Exploring transient states of PAmKate to enable improved cryogenic single-molecule imaging.

Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nano-scale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) which photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remains a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find a transient state forms with a lifetime of ~2 ms which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for study of other FPs at cryogenic temperatures.

Engineered immunomodulatory extracellular vesicles derived from epithelial cells acquire capacity for positive and negative T cell co-stimulation in cancer and autoimmunity.

Extracellular vesicles (EVs) are generated by all cells and systemic administration of allogenic EVs derived from epithelial and mesenchymal cells have been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cells derived EVs can be modified to acquire the capacity to induce immune response, we engineered 293T EVs to harbor the immunomodulatory CD80, OX40L and PD-L1 molecules. We demonstrated abundant levels of these proteins on the engineered cells and EVs. Functionally, the engineered EVs efficiently elicit positive and negative co-stimulation in human and murine T cells. In the setting of cancer and auto-immune hepatitis, the engineered EVs modulate T cell functions and alter disease progression. Moreover, OX40L EVs provide additional benefit to anti-CTLA-4 treatment in melanoma-bearing mice. Our work provides evidence that epithelial cell derived EVs can be engineered to induce immune responses with translational potential to modulate T cell functions in distinct pathological settings.

Perfect Chirality with Imperfect Polarization.

Unidirectional (chiral) emission of light from a circular dipole emitter into a waveguide is only possible at points of perfect circular polarization (C points), with elliptical polarizations yielding a lower directional contrast. However, there is no need to restrict engineered systems to circular dipoles, and with an appropriate choice of dipole unidirectional emission is possible for any elliptical polarization. Using elliptical dipoles, rather than circular, typically increases the size of the area suitable for chiral interactions (in an exemplary mode by a factor ∼30), while simultaneously increasing coupling efficiencies. We propose illustrative schemes to engineer the necessary elliptical transitions in both atomic systems and quantum dots.

Protection against SARS-CoV-2 by BCG vaccination is not supported by epidemiological analyses.

The Bacillus Calmette-Guerin (BCG) vaccine provides protection against tuberculosis (TB), and is thought to provide protection against non-TB infectious diseases. BCG vaccination has recently been proposed as a strategy to prevent infection with SARS-CoV-2 (CoV-2) to combat the COVID-19 outbreak, supported by its potential to boost innate immunity and initial epidemiological analyses which observed reduced severity of COVID-19 in countries with universal BCG vaccination policies. Seventeen clinical trials are currently registered to inform on the benefits of BCG vaccinations upon exposure to CoV-2. Numerous epidemiological analyses showed a correlation between incidence of COVID-19 and BCG vaccination policies. These studies were not systematically corrected for confounding variables. We observed that after correction for confounding variables, most notably testing rates, there was no association between BCG vaccination policy and COVD-19 spread rate or percent mortality. Moreover, we found variables describing co-morbidities, including cardiovascular death rate and smoking prevalence, were significantly associated COVID-19 spread rate and percent mortality, respectively. While reporting biases may confound our observations, our epidemiological findings do not provide evidence to correlate overall BCG vaccination policy with the spread of CoV-2 and its associated mortality.

Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity.

Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies against the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological ELISA using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 reverse transcription PCR-confirmed, SARS-CoV-2-hospitalized patients, as well as 464 healthy and non-COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies against the 2 different viral proteins showed a moderate correlation. Antibodies against N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.9% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD-binding antibodies exhibited neutralizing capacity, but only 74% of N-protein-positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein-binding antibodies does not always correlate with presence of S-RBD-neutralizing antibodies and caution against the extensive use of N-protein-based serology testing for determination of potential COVID-19 immunity.

Photon Statistics of Filtered Resonance Fluorescence.

Spectral filtering of resonance fluorescence is widely employed to improve single photon purity and indistinguishability by removing unwanted backgrounds. For filter bandwidths approaching the emitter linewidth, complex behavior is predicted due to preferential transmission of components with differing photon statistics. We probe this regime using a Purcell-enhanced quantum dot in both weak and strong excitation limits, finding excellent agreement with an extended sensor theory model. By changing only the filter width, the photon statistics can be transformed between antibunched, bunched, or Poissonian. Our results verify that strong antibunching and a subnatural linewidth cannot simultaneously be observed, providing new insight into the nature of coherent scattering.

Phonon-Induced Optical Dephasing in Single Organic Molecules.

