Various ubiquitin plasmids from Michael Rape lab, including but not limited to: pCS2-no his-ubiquitin wtpCS2-no his-ubiquitin all RpET30a-ubiquitin (no tag)pET30a-ubiquitin K0 (no tag) 

Volumetric additive manufacturing and vat-polymerization 3D printing methods rapidly solidify freeform objects via photopolymerization, but problematically raises the local temperature in addition to degree-of-conversion (DOC). The generated heat can critically affect the printing process as it can auto-accelerate the polymerization reaction, trigger convection flows, and cause optical aberrations. Therefore, temperature measurement alongside conversion state monitoring is crucial for devising mitigation strategies and implementing process control. Traditional infrared imaging suffers from multiple drawbacks such as limited transmission of measurement signal, material-dependent absorptions, and high background signals emitted by other objects. Consequently, a viable temperature and DOC monitoring method for volumetric 3D printing doesn’t exist.To address this opportunity, UC Berkeley researchers have developed a tomographic imaging technique that detects the spatiotemporal evolution of temperature and DOC during volumetric printing. The invention lays foundations for the development of volumetric measurement systems that uniquely resolve both temperature and DOC in volumetric printing.This novel Berkeley measurement system is envisaged as an integral tool for existing manufacturing technologies, such as computed axial lithography (CAL, Tech ID #28754), and as a new research tool for commercial biomanufacturing, general fluid dynamics, and more.

Classical methodologies for examining phage gene function, including UV/random mutagenesis and amber mutation, are difficult to assay efficiently on a genome-wide scale. Additionally, there are notable challenges in targeting phage genes with Cas9/12, such as epigenetic modifications, physical sequestration in the nucleus, absence of DNA genomes or intermediates in RNA phages, and efficient ligation/recombination processes. The limitation of current tools is also evident in failed attempts to apply transposon libraries in virulent phages, further underscoring the necessity for innovative approaches in phage functional genomics. UC Berkeley researchers made the surprising discovery that catalytically inactivated Cas13 (dCas13) in complex with a guide RNA can bind to and modulate activity of viral target RNAs. Viruses have evolved numerous and diverse strategies to protect their genomes from host defenses, including encoding their genomes across several Baltimore classes (e.g., dsDNA, dsRNA, ssDNA, and ssRNA), employing diverse genome modification strategies, and employing advanced genome compartmentalization strategies. These protective strategies have severely limited the applicability and effectiveness of previously existing approaches. Thus, this invention provides methods and compositions for modulating the activity of a viral target RNA.

UC Berkeley researchers have developed several methods that take advantage of all of the information contained in ion signals in charge detection mass spectrometry (CDMS). Unlike most conventional types of mass spectrometry (MS), which rely on mass-to-charge ratio (m/z) measurements of ensembles of ions, CDMS instead makes direct measurements of the mass of individual ions. CDMS has recently gained significant popularity in the analysis of large biomolecules, nanoparticles, and nanodroplets because it is one of very few methods that can characterize these analytes. State-of-the-art CDMS instruments incorporate ion traps and signals from individual trapped ions are used to find the mass, charge, and energy of these ions. Previously used techniques have used Fourier transform (FT)-based analyses, but only use the fundamental and/or second harmonic frequency and amplitude as the basis of the measurement. The significant additional information contained in the higher order harmonic frequencies and amplitudes of the ion signal is fully utilized in the novel methods comprising this invention and large improvements in measurement uncertainties are realized as a result. 

Hyperspectral imaging, the practice of capturing detailed spectral (color) information from the output of an optical instrument such as a microscope or telescope, is useful in biological and astronomical research and in manufacturing. In addition to being bulky and expensive, existing hyperspectral imagers typically require scanning across a specimen, limiting temporal resolution and preventing dynamic objects from being effectively imaged. Snapshot methods which eliminate scanning are limited by a tradeoff between spatial and spectral resolution.In order to address these problems, researchers at UC Berkeley have developed a hyperspectral imager which can be attached to the output of any benchtop microscope. The imager is compact (about 6-inches), and can achieve a higher spatial resolution than traditional snapshot imagers. Additionally, this imager needs only one exposure to collect measurements for an arbitrary number of spectral filters, giving it unprecedented spectral resolution.

