Small intestine and colon tissue-resident memory CD8+ T cells exhibit molecular heterogeneity and differential dependence on Eomes.

Tissue-resident memory CD8+ T (TRM) cells are a subset of memory T cells that play a critical role in limiting early pathogen spread and controlling infection. TRM cells exhibit differences across tissues, but their potential heterogeneity among distinct anatomic compartments within the small intestine and colon has not been well recognized. Here, by analyzing TRM cells from the lamina propria and epithelial compartments of the small intestine and colon, we showed that intestinal TRM cells exhibited distinctive patterns of cytokine and granzyme expression along with substantial transcriptional, epigenetic, and functional heterogeneity. The T-box transcription factor Eomes, which represses TRM cell formation in some tissues, exhibited unexpected context-specific regulatory roles in supporting the maintenance of established TRM cells in the small intestine, but not in the colon. Taken together, these data provide previously unappreciated insights into the heterogeneity and differential requirements for the formation vs. maintenance of intestinal TRM cells.

Global identification of modular cullin-RING ligase substrates.

Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.

The C-degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes.

Protein termini are determinants of protein stability. Proteins bearing degradation signals, or degrons, at their amino- or carboxyl-termini are eliminated by the N- or C-degron pathways, respectively. We aimed to elucidate the function of C-degron pathways and to unveil how normal proteomes are exempt from C-degron pathway-mediated destruction. Our data reveal that C-degron pathways remove mislocalized cellular proteins and cleavage products of deubiquitinating enzymes. Furthermore, the C-degron and N-degron pathways cooperate in protein removal. Proteome analysis revealed a shortfall in normal proteins targeted by C-degron pathways, but not of defective proteins, suggesting proteolysis-based immunity as a constraint for protein evolution/selection. Our work highlights the importance of protein termini for protein quality surveillance, and the relationship between the functional proteome and protein degradation pathways.

100-kHz carbon monoxide TP-PLIF imaging using ultra-narrow-linewidth burst-mode OPO.

A 100-kHz rate two-photon planar laser-induced fluorescence (TP-PLIF) imaging of carbon monoxide (CO) is successfully demonstrated utilizing a narrow-linewidth optical parametric oscillator (OPO) generating light at ∼230.1 nm. A specially designed injection-seeded burst-mode OPO was constructed and characterized for this purpose. This OPO efficiently converts the 355-nm output of a high-energy nanosecond burst-mode laser to ∼230.1 nm following parametric splitting and mixing processes. Generation of an ultra-narrow-linewidth 230.1 nm laser pulse is crucial for effectively exciting CO via a two-photon process from the ground X1Σ+ to the B1Σ+ electronic state-enabling PLIF imaging over a large area. The experimental setup is capable of tracking high-speed flow structures of a CO/N2 mixture, showcasing detection speeds 100 times greater than those achieved with previous femtosecond laser sources. This substantial increase in repetition rate will allow time-resolved CO-TP-PLIF measurements in highly dynamic hypersonic boundary layers and detonation-driven combustion processes for revealing chemical kinetics and turbulent aerodynamics.

Long-lived nitric oxide molecular tagging velocimetry with 1 + 1 REMPI.

The successful demonstration of long-lived nitric oxide (NO) fluorescence for molecular tagging velocimetry (MTV) measurements is described in this Letter. Using 1 + 1 resonance-enhanced multiphoton ionization (REMPI) of NO at a wavelength near 226 nm, targeting the overlapping Q1(7) and Q21(7) lines of the A-X (0, 0) electronic system, the lifetime of the NO MTV signal was observed to be approximately 8.6 µs within a 100-Torr cell containing 2% NO in nitrogen. This is in stark contrast to the commonly reported single photon NO fluorescence, which has a much shorter calculated lifetime of approximately 43 ns at this pressure and NO volume fraction. While the shorter lifetime fluorescence can be useful for molecular tagging velocimetry with single laser excitation within very high-speed flows at some thermodynamic conditions, the longer lived fluorescence shows the potential for an order of magnitude more accurate and precise velocimetry, particularly within lower speed regions of hypersonic flow fields such as wakes and boundary layers. The physical mechanism responsible for the generation of this long-lived signal is detailed. Furthermore, the effectiveness of this technique is showcased in a high-speed jet flow, where it is employed for precise flow velocity measurements.

