Exonuclease-enhanced prime editors.

Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2.

A reversible state of hypometabolism in a human cellular model of sporadic Parkinson's disease.

Sporadic Parkinson's Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism.

Genetically encoded barcodes for correlative volume electron microscopy.

While genetically encoded reporters are common for fluorescence microscopy, equivalent multiplexable gene reporters for electron microscopy (EM) are still scarce. Here, by installing a variable number of fixation-stable metal-interacting moieties in the lumen of encapsulin nanocompartments of different sizes, we developed a suite of spherically symmetric and concentric barcodes (EMcapsulins) that are readable by standard EM techniques. Six classes of EMcapsulins could be automatically segmented and differentiated.

Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs.

Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest.

Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages.

Photoacoustic Neuroimaging - Perspectives on a Maturing Imaging Technique and its Applications in Neuroscience.

A prominent goal of neuroscience is to improve our understanding of how brain structure and activity interact to produce perception, emotion, behavior, and cognition. The brain's network activity is inherently organized in distinct spatiotemporal patterns that span scales from nanometer-sized synapses to meter-long nerve fibers and millisecond intervals between electrical signals to decades of memory storage. There is currently no single imaging method that alone can provide all the relevant information, but intelligent combinations of complementary techniques can be effective.

Non-invasive and high-throughput interrogation of exon-specific isoform expression.

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing.

Noninvasive visualization of electrical conductivity in tissues at the micrometer scale.

Despite its importance in regulating cellular or tissue function, electrical conductivity can only be visualized in tissue indirectly as voltage potentials using fluorescent techniques, or directly with radio waves. These either requires invasive procedures like genetic modification or suffers from limited resolution. Here, we introduce radio-frequency thermoacoustic mesoscopy (RThAM) for the noninvasive imaging of conductivity by exploiting the direct absorption of near-field ultrashort radio-frequency pulses to stimulate the emission of broadband ultrasound waves.

Genetically Encoded Self-Assembling Iron Oxide Nanoparticles as a Possible Platform for Cancer-Cell Tracking.

The study of growth and possible metastasis in animal models of tumors would benefit from reliable cell labels for noninvasive whole-organism imaging techniques such as magnetic resonance imaging. Genetically encoded cell-tracking reporters have the advantage that they are contrast-selective for viable cells with intact protein expression machinery. Besides, these reporters do not suffer from dilution during cell division.

Genetically encodable materials for non-invasive biological imaging.

Many questions in basic biology and medicine require the ability to visualize the function of specific cells and molecules inside living organisms. In this context, technologies such as ultrasound, optoacoustics and magnetic resonance provide non-invasive imaging access to deep-tissue regions, as used in many laboratories and clinics to visualize anatomy and physiology. In addition, recent work has enabled these technologies to image the location and function of specific cells and molecules inside the body by coupling the physics of sound waves, nuclear spins and light absorption to unique protein-based materials.

Structural perspective on revealing heat dissipation behavior of CoFeO-Pd nanohybrids: great promise for magnetic fluid hyperthermia.

Loss mechanisms in fluid heating of cobalt ferrite (CFO) nanoparticles and CFO-Pd heterodimer colloidal suspensions are investigated as a function of particle size, fluid concentration and magnetic field amplitude. The specific absorption rate (SAR) is found to vary with increasing particle size due to a change in dominant heating mechanism from susceptibility to hysteresis and frictional loss. The maximum SAR is obtained for particle diameters of 11-15 nm as a result of synergistic contributions of susceptibility loss, including Néel and Brownian relaxation and especially hysteresis loss, thereby validating the applicability of linear response theory to superparamagnetic CFO nanoparticles.

Nucleus size and DNA accessibility are linked to the regulation of paraspeckle formation in cellular differentiation.

Background: Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo.

Results: As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages.

Artifact-free deconvolution in light field microscopy.

The sampling patterns of the light field microscope (LFM) are highly depth-dependent, which implies non-uniform recoverable lateral resolution across depth. Moreover, reconstructions using state-of-the-art approaches suffer from strong artifacts at axial ranges, where the LFM samples the light field at a coarse rate. In this work, we analyze the sampling patterns of the LFM, and introduce a flexible light field point spread function model (LFPSF) to cope with arbitrary LFM designs.

