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Scientists at UC Irvine have developed a sensitive, customizable, and user-friendly sensor for (1) strain detection as a result of cellular movement, (2) micro-fluidic device pressure detection, and (3) real-time monitoring of valve statuses in microfluidic chips. This research tool will provide new insights regarding cellular biophysics.

Researchers at UC Irvine have developed an organoid culture system capable of generating three-dimensional molecular gradients. This recapitulates in vivo tissue development more accurately than current two-dimensional organoid culture systems and will allow scientists to study human-specific disease mechanisms in native tissue.

During initial stages of development, the human brain self assembles from a vast network of billions of neurons into a system capable of sophisticated cognitive behaviors. The human brain maintains these capabilities over a lifetime of homeostasis, and neuroscience helps us explore the brain’s capabilities. The pace of progress in neuroscience depends on experimental toolkits available to researchers. New tools are required to explore new forms of experiments and to achieve better statistical certainty.Significant challenges remain in modern neuroscience in terms of unifying processes at the macroscopic and microscopic scale. Recently, brain organoids, three-dimensional neural tissue structures generated from human stem cells, are being used to model neural development and connectivity. Organoids are more realistic than two-dimensional cultures, recapitulating the brain, which is inherently three-dimensional. While progress has been made studying large-scale brain patterns or behaviors, as well as understanding the brain at a cellular level, it’s still unclear how smaller neural interactions (e.g., on the order of 10,000 cells) create meaningful cognition. Furthermore, systems for interrogation, observation, and data acquisition for such in vitro cultures, in addition to streaming data online to link with these analysis infrastructures, remains a challenge.

During In the last few decades, the use of miniaturized electrochemical devices has grown rapidly and found diverse applications in scientific and consumer products. The process of developing specialized electrochemical devices is often time-consuming and expensive. Experimental setups involving electrochemistry often use specialized measurement equipment such as a potentiostat. A potentiostat is an analytical instrument that controls the voltage and current between two or more electrodes in a cell. The accuracy, precision, and flexibility of applying or measuring voltages and currents depends on the quality and design of the electronic hardware, which for commercially available potentiostats, often correlate with the device’s cost and architecture. Consequently, one of the challenges faced by today’s electrochemical research community is how to perform modern experimental designs with expensive, asynchronous, and inflexible potentiostats.

In cells, DNA is organized by wrapping DNA strands around histone proteins, creating protein-DNA complexes called nucleosomes which comprise the basic unit of chromatin. Chromatin is associated with regions of low gene expression, as compacted DNA is inaccessible to proteins that would promote transcription. Conversely, regions in the DNA not bound by histones experience higher gene expression, as this DNA is readily available to be transcribed.  Nucleosomes are not uniformly positioned on a DNA molecule, and they change based on factors like which genes are expressed during different cellular processes. It is beneficial to understand where nucleosomes are positioned, as this can provide insight into how genes are regulated, or how factors like epigenetic modifications or chromatin structure affect this accessibility and can additionally illuminate gene expression patterns in disease for designing therapies. Nucleosome profiling is a technique used to study the positions of nucleosomes along a DNA molecule. Typically, histones are crosslinked to DNA, then the DNA is fragmented and digested leaving only regions protected by nucleosomes left for short-read sequencing. However, this fragmentation only reveals nucleosome positioning at the resolution of a few hundred base pairs, leaving the larger genomic context of these nucleosome positions to be desired. To address this, researchers at UC Santa Cruz developed Add-SEQ, a pipeline using long-read nanopore sequencing to map nucleosomes across long stretches > 10 kb of single DNA molecules.

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.

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.

Novel system for imaging of specimens in high refractive index solutions on the Zeiss Z.1 fluorescence light sheet microscope. System will allow for deep imaging of large and intact biological samples (i.e. mouse brain) for enhanced optical resolution and faster imaging speed.

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.

Researchers at the University of California, Davis have developed a method to stop bacterial growth while maintaining desirable metabolic functions for therapeutic and biotechnological applications.

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.

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.

As an important tool for electrophysiological recordings, neural electrodes implanted on the brain surface have been instrumental in basic neuroscience research to study large-scale neural dynamics in various cognitive processes, such as sensorimotor processing as well as learning and memory. In clinical settings, neural recordings have been adopted as a standard tool to monitor the brain activity in epilepsy patients before surgery for detection and localization of epileptogenic zones initiating seizures and functional cortical mapping. Neural activity recorded from the brain surface exhibits rich information content about the collective neural activities reflecting the cognitive states and brain functions. For the interpretation of surface potentials in terms of their neural correlates, most research has focused on local neural activities.   From basic neuroscience research to clinical treatments and neural engineering, electrocorticography (ECoG) has been widely used to record surface potentials to evaluate brain function and develop neuroprosthetic devices. However, the requirement of invasive surgeries for implanting ECoG arrays significantly limits the coverage of different cortical regions, preventing simultaneous recordings from spatially distributed cortical networks. However, this rich information content of surface potentials encoded for the large-scale cortical activity remains unexploited and little is known on how local surface potentials are correlated with the spontaneous neural activities of distributed large-scale cortical networks. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0in; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

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Researchers at the University of California, Davis have developed a deep learning simulation model to predict mutated T-cell receptor affinity and avidity for immunotherapy applications.

The inventors propose a novel framework generating new molecules that potentially inhibit the Mpro protein, the main protease of SARS-COV-2. The technology combines deep reinforcement learning (RL) with real-time molecular docking on the 3d structure of Mpro using AutoDock Vina, an open-source program for doing molecular docking. A second second docking software, Glide, was used to validate the generated molecules. The AutoDock and Glide docking softwares showed consensus on 41 molecules as potential potent Mpro inhibitors that were sufficiently easy to synthesize. The inventors show that this method samples the drug chemical space efficiently, covering a much broader space than molecules submitted to the COVID moonshot project, and the molecules have the correct shape and non-bonded interactions to fit into the binding pocket. Moreover, this approach only relies on the structure of the target protein, which means it can be easily adapted for future development of other inhibitors.

Researchers at the University of California, Davis have developed a programmable machine that shakes and repeatedly inverts large numbers of small containers - such as vials and flasks – in order to mix high-viscosity fluids.

Prof. Quan Cheng and colleagues from the University of California, Riverside have developed aluminum (Al) microchips for highly sensitive SPR detection of bioanalytical targets. This technology allows for determination of binding kinetics of drug targets and disease marker detection. In addition to applications for SPR, these Al microchips enable other surface-based techniques such as enhanced Raman spectroscopy and MALDI-MS for direct pathogen identification. Compared to traditional gold substrates, Al has a broad range of advantages. It is more plasmonically active, leading to high optical sensitivity, and it is chemically flexible for design of various analytical platforms. Al also has several manufacturing benefits that make it commercially appealing when compared to gold, such as higher abundance, lower cost, and simple integration into existing manufacturing processes such as CMOS. Fig 1: (Top) Fabrication of aluminum microchips. (Bottom) Aluminum demonstrates a high theoretical and practical plasmonic activity correlating to a higher detection sensitivity for biological targets.  

Prof. Quan Cheng and colleagues from the University of California, Riverside have developed a gold microchip consisting of a nanoscale film fabricated on a gold substrate for highly effective, matrix-free laser desorption ionization mass spectrometry (LDI-MS) analysis of lipids. This technology allows for effective analysis of low mass metabolites without the need for time consuming extraction methods. The microchip also enhances fluorescent signal through metal enhanced fluorescence (MEF) allowing single cells to be located easily and improves ionization of lipids. Fig 1: Gold microchips enable localization of cells with MEF and efficient ionization of lipid species. The lipid fingerprint can then be used to trace changes caused by toxicants or identify microbial species present.