Publications

Supplementary Figure 1B from Chemotherapy-Induced Genotoxic Stress Promotes Sensitivity to Natural Killer Cell Cytotoxicity by Enabling Missing-Self Recognition

The living cell possesses extraordinary molecular and biochemical mechanisms by which to recognize and efficiently remove foreign, damaged, or denatured proteins. This essential function has been a barrier to the overexpression of recombinant proteins in most expression systems. A notable exception is the overexpression in <i>E. coli</i> of recombinant proteins, most of which, however, end-up as "inclusion bodies", <i>i.e.</i>, cytoplasmic aggregates of proteins that are inaccessible to the cell's proteasome. "Fusion constructs as protein overexpression vectors" proved to be unparalleled in their ability to cause substantial accumulation of recombinant proteins from plants, animals, and bacteria, as soluble proteins in unicellular cyanobacteria. Recombinant protein levels in the range of 10-20% of the total cellular protein can be achieved. The present work investigated this unique property in the context of recombinant protein stability in <i>Synechocystis</i> sp. PCC 6803 by developing and applying an <i>in vivo</i> cellular tobacco etch virus cleavage system with the objective of separating the target heterologous proteins from their fusion leader sequences. The work provides new insights about the overexpression, cellular stability, and exploitation of transgenes with commercial interest, highly expressed in a cyanobacterial biofactory. The results support the notion that eukaryotic plant- and animal-origin recombinant proteins are unstable, when free in the cyanobacterial cytosol but stable when in a fusion configuration with a highly expressed cyanobacterial native or heterologous protein. Included in this analysis are recombinant proteins of the plant isoprenoid biosynthetic pathway (isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase), the human interferon protein, as well as prokaryotic proteins (tetanus toxin fragment C and the antibiotic resistance genes kanamycin and chloramphenicol). The future success of synthetic biology approaches with cyanobacteria and other systems would require overexpression of pathway enzymes to attain product volume, and the work reported in this paper sets the foundation for such recombinant pathway enzyme overexpression.

The types of the immune responses generated against an antigen are determined by the intracellular fate of the antigen. Endogenous antigens are processed in the cytoplasm and initiate cytotoxic T lymphocyte (CTL) activation. In contrast, exogenous antigens are degraded in the lysosome (or phagolysosome) of antigen presenting cells (APCs), and induce antibody-mediated immune responses and assist CTL activation. Therefore, maximizing a desired response by controlling delivery pathways is indispensable in vaccine development and immunotherapy. New cleavable microparticles have been prepared for use as protein-based vaccine carriers by polymerizing water soluble monomers including a newly developed aliphatic monomer with a pendant primary amine group and a cleavable acetal linkage with a wholly aliphatic cleavable acetal cross-linker. Incorporation of the cleavable amine monomer in the polymerization mixture increased the encapsulation efficiency of a model antigen, ovalbumin. Ex vivo assays showed that the composition of the particles greatly affected the magnitude and the pathway of antigen presentations, which determine the type of immune responses. The degradable particles synthesized with the new cross-linker enhanced MHC I antigen presentation 2-3-fold over nondegradable particles. It was also found that, by adding 10% cationic cleavable monomers to the microparticles, MHC I restricted antigen presentation was enhanced ca. 75 times over that achieved with nondegradable particles. The microparticles introduced in this study can be further used for targeting and gene delivery due to functionalizable and cleavable cationic monomers in addition to degradability.

How might the computing and communications world be materially different in 10 to 15 years, and how might we define a research agenda that would get us to that world?

Printed antennas exemplified by the microstrip patch antenna offer an attractive solution to compact, conformal and low cost design of modem wireless communications equipment, RF sensors and radar systems. Recent applications have pushed the frequency well into the ram-wave region even in the commercial arena as evidenced by the worldwide race to develop advanced collision warning radar systems for automobiles at the 76 GHz band.[1] Microstrip-based planar antennas fabricated on a substrate with a high dielectric constant (Si, GaAs and InP) are strongly preferred for easy integration with the MMIC RF front-end circuitry. However, it is well known that patch antennas on high dielectric constant substrates are highly inefficient radiators due to surface wave losses and have very narrow frequency bandwidth (approximately one to two percent). This situation becomes extremely severe as applications move to higher frequencies, resulting in patch antennas with reduced gain and efficiency as well as an unacceptably high level of cross polarization and mutual coupling within an array environment. Therefore, much effort has been made recently to realize high efficiency patch antennas on high permittivity substrates, including using the latest micromachining technology.[2,3]

Abstract A common feature of hematopoietic activating immunoreceptors resides in their association at the cell surface with transmembrane signaling adaptors. Several adaptors, such as the CD3 molecules, FcRγ and KARAP/DAP12, harbor intracytoplasmic immunoreceptor tyrosine‐based activation motifs (ITAM) that activate Syk‐family protein tyrosine kinases. In contrast, another transmembrane adaptor, DAP10, bears a YxxM motif that delivers signals by activation of lipid kinase pathways. We show here that the human signal‐regulatory protein SIRPβ1 can associate with both DAP10 and KARAP/DAP12 in a model of RBL‐2H3 cell transfectants. In association with KARAP/DAP12, SIRPβ1 complexes are capable of inducing serotonin release and tumor necrosis factor (TNF) secretion. By contrast,in the absence of KARAP/DAP12, engagement of SIRPβ1:DAP10 complexes does not lead to detectable serotonin release or TNF secretion by RBL‐2H3 transfectants. However, triggering of SIRPβ1:DAP10 complexes co‐stimulates RBL‐2H3 effector function induced by sub‐optimal stimulation of the endogenous FcϵRI complex. Therefore, we report here a cellular model in which the association of a cell surface receptor with various signaling adaptors dictates the co‐stimulatory or the direct stimulatory properties of the complex.

Abstract NKG2D is one of the most important activating receptors expressed by NK cells in terms of tumor cell recognition. NKG2D binds to different ligands, and triggers cytolysis and cytokine release by NK cells. Certain human NKG2D ligands can be shed and are thought to inhibit NK-mediated surveillance of cancer. Here we showed that a mouse NKG2D ligand, MULT1, is cleaved efficiently from cells, and accumulates in the serum of tumors and inflammatory diseases. Cleaved MULT1 binds to NKG2D with high affinity (~10 nM). Remarkably, an engineered form of the shed MULT1, when secreted by tumor cells lacking NKG2D ligands, causes tumor rejection, rather than inhibiting it. Rejection is mediated by NK cells and in some cases T cells. Provision of recombinant MULT1 along with B16 melanoma cells resulted in induction of NK function ex vivo and tumor rejection in vivo. We propose a model in which expression of NKG2D ligands by non-tumor cells, persistently stimulate NK cells, resulting in desensitization of the NK cells; interruption of the interaction by soluble MULT1 restores NK cell functionality, causing tumor elimination. Consistent with this model, Rag2-/- mice which also lack NKG2D show higher NK activity than Rag2-/- mice, resulting in better B16 tumor rejection. Our study represents the first instance of NKG2D ligand shedding in the mouse, and provides the first evidence that such ligands are immunostimulatory, reversing the conventional understanding of soluble NKG2D ligands.

The theory- and evidence-based reflective R2C2 Facilitated Feedback Model appears stable and helpful for physicians in facilitating their reflection on and use of formal performance assessment feedback.