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At least 361 records · Page 20

In vivo human T cell engineering with enveloped delivery vehicles

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR–Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody–antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

42 ENGINEERING↗

Single-molecule live-cell RNA imaging with CRISPR–Csm

Abstract Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR–Csm complex with multiplexed guide RNAs for direct and efficient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual nativeNOTCH2andMAP1Btranscripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation ofNOTCH2mRNA at the endoplasmic reticulum and directional transport ofMAP1BmRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

Biotechnology & Applied Microbiology↗

Label-free nanoscopy of cell metabolism by ultrasensitive reweighted visible stimulated Raman scattering

Nanoscopic imaging of cell metabolism is hindered by the incompatibility of small metabolites with fluorescent dyes and the limited resolution of imaging mass spectrometry. We present ultrasensitive reweighted visible stimulated Raman scattering (URV-SRS), a label-free vibrational nanoscopy technique that enables multiplexed detection of metabolic nanostructures within cells. We developed an extensively chirped spectral focusing visible SRS microscope that achieves a detection limit of 4,000 molecules and introduced a self-supervised learning-based denoiser to robustly suppress non-independent SRS noise by over 7.2 dB. The instrumentation-based signal enhancement and computation-based noise suppression synergistically improved the detection sensitivity by 50 times over near-infrared SRS. Leveraging this enhanced sensitivity, we further pushed the resolution to nanoscopic levels by introducing Fourier reweighting to amplify sub-100 nm spatial frequencies previously overwhelmed by noise. Validated by Fourier ring correlation, URV-SRS achieves a lateral resolution of 86 nm in cellular imaging. Here, we applied URV-SRS to elucidate the reprogramming of metabolic nanostructures associated with virus replication in Vero E6 host cells and to compositionally delineate subcellular fatty acid synthesis in engineered Escherichia coli, demonstrating its capability towards nanoscopic spatial metabolomics.

59 BASIC BIOLOGICAL SCIENCES↗

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients in the DRC enrolled from 2017 to 2018

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 ( pfhrp2/3 ) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp 2/3 deletions. Samples were assayed for HRP2, pan- Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

60 APPLIED LIFE SCIENCES↗

Optimal reference genes for RNA tissue analysis in small animal models of hemorrhagic fever viruses

Abstract Reverse-transcription quantitative polymerase chain reaction assays are frequently used to evaluate gene expression in animal model studies. Data analyses depend on normalization using a suitable reference gene (RG) to minimize effects of variation due to sample collection, sample processing, or experimental set-up. Here, we investigated the suitability of nine potential RGs in laboratory animals commonly used to study viral hemorrhagic fever infection. Using tissues (liver, spleen, gonad [ovary or testis], kidney, heart, lung, eye, brain, and blood) collected from naïve animals and those infected with Crimean–Congo hemorrhagic fever (mice), Nipah (hamsters), or Lassa (guinea pigs) viruses, optimal species-specific RGs were identified based on five web-based algorithms to assess RG stability. Notably, the Ppia RG demonstrated stability across all rodent tissues tested. Optimal RG pairs that include Ppia were determined for each rodent species ( Ppia and Gusb for mice; Ppia and Hrpt for hamsters; and Ppia and Gapdh for guinea pigs). These RG pair assays were multiplexed with viral targets to improve assay turnaround time and economize sample usage. Finally, a pan-rodent Ppia assay capable of detecting Ppia across multiple rodent species was developed and successfully used in ecological investigations of field-caught rodents, further supporting its pan-species utility.

Davies, Katherine A.↗

Potentiating antibiotic efficacy via perturbation of non-essential gene expression

Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations in Escherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization of E. coli without a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing a Salmonella enterica intracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies, csgD, fnr, recA and acrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates of Klebsiella pneumoniae and E. coli showed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.

60 APPLIED LIFE SCIENCES↗

Long-range cooperative resonances in rare-earth ion arrays inside photonic resonators

Engineering arrays of active optical centers to control the interaction Hamiltonian between light and matter has been the subject of intense research recently. Collective interaction of atomic arrays with optical photons can give rise to directionally enhanced absorption or emission, which enables engineering of broadband and strong atom-photon interfaces. Here, we report on the observation of long-range cooperative resonances in an array of rare-earth ions controllably implanted into a solid-state lithium niobate micro-ring resonator. We show that cooperative effects can be observed in an ordered ion array extended far beyond the light’s wavelength. We observe enhanced emission from both cavity-induced Purcell enhancement and array-induced collective resonances at cryogenic temperatures. Engineering collective resonances as a paradigm for enhanced light-matter interactions can enable suppression of free-space spontaneous emission. The multi-functionality of lithium niobate hosting rare-earth ions can open possibilities of quantum photonic device engineering for scalable and multiplexed quantum networks.