We present a joint experiment-theory analysis of the temperature-dependent emission spectra, zero-phonon linewidth, and second-order correlation function of light emitted from a single organic molecule. We observe spectra with a zero-phonon line together with several additional sharp peaks, broad phonon sidebands, and a strongly temperature dependent homogeneous broadening. Our model includes both localized vibrational modes of the molecule and a thermal phonon bath, which we include nonperturbatively, and is able to capture all observed features. For resonant driving we measure Rabi oscillations that become increasingly damped with temperature, which our model naturally reproduces. Our results constitute an essential characterization of the photon coherence of molecules, paving the way to their use in future quantum information applications.

Identification of IgG antibody response to SARS-CoV-2 spike protein and its receptor binding domain does not predict rapid recovery from COVID-19

Diagnostic testing and evaluation of patient immunity against the novel severe acute respiratory syndrome (SARS) corona virus that emerged last year (SARS-CoV-2) are essential for health and economic crisis recovery of the world. It is suggested that potential acquired immunity against SARS-CoV-2 from prior exposure may be determined by detecting the presence of circulating IgG antibodies against viral antigens, such as the spike glycoprotein and its receptor binding domain (RBD). Testing our asymptomatic population for evidence of COVID-19 immunity would also offer valuable epidemiologic data to aid health care policies and health care management. Currently, there are over 100 antibody tests that are being used around the world without approval from the FDA or similar regulatory bodies, and they are mostly for rapid and qualitative assessment, with different degrees of error rates. ELISA-based testing for sensitive and rigorous quantitative assessment of SARS-CoV-2 antibodies can potentially offer mechanistic insights into the COVID-19 disease and aid communities uniquely challenged by limited financial resources and access to commercial testing products. Employing recombinant SARS-CoV-2 RBD and spike protein generated in the laboratory, we devised a quantitative ELISA for the detection of circulating serum antibodies. Serum from twenty SARS-CoV-2 RT-PCR confirmed COVID-19 hospitalized patients were used to detect circulating IgG titers against SARS-CoV-2 spike protein and RBD. Quantitative detection of IgG antibodies to the spike glycoprotein or the RBD in patient samples was not always associated with faster recovery, compared to patients with borderline antibody response to the RBD. One patient who did not develop antibodies to the RBD completely recovered from COVID-19. In surveying 99 healthy donor samples (procured between 2017-February 2020), we detected RBD antibodies in one donor from February 2020 collection with three others exhibiting antibodies to the spike protein but not the RBD. Collectively, our study suggests that more rigorous and quantitative analysis, employing large scale samples sets, is required to determine whether antibodies to SARS-CoV-2 spike protein or RBD is associated with protection from COVID-19 disease. It is also conceivable that humoral response to SARS-CoV-2 spike protein or RBD works in association with adaptive T cell response to determine clinical sequela and severity of COVID-19 disease.

Randomized trial of a patient education tool about leiomyoma.

OBJECTIVE: Uterine leiomyomata are a frequent indication for women seeking gynecologic care [1]. The objective of our study was to assess whether patient knowledge about leiomyomata, anxiety, or satisfaction with counseling differed in patients who received multimedia counseling versus standard counseling. METHODS: Women with leiomyomata who presented to the gynecology clinic at a single institution were randomized to standard counseling or multimedia counseling using the drawMD OB/GYN iPad™ application. Participants completed a pre-counseling questionnaire, received the designated method of counseling, and completed a post-counseling questionnaire. Outcomes of the study included assessment of patient knowledge, satisfaction, and anxiety. RESULTS: Seventy-two participants were randomized. There was no significant difference in post-counseling anxiety between the groups (p = 0.86). For both groups, anxiety significantly improved after counseling. Both groups were satisfied with the counseling they received, however, there was no difference between groups. Participants in both groups significantly improved their knowledge about fibroids post-counseling. CONCLUSION: Counseling of patients with leiomyomata improves patient satisfaction and knowledge. The addition of a multimedia tool may or may not enhance patient counseling. PRACTICE IMPLICATIONS: This is the first prospective, randomized controlled trial evaluating the impact of a multimedia tool on patient education and counseling for patients with leiomyomata.

Light Scattering from Solid-State Quantum Emitters: Beyond the Atomic Picture.

Coherent scattering of light by a single quantum emitter is a fundamental process at the heart of many proposed quantum technologies. Unlike atomic systems, solid-state emitters couple to their host lattice by phonons. Using a quantum dot in an optical nanocavity, we resolve these interactions in both time and frequency domains, going beyond the atomic picture to develop a comprehensive model of light scattering from solid-state emitters. We find that even in the presence of a low-Q cavity with high Purcell enhancement, phonon coupling leads to a sideband that is completely insensitive to excitation conditions and to a nonmonotonic relationship between laser detuning and coherent fraction, both of which are major deviations from atomlike behavior.