Alternative Splicing Mediated by RNA-Binding Protein RBM24 Facilitates Cardiac Myofibrillogenesis in a Differentiation Stage-Specific Manner.

BACKGROUND: Mutations in genes encoding sarcomeric proteins lead to failures in sarcomere assembly, the building blocks of contracting muscles, resulting in cardiomyopathies that are a leading cause of morbidity and mortality worldwide. Splicing variants of sarcomeric proteins are crucial at different stages of myofibrillogenesis, accounting for sarcomeric structural integrity. RBM24 (RNA-binding motif protein 24) is known as a tissue-specific splicing regulator that plays an essential role in cardiogenesis. However, it had been unclear if the developmental stage-specific alternative splicing facilitated by RBM24 contributes to sarcomere assembly and cardiogenesis. Our aim is to study the molecular mechanism by which RBM24 regulates cardiogenesis and sarcomere assembly in a temporal-dependent manner. METHODS: We ablated RBM24 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques. RESULTS: Although RBM24-/- hESCs still differentiated into sarcomere-hosting cardiomyocytes, they exhibited disrupted sarcomeric structures with punctate Z-lines due to impaired myosin replacement during early myofibrillogenesis. Transcriptomics revealed >4000 genes regulated by RBM24. Among them, core myofibrillogenesis proteins (eg, ACTN2 [α-actinin 2], TTN [titin], and MYH10 [non-muscle myosin IIB]) were misspliced. Consequently, MYH6 (muscle myosin II) cannot replace nonmuscle myosin MYH10, leading to myofibrillogenesis arrest at the early premyofibril stage and causing disrupted sarcomeres. Intriguingly, we found that the ABD (actin-binding domain; encoded by exon 6) of the Z-line anchor protein ACTN2 is predominantly excluded from early cardiac differentiation, whereas it is consistently included in human adult heart. CRISPR/Cas9-mediated deletion of exon 6 from ACTN2 in hESCs, as well as forced expression of full-length ACTN2 in RBM24-/- hESCs, further corroborated that inclusion of exon 6 is critical for sarcomere assembly. Overall, we have demonstrated that RBM24-facilitated inclusion of exon 6 in ACTN2 at distinct stages of cardiac differentiation is evolutionarily conserved and crucial to sarcomere assembly and integrity. CONCLUSIONS: RBM24 acts as a master regulator to modulate the temporal dynamics of core myofibrillogenesis genes and thereby orchestrates sarcomere organization.

Multiline molecular tagging velocimetry of nitric oxide at 100 kHz using an injection-seeded burst-mode OPO.

An injection-seeded, burst-mode optical parametric oscillator (OPO) operating at a repetition rate of 100 kHz is used to demonstrate the multiline molecular tagging velocimetry of an underexpanded jet using nitric oxide fluorescence. The very narrow linewidth of the OPO system, along with the relatively high pulse energies of the burst-mode system, enables efficient single-photon excitation of nitric oxide along multiple laser beam lines at a high repetition rate. Simultaneous one-dimensional velocity profile measurements were obtained of an underexpanded jet system at six different locations using a reference initial image and single-shot delayed images. A methodology for calculating the uncertainty of single-shot velocity is also described. Mean and root-mean-square velocity profiles are obtained at multiple locations simultaneously over a sampling time of 1 ms. The high-repetition-rate velocity measurements also appear to capture the onset of velocity oscillations and has the potential to reveal velocity frequency content occurring in the tens of kHz. The demonstrated velocimetry technique could be paired with other emerging burst-mode laser capabilities for a quantitative multiparameter gas property or multicomponent gas velocity measurements for supersonic and hypersonic flows, especially within ground test facilities that are limited to very short run durations.

Recent progress in high-speed laser diagnostics for hypersonic flows [Invited].