42 ENGINEERING↗

Direct time-resolved detection and quantification of key reactive intermediates in diethyl ether oxidation at T = 450–600 K

High-pressure multiplexed photoionization mass spectrometry (MPIMS) with tunable vacuum ultraviolet (VUV) ionization radiation from the Lawrence Berkeley Labs Advanced Light Source is used to investigate the oxidation of diethyl ether (DEE). Kinetics and photoionization (PI) spectra are simultaneously measured for the species formed. Several stable products from DEE oxidation are identified and quantified using reference PI cross-sections. In addition, we directly detect and quantify three key chemical intermediates: peroxy (ROO˙), hydroperoxyalkyl peroxy (˙OOQOOH), and ketohydroperoxide (HOOP[double bond, length as m-dash]O, KHP). These intermediates undergo dissociative ionization (DI) into smaller fragments, making their identification by mass spectrometry challenging. Furthermore, with the aid of quantum chemical calculations, we identify the DI channels of these key chemical species and quantify their time-resolved concentrations from the overall carbon atom balance at T = 450 K and P = 7500 torr. This allows the determination of the absolute PI cross-sections of ROO˙, ˙OOQOOH, and KHP into each DI channel directly from experiment. The PI cross-sections in turn enable the quantification of ROO˙, ˙OOQOOH, and KHP from DEE oxidation over a range of experimental conditions that reveal the effects of pressure, O 2 concentration, and temperature on the competition among radical decomposition and second O 2 addition pathways.

42 ENGINEERING↗

Formic acid catalyzed isomerization and adduct formation of an isoprene-derived Criegee intermediate: experiment and theory

Isoprene is the most abundant non-methane hydrocarbon emitted into the Earth's atmosphere. Ozonolysis is an important atmospheric sink for isoprene, which generates reactive carbonyl oxide species (R 1 R 2 C[double bond, length as m-dash]O + O – ) known as Criegee intermediates. This study focuses on characterizing the catalyzed isomerization and adduct formation pathways for the reaction between formic acid and methyl vinyl ketone oxide (MVK-oxide), a four-carbon unsaturated Criegee intermediate generated from isoprene ozonolysis. syn-MVK-oxide undergoes intramolecular 1,4 H-atom transfer to form a substituted vinyl hydroperoxide intermediate, 2-hydroperoxybuta-1,3-diene (HPBD), which subsequently decomposes to hydroxyl and vinoxylic radical products. In this paper, we report direct observation of HPBD generated by formic acid catalyzed isomerization of MVK-oxide under thermal conditions (298 K, 10 torr) using multiplexed photoionization mass spectrometry. The acid catalyzed isomerization of MVK-oxide proceeds by a double hydrogen-bonded interaction followed by a concerted H-atom transfer via submerged barriers to produce HPBD and regenerate formic acid. The analogous isomerization pathway catalyzed with deuterated formic acid (D2-formic acid) enables migration of a D atom to yield partially deuterated HPBD (DPBD), which is identified by its distinct mass (m/z 87) and photoionization threshold. In addition, bimolecular reaction of MVK-oxide with D 2 -formic acid forms a functionalized hydroperoxide adduct, which is the dominant product channel, and is compared to a previous bimolecular reaction study with normal formic acid. Complementary high-level theoretical calculations are performed to further investigate the reaction pathways and kinetics.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Primary photodissociation mechanisms of pyruvic acid on S 1 : observation of methylhydroxycarbene and its chemical reaction in the gas phase

Pyruvic acid, a representative alpha-keto carboxylic acid, is one of the few organic molecules destroyed in the troposphere by solar radiation rather than by reactions with free radicals. To date, only its stable final products were identified, often with contribution from secondary chemistry, making it difficult to elucidate photodissociation mechanisms following excitation to the lowest singlet excited-state (S 1 ) and the role of the internal hydrogen bond in the most-stable Tc conformer. Using multiplexed photoionization mass spectrometry we report the first direct experimental evidence, via the observation of singlet methylhydroxycarbene (MHC) following 351 nm excitation, supporting the decarboxylation mechanism previously proposed. Decarboxylation to MHC + CO 2 represents 97–100% of product branching at 351 nm. We observe vinyl alcohol and acetaldehyde, which we attribute to isomerization of MHC. We also observe a 3 ± 2% yield of the Norrish Type I photoproducts CH 3 CO + DOCO, but only from d 1 -pyruvic acid. At 4 Torr pressure, we measure a photodissociation quantum yield of $1.0^{+0}_{–0.4}$, consistent with IUPAC recommendations. However, our measured product branching fractions disagree with IUPAC. In light of previous calculations, these results support a mechanism in which hydrogen transfer on the S 1 excited state occurs at least partially by tunneling, in competition with intersystem crossing to the T 1 state. Here, we present the first evidence of a bimolecular reaction of MHC in the gas phase, where MHC reacts with pyruvic acid to produce a C 4 H 8 O 2 product. This observation implies that some MHC produced from pyruvic acid in Earth's troposphere will be stabilized and participate in chemical reactions with O 2 and H 2 O, and should be considered in atmospheric modeling.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Selectively accessing the hotspots of optical nanoantennas by self-aligned dry laser ablation