UC Researchers have the following U2 OS cell linesU2OS cell lines (3 clones) TFEB N-terminal Halo-TFEB: #E5, #C8, #G12           U2OS cell lines (3 clones) TFE3 N-terminal Halo-TFE3: #B9 #C3, #E1 U2OS cell lines (2 clones) TFE3 C-terminal Halo-TFE3: #A9, #D2 U2OS stable cell line L30 3xF-Halo-GDGAGLIN-TFEB U2OS stable cell line EF1a 3xF-Halo-GDGAGLIN-TFEB  

Plasma medicine has emerged as a promising approach for treatment of biofilm-related and virus infections, assistance in cancer treatment, and treatment of wounds and skin diseases. However, an important challenge arises with the need to adapt control policies, often only determined after each treatment and using limited observations of therapeutic effects. Control policy adaptation that accounts for the variable characteristics of plasma and of target surfaces across different subjects and treatment scenarios is needed. Personalized, point-of-care plasma medicine can only advance efficaciously with new control policy strategies.To address this opportunity, UC Berkeley researchers have developed a novel control scheme for tailored and personalized plasma treatment of surfaces. The approach draws from concepts in deep learning, Bayesian optimization and embedded control. The approach has been demonstrated in experiments on a cold atmospheric plasma jet, with prototypical applications in plasma medicine.

RNA-mediated adaptive immune systems in bacteria and archaea rely on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genomic loci and CRISPR associated (Cas) proteins that function together to provide protection from invading viruses and plasmids. Genome editing can be carried out using a CRISPR-Cas system comprising a CRISPR-Cas effector polypeptide and a guide nucleic acid, such as a guide RNA. However, unintended chromosomal abnormalities following on-target genome editing, such as chromosome loss, are potential concerns for genome editing. UC Berkeley researchers and others have developed a method to modulate the expression levels of the DNA damage response factor p53 in order to mitigate chromosomal abnormalities that occur after genome editing by nucleases like Cas9. The invention provides treatment methods by generating a modified cell and then administering the modified cell to an individual in need thereof and compositions having a CRISPR-Cas effector polypeptide, a guide nucleic acid, and an agent that increases the level of a p53 polypeptide in a mammalian cell.

Metamaterials are constructed from regular patterns of simpler constituents known as unit cells. These engineered metamaterials can exhibit exotic mechanical properties not found in naturally occurring materials, and accordingly they have the potential for use in a variety of applications from running shoe soles to automobile crumple zones to airplane wings. Practical design using metamaterials requires the specification of the desired mechanical properties based on understanding the precise unit cell structure and repeating pattern. Traditional design approaches, however, are often unable to take advantage of the full range of possible stress-strain relationships, as they are hampered by significant nonlinear behavior, process-dependent manufacturing errors, and the interplay between multiple competing design objectives. To solve these problems, researchers at UC Berkeley have developed a machine learning algorithm in which designers input a desired stress-strain curve that encodes the mechanical properties of a material. Within seconds, the algorithm outputs the digital design of a metamaterial that, once printed, fully encapsulates the desired properties from the inputted stress-strain curve. This algorithm produces results with a fidelity to the desired curve in excess of 90%, and can reproduce a variety of complex phenomena completely inaccessible to existing methods.

DP-L4056 is a prophage-cured strain of Listeria monocytogenes based on wild-type strain 10403S. A prophage is a bacteriophage genome that is integrated into a bacterial genome. It remains latent until activation by an external factor, and activation leads to production of new bacteriophage particles that lyse the bacterial cell and spread. Curing the prophages in Listeria monocytogenes strain 10403S, which is ubiquitous in the microbiology community as a wild-type reference strain, allows for more predictable engineering and performance of Listeria monocytogenes.

