Applicability of multi-objective optimization in classroom acoustics design using analytical and geometrical acoustic models

The purpose of this study is to improve the acoustic quality of classrooms for a better teaching-learning process by the development of a routine which aids in the optimization of typology, extension and position of acoustic materials. It is a tool addressed to architects, building designers and professionals alike that are involved in the planning, construction and renovation of rooms. One classroom in Turin has been selected for this study and a basic geometric model has been built in Grasshopper, that serves as the environment for parametric investigation and improvement of acoustics parameters. Reverberation time and STI, which are considered as the most important descriptors in classroom acoustics have been determined using theoretical calculations (Sabine, Eyring and Barron&Lee theories) and geometrical acoustic (GA) simulations (Pachyderm). The latter allows to take into account the scattering properties of surfaces and different combinations of all the acoustic materials Finally, Octopus has been used to perform multi‐objective optimization runs considering as objectives the acoustic parameters and the acoustic design/renovation costs. The algorithm has been developed in order to allow to choose different optimization sets depending on the material or the type of acoustic treatment to optimize. Said model provides, essential information on the acoustic quality of the classroom and recommendations on how to increase it by improving teaching-learning activities; which would normally be time-consuming. The results show that the GA simulations and theoretical calculations are compatible for the solutions without scattering properties. However, it needs further development in order to extend its application field in fact right now operating on the Grasshopper canvas would require significant knowledge on the designer?s behalf concerning parametric design and acoustics

690. Permanent Epigenetic Silencing of Human Genes With Artificial Transcriptional Repressors

There are several diseases whereby the goal of gene therapy is to silence rather than replace a gene function. Paradigmatic examples are diseases caused by a dominant negative mutation or those in which silencing of a host gene confers resistance to a pathogen or compensates the function of the missing gene. Yet, gene silencing can be used to enhance efficacy of cell therapy and for biotechnological applications. Until now, two technologies have been used to silence gene expression, namely RNA interference with short harping RNAs (shRNA) and gene disruption with Artificial Nucleases (ANs). Although some promising pre-clinical and clinical data have been already obtained, the low efficiency of knock-down with shRNA and of biallelic disruption with ANs may limit efficacy of these treatments, especially when residual gene activity can exert a biological function. To overcome this issue, we have developed a novel modality of gene silencing that exploits endogenous epigenetic mechanisms to convey robust and heritable states of repression at the desired target gene. We have generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of a chromatinmodifying enzyme involved in silencing of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in human cell lines and primary cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains that, when transiently co-assembled on the promoter of the reporter cassette, fully abrogated transgene expression in up to 90% of treated cells. Importantly, silencing was maintained for more than 250 days in cultured cell lines, was resistant to in vitro differentiation or metabolic activation of primary cells, and was confined to the reporter cassette. Silencing was associated with high levels of de novo DNA methylation at the targeted locus and was dependent on this epigenetic mark for its propagation. Finally, transient transfection of 3 ATRs targeted to the promoter region of the Beta-2-microglobulin (B2M) gene resulted in the loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of treated cells. This phenotype was associated with a switch in the epigenetic and transcriptional state of the constitutively active B2M gene, which became highly decorated with DNA methylation and deprived of RNA PolII and of its transcript. Of note, silencing was resistant to IFN-γ treatment, a potent B2M inducer. Overall, these data provide the first demonstration of efficient and stable silencing of an endogenous gene upon transient delivery of ATRs. This result was made possible by repurposing the machinery involved in silencing of ERVs, which instructs self-sustaining repressive epigenetic states on the gene of interest. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for the treatment of several diseases. (LN & AL co-authorship

729 inheritable silencing of endogenous gene by hit and run targeted epigenetic editing