Plasmonic nanostructures serve as optical antennas for concentrating the energy of incoming light in localized hotspots close to their surface. By positioning nanoemitters in the antenna hotspots, energy transfer is enabled, leading to novel hybrid antenna-emitter-systems, where the antenna can be used to manipulate the optical properties of the nano-objects. The challenge remains how to precisely position emitters within the hotspots. We report a self-aligned process based on dry laser ablation of a calixarene that enables the attachment of molecules within the electromagnetic hotspots at the tips of gold nanocones. Within the laser focus, the ablation threshold is exceeded in nanoscale volumes, leading to selective access of the hotspot areas. A first indication of the site-selective functionalization process is given by attaching fluorescently labelled proteins to the nanocones. In a second example, Raman-active molecules are selectively attached only to nanocones that were previously exposed in the laser focus, which is verified by surface enhanced Raman spectroscopy. Enabling selective functionalization is an important prerequisite e.g. for preparing single photon sources for quantum optical technologies, or multiplexed Raman sensing platforms.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Time-resolved quantification of key species and mechanistic insights in low-temperature tetrahydrofuran oxidation

Here, we investigate the kinetics and report the time-resolved concentrations of key chemical species in the oxidation of tetrahydrofuran (THF) at 7500 torr and 450–675 K. Experiments are carried out using high-pressure multiplexed photoionization mass spectrometry (MPIMS) combined with tunable vacuum ultraviolet radiation from the Berkely Lab Advanced Light Source. Intermediates and products are quantified using reference photoionization (PI) cross sections, when available, and constrained by a global carbon balance tracking approach at all experimental temperatures simultaneously for the species without reference cross sections. From carbon balancing, we determine time-resolved concentrations for the ROO˙ and ˙OOQOOH radical intermediates, butanedial, and the combined concentration of ketohydroperoxide (KHP) and unsaturated hydroperoxide (UHP) products stemming from the ˙QOOH + O 2 reaction. Furthermore, we quantify a product that we tentatively assign as fumaraldehyde, which arises from UHP decomposition via H 2 O or ˙OH + H loss. The experimentally derived species concentrations are compared with model predictions using the most recent literature THF oxidation mechanism of Fenard et al., (Combust. Flame, 2018, 191, 252–269). Our results indicate that the literature mechanism significantly overestimates THF consumption and the UHP + KHP concentration at our conditions. The model predictions are sensitive to the rate coefficient for the ROO˙ isomerization to ˙QOOH, which is the gateway for radical chain propagating and branching pathways. Comparisons with our recent results for cyclopentane (Demireva et al., Combust. Flame, 2023, 257, 112506) provide insights into the effect of the ether group on reactivity and highlight the need to determine accurate rate coefficients of ROO˙ isomerization and subsequent reactions.

Demireva, Maria↗

Thermal kinetic inductance detectors for millimeter-wave detection

Thermal Kinetic-Inductance Detectors (TKIDs) combine the excellent noise performance of traditional bolometers with a radio frequency multiplexing architecture that enables the large detector counts needed for the next generation of millimeter-wave instruments. In this paper, we first discuss the expected noise sources in TKIDs and derive the limits where the phonon noise contribution dominates over the other detector noise terms: generation–recombination, amplifier, and two-level system noise. Second, we characterize aluminum TKIDs in a dark environment. We present measurements of TKID resonators with quality factors of about 10 5 at 80 mK. We also discuss the bolometer thermal conductance, heat capacity, and time constants. These were measured by the use of a resistor on the thermal island to excite the bolometers. These dark aluminum TKIDs demonstrate a noise equivalent power, NEP = 2 × 10 − 17 W / Hz , with a 1 / f knee at 0.1 Hz, which provides background noise limited performance for ground-based telescopes observing at 150 GHz.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Looking at the bigger picture: Identifying the photoproducts of pyruvic acid at 193 nm