Single-cell analyses are revolutionizing biomedicine and biology, with genomics (DNA) and transcriptomics (RNA) tools leading the way. At the protein-level, single-cell analyses are limited to mass spectrometry and immunoassays. Neither assay provides comprehensive coverage of proteome for single cells, missing key protein forms (called isoforms).  UC Berkeley researchers have developed a hybrid droplet-electrophoresis device, termed “DropBlot”, to detect proteins from patient-derived tissue biospecimens relevant to clinical medicine and pathology. The DropBlot takes advantage of water-in-oil (W/O) droplets to encapsulate single cells derived from chemically fixed tissues, thus providing a picoliter-volume reaction chamber in which said cells are lysed and subjected to harsh lysis conditions (100ºC, 2 hours), as needed for fixed cells. We report an all-in-one microdevice to facilitate cell-laden droplet loading with >98% microwell occupancy. Droplets remain intact under the electric field and protein isoforms are shown to electromigrate out of the droplet and into a microfluidic separation channel where protein sizing takes place via the action of electrophoresis in a photoactive polyacrylamide (PA) gel. DropBlot has been successfully applied to live and fixed cancer cell lines and resolved proteins with high sensitivity.

Manipulation of eukaryotic genomes, particularly the integration of multi-kilobase DNAsequences, remains challenging and limits the rapidly growing fields of synthetic biology andcell engineering. Large serine recombinases (LSRs) are enzymes that recognize specific targetsequences on a DNA donor sequence and DNA target sequence to catalyze a recombinationreaction that results in the insertion of a DNA donor in a sequence-specific manner. Genomeediting can be carried out using an LSR system and a DNA donor nucleic acid, such as a plasmidor double-stranded DNA. However, in a human genome, these systems can exhibit variableefficiency and specificity.In this invention, UC Berkeley researchers and others have developed optimized compositionsto significantly increase the efficiency and specificity of LSRs to target a specific genomic locusin human cells. Via fusion to additional protein systems, this engineered composition retainsthe simplicity of a single protein for gene delivery. The invention also encompasses use in invivo or ex vivo gene therapy and the creation of modified cell lines or transgenic animals.

The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets. The programmable nature of these systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation. There is a need in the art for additional CRISPR-Cas systems with improved cleavage and manipulation under a variety of conditions and ones that are particularly thermostable under those conditions. UCB researchers created a set of efficient CRISPR-Cas9 proteins from a thermostable Cas9 from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) through directed evolution. The gene editing activity of the evolved mutant proteins was improved by up to four orders of magnitude compared to the wild-type GeoCas9. The researchers showed that the gene editors based on the evolved GeoCas9 can be effectively assembled into lipid nanoparticles (LNP) for the rapid delivery to different cell lines in vitro as well as different organs or tissues in vivo. The LNP-based delivery strategy could also be extended to other gene editors.  

CRISPR-Cas systems comprise a CRISPR-associated (Cas) effector polypeptide and a guide nucleic acid. Such CRISPR-Cas systems can bind to and modify a targeted nucleic acid. The programmable nature of these CRISPR-Cas effector systems has facilitated their use as a versatile technology for use in, e.g., gene editing.   UC Berkeley researchers have discovered new CRISPR-Cas effector Cas12L/Cas Lambda/Casλ polypeptides and methods of modifying a target nucleic acid using a Cas12L/Cas Lambda polypeptide.

Human diseases that follow a dominant negative inheritance pattern present a great challenge for treatment using gene therapy methods. In such cases, a copy of an allele is inherited from each parent: one is a pathogenic allele causing a disease phenotype (e.g., by exerting a toxic, gain-of-function effect) and the other is a wild-type (non-pathogenic) allele. Allele-specific targeting is especially important when the wild-type allele is crucial to normal function, e.g., the wild-type allele encodes a protein whose function is critical. There is therefore a need for compositions and methods of allele-specific gene editing.   UC Berkeley researchers have created methods and systems for reducing the level of an RNA transcript from a target nucleic acid in an allele-specific manner. Such systems and methods can be used to treat a disease that results from or is caused by a toxic gain-of-function protein.   

