Accuracy of Dysphagia Screening by Non-clinical Research Staff in the Emergency Department.

Although the emergency department (ED) is the initial care setting for the majority of older adults requiring hospital admission, there is a paucity of ED-based dysphagia research in this at-risk population. This is driven by barriers to dysphagia evaluation in this complex care environment. Therefore, we assessed the reliability of trained, non-clinical ED research staff in administering dysphagia screening tools compared to trained speech pathologists (SLPs). We also aimed to determine perceptual screening discrepancies (e.g. voice change) between clinical and non-clinical staff. Forty-two older adults with suspected pneumonia were recruited during an ED visit and underwent dysphagia (Toronto Bedside Swallow Screening Tool; TOR-BSST©) and aspiration (3-oz water swallow test; 3-oz WST) screening by trained non-clinical research staff. Audio-recordings of screenings were re-rated post-hoc by trained, blinded SLPs with discrepancies resolved via consensus. Cohen's kappa (unweighted) revealed moderate agreement in pass/fail ratings between clinical and non-clinical staff for both the TOR-BSST© (k = 0.75) and the 3 oz WST (k = 0.66) corresponding to excellent sensitivity and good specificity for both the TOR-BSST (SN = 94%, SP = 85%) and the 3 oz WST (SN = 90%, SP = 81%). Further analysis of TOR-BSST perceptual parameters revealed that most discrepancies between clinicians and non-clinicians resulted from over-diagnosis of change in vocal quality (53%). These results support the feasibility of non-clinical research staff administering screening tools for dysphagia and aspiration in the ED. Dysphagia screening may not necessitate clinical staff involvement, which may improve feasibility of large-scale ED research. Future training of research staff should focus on perceptual assessment of vocal quality.

Imaging HIV-1 Nuclear Import, Uncoating, and Proviral Transcription.

Live-cell imaging has become a powerful tool for dissecting the behavior of viral complexes during HIV-1 infection with high temporal and spatial resolution. Very few HIV-1 particles in a viral population are infectious and successfully complete replication (~1/50). Single-particle live-cell imaging enables the study of these rare infectious viral particles, which cannot be accomplished in biochemical assays that measure the average property of the entire viral population, most of which are not infectious. The timing and location of many events in the early stage of the HIV-1 life cycle, including nuclear import, uncoating, and integration, have only recently been elucidated. Live-cell imaging also provides a valuable approach to study interactions of viral and host factors in distinct cellular compartments and at specific stages of viral replication. Successful live-cell imaging experiments require careful consideration of the fluorescent labeling method used and avoid or minimize its potential impact on normal viral replication and produce misleading results. Ideally, it is beneficial to utilize multiple virus labeling strategies and compare the results to ensure that the virion labeling did not adversely influence the viral replication step that is under investigation. Another potential benefit of using different labeling strategies is that they can provide information about the state of the viral complexes. Here, we describe our methods that utilize multiple fluorescent protein labeling approaches to visualize and quantify important events in the HIV-1 life cycle, including docking HIV-1 particles with the nuclear envelope (NE) and their nuclear import, uncoating, and proviral transcription.

Single-Virion Analysis: A Method to Visualize HIV-1 Particle Content Using Fluorescence Microscopy.

HIV-1 virions incorporate viral RNA, cellular RNAs, and proteins during the assembly process. Some of these components, such as the viral RNA genome and viral proteins, are essential for viral replication, whereas others, such as host innate immune proteins, can inhibit virus replication. Therefore, analyzing the virion content is an integral part of studying HIV-1 replication. Traditionally, virion contents have been examined using biochemical assays, which can provide information on the presence or absence of the molecule of interest but not its distribution in the virion population. Here, we describe a method, single-virion analysis, that directly examines the presence of molecules of interest in individual viral particles using fluorescence microscopy. Thus, this method can detect both the presence and the distribution of molecules of interest in the virion population. Single-virion analysis was first developed to study HIV-1 RNA genome packaging. In this assay, HIV-1 unspliced RNA is labeled with a fluorescently tagged RNA-binding protein (protein A) and some of the Gag proteins are labeled with a different fluorescent protein (protein B). Using fluorescence microscopy, HIV-1 particles can be identified by the fluorescent protein B signal and the presence of unspliced HIV-1 RNA can be identified by the fluorescent protein A signal. Therefore, the proportions of particles that contain unspliced RNA can be determined by the fraction of Gag particles that also have a colocalized RNA signal. By tagging the molecule of interest with fluorescent proteins, single-virion analysis can be easily adapted to study the incorporation of other viral or host cell molecules into particles. Indeed, this method has been adapted to examine the proportion of HIV-1 particles that contain APOBEC3 proteins and the fraction of particles that contain a modified Gag protein. Therefore, single-virion analysis is a flexible method to study the nucleic acid and protein content of HIV-1 particles.

