptcy. The consequences of an event of default vary by agreement; penalty payments or a change in the payment priorities of the defaulting counterparty (“flip clause”) are common. A flip clause is a provision which changes the party’s payment priority for the proceeds from the liquidation of the collateral underlying the swap. Whether a flip clause is covered by the Safe Harbor Provision is the subject of this memo. There are three cases involving the Lehman Bros. bankruptcy that are of particular importance to this issue. In the immediate fallout from the 2008 financial crisis, and the collapse of the swap market, Judge Andrew J. Peck decided two cases related to the early termination of swap agreements as a result of Lehman’s bankruptcy. Judge Peck found the penalty payments were covered by the Safe Harbor Provision but the flip clauses were not because a change in payment priority created different rights than those the defaulting party held before the bankruptcy filing. Judge Peck retired in 2014 and in 2016 Judge Shelley C. Chapman decided another case from the Lehman Bros bankruptcy. Judge Chapman applied a plain reading of the Safe Harbor Provision to hold flip clauses were covered as a part of the “liquidation, termination, or acceleration of the agreement.” This memorandum addresses the issue of whether the safe harbor provisions set forth in the Code cover flip clauses triggered by the early termination of a swap agreement. Part I addresses Judge Peck’s narrow reading of the Safe Harbor Provision in the aftermath of the financial crisis. Part II notes cases that addressed safe harbor provisions in between Judge Peck and Judge Chapman’s rulings. Part III discusses Judge Chapman’s plain reading of the Safe Harbor Provision. Part IV summarizes the case that dealt with another of the Code’s safe harbor provisions

Design, synthesis and biological evaluation of 1,3-dihydroxyxanthone derivatives: Effective agents against acetylcholinesterase

The present work concerns the rational design and development of new inhibitors of acetylcholinesterase (AChE) based on the privileged xanthone scaffold. In order to understand and rationalize the mode of action of these target structures a theoretical study was initially conducted. From the results of rational design, a new variety of amphiphilic xanthone derivatives were synthesized, structurally characterized and evaluated as potential anti-Alzheimer agents. The results showed that most of the synthesized compounds exhibited high AChE inhibitory activity at the micromolar range (IC50, 0.46–12.09 μM). The synthetic xanthone 11 showed the best inhibitory effect on AChE and a molecular modeling study revealed that 11 targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Therefore, this compound could be considered as a potential lead compound towards new drugs for the treatment of Alzheimer’s disease.Fil: Menéndez, Cintia Anabella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Biscussi, Brunella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Accordino, Sebastian Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Murray, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Gerbino, Darío César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Appignanesi, Gustavo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; Argentin

Size dependence of dynamic fluctuations in liquid and supercooled water

We study the evolution of dynamic fluctuations averaged over different space lengths and time scales to characterize spatially and temporally heterogeneous behavior of TIP4P/2005 water in liquid and supercooled states. Analyzing a 250 000 molecules simulated system, we provide evidence of the existence, upon supercooling, of a significant enhancement of spatially localized dynamic fluctuations stemming from regions of correlated mobile molecules. We show that both the magnitude of the departure from the value expected for the system-size dependence of an uncorrelated system and the system size at which such a trivial regime is finally recovered clearly increase upon supercooling. This provides a means to estimate an upper limit to the maximum length scale of influence of the regions of correlated mobile molecules. Notably, such an upper limit grows two orders of magnitude on cooling, reaching a value corresponding to a few thousand molecules at the lowest investigated temperature.Fil: Montes de Oca, Joan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Accordino, Sebastián R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Appignanesi, Gustavo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Handle, Philip H.. Universidad de Innsbruck; AustriaFil: Sciortino, Francesco. Università degli studi di Roma "La Sapienza"; Itali

Structural features of high-local-density water molecules: Insights from structure indicators based on the translational order between the first two molecular shells