Vibrational enhancement of quadrature squeezing and phase sensitivity in resonance fluorescence.

Vibrational environments are commonly considered to be detrimental to the optical emission properties of solid-state and molecular systems, limiting their performance within quantum information protocols. Given that such environments arise naturally it is important to ask whether they can instead be turned to our advantage. Here we show that vibrational interactions can be harnessed within resonance fluorescence to generate optical states with a higher degree of quadrature squeezing than in isolated atomic systems. Considering the example of a driven quantum dot coupled to phonons, we demonstrate that it is feasible to surpass the maximum level of squeezing theoretically obtainable in an isolated atomic system and indeed come close to saturating the fundamental upper bound on squeezing from a two-level emitter. We analyse the performance of these vibrationally-enhanced squeezed states in a phase estimation protocol, finding that for the same photon flux, they can outperform the single mode squeezed vacuum state.

Probing Electron-Phonon Interaction through Two-Photon Interference in Resonantly Driven Semiconductor Quantum Dots.

We investigate the temperature dependence of photon coherence properties through two-photon interference (TPI) measurements from a single quantum dot (QD) under resonant excitation. We show that the loss of indistinguishability is related only to the electron-phonon coupling and is not affected by spectral diffusion. Through these measurements and a complementary microscopic theory, we identify two independent separate decoherence processes, both of which are associated with phonons. Below 10 K, we find that the relaxation of the vibrational lattice is the dominant contribution to the loss of TPI visibility. This process is non-Markovian in nature and corresponds to real phonon transitions resulting in a broad phonon sideband in the QD emission spectra. Above 10 K, virtual phonon transitions to higher lying excited states in the QD become the dominant dephasing mechanism, this leads to a broadening of the zero phonon line, and a corresponding rapid decay in the visibility. The microscopic theory we develop provides analytic expressions for the dephasing rates for both virtual phonon scattering and non-Markovian lattice relaxation.

A Cooking Intervention to Increase Vegetable Consumption by Parents With Children Enrolled in an Early Head Start Home Visiting Program: A Pilot Study in Portland, Oregon, 2013-2014.

INTRODUCTION: Cooking interventions may improve diet quality. Most cooking interventions are delivered in group settings. Home visiting programs may be an appropriate mechanism for delivering such interventions to low-income families with young children. We conducted a pilot study to test the feasibility of using a cooking intervention delivered by home visitors to improve attitudes and behaviors related to vegetable consumption by low-income parents with children enrolled in a home visiting program. METHODS: We invited 121 parents with children enrolled in an Early Head Start Home Visiting program in Portland, Oregon, to participate. During 2013-2014, each month for 8 months, home visitors (n = 14) implemented 1 cooking activity plus 1 complementary activity focused on 12 vegetables. We collected pre- and post-intervention data on participants' cooking confidence and whether they tried and liked the selected vegetables. We also measured fidelity to protocol and home visitors' perception of intervention usability. RESULTS: Of 104 participants, 58 provided pre- and post-intervention data. We observed a significant increase in confidence in baking, roasting or grilling vegetables; cooking 6 of 10 vegetables; and trying 7 of 12 vegetables. Nearly all respondents participated in the monthly cooking activity (96%) and complementary activity (94%). Twelve of 14 home visitors reported that the intervention was acceptable, feasible, and easy to understand, and needed systems supports to implement. CONCLUSION: Cooking interventions may be a feasible approach to improving attitudes and behaviors related to vegetable consumption by low-income families with young children. Additional research is needed to assess the impact of such interventions on vegetable consumption.

A broadband tapered nanocavity for efficient nonclassical light emission.

We present the design of a tapered nanocavity, obtained by sandwiching a photonic wire section between a planar gold reflector and a few-period Bragg mirror integrated into the tapered wire. Thanks to its ultrasmall mode volume (0.71 λ3/n3), this hybrid nanocavity largely enhances the spontaneous emission rate of an embedded quantum dot (Purcell factor: 6), while offering a wide operation bandwidth (full-width half-maximum: 20 nm). In addition, the top tapered section shapes the cavity far-field emission into a very directive output beam, with a Gaussian spatial profile. For realistic taper dimensions, a total outcoupling efficiency to a Gaussian beam of 0.8 is predicted. Envisioned applications include bright sources of non-classical states of light, such as widely tunable sources of indistinguishable single photons and polarization-entangled photon pairs.