HIV-1 uncoating requires long double-stranded reverse transcription products.

HIV-1 cores, which contain the viral genome and replication machinery, must disassemble (uncoat) during viral replication. However, the viral and host factors that trigger uncoating remain unidentified. Recent studies show that infectious cores enter the nucleus and uncoat near the site of integration. Here, we show that efficient uncoating of nuclear cores requires synthesis of a double-stranded DNA (dsDNA) genome >3.5 kb and that the efficiency of uncoating correlates with genome size. Core disruption by capsid inhibitors releases viral DNA, some of which integrates. However, most of the viral DNA is degraded, indicating that the intact core safeguards viral DNA. Atomic force microscopy and core content estimation reveal that synthesis of full-length genomic dsDNA induces substantial internal strain on the core to promote uncoating. We conclude that HIV-1 cores protect viral DNA from degradation by host factors and that synthesis of long double-stranded reverse transcription products is required to trigger efficient HIV-1 uncoating.

Deglutologist Practices and Perceptions of the Penetration-Aspiration Scale: A Survey Study.

Successful dysphagia management requires accurate, succinct diagnosis and characterization of swallowing safety impairments. However, the Penetration-Aspiration Scale (PAS) remains the only available tool developed exclusively for assessment of airway protection. To best support efforts to advance the field's understanding of swallowing safety, it is crucial to understand current clinician practice patterns, perceptions, and accuracy regarding the PAS. A 46-item survey was developed and distributed to deglutologists internationally examining: (1) Demographics; (2) Scale Practices; (3) Swallowing Safety Priorities; (4) Scale Perceptions; and (5) Accuracy. The first four sections consisted of questionnaires. In the optional fifth section, respondents were asked to score five videos of swallows collected via videofluoroscopy and previously PAS-scored by two trained raters. In total, 335 responses were analyzed. The majority of respondents self-reported PAS training (84%); 90% of untrained respondents were receptive to training. Respondents reported using the PAS "always" (40%) or "frequently" (29%), and that the PAS carries "a great deal of" weight in assessment (40%). Reported application of the PAS was heterogeneous, with the most common approach being "single worst score per unique presentation" (45%). Most respondents (64%) prioritized a parameter not captured by the PAS. Untrained respondents were significantly more confident with PAS ratings than trained respondents (X2 = 7.47; p = 0.006). Of 1460 PAS ratings provided, 364 of them were accurate (25%) when compared to ratings by trained lab members. Results of this survey reflect ubiquitous use of the PAS, unmet needs for assessment of swallowing safety, low accuracy despite generally high confidence, and heterogenous training that does not correspond to confidence. This emphasizes the need for additional training in clinical application of the PAS as well as development of novel metrics to optimize assessments of swallowing safety.

Use of deep learning to segment bolus during videofluoroscopic swallow studies.

Anatomical segmentations generated using artificial intelligence (AI) have the potential to significantly improve video fluoroscopic swallow study (VFS) analysis. AI segments allow for various metrics to be determined without additional time constraints streamlining and creating new opportunities for analysis. While the opportunity is vast, it is important to understand the challenges and limitations of the underlying AI task. This work evaluates a bolus segmentation network. The first swallow of thin or liquid bolus from 80 unique patients were manually contoured from bolus first seen in the oral cavity to end of swallow motion. The data was split into a 75/25 training and validation set and a 4-fold cross validation was done. A U-Net architecture along with variations were tested with the dice coefficient as the loss function and overall performance metric. The average validation set resulted in a dice coefficient of 0.67. Additional analysis to characterize the variability of images and performance on sub intervals was conducted indicating high variability among the processes required for training the network. It was found that bolus in the oral cavity consistently degrades performance due to misclassification of teeth and unimportant residue. The dice coefficients dependence on structure size can have substantial effects on the reported value. This work shows the efficacy of bolus segmentation and identifies key areas that are detriments to the performance of the network.