The recent progress in high-speed (≥100k H z) laser diagnostics for hypersonic flows is reviewed. Owing to the ultrahigh flow speed, a laser frequency of 100 kHz or higher is required for hypersonic diagnostics. Here, two main laser diagnostic techniques are discussed: focused laser differential interferometry (FLDI) and pulse-burst laser-based diagnostics. Single- and multiple-point FLDI measurements have been widely applied to hypersonic flows for flow velocity and density fluctuation measurements. The progress of pulse-burst laser-based hypersonic diagnostics, including flow velocity measurements and 2D flow visualization, is also discussed.

Laser Applications to Chemical, Security, and Environmental Analysis: introduction to the feature issue.

The eighteenth topical meeting on Laser Applications to Chemical, Security, and Environmental Analysis (LACSEA) was held in Vancouver, Canada from 11-15 July 2022, as part of the Optica Optical Sensors and Sensing Congress in a hybrid format allowing on-site and online attendance. The meeting featured a broad range of distinguished papers focusing on recent advances in laser and optical spectroscopy. A total of 52 contributed and invited papers were presented during the meeting, including topics such as photo-acoustic spectroscopy, imaging, non-linear technologies, frequency combs, remote sensing, environmental monitoring, aerosols, combustion diagnostics, hypersonic flow diagnostics, nuclear diagnostics, fs/ps applications, and machine learning and computational sensing.

Mach 18 flow velocimetry with 100-kHz KTV and PLEET in AEDC Tunnel 9.

Krypton Tagging Velocimetry (KTV) and Picosecond Laser Electronic Excitation Tagging (PLEET) velocimetry at a 100-kHz rate were demonstrated in Mach 18 flow conditions at the Arnold Engineering Development Center (AEDC) Tunnel 9 employing a burst-mode laser system and a custom optical parametric oscillator (OPO). The measured freestream flow velocities from both KTV and PLEET agreed well with the theoretical calculation. The increase in repetition rate provides better capability to perform time-resolved velocimetry measurements in hypersonic flow environments.

High-repetition-rate krypton tagging velocimetry in Mach-6 hypersonic flows.

A 100 kHz krypton (Kr) tagging velocimetry (KTV) technique was demonstrated in a Mach-6 Ludwieg tube using a burst-mode laser-pumped optical parametric oscillator system. The single-beam KTV scheme at 212 nm produced an insufficient signal in this large hypersonic wind tunnel because of its low Kr seeding (≤5%), low static pressure (∼2.5torr), and long working distance (∼1m). To overcome these issues, a new scheme using two excitation beams was developed to enhance KTV performance. A 355 nm laser beam was combined with the 212 nm beam to promote efficient two-photon Kr excitation at 212 nm, and increase the probability of 2 + 1 resonant-enhanced multiphoton ionization by adding a 355 nm beam. A signal enhancement of approximately six times was obtained. Using this two-excitation beam approach, strong long-lasting KTV was successfully demonstrated at a 100 kHz repetition rate in a Mach-6 flow.

The Mutation Hotspots at UGT1A Locus May Be Associated with Gilbert's Syndrome Affecting the Taiwanese Population.

Gilbert's syndrome is mainly diagnosed through genetic analysis and is primarily detected through a mutation in the promoter region of the UGT1A1 gene. However, most of the research has been conducted on Caucasian populations. In this study, we studied the Han population in Taiwan to investigate the possibility of other mutations that could cause Gilbert's syndrome. This study comprised a test group of 45 Taiwanese individuals with Gilbert's syndrome and 180 healthy Taiwanese individuals as a control group. We extracted DNA from the blood samples and then used Axiom Genome-Wide TWB 2.0 array plates for genotyping. Out of 302,771 single nucleotide polymorphisms (SNPs) from 225 subjects, we detected 57 SNPs with the most significant shift in allele frequency; 27 SNPs among them were located in the UGT1A region. Most of the detected SNPs highly correlated with each other and are located near the first exon of UGT1A1, UGT1A3, UGT1A6, and UGT1A7. We used these SNPs as an input for the machine learning algorithms and developed prediction models. Our study reveals a good association between the 27 SNPs detected and Gilbert's syndrome. Hence, this study provides a reference for diagnosing Gilbert's syndrome in the Taiwanese population in the future.

