Generation of hypoxia-sensing chimeric antigen receptor T cells

Exploiting hypoxia in solid malignancies to restrict expression of chimeric antigen receptors (CARs) on engineered T cells to the tumor microenvironment overcomes the risk of on-target off-tumor toxicity and minimizes tonic signaling, which promotes CAR T cell exhaustion. This protocol summarizes the synthetic biology underlying the development of a stringent oxygen-sensitive CAR for in vitro and in vivo preclinical characterization. For complete details on the use and execution of this protocol, please refer to Kosti et al. (2021)

Quantum state engineering assisted by entanglement

We suggest a general scheme for quantum state engineering based on conditional measurements carried out on entangled twin-beam of radiation. Realistic detection schemes such as {\sc on/off} photodetection, homodyne detection and joint measurement of two-mode quadratures are analyzed in details. Imperfections of the apparatuses, such as nonunit quantum efficiency and finite resolution, are taken into account. We show that conditional {\sc on/off} photodetection provides a reliable scheme to verify nonclassicality, whereas conditional homodyning represents a tunable and robust source of squeezed light. We also describe optical teleportation as a conditional measurement, and evaluate the degrading effects of finite amount of entanglement, decoherence due to losses, and nonunit quantum efficiency.Comment: Some pics with low resolution. Originals at http://www.qubit.i

Phagosomal Proteins of \u3ci\u3eDictyostelium discoideum\u3c/i\u3e

In recognizing food particles, Dictyostelium cell-surface molecules initiate cytoskeletal rearrangements that result in phagosome formation. After feeding D. discoideum cells latex beads, early phagosomes were isolated on sucrose step gradients. Protein analyses of these vesicles showed that they contained glycoproteins and surface-labeled species corresponding to integral plasma membrane proteins. Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa-actin bundling protein. As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H+-ATPase that was detected by immunoblotting. Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete. The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others. Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis

Consistent Video Filtering for Camera Arrays

International audienceVisual formats have advanced beyond single-view images and videos: 3D movies are commonplace, researchers have developed multi-view navigation systems, and VR is helping to push light field cameras to mass market. However, editing tools for these media are still nascent, and even simple filtering operations like color correction or stylization are problematic: naively applying image filters per frame or per view rarely produces satisfying results due to time and space inconsistencies. Our method preserves and stabilizes filter effects while being agnostic to the inner working of the filter. It captures filter effects in the gradient domain, then uses \emph{input} frame gradients as a reference to impose temporal and spatial consistency. Our least-squares formulation adds minimal overhead compared to naive data processing. Further, when filter cost is high, we introduce a filter transfer strategy that reduces the number of per-frame filtering computations by an order of magnitude, with only a small reduction in visual quality. We demonstrate our algorithm on several camera array formats including stereo videos, light fields, and wide baselines

Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase

Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings: This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance: Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes

Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings: This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance: Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes

A framework for non-intrusive load monitoring and diagnostics

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2006.Includes bibliographical references (p. 259-260).The widespread use of electrical and electromechanical systems places increasing demands on monitoring and diagnostic techniques. The non-intrusive load monitor (NILM) provides a low-cost, low-maintenance way to perform this monitoring and diagnostics from a centralized location. This work critically evaluates the current state of the NILM hardware and software in order to develop new techniques and a new hardware and software framework in which to better apply the NILM to real-world systems. New diagnostic indicators are developed on the USCGC SENECA using an improved hardware and software platform. A database-driven framework with the flexibility to create and implement these and future diagnostic indicators is presented.by James Paris.M.Eng

A comprehensive system for non-intrusive load monitoring and diagnostics

Thesis (Ph. D.)--Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references (pages 603-612).Energy monitoring and smart grid applications have rapidly developed into a multi-billion dollar market. The continued growth and utility of monitoring technologies is predicated upon the ability to economically extract actionable information from acquired data streams. One of the largest roadblocks to effective analytics arises from the disparities of scale inherent in all aspects of data collection and processing. Managing these multifaceted dynamic range issues is crucial to the success of load monitoring and smart grid technology. This thesis presents NilmDB, a comprehensive framework for energy monitoring applications. The NilmDB management system is a network-enabled database that supports efficient storage, retrieval, and processing of vast, timestamped data sets. It allows a flexible and powerful separation between on-site, high-bandwidth processing operations and off-site, low-bandwidth control and visualization. Specific analysis can be performed as data is acquired, or retroactively as needed, using short filter scripts written in Python and transferred to the monitor. The NilmDB framework is used to implement a spectral envelope preprocessor, an integral part of many non-intrusive load monitoring workflows that extracts relevant harmonic information and provides significant data reduction. A robust approach to spectral envelope calculation is presented using a 4-parameter sinusoid fit. A new physically-windowed sensor architecture for improving the dynamic range of non-intrusive data acquisition is also presented and demonstrated. The hardware architecture utilizes digital techniques and physical cancellation to track a large-scale main signal while maintaining the ability to capture small-scale variations.by James Paris.Ph.D