The Impact of Modifiable Preoral Factors on Swallowing and Nutritional Outcomes in Healthy Adults: A Scoping Review.

PURPOSE: Swallowing has previously been characterized as consisting of four phases; however, it has become apparent that these four phases are not truly discrete and may be influenced by factors occurring prior to bolus entrance into the oral cavity (i.e., preoral factors). Still, the relationship between these factors and swallowing remains poorly understood. The aim of this review was to synthesize and characterize the literature pertaining to the influence of preoral factors on swallowing and nutritional outcomes in healthy individuals. METHOD: We performed a scoping review, searching the databases of PubMed, CINAHL, Cochrane, and Scopus. Search terms included those related to swallowing, experience of preoral factors, and exclusionary terminology to reduce animal and pediatric literature. Our initial search revealed 5,560 unique articles, of which 153 met our inclusionary criteria and were accepted into the review. RESULTS: Of the accepted articles, 78% were focused exclusively on nutritional outcomes, 17% were focused on both swallowing and nutritional outcomes, and 5% were focused on solely swallowing outcomes. Of the preoral factors examined, 99% were exteroceptive in nature (17% olfactory, 44% visual, 21% auditory, 7% tactile, 11% other), while 1% were proprioceptive in nature. CONCLUSIONS: This review supports the influence of preoral factors on swallowing and nutritional outcomes. However, there is a large emphasis on the visual modality and on nutritional outcomes. Nearly none of the literature found in this review directly measured swallowing safety, efficiency, or physiology. Future work will benefit from a larger focus on proprioceptive preoral factors as they relate to swallowing outcomes.

Dysphagia in Parkinson Disease: Part I - Pathophysiology and Diagnostic Practices.

Purpose of Review: Dysphagia affects the majority of individuals with Parkinson disease (PD) and is not typically diagnosed until later in disease progression. This review will cover the current understanding of PD pathophysiology, and provides an overview of dysphagia in PD including diagnostic practices, gaps in knowledge, and future directions. Recent Findings: Many non-motor and other motor signs of PD appear in the prodrome prior to the manifestation of hall- mark signs and diagnosis. While dysphagia often presents already in the prodrome, it is not routinely addressed in standard neurology examinations. Summary: Dysphagia in PD can result in compromised efficiency and safety of swallowing, which significantly contributes to malnutrition and dehydration, decrease quality of life, and increase mortality. The heterogeneous clinical presentation of PD complicates diagnostic procedures which often leads to delayed treatment. Research has advanced our knowledge of mechanisms underlying PD, but dysphagia is still largely understudied, especially in the prodromal stage.

The capsid lattice engages a bipartite NUP153 motif to mediate nuclear entry of HIV-1 cores.

Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies in vitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.

Entangled Photon Spectroscopy.

The enhanced interest in quantum-related phenomena has provided new opportunities for chemists to push the limits of detection and analysis of chemical processes. As some have called this the second quantum revolution, a time has come to apply the rules learned from previous research in quantum phenomena toward new methods and technologies important to chemists. While there has been great interest recently in quantum information science (QIS), the quest to understand how nonclassical states of light interact with matter has been ongoing for more than two decades. Our entry into this field started around this time with the use of materials to produce nonclassical states of light. Here, the process of multiphoton absorption led to photon-number squeezed states of light, where the photon statistics are sub-Poissonian. In addition to the great interest in generating squeezed states of light, there was also interest in the formation of entangled states of light. While much of the effort is still in foundational physics, there are numerous new avenues as to how quantum entanglement can be applied to spectroscopy, imaging, and sensing. These opportunities could have a large impact on the chemical community for a broad spectrum of applications.In this Account, we discuss the use of entangled (or quantum) light for spectroscopy as well as applications in microscopy and interferometry. The potential benefits of the use of quantum light are discussed in detail. From the first experiments in porphyrin dendrimer systems by Dr. Dong-Ik Lee in our group to the measurements of the entangled two photon absorption cross sections of biological systems such as flavoproteins, the usefulness of entangled light for spectroscopy has been illustrated. These early measurements led the way to more advanced measurements of the unique characteristics of both entangled light and the entangled photon absorption cross-section, which provides new control knobs for manipulating excited states in molecules.The first reports of fluorescence-induced entangled processes were in organic chromophores where the entangled photon cross-section was measured. These results would later have widespread impact in applications such as entangled two-photon microscopy. From our design, construction and implementation of a quantum entangled photon excited microscope, important imaging capabilities were achieved at an unprecedented low excitation intensity of 107 photons/s, which is 6 orders of magnitude lower than the excitation level for the classical two-photon image. New reports have also illustrated an advantage of nonclassical light in Raman imaging as well.From a standpoint of more precise measurements, the use of entangled photons in quantum interferometry may offer new opportunities for chemistry research. Experiments that combine molecular spectroscopy and quantum interferometry, by utilizing the correlations of entangled photons in a Hong-Ou-Mandel (HOM) interferometer, have been carried out. The initial experiment showed that the HOM signal is sensitive to the presence of a resonant organic sample placed in one arm of the interferometer. In addition, parameters such as the dephasing time have been obtained with the opportunity for even more advanced phenomenology in the future.