The two-liquids scenario for liquid water assumes the existence of two competing preferential local molecular structural states characterized by either low or high local density. While the former is expected to present good local order thus involving privileged structures, the latter is usually regarded as conforming a high-entropy unstructured state. A main difference in the local arrangement of such "classes" of water molecules can be inferred from the degree of translational order between the first and second molecular shells. This is so, since the low-local-density molecules present a clear gap between the first two shells while in the case of the high-local-density ones, one or more molecules from the second shell have collapsed toward the first one, thus populating the intershell region. Some structural indicators, like the widely employed local structure index and the recently introduced ζ index, have been devised precisely on the basis of this observation, being successful in detecting well-structured low-local-density molecules. However, the nature of the high-local-density state has been mainly disregarded over the years. In this work we employ molecular dynamics simulations for two water models (the extended simple point charge model and the five-site model) at the liquid and supercooled regimes combined with the inherent dynamics approach (energy minimizations of the instantaneous configurations) in order to both rationalize the detailed structural and topological information that these indicators provide and to advance in our understanding of the high-density state.Fil: Montes de Oca, Joan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Accordino, Sebastián R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Verde, Alejandro Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Alarcón, Laureano M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Appignanesi, Gustavo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; Argentin

Studio e sviluppo di descrittori locali per nuvole di punti basati su proprietà geometriche

In questo lavoro sono stati proposti due nuovi descrittori per cloud point: ReSHOT e KPL-Descriptor. Inoltre sono state testate delle idee per migliorare le performance di tutta la pipeline di feature matching. Il lavoro comprende una fase di comparazione con i descrittori preesistenti

Comparing the performance of two structural indicators for different water models while seeking for connections between structure and dynamics in the glassy regime

In this work, we compare the performance of two structural indicators based on the degree of translational order up to the second coordination shell in three water models: SPC/E, TIP4P/2005, and TIP5P. Beyond directly contrasting their distributions for different temperatures to evidence their usefulness in estimating the fraction of structured and unstructured molecules and, when possible, their classification capability, we also correlate them with an indirect measure of structural constraint: the dynamic propensity. Furthermore, this procedure enables us to show the existence of evident correlations between structural and dynamical information. More specifically, we find that locally structured molecules display a preference for low dynamic propensity values and, more conspicuously, that locally unstructured molecules are extremely subject to high dynamic propensity. This result is particularly relevant for the supercooled regime where the establishment of firm links between the structure and dynamics has remained rather elusive since the occurrence of dynamics that vary in orders of magnitude upon supercooling usually contrast with barely noticeable overall structural changes.Fil: Verde, Alejandro Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Montes de Oca, Joan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Accordino, Sebastián R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Alarcón, Laureano M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Appignanesi, Gustavo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; Argentin

Wrapping Effects within a Proposed Function-Rescue Strategy for the Y220C Oncogenic Mutation of Protein p53

Soluble proteins must protect their structural integrity from water attack by wrapping interactions which imply the clustering of nonpolar residues around the backbone hydrogen bonds. Thus, poorly wrapped hydrogen bonds constitute defects which have been identified as promoters of protein associations since they favor the removal of hydrating molecules. More specifically, a recent study of our group has shown that wrapping interactions allow the successful identification of protein binding hot spots. Additionally, we have also shown that drugs disruptive of protein-protein interfaces tend to mimic the wrapping behavior of the protein they replace. Within this context, in this work we study wrapping three body interactions related to the oncogenic Y220C mutation of the tumor suppressor protein p53. Our computational results rationalize the oncogenic nature of the Y220C mutation, explain the binding of a drug-like molecule already designed to restore the function of p53 and provide clues to help improve this function-rescue strategy and to apply in other drug design or re-engineering techniques.Fil: Accordino, Sebastián Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Química del Sur; Argentina.;Fil: Rodríguez Fris, Jorge Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Química del Sur; Argentina.;Fil: Appignanesi, Gustavo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Química del Sur; Argentina.

Quality of working life systems in municipalities : a lasting innovation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Urban Studies, 1987.Bibliography: v. 2, leaves 385-397.by John Joseph Accordino.Ph.D