In this paper, we present a novel charge-free fluorescence-switchable near-infrared (IR) dye based on merocyanine for target specific imaging. In contrast to the typical bathochromic shift approach by extending πconjugation, the bathochromic shift of our merocyanine dye to the near-IR region is due to an unusual S-cis diene conformer. This is the first example where a fluorescent dye adopts the stable S-cis conformation. In addition to the novel bathochromic shift mechanism, the dye exhibits fluorescence-switchable properties in response to polarity and viscosity. By incorporating a protein-specific ligand to the dye, the probes (for SNAP-tag and hCAII proteins) exhibited dramatic fluorescence increase (up to 300-fold) upon binding with its target protein. The large fluorescence enhancement, near-IR absorption/ emission, and charge-free scaffold enabled no-wash and site-specific imaging of target proteins in living cells and in vivo with minimum background fluorescence. We believe that our unconventional approach for a near-IR dye with the S-cis diene conformation can lead to new strategies for the design of near-IR dyes.
The streptavidin–biotin controlled binding probe has several advantages for the detection of enzymes and reactive small molecules, such as minimal background, multiple signal amplification steps, and wide selection of the optimal dyes for detection.
Network I/O virtualization plays an important role in cloud computing. This paper addresses the system-wide virtualization issues of TCP/IP Offload Engine (TOE) and presents the architectural designs. We identify three critical factors that affect the performance of a TOE: I/O virtualization architectures, quality of service (QoS), and virtual machine monitor (VMM) scheduler. In our device emulation based TOE, the VMM manages the socket connections in the TOE directly and thus can eliminate packet copy and demultiplexing overheads as appeared in the virtualization of a layer 2 network card. To further reduce hypervisor intervention, the direct I/O access architecture provides the per VM-based physical control interface that helps removing most of the VMM interventions. The direct I/O access architecture out-performs the device emulation architecture as large as 30 percent, or achieves 80 percent of the native 10 Gbit/s TOE system. To continue serving the TOE commands for a VM, no matter the VM is idle or switched out by the VMM, we decouple the TOE I/O command dispatcher from the VMM scheduler. We found that a VMM scheduler with preemptive I/O scheduling and a programmable I/O command dispatcher with deficit weighted round robin (DWRR) policy are able to ensure service fairness and at the same time maximize the TOE utilization.
Currently most fluorogenic probes are developed for the analysis of enzymes, where a bond breaking or rearrangement reaction is required to transform a nonfluorescent enzymatic substrate into a fluorescent product. However, this approach cannot be used for proteins that do not possess enzymatic activities. In this article, we show that fluorogenic probes with a self-immolative difluorophenyl ester linker can mimic the bond disassembly processes of fluorogenic enzyme substrates for the rapid analysis of nonenzymatic proteins. Although numerous self-immolative reagents have shown promising applications in sensors, drug delivery systems, and material chemistry, all of them are triggered by either enzymes or small reactive molecules. In our strategy, the probe binds to the protein via a specific protein–ligand interaction, inducing a chemical reaction between the self-immolative linker and an amino acid of the protein, thereby triggering a cascade reaction that leads to the activation and release of the fluorogenic reporter. In contrast, a phenyl ester linker without the difluoro substituent cannot be triggered to release the fluorogenic reporter. With this probe design, live-cell imaging of extracellular and intracellular endogenous tumor marker proteins can be achieved with high selectivity and sensitivity.
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