10 kHz two-color OH PLIF thermometry using a single burst-mode OPO.

10-kHz hydroxyl radical (OH) two-color planar laser-induced fluorescence (TC-PLIF) thermometry was demonstrated with a single burst-mode optical parametric oscillator (OPO) and a single camera. A fast, dual-wavelength switched seed laser enabled a high-energy, high-repetition-rate burst-mode laser to generate two 10-kHz pulse trains at wavelengths of ${\sim}{354.8}\;{\rm nm}$. The two pulse trains are colinear with 3 µs time interval between the pulse pairs. The injection-seeded OPO efficiently converts the burst-mode laser output to 285.62 and 285.67 nm to excite the ${Q}_2({12})$ and ${P}_1({8})$ OH transitions. PLIF images were collected from each of the two excitation transitions, and intensity ratios from the images were used to determine local temperatures. The development of fast, dual-wavelength switching, burst-mode OPO technology significantly reduces the experimental complexity of the high-speed TC-PLIF thermometry and simplifies its implementation in harsh combustion and flow test facilities.

Burst-mode 100 kHz N2 ps-CARS flame thermometry with concurrent nonresonant background referencing.

A burst-mode nitrogen (N2) picosecond vibrational coherent anti-Stokes Raman scattering (ps-VCARS) system is presented for accurate flame thermometry at 100 kHz repetition rate. A frequency-tripled ps burst-mode laser is used to pump a custom optical parametric generator/amplifier to produce 607 nm broadband Stokes pulses with 120cm-1 bandwidth, along with a narrowband 532 nm pump/probe beam. A simultaneous shot-to-shot nonresonant background (NRB) measurement is implemented to account for Stokes spectral profile and beam overlap fluctuations. The 100 kHz ps-VCARS data are benchmarked in a near-adiabatic CH4/air Hencken calibration flame with an accuracy of 1.5% and precision of 4.7% up to peak flame temperatures. The use of N2 VCARS and simultaneous NRB measurements enables high-speed thermometry for a wide range of fuels and combustion applications.

Office of Management and Budget Racial/Ethnic Categories in Mortality Research: A Framework for Including the Voices of Racialized Communities.

Since its founding, the US government has sorted people into racial/ethnic categories for the purpose of allowing or disallowing their access to social services and protections. The current Office of Management and Budget racial/ethnic categories originated in a dominant racial narrative that assumed a binary biological difference between Whites and non-Whites, with a hard-edged separation between them. There is debate about their continued use in researching group differences in mortality profiles and health outcomes: should we use them with modifications, cease using them entirely, or develop a new epistemology of human similarities and differences? This essay offers a research framework for including in these debates the daily lived experiences of the 110 million racialized non-White Americans whose lived experiences are the legacy of historically limited access to society's services and protections. The experience of Latinos in California is used to illustrate the major elements of this framework that may have an effect on mortality and health outcomes: a subaltern fuzzy-edged multivalent racial narrative, agency, voice, and community and cultural resilience.

Emissions in short-gated ns/ps/fs-LIBS for fuel-to-air ratio measurements in methane-air flames.

A study of short-gated 10 nanosecond (ns), 100 picosecond (ps), and 100 femtosecond (fs) laser induced breakdown spectroscopy (LIBS) was conducted for fuel-to-air ratio (FAR) measurements in an atmospheric Hencken flame. The intent of the work is to understand which emission lines are available near the optical range in each pulse width regime and which emission ratios may be favorable for generating equivalence ratio calibration curves. The emission spectra in the range of 550-800 nm for ns-LIBS and ps-LIBS are mostly similar with slightly elevated atomic oxygen lines by ps-LIBS. Spectra from fs-LIBS show the lowest continuum background and prominent individual atomic lines, though have significantly weaker ionic emission from nitrogen. A qualitative explanation based on assumed local thermodynamic equilibrium and electron temperatures calculated by the ${{\rm{N}}_{\rm{II}}}({{565}}\;{\rm{nm}})$ and ${{\rm{N}}_{\rm{II}}}({{594}}\;{\rm{nm}})$ emissions is presented. In studying line emission ratios for FAR calculation, it is found that ${{\rm{H}}_\alpha}({{656}}\;{\rm{nm}})/{{\rm{N}}_{\rm{II}}}({{568}}\;{\rm{nm}})$ is best for FAR measurements with ns-LIBS and remains viable for ps-LIBS, while ${{\rm{H}}_\alpha}({{656}}\;{\rm{nm}})/{{\rm{O}}_{\rm I}}({{777}}\;{\rm{nm}})$ is optimal for the ps-LIBS and fs-LIBS cases. Due to low continuum background and short time delay for spectra collection, fs-LIBS is very promising for high-speed FAR measurements using short-gated LIBS.