Intranuclear Positions of HIV-1 Proviruses Are Dynamic and Do Not Correlate with Transcriptional Activity.

The relationship between spatiotemporal distribution of HIV-1 proviruses and their transcriptional activity is not well understood. To elucidate the intranuclear positions of transcriptionally active HIV-1 proviruses, we utilized an RNA fluorescence in situ hybridization assay and RNA stem loops that bind to fluorescently labeled bacterial protein (Bgl-mCherry) to specifically detect HIV-1 transcription sites. Initially, transcriptionally active wild-type proviruses were located closer to the nuclear envelope (NE) than expected by random chance in HeLa (∼1.4 µm) and CEM-SS T cells (∼0.9 µm). Disrupting interactions between HIV-1 capsid and host cleavage and polyadenylation specificity factor (CPSF6) resulted in localization of proviruses to lamina-associated domains (LADs) adjacent to the NE in HeLa cells (∼0.9 - 1.0 µm); however, in CEM-SS T cells, there was little or no shift toward the NE (∼0.9 µm), indicating cell-type differences in the locations of transcriptionally active proviruses. The distance from the NE was not correlated with transcriptional activity, and transcriptionally active proviruses were randomly distributed throughout the HeLa cell after several cell divisions, indicating that the intranuclear locations of the chromosomal sites of integration are dynamic. After nuclear import HIV-1 cores colocalized with nuclear speckles, nuclear domains enriched in pre-mRNA splicing factors, but transcriptionally active proviruses detected 20 h after infection were mostly located outside but near nuclear speckles, suggesting a dynamic relationship between the speckles and integration sites. Overall, these studies establish that the nuclear distribution of HIV-1 proviruses is dynamic and the distance between HIV-1 proviruses and the NE does not correlate with transcriptional activity. IMPORTANCE HIV-1 integrates its genomic DNA into the chromosomes of the infected cell, but how it selects the site of integration and the impact of their location in the 3-dimensional nuclear space is not well understood. Here, we examined the nuclear locations of proviruses 1 and 5 days after infection and found that integration sites are first located near the nuclear envelope but become randomly distributed throughout the nucleus after a few cell divisions, indicating that the locations of the chromosomal sites of integration that harbor transcriptionally active proviruses are dynamic. We also found that the distance from the nuclear envelope to the integration site is cell-type dependent and does not correlate with proviral transcription activity. Finally, we observed that HIV-1 cores were localized to nuclear speckles shortly after nuclear import, but transcriptionally active proviruses were located adjacent to nuclear speckles. Overall, these studies provide insights into HIV-1 integration site selection and their effect on transcription activities.

The rate of use of Veterans Affairs chiropractic care: a 5-year analysis.