Gene silencing holds great promise for the treatment of several diseases and can be exploited to investigate gene function and activity of the regulatory genome. Here, we develop a novel modality of gene silencing that exploits epigenetics to achieve stable and highly efficient repression of target genes. To this end, we generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of chromatin-modifying enzymes involved in silencing process of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains (namely KRAB, DNMT3A and DNMT3L) that, when transiently co-assembled on the promoter of the reporter cassette, recreate a powerful embryonic-specific repressive complex capable of inducing full and long-term (>150 days) silencing of transgene expression in up to 90% of the cells. The ATR-induced silencing was cell type and locus independent, and resistant to metabolic activation of the cells. Importantly, these findings were holding true also for endogenous genes embedded in their natural chromatin context, as shown for the highly and ubiquitously expressed B2M gene. Here, transient co-delivery of TALE-based ATRs resulted in loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of the cells. This phenotype was associated with a drastic switch in the epigenetic and transcriptional state of the constitutively active B2M promoter, which become highly decorated with de novo DNA methylation and deprived of RNAP II. Importantly, silencing was sharply confined to the targeted gene and resistant to INF-γ, a potent natural activator of B2M. We further extended these studies by showing that our silencing approach is portable to the CRISPR/dCas9 DNA binding technology. In this setting, comparable levels of B2M silencing (up to 80%) were achieved using either pools or even individual sgRNAs coupled to dCas9-based ATRs. Yet, adoption of this technology allowed performing simultaneous, highly efficient multiplex gene silencing within the same cell, as shown for B2M, IFNAR1 and VEGFA. Finally, we assessed resistance of the silenced gene to activity of potent artificial transcription activators and chromatin remodelers, and found that only targeted DNA demethylation was able to reawaken the silent gene. This allowed performing iterative cycles of silencing and reactivation of the same gene in the same cell population. Overall, these data provide the first demonstration of efficient and stable epigenetic silencing of endogenous genes upon transient delivery of ATRs. This was accomplished by repurposing the ERVs silencing machinery, which instructs self-sustaining repressive epigenetic states to the target gene. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for both basic and translational research

Performance comparison between the Click Modular Router and the NetFPGA

It is possible to forward minimum-sized packets at rates of hundreds of Mbps using commodity hardware and Linux. We had a preference for the Click Modular Router platform due its flexibility and the fact that it claimed to have equal or higher performance than native forwarding if used with its polling drivers. Moreover, the NetFPGA is an open networking platform accelerator that enables researchers and instructors to build working prototypes of high-speed, hardware-accelerated networking systems. NetFPGA reference designs comprised in the system include an IPv4 router, an Ethernet switch, a four-port NIC, and SCONE (Software Component of NetFPGA). Researchers have used the platform to build advanced network flow processing systems. We have followed the RFC1242 - Benchmarking Terminology for Network Interconnection Devices - and the RFC2544 - Benchmarking Methodology for Network Interconnection Devices - in order to define the specific set of tests to use to describe the performance characteristics of the two routers. We have also shown a test comparison between the NetFPGA and the Click router about a file transfer using the FTP and the HTTP protocol.Overall, the NetFPGA router performance outperforms the Click router performance

NetFPGA Hardware Modules for Input, Output and EWMA Bit-Rate Computation

NetFPGA is a hardware board that it is becoming increasingly popular in various research areas. It is a hardware customizable router and it can be used to study, implement and test new protocols and techniques directly in hardware. It allows researchers to experience a more real experiment environment. In this paper we present a work about the design and development of four new modules built on top of the NetFPGA Reference Router design. In particular, they compute the input and output bit rate run time and provide an estimation of the input bit rate based on an EWMA filter. Moreover we extended the rate limiter module which is embedded within the output queues in order to test our improved Reference Router. Along the paper we explain in detail each module as far as the architecture and the implementation are concerned. Furthermore, we created a testing environment which show the effectiveness and effciency of our module

A P2P Platform for real-time multicast video streaming leveraging on scalable multiple descriptions to cope with bandwidth fluctuations

In the immediate future video distribution applications will increase their diffusion thanks tothe ever-increasing user capabilities and improvements in the Internet access speed and performance.The target of this paper is to propose a content delivery system for real-time streaming services based ona peer-to-peer approach that exploits multicast overlay organization of the peers to address thechallenges due to bandwidth heterogeneity. To improve reliability and flexibility, video is coded using ascalable multiple description approach that allows delivery of sub-streams over multiple trees andallows rate adaptation along the trees as the available bandwidth changes. Moreover, we have deployeda new algorithm for tree-based topology management of the overlay network. In fact, tree based overlaynetworks better perform in terms of end-to-end delay and ordered delivery of video flow packets withrespect to mesh based ones. We also show with a case study that the proposed system works better thansimilar systems using only either multicast or multiple trees