Here, photodissociation of pyruvic acid (PA) was studied in the gas-phase at 193 nm using two complementary techniques. The time-sliced velocity map imaging arrangement was used to determine kinetic energy release distributions of fragments and estimate dissociation timescales. The multiplexed photoionization mass spectrometer setup was used to identify and quantify photoproducts, including isomers and free radicals, by their mass-to-charge ratios, photoionization spectra, and kinetic time profiles. Using these two techniques, it is possible to observe the major dissociation products of PA photodissociation: CO 2 , CO, H, OH, HCO, CH 2 CO, CH 3 CO, and CH 3 . Acetaldehyde and vinyl alcohol are minor primary photoproducts at 193 nm, but products that are known to arise from their unimolecular dissociation, such as HCO, H 2 CO, and CH 4 , are identified and quantified. A multivariate analysis that takes into account the yields of the observed products and assumes a set of feasible primary dissociation reactions provides a reasonable description of the photoinitiated chemistry of PA despite the necessary simplifications caused by the complexity of the dissociation. These experiments offer the first comprehensive description of the dissociation pathways of PA initiated on the S 3 excited state. Most of the observed products and yields are rationalized on the basis of three reaction mechanisms: (i) decarboxylation terminating in CO 2 + other primary products (~50%); (ii) Norrish type I dissociation typical of carbonyls (~30%); and (iii) O—H and C—H bond fission reactions generating the H atom (~10%). The analysis shows that most of the dissociation reactions create more than two products. This observation is not surprising considering the high excitation energy (~51 800 cm –1 ) and fairly low energy required for dissociation of PA. We find that two-body fragmentation processes yielding CO 2 are minor, and the expected, unstable primary co-fragment, methylhydroxycarbene, is not observed because it probably undergoes fast secondary dissociation and/or isomerization. Norrish type I dissociation pathways generate OH and only small yields of CH 3 CO and HOCO, which have low dissociation energies and further decompose via three-body fragmentation processes. Experiments with d 1 -PA (CH 3 COCOOD) support the interpretations. The dissociation on S 3 is fast, as indicated by the products’ recoil angular anisotropy, but the roles of internal conversion and intersystem crossing to lower states are yet to be determined.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Fast and flexible control of beam longitudinal phase space for high-repetition-rate x-ray free-electron lasers

The wide-ranging requirements for the photon properties from multiple beamlines in superconducting based free-electron lasers (FELs) demand more challenging beam manipulation techniques. Shot-by-shot control of electron beam bunch length and peak current at high repetition rate up to megahertz is highly desired. In this paper, we present a comprehensive study of a method based on a 2-m-long normal conducting radio-frequency cavity to achieve fast and flexible control of beam compression and realize the full potential of the facility, including theoretical analysis, beam dynamics simulations, and conceptual cavity design for the Linac Coherent Light Source II and its high-energy upgrade. We illustrate the physical mechanism of the chirping cavity on the control of the final beam compression and propose methods to lower the requirements for the cavity parameters. The application of this method will allow tailored photon properties of individual beamlines to optimize their performance and drastically improve the multiplexing capabilities of a high-repetition rate FEL facility.

42 ENGINEERING↗

Overview of the Langmuir probe system on the Mega Ampere Spherical Tokamak (MAST) Upgrade

A detailed description of the Langmuir probe system on Mega Ampere Spherical Tokamak Upgrade is presented. The system features 850 tile-embedded probes and 40 bespoke electronic modules that each have the capability to drive and acquire data from up to 16 probes in a time-multiplexed manner. The system provides spatiotemporal-resolved measurements (1 cm and ~1 ms, respectively) in the divertor region of ion saturation current, electron temperature, and floating potential. The standard interpretation of current-voltage (IV) characteristics is to apply a four-parameter fit, based on unmagnetized probe theory, which includes a linear model for the ion saturation region. In conclusion, to mitigate the effect of the magnetic field, analysis is restricted to the region of the IV characteristic, which is sensitive to only the tail of the electron energy distribution function.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

High-sensitivity electronic Stark spectrometer featuring a laser-driven light source

We report developmental details of a high-sensitivity Stark absorption spectrometer featuring a laser-driven light source. The light source exhibits intensity fluctuations of ~0.3% over timescales ranging from 1 min to 12 h, minimal drift (≤0.1%/h), and very little 1/f noise at frequencies greater than 200 Hz, which are comparable to or better than an arc-driven light source. Additional features of the spectrometer include balanced detection with multiplex sampling, which yielded lower noise in A, and constant wavelength or wavenumber (energy) spectral bandpass modes. We achieve noise amplitudes of ~7 × 10 –4 and ~6 × 10 –6 in measurements of single A and ΔA spectra (with 92 data points) taking ~7 and ~19 min, respectively.

47 OTHER INSTRUMENTATION↗

Interframe-tunable ultrafast differential-displacement holography

Here we describe the details of a digital holographic microscopy diagnostic capable of quantifying both the topography and velocity of a km/s object with adjustable temporal sensitivity. This technique involves spatially multiplexing a double pulse reflected from a target with reference beams of precisely known temporal separation.

47 OTHER INSTRUMENTATION↗