BACKGROUND: The US Department of Veterans Affairs (VA) has initiated various approaches to provide chiropractic care to Veterans. Prior work has shown substantial increase in use of VA chiropractic care between fiscal years (FY) 2005-2016. However, the extent of the availability of these services to the Veteran population remains unclear. The purpose of this study was to analyze the rate of Veteran use of VA chiropractic services, both from on-site care at VA facilities and VA purchased care from community care providers. This study analyzed facility characteristics associated with chiropractic use by both care delivery mechanisms (on-site and in the community). METHODS: Cross-sectional analyses of administrative data were conducted for FY 2014-2019. Data were obtained from VA's Corporate Data Warehouse. The variables extracted included number of unique Veterans receiving VA chiropractic care on-site and in the community, total Veteran population of the VA facilities, size of the VA chiropractic workforce (measured as Full-Time Equivalent, FTE), and facility characteristics (geographic region and the facility complexity). Descriptive statistics, mixed model, and multivariant models were used to analyze data. RESULTS: Use of VA chiropractic care increased over the six-year period for both on-site and community care. National average for on-site use of the population was 1.27% in FY14 and 1.48% in FY19. Community care use was 0.29% and 1.76% for the same years. Use at individual facilities varied widely in each FY. Factors such as chiropractor FTE, geographic locations, and the complexity of the VA facility are associated with use of chiropractic services. CONCLUSION: The VA has expanded the non-pharmacologic treatments available to Veterans by providing chiropractic services, yet chiropractic use remains low compared to other US populations. As Veterans have a high prevalence of pain and musculoskeletal conditions, continued work to assess and achieve the optimal levels of chiropractic use in this population is warranted.

The Relationship Between Menopause and Dysphagia: A Scoping Review.

Purpose: Menopause marks the end of fertility and rapid decline of ovarian hormones in the female body, which corresponds to a myriad of changes to bodily systems, including the upper aerodigestive tract. Despite substantial evidence that menopause negatively impacts oral health, bones, and skeletal muscles, little research has examined these effects as they relate to swallowing. The purpose of this scoping review was to compile and summarize the existing literature investigating the relationship between menopause and swallowing-related structures and physiology. Methods: Search terms were selected for three databases (PubMed, Scopus, and CINAHL) to gather relevant literature evaluating the relationship between menopause and swallowing-related anatomy as well as swallowing functions in both human and animal models. Relevant articles were reviewed, collated, and summarized to synthesize findings, identify gaps in the literature, and provide suggestions for future directions. Results: This scoping review yielded 204 studies with the majority of these studies relating to one or more of the following categories: oral health, saliva, mandibular structures, and taste. Common oral symptoms reported in the literature included xerostomia, hyposalivation, tooth decay, inflammation of oral mucosa, and oral pain. Although literature supports that menopause adversely affects oral health, saliva, mandibular structures, and alters taste, a dearth of information was evident regarding how these hormone-dependent changes can adversely affect swallowing. Conclusions: The relationship between menopause and swallowing has been overlooked by field of speech-language pathology. By identifying the major gaps in the literature, these results will inform future investigations evaluating relationships among ovarian hormones and swallowing.

Enhancing Entangled Two-Photon Absorption for Picosecond Quantum Spectroscopy.

Entangled two-photon absorption (ETPA) is known to create photoinduced transitions with extremely low light intensity, reducing the risk of phototoxicity compared to classical two-photon absorption. Previous works have predicted the ETPA cross-section, σe, to vary inversely with the product of entanglement time (Te) and entanglement area (Ae), i.e., σe ∼ 1/AeTe. The decreasing σe with increasing Te has limited ETPA to fs-scale Te, while ETPA applications for ps-scale spectroscopy have been unexplored. However, we show that spectral-spatial coupling, which reduces Ae as the SPDC bandwidth (σf) decreases, plays a significant role in determining σe when Te > ∼100 fs. We experimentally measured σe for zinc tetraphenylporphyrin at several σf values. For type-I ETPA, σe increases as σf decreases down to 0.1 ps-1. For type-II SPDC, σe is constant for a wide range of σf. With a theoretical analysis of the data, the maximum type-I σe would occur at σf = 0.1 ps-1 (Te = 10 ps). At this maximum, σe is 1 order of magnitude larger than fs-scale σe and 3 orders of magnitude larger than previous predictions of ps-scale σe. By utilizing this spectral-spatial coupling, narrowband type-I ETPA provides a new opportunity to increase the efficiency of measuring nonlinear optical signals and to control photochemical reactions requiring ps temporal precision.

Visual Modeling: A Socialization-Based Intervention to Improve Nutritional Intake Among Nursing Home Residents.