The inventors have developed a novel 3D printing technique, named Non-Planar Granular 3D Printing (NGP), which selectively deposits a liquid binder into granular particles, enabling rapid fabrication of complex 3-dimensional objects. For this new method, an industrial robotic arm is equipped with a dispenser attached to a long metal needle, called a liquid deposition end-effector, and a container of granular particles, such as sand, beads, or powders. The needle moves freely as it injects the binding liquid into the granular material. Like other 3D printing methods, NGP can use a CAD 3D model and conventional slicing software to produce a robotic toolpath following a desired height and width. However, the advantage of the process lies in its ability to 3D print objects non-planarly, by moving the extruder’s dispensing tip freely within the granular medium. The selective application of the binding liquid causes the particles to bond together, forming parts of the 3D printed object. Meanwhile, the loose particles remaining in the container temporarily support the weight of the wet particles while they cure. This unique approach enables the creation of complex geometric forms without the need for supporting structures that are typical in traditional 3D printing methods, thereby eliminating material waste typically associated with such processes. After the completion of the process, and the binding material has cured, the hard objects can be easily extracted from the container, leaving behind the remaining loose particles, which can be repeatedly re-used.   

Methyl-CpG-binding-protein 2 (MeCP2) is a nuclear protein expressed in all cell types, especially neurons. Mutations in the MECP2 gene cause Rett syndrome (RTT), an incurable neurological disorder that disproportionately affects young girls. Strategies to restore MeCP2 expression phenotypically reverse RTT-like symptoms in male and female MeCP2-deficient mice, suggesting that direct nuclear delivery of functional MeCP2 could restore MeCP2 activity.The inventors have discovered that ZF-tMeCP2, a conjugate of MeCP2(aa13-71, 313-484) and the cell-permeant mini-protein ZF5.3, binds DNA in a methylation-dependent manner and reaches the nucleus of model cell lines intact at concentrations above 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT co-repressor complex and selectively represses transcription from methylated promoters. Efficient nuclear delivery of ZF-tMeCP2 relies on a unique endosomal escape portal provided by HOPS-dependent endosomal fusion.In a comparative evaluation, the inventors observed the Tat conjugate of MeCP2 (Tat-tMeCP2) (1) degrades within the nucleus, (2) is not selective for methylated promoters, and (3) traffics in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of protein-derived therapeutics.

Type III CRISPR-Cas systems recognize and degrade RNA molecules using an RNA-guided mechanism that occurs widely in microbes for adaptive immunity against viruses. The inventors have demonstrated that this multi-protein system can be leveraged for programmable RNA knockdown of both nuclear and cytoplasmic transcripts in mammalian cells. Using single-vector delivery of the S. thermophilus Csm complex, RNA knockdown was achieved with high efficiency (90-99%) and minimal off-targets, outperforming existing technologies of shRNA- and Cas13-mediated knockdown. Furthermore, unlike Cas13, Csm is devoid of trans-cleavage activity and thus does not induce non-specific transcriptome-wide degradation and cytotoxicity. Catalytically inactivated Csm can also be used for programmable RNA-binding, which the inventors exploit for live-cell RNA imaging. This work demonstrates the feasibility and efficacy of multi-subunit CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.