Modelling exciton-phonon interactions in optically driven quantum dots.

We provide a self-contained review of master equation approaches to modelling phonon effects in optically driven self-assembled quantum dots. Coupling of the (quasi) two-level excitonic system to phonons leads to dissipation and dephasing, the rates of which depend on the excitation conditions, intrinsic properties of the QD sample, and its temperature. We describe several techniques, which include weak-coupling master equations that are perturbative in the exciton-phonon coupling, as well as those based on the polaron transformation that can remain valid for strong phonon interactions. We additionally consider the role of phonons in altering the optical emission characteristics of quantum dot devices, outlining how we must modify standard quantum optics treatments to account for the presence of the solid-state environment.

Memory Th1 Cells Are Protective in Invasive Staphylococcus aureus Infection.

Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans.

Protein A is released into the Staphylococcus aureus culture supernatant with an unprocessed sorting signal.

The immunoglobulin binding protein A (SpA) of Staphylococcus aureus is synthesized as a precursor with a C-terminal sorting signal. The sortase A enzyme mediates covalent attachment to peptidoglycan so that SpA is displayed on the surface of the bacterium. Protein A is also found in the extracellular medium, but the processes involved in its release are not fully understood. Here, we show that a portion of SpA is released into the supernatant with an intact sorting signal, indicating that it has not been processed by sortase A. Release of SpA was reduced when the native sorting signal of SpA was replaced with the corresponding region of another sortase-anchored protein (SdrE). Similarly, a reporter protein fused to the sorting signal of SpA was released to a greater extent than the same polypeptide fused to the SdrE sorting signal. Released SpA protected bacteria from killing in human blood, indicating that it contributes to immune evasion.

Factors associated with increased healthcare costs in Medicare Advantage patients with type 2 diabetes enrolled in a large representative health insurance plan in the US.

AIM: The objective of this study was to apply quantile regression (QR) methodology to a population from a large representative health insurance plan with known skewed healthcare utilization attributes, co-morbidities, and costs in order to identify predictors of increased healthcare costs. Further, this study provides comparison of the results to those obtained using ordinary least squares (OLS) regression methodology. METHODS: Members diagnosed with Type 2 Diabetes and with 24 months of continuous enrollment were included. Baseline patient demographic, clinical, consumer/behavioural, and cost characteristics were quantified. Quantile regression was used to model the relationship between the baseline characteristics and total healthcare costs during the follow-up 12 month period. RESULTS: The sample included 83,705 patients (mean age = 70.6 years, 48% male) residing primarily in the southern US (78.1%); 81.2% of subjects were on oral-only anti-diabetic therapy. Co-morbid conditions included nephropathy (43.5%), peripheral artery disease (26.4%), and retinopathy (18.0%). Variables with the strongest relationship with costs during the follow-up period included outpatient visits, ER visits, inpatient visits, and Diabetes Complications Severity Index score during the baseline period. In the top cost quantiles, each additional glycohemoglobin (HbA1c) test was associated with cost savings ($1400 in the 98th percentile). Stage 4 and Stage 5 chronic kidney disease were associated with an incremental cost increase of $33,131 and $106,975 relative to Stage 1 or no CKD in the 98th percentile ($US). CONCLUSIONS: These results demonstrate that QR provides additional insight compared to traditional OLS regression modeling, and may be more useful for informing resource allocation to patients most likely to benefit from interventions. This study highlights that the impact of clinical and demographic characteristics on the economic burden of the disease vary across the continuum of healthcare costs. Understanding factors that drive costs on an individual patient level provide important insights that will help in ameliorating the clinical, humanistic, and economic burden of diabetes.

Temperature-dependent Mollow triplet spectra from a single quantum dot: Rabi frequency renormalization and sideband linewidth insensitivity.

We investigate temperature-dependent resonance fluorescence spectra obtained from a single self-assembled quantum dot. A decrease of the Mollow triplet sideband splitting is observed with increasing temperature, an effect we attribute to a phonon-induced renormalization of the driven dot Rabi frequency. We also present first evidence for a nonperturbative regime of phonon coupling, in which the expected linear increase in sideband linewidth as a function of temperature is canceled by the corresponding reduction in Rabi frequency. These results indicate that dephasing in semiconductor quantum dots may be less sensitive to changes in temperature than expected from a standard weak-coupling analysis of phonon effects.