637. Targeting of Myeloid Leukemia by IL-10-Engineered Human CD4+ Tr1 Cells

T regulatory type 1 (Tr1) cells, characterized by the co-expression of CD49b and LAG-3 and the ability to secrete high amounts of IL-10, control immune responses by IL-10 and TGF-beta production and by killing of myeloid cells via a Granzyme B-dependent mechanism. Tr1 cells are induced in vitro in the presence of recombinant human IL-10 or tolerogenic dendritic cells secreting high amounts of IL-10 (DC-10). Proof-of-principle clinical trials suggest that Tr1 cells can modulate Graft-versus Host Disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, their ability to mediate anti-leukemic activity or their effects of Graft versus Leukemia is largely unknown. We previously showed that enforced IL-10 expression converts human CD4+ T cells into Tr1-like (CD4IL-10) cells that suppress effector T cells in vitro and prevent xenogeneic-GvHD in humanized models. We now demonstrate that these CD4IL-10 cells selectively kill myeloid cell lines and myeloid blasts in vitro in HLA-class I-dependent but antigen-independent manner. Moreover, cytotoxic activity of CD4IL-10 cells is Granzyme B-dependent, is specific for CD13+ cells, and requires CD54 and CD112 expression on target cell lines or primary leukemic blast. Adoptive transfer of CD4IL-10 cells in humanized models mediates direct anti-leukemic activity, and does not compromise the anti-leukemic effect of allogeneic T cells while inhibits xeno-GvHD. These findings provide a strong rationale for designing personalized immunotherapy approaches using CD4IL-10 cells after allo-HSCT to cure myeloid malignancies

Phase diagram of QCD with four quark flavors at finite temperature and baryon density

We analyze the phase diagram of QCD with four staggered flavors in the (mu, T) plane using a method recently proposed by us. We explore the region T > 0.7 Tc and mu <1.4 Tc, where Tc is the transition temperature at zero baryon density, and find a first order transition line. Our results are quantitatively compatible with those obtained with the imaginary chemical potential approach and the double reweighting method, in the region where these approaches are reliable, T > 0.9 Tc and mu < Tc. But, in addition, our method allows us to extend the transition line to lower temperatures and higher chemical potentials.Comment: 14 pages, 8 figures. Comments and new data added. Version to be published in Nuclear Physics

6. Targeted Genome Editing of Cell Lines for Improved and Scalable Production of Lentiviral Vectors for Human Gene Therapy

Lentiviral vectors (LVs) represent efficient and versatile vehicles for gene therapy. The manufacturing of clinical-grade LVs relies on transient transfection of vector components. This method is labor and cost intensive and becomes challenging when facing the need of scale-up and standardization. The development of stable LV producer cell lines will greatly facilitate overcoming these hurdles. We have generated an inducible LV packaging cell line, carrying the genes encoding for third-generation vector components stably integrated in the genome under the control of tetracycline-regulated promoters. In order to minimize the immunogenicity of LVs for in vivo administration, we set out to remove the highly polymorphic and antigenic class-I major histocompatibility complex (MHC-I) expressed on LV packaging cells and subsequently incorporated on the LV envelope. We performed genetic disruption of the β-2 microglobulin (B2M) gene, a required component for the assembly and trafficking of the MHC-I to the plasma membrane in LV producer cells, exploiting the RNA-guided Cas9 nuclease. We generated B2M-negative cells devoid of surface-exposed MHC-I, which retain the ability to produce LVs. In order to insert the LV genome of interest in the packaging cell line, we performed site-specific integration in predetermined loci of the genome of these cells, chosen for robust expression, exploiting artificial nucleases and homology-directed repair. In several independent iterations of this process, we generated producer cell lines both for LV expressing marker genes and a therapeutic gene, i.e. coagulation factor IX (FIX), the gene mutated in hemophilia B. We show that these LV producer cells are stable in culture and can produce several liters of LV-containing conditioned medium. These LVs have comparable or only slightly lower infectious titer and specific infectivity than LVs produced by state-of-the-art transient transfection process and can transduce therapeutically relevant target cells, such as hematopoietic stem/progenitor cells and T lymphocytes to high efficiency. Moreover, we intravenously administered FIX-expressing LVs produced by the cell line to hemophilia B mice and established therapeutic levels of circulating FIX. These data indicate that site-specific integration is an efficient, rapid and reproducible method to generate LV producer cells, starting from a universal stable inducible LV packaging cell line. Overall, we provide evidence that rationally designed targeted genome engineering can be used to improve the quality, safety and sustainability of LV production for clinical use