The inventors have developed an engineering approach to identify novel and nonobvious membrane-associated accessory protein (MAAP) sequence variants that confer increased Adeno-associated virus (AAV) secretion during packaging. The technique is based upon the iterative process of sequence diversification and selection of functional gene variants known as directed evolution. First, the inventors generated a library of more than 1E6 MAAP variants. The variants were subjected to five rounds of packaging into an AAV2 capsid for which MAAP expression was inactivated without altering the viral protein VP1 open reading frame (ORF) (AAV2-MAAP-null). Among each iterative packaging round, the inventors observed a progressive increase in both the overall titer and ratio of secreted vector genomes conferred by the bulk selected MAAP library population. Next-generation sequencing uncovered common mutational features that were enriched up to over 10,000-fold on the amino acid level. Individual MAAP variants were isolated and systematically tested for effect on recombinant AAV2-MAAP-null packaging in HEK293 cells. The inventors predict that this work may be applicable to increasing per-cell AAV output in industrial settings, potentially reducing global costs and increasing functional vector recovery in downstream manufacturing processes.BACKGROUNDParvoviruses are small, single-stranded DNA viruses that are ubiquitously found in many animal species. AAV is a prototypic dependoparvovirus whose replication cycle requires the function of helper genes from larger co-infected viruses such as Adenoviruses or Herpesviruses. The natural genome of AAV contains ~4.7 kb of ssDNA that encodes up to ten known proteins in a highly overlapped fashion. The rep gene encodes four protein products named based on their molecular weight: Rep72 and Rep68 facilitate genomic replication, whereas Rep52, and Rep40 play essential roles in loading nascent ssDNA genomes into assembled capsids. Downstream of rep lies the cap gene, which encodes three known protein products off of overlapping reading frames: VP1, VP2, and VP3 are structural proteins that assemble to form the capsid, the assembly activating protein (AAP) targets VP proteins to the nucleus and is involved in capsid assembly. The most recently discovered AAV-encoded gene is the membrane-associated accessory protein (MAAP). MAAP is encoded by an alternative ORF in the AAV cap gene that is found in all presently reported natural serotypes. Gene delivery by recombinant AAV (rAAV) have shown significant success in both research and clinical gene therapy applications. In the rAAV system, Rep and Cap are removed from between AAV’s 5’ and 3’ inverted terminal repeats (ITRs) and provided in trans. Instead, a transgene of interest is inserted between the ITRs and subsequently packaged into the nascent AAV capsids. However, manufacturing quantities of good manufacturing practice (GMP)-grade rAAVs necessary to achieve current and projected dosing requirements–particularly in a clinical context–presents a significant hurdle to expanding rAAV-based gene therapies. Recently, evidence has emerged supporting a functional role of MAAP in AAV egress. This led to the hypothesis that MAAP could be engineered to facilitate increased levels of secreted AAV produced from HEK293 cells. 

New chemistries are emerging for the direct attachment of complex molecules to cell surfaces. Chemistries that modify cells must perform under a narrow set of conditions in order to maintain cell viability. They must proceed in buffered aqueous media at the optimal physiological pH—typically pH 7.4—and within a temperature range of 4 – 37 ºC. Furthermore, these reactions must have sufficiently rapid kinetics to achieve high conversion even when confronted with the limits of surface diffusion characteristics. Due to these requirements, few chemistries exist that can attach molecules and proteins to live cells.  There is a need for improved methods of attaching proteins to living cells.   UC researchers have developed a convenient enzymatic strategy for the modification of cell surfaces for targeted immunotherapy applications.  

The inventors have developed a method of mutagenizing user-defined regions of cytoplasmic DNA using a single guide RNA (sgRNA) or combinations of sgRNAs and a highly engineered fusion polypeptide comprising: a nuclear export sequence (NES)-containing, engineered nuclear localization sequence (NLS)-lacking, enzymatically active, RNA-guided endonuclease that introduces a single-stranded break in cytoplasmic DNA, and an error-prone DNA polymerase. This novel technology encompasses and provides evidence for the use of RNA-guided nucleases with relaxed PAM requirements, which are particularly useful for AT-rich targets such as the vaccinia virus genome. The inventors show that the truncation of up to several base pairs from the PAM-distal template binding region of the sgRNAs significantly increases the functional activity and specificity of the targeted mutagenesis complex. Moreover, the invention describes specific methods for the use of this technology to edit cytoplasmically replicating viruses with large DNA genomes, using poxviruses as a model system. The novel editing platform and methods selectively and continuously accelerate diversification of user-defined sites in the vaccinia genome during infection, while retaining most library members, due to significantly lowering deleterious off-target mutations. BACKGROUND Nucleocytoplasmic large DNA viruses (NCLDVs) are a group of viruses that harbor large (150 kbp - 1.2 mbp) double-stranded DNA genomes and replicate in the cytoplasm of eukaryotic cells. An example of an NCLDV that has historically been among the most prominent tools in human health is vaccinia, a poxvirus. Hundreds of millions of humans have been intentionally inoculated with vaccinia as part of a global effort to eliminate smallpox, which was declared eradicated in 1980.Vaccinia and some other poxviruses remain highly scientifically relevant in the post-eradication world. They are useful as vaccines against deadly poxvirus outbreaks that could potentially arise from natural spillover, bioterrorism, or biowarfare, as well as due to their therapeutic promise as oncolytic agents to selectively deliver anti-cancer transgenes and recruit adaptive immunity while leaving healthy cells unharmed. Directed evolution is a powerful engineering technique for evolving new phenotypes that are beneficial for biotechnological applications but for which there may have never been a selective pressure to evolve in nature. Both natural and directed evolution depend upon generation of genetic diversity, followed by a selective pressure. While natural evolution generates genetic diversity randomly and throughout the entirety of the genome, directed evolution ideally focuses mutations within specific genomic windows connected to the phenotype that one wishes to engineer. However, there is a need in the art for compositions and methods for mutagenizing a target DNA in the cytoplasm of mammalian cells. NCLDVs, which either partially or entirely express their own replicative and translational machinery independent of the nucleus, are difficult, and in many cases impossible, to produce from plasmid DNA in cells. Thus, NCLDVs are not amenable to standard in vitro molecular diversification strategies.  

