Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

Prevention of tuberculosis in macaques after intravenous BCG immunization.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide1. The only available vaccine, BCG (Bacillus Calmette-Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission1,2. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography-computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.

The Effect of the Consent Process on Patient Satisfaction With Pain Management: A Randomized Controlled Trial.

STUDY OBJECTIVE: We aim to determine whether the timing and context of informed consent affects the subjective outcome of patient satisfaction with pain management. METHODS: We conducted a randomized controlled trial in a single emergency department (ED). Patients aged 18 years or older with a triage pain score of greater than or equal to 4 provided consent to participate in a pain management study. They were randomized to consent in the ED or at follow-up. All patients were followed up at 48 hours post-ED discharge. Patients who consented at follow-up were unaware of the study until cold called. The primary outcome was patient satisfaction with pain management. Secondary analyses examined effects on follow-up and participation rates. Variables associated with patients' being very satisfied were determined with multivariate logistic regression. RESULTS: Outcome data were obtained on 655 of 825 patients enrolled (79.4%). Patients who provided consent at follow-up were less likely to be very satisfied compared with those who consented in the ED (difference in proportions 11.5%; 95% confidence interval 3.5 to 19.4). Follow-up and participation rates did not differ between the groups. Patients who received pain advice and adequate analgesia (both as defined in this study) were more likely to be very satisfied (odds ratio 5.18, 95% confidence interval 2.82 to 9.52; and odds ratio 1.54, 95% confidence interval 1.07 to 2.22, respectively). CONCLUSION: The timing and context of informed consent significantly affect the subjective outcome of patient satisfaction, and this should be considered during study design. Clinicians should strive to provide pain advice and adequate analgesia to maximize their patients' satisfaction.

A Network of Sputum MicroRNAs Is Associated with Neutrophilic Airway Inflammation in Asthma.

Rationale: MicroRNAs are potent regulators of biologic systems that are critical to tissue homeostasis. Individual microRNAs have been identified in airway samples. However, a systems analysis of the microRNA-mRNA networks present in the sputum that contribute to airway inflammation in asthma has not been published.Objectives: Identify microRNA and mRNA networks in the sputum of patients with asthma.Methods: We conducted a genome-wide analysis of microRNA and mRNA in the sputum from patients with asthma and correlated expression with clinical phenotypes. Weighted gene correlation network analysis was implemented to identify microRNA networks (modules) that significantly correlate with clinical features of asthma and mRNA expression networks. MicroRNA expression in peripheral blood neutrophils and lymphocytes and in situ hybridization of the sputum were used to identify the cellular sources of microRNAs. MicroRNA expression obtained before and after ozone exposure was also used to identify changes associated with neutrophil counts in the airway.Measurements and Main Results: Six microRNA modules were associated with clinical features of asthma. A single module (nely) was associated with a history of hospitalizations, lung function impairment, and numbers of neutrophils and lymphocytes in the sputum. Of the 12 microRNAs in the nely module, hsa-miR-223-3p was the highest expressed microRNA in neutrophils and was associated with increased neutrophil counts in the sputum in response to ozone exposure. Multiple microRNAs in the nely module correlated with two mRNA modules enriched for TLR (Toll-like receptor) and T-helper cell type 17 (Th17) signaling and endoplasmic reticulum stress. hsa-miR-223-3p was a key regulator of the TLR and Th17 pathways in the sputum of subjects with asthma.Conclusions: This study of sputum microRNA and mRNA expression from patients with asthma demonstrates the existence of microRNA networks and genes that are associated with features of asthma severity. Among these, hsa-miR-223-3p, a neutrophil-derived microRNA, regulates TLR/Th17 signaling and endoplasmic reticulum stress.

Low Levels of T Cell Exhaustion in Tuberculous Lung Granulomas.

The hallmarks of pulmonary Mycobacterium tuberculosis infection are lung granulomas. These organized structures are composed of host immune cells whose purpose is to contain or clear infection, creating a complex hub of immune and bacterial cell activity, as well as limiting pathology in the lungs. Yet, given cellular activity and the potential for frequent interactions between host immune cells and M. tuberculosis-infected cells, we observed a surprisingly low quantity of cytokine-producing T cells (<10% of granuloma T cells) in