Laser applications to chemical, security, and environmental analysis: introduction to the feature issue.

This Applied Optics feature issue on laser applications to chemical, security, and environmental analysis (LACSEA) highlights papers presented at the LACSEA 2020 Seventeenth Topical Meeting sponsored by The Optical Society (OSA).

Compact fiber-coupled UV-NIR hyperspectral imaging sensor for characterizing ultra-high temperature ceramic materials.

A compact fiber-coupled hyperspectral imaging sensor (HSIS) operating within the range of ultraviolet to near-infrared (UV-NIR) wavelengths is designed and developed for the remote recording of two-dimensional (2D) spectrally resolved thermal radiation and chemiluminescent emission from ultra-high-temperature ceramics (UHTCs). Using simulations, the entire system is optimized to improve the collection efficiency and minimize aberrations. The design, construction, and characterization of the HSIS sensor are discussed in detail. We present the 2D spectrally resolved measurements of the simultaneous thermal radiation and BO2∗ chemiluminescent emission from a commonly used UHTC (HfB2-SiC) material under high-heat-flux conditions. Our results show that BO2∗ chemiluminescence corresponds directly to material ablation and can be used to track the formation of the protective heat-resistant glass/oxide layer. Furthermore, the temperature measurements demonstrate the heat distribution properties of the sample and indicate the locations at which BO2∗ chemiluminescence is possible. These results highlight the application prospects of the compact fiber-coupled HSIS for high-temperature material characterization in practical arc-jet facilities with limited optical access.

100 kHz krypton-based flow tagging velocimetry in a high-speed flow.

Krypton (Kr)-based tagging velocimetry is demonstrated in a Kr/N2 jet at 100 kHz repetition rate using a custom-built burst-mode laser and optical parametric oscillator (OPO) system. At this repetition rate, the wavelength-tunable, narrow linewidth laser platform can generate up to 7 mJ/pulse at resonant Kr two-photon-excitation wavelengths. Following a comprehensive study, we have identified the 212.56 nm two-photon-excitation transition as ideal for efficient Kr-based velocimetry, producing a long-lived (∼40µs) fluorescence signal from single-laser-pulse tagging that is readily amenable to velocity tracking without the need for a second "read" laser pulse. This long-lived fluorescence signal is found to emanate from N2-rather than from Kr-following efficient energy transfer. Successful flow velocity tracking is demonstrated at multiple locations in a high-speed Kr/N2 jet flow. The 100 kHz repetition rate provides the ability to perform time-resolved velocimetry measurements in high-speed and even hypersonic flow environments, where standard velocimetry approaches are insufficient to capture the relevant dynamics.

10 kHz 2D thermometry in turbulent reacting flows using two-color OH planar laser-induced fluorescence.

10 kHz two-color OH planar laser-induced fluorescence (PLIF) thermometry was demonstrated in both laminar Hencken flames and turbulent premixed jet flames using two injection-seeded optical parametric oscillators (OPOs) pumped by a high-speed three-legged burst-mode laser. The two burst-mode OPOs generate ∼5mJ/pulse at 282 nm and 286 nm to excite the Q1(5) and Q1(14) transitions of the A2Σ+←X2Π (1,0) system of OH, respectively. PLIF images were collected simultaneously from each of the two transitions and ratios of intensities from the two images were used to determine local temperatures. Analyses of flame curvature, temperature, and the correlation in time of these two quantities are also discussed. The results from this work are promising for the use of this technique in more complex flow environments and at, potentially, even higher repetition rate.