Purpose Malnutrition is a widespread, dangerous, and costly condition among institutionalized older adults and can be both a contributor to and consequence of dysphagia for individuals with cognitive impairment. However, interventions to maximize intake in individuals with dementia are limited and frequently problematic, with negative implications for independence and quality of life. The goal of this study was to examine a novel, socialization-grounded intervention based on visual modeling, utilizing the theoretical underpinnings of motor resonance and mimicry. Method To examine the impact of environment on intake, data were collected from four nursing home residents (M age = 83.5 years, SD = 4.2; three women) with dementia. Weight of food and liquid intake was measured across 15 meals and three different mealtime conditions: the "baseline condition" in which the individual ate alone, the "watch condition" in which the individual ate in the company of a "mealtime buddy," and the "eat" condition in which the individual consumed a meal while the "mealtime buddy" did the same. Results Data visualization supported a weak functional relation between eating environment and amount of intake consumed across participants. Log response ratio estimates suggested a trend for increased weight of food consumed during the eat condition as compared to baseline and the eat condition as compared to the watch condition for some participants. Conclusions These results preliminarily support the benefit of a visual model for increased consumption in some individuals with dementia. The presence and magnitude of the effect across conditions varied based on individual-level factors, such as cognitive status, which has implications for implementation. Overall, this study provides initial proof of concept regarding the use of visual modeling as an intervention approach, laying the foundation for larger scale future studies.

HIV-1 cores retain their integrity until minutes before uncoating in the nucleus.

We recently reported that HIV-1 cores that retained >94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site <1.5 h before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear. Here, we utilized a previously reported vector in which green fluorescent protein is inserted in HIV-1 Gag (iGFP); proteolytic processing efficiently releases GFP, some of which remains trapped inside capsids and serves as a fluid phase content marker that is released when the capsids lose their integrity. We found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker ∼1 to 3 min before loss of capsid-associated mRuby-tagged cleavage and polyadenylation specificity factor 6 (mRuby-CPSF6). In contrast, loss of GFP fused to CA and mRuby-CPSF6 occurred simultaneously, indicating that viral cores retain their integrity until just minutes before uncoating. Our results indicate that HIV-1 evolved to retain its capsid integrity and maintain a separation between macromolecules in the viral core and the nuclear environment until uncoating occurs just before integration. These observations imply that intact HIV-1 capsids are imported through nuclear pores; that reverse transcription occurs in an intact capsid; and that interactions between the preintegration complex and LEDGF/p75, and possibly other host factors that facilitate integration, must occur during the short time period between loss of capsid integrity and integration.

Efficient Modeling of Organic Chromophores for Entangled Two-Photon Absorption.

The use of a nonclassical light source for studying molecular electronic structure has been of great interest in many applications. Here we report a theoretical study of entangled two-photon absorption (ETPA) in organic chromophores, and we provide new insight into the quantitative relation between ETPA and the corresponding unentangled TPA based on the significantly different line widths associated with entangled and unentangled processes. A sum-over-states approach is used to obtain classical TPA and ETPA cross sections and to explore the contribution of each electronic state to the ETPA process. The transition moments and energies needed for this calculation were obtained from a second linear-response (SLR) TDDFT method [J. Chem. Phys., 2016, 144, 204105], which enables the treatment of relatively large polythiophene dendrimers that serve as two-photon absorbers. In addition, the SLR calculations provide estimates of the excited state radiative line width, which we relate to the entangled two-photon density of states using a quantum electrodynamic analysis. This analysis shows that for the dendrimers being studied, the line width for ETPA is orders of magnitude narrower than for TPA, corresponding to highly entangled photons with a large Schmidt number. The calculated cross sections are in good agreement with the experimentally reported values. We also carried out a state-resolved analysis to unveil pathways for the ETPA process, and these demonstrate significant interference behavior. We emphasize that the use of entangled photons in TPA process plays a critical role in probing the detailed electronic structure of a molecule by probing light-matter interference nature in the quantum limit.

HIV-1 uncoats in the nucleus near sites of integration.

HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat <1.5 h before integration near (<1.5 µm) their genomic integration sites. These results fundamentally change our current understanding of HIV-1 postentry replication events including mechanisms of nuclear import, uncoating, reverse transcription, integration, and evasion of innate immunity.