The inventors have developed a scalable laser aperture that emits light perpendicular to the surface. The aperture can, in principal, scale to arbitrarily large sizes, offering a universal architecture for systems in need of small, intermediate, or high power. The technology is based on photonic crystal apertures, nanostructured apertures that exhibit a quasi-linear dispersion at the center of the Brillouin zone together with a mode-dependent loss controlled by the cavity boundaries, modes, and crystal truncation. Open Dirac cavities protect the fundamental mode and couple higher order modes to lossy bands of the photonic structure. The technology was developed with an open-Dirac electromagnetic aperture, known as a Berkeley Surface Emitting Laser (BKSEL).  The inventors demonstrate a subtle cavity-mode-dependent scaling of losses. For cavities with a quadratic dispersion, detuned from the Dirac singularity, the complex frequencies converge towards each other based on cavity size. While the convergence of the real parts of cavity modes towards each other is delayed, going quickly to zero, the normalized complex free-spectral range converge towards a constant solely governed by the loss rate of Bloch bands. The inventors show that this unique scaling of the complex frequency of cavity modes in open-Dirac electromagnetic apertures guarantees single-mode operation of large cavities. The technology demonstrates scaled up single-mode lasing, and confirmed from far-field measurements. By eliminating limits on electromagnetic aperture size, the technology will enable groundbreaking applications for devices of all sizes, operating at any power level. BACKGROUND Single aperture cavities are bounded by higher order transverse modes, fundamentally limiting the power emitted by single-mode lasers, as well as the brightness of quantum light sources. Electromagnetic apertures support cavity modes that rapidly become arbitrarily close with the size of the aperture. The free-spectral range of existing electromagnetic apertures goes to zero when the size of the aperture increases. As a result, scale-invariant apertures or lasers has remained elusive until now.  Surface-emitting lasers have advantages in scalability over commercially widespread vertical-cavity surface-emitting lasers (VCSELs). When a photonic crystal is truncated to a finite cavity, the continuous bands break up into discrete cavity modes. These higher order modes compete with the fundamental lasing mode and the device becomes more susceptible to multimode lasing response as the cavity size increases. 

The inventors engineered a set of LbCas12a mutants through rational design and directed evolution. The engineered mutants can function as efficient genome editors with minimized trans-activity.

These are mice on the BALB/cAnNCrl background that lack functional STING protein. A 5 bp deletion (GCCTC; nucleotides 402-406 of NM_001289591.1) was introduced to exon 3 of the mouse Sting1 gene via CRISPR/Cas9 methodologies 

Gene targeted B6 strain mice in which the Klrk1 gene, which encodes the NKG2D immunoreceptor, is inactivated. On the C57Bl/6J genetic background.  Strain name: B6.Cg-Klrk1tm1Dhr  The inventors provided the first characterization of NKG2D-deficient mice, including evidence that NKG2D was not necessary for NK cell development but was critical for immunosurveillance of epithelial and lymphoid malignancies in two transgenic models of de novo tumorigenesis. In both models, the inventors detected NKG2D ligands on the tumor cell surface ex vivo, providing needed evidence for ligand expression by primary tumors.  Ligands for the NKG2D stimulatory receptor are frequently upregulated on tumor lines, rendering them sensitive to natural killer (NK) cells, but this mouse represents the first spontaneous cancer model addressing the role of NKG2D in tumor surveillance.