Iron-Sequestering Nanocompartments as Multiplexed Electron Microscopy Gene Reporters.

Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of (Qt), which in combination with the recently characterized encapsulin system from (Mx) enables multiplexed gene reporter imaging conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with = 4 icosahedral symmetry and a diameter of ∼43 nm harboring two putative pore regions at the 5-fold and 3-fold axes.

Whole-Cell Photoacoustic Sensor Based on Pigment Relocalization.

Photoacoustic (optoacoustic) imaging can extract molecular information with deeper tissue penetration than possible by fluorescence microscopy techniques. However, there is currently still a lack of robust genetically controlled contrast agents and molecular sensors that can dynamically detect biological analytes of interest with photoacoustics. In a biomimetic approach, we took inspiration from cuttlefish who can change their color by relocalizing pigment-filled organelles in so-called chromatophore cells under neurohumoral control.

Author Correction: Zebrafish and medaka offer insights into the neurobehavioral correlates of vertebrate magnetoreception.

Text for Correction.

Bacterial encapsulins as orthogonal compartments for mammalian cell engineering.

We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT).

Zebrafish and medaka offer insights into the neurobehavioral correlates of vertebrate magnetoreception.

An impediment to a mechanistic understanding of how some species sense the geomagnetic field ("magnetoreception") is the lack of vertebrate genetic models that exhibit well-characterized magnetoreceptive behavior and are amenable to whole-brain analysis. We investigated the genetic model organisms zebrafish and medaka, whose young stages are transparent and optically accessible. In an unfamiliar environment, adult fish orient according to the directional change of a magnetic field even in darkness.

NeuBtracker-imaging neurobehavioral dynamics in freely behaving fish.

A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.

Calcium Sensor for Photoacoustic Imaging.

We introduce a selective and cell-permeable calcium sensor for photoacoustics (CaSPA), a versatile imaging technique that allows for fast volumetric mapping of photoabsorbing molecules with deep tissue penetration. To optimize for Ca-dependent photoacoustic signal changes, we synthesized a selective metallochromic sensor with high extinction coefficient, low quantum yield, and high photobleaching resistance. Micromolar concentrations of Ca lead to a robust blueshift of the absorbance of CaSPA, which translated into an accompanying decrease of the peak photoacoustic signal.

Microfluidic sorting of intrinsically magnetic cells under visual control.

Magnetic cell sorting provides a valuable complementary mechanism to fluorescent techniques, especially if its parameters can be fine-tuned. In addition, there has recently been growing interest in studying naturally occurring magnetic cells and genetic engineering of cells to render them magnetic in order to control molecular processes via magnetic fields. For such approaches, contamination-free magnetic separation is an essential capability.

Functional optoacoustic neuro-tomography for scalable whole-brain monitoring of calcium indicators.

Non-invasive observation of spatiotemporal activity of large neural populations distributed over entire brains is a longstanding goal of neuroscience. We developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains. It can record 100 volumetric frames per second across scalable fields of view ranging between 50 and 1000 mm with respective spatial resolution of 35-200 μm.

Near-Infrared Photoacoustic Imaging Probe Responsive to Calcium.

Photoacoustic imaging (PAI) is an attractive imaging modality that can volumetrically map the distribution of photoabsorbing molecules with deeper tissue penetration than multiphoton microscopy. To enable dynamic sensing of divalent cations via PAI, we have engineered a new reversible near-infrared probe that is more sensitive to calcium as compared to other biologically relevant cations. The metallochromic compound showed a strong reduction of its peak absorbance at 765 nm upon addition of calcium ions that was translated into robust signal changes in photoacoustic images.

Hyperpolarized Multi-Metal C-Sensors for Magnetic Resonance Imaging.

We introduce hyperpolarizable C-labeled probes that identify multiple biologically important divalent metals via metal-specific chemical shifts. These features enable NMR measurements of calcium concentrations in human serum in the presence of magnesium. In addition, signal enhancement through dynamic nuclear polarization (DNP) increases the sensitivity of metal detection to afford measuring micromolar concentrations of calcium as well as simultaneous multi-metal detection by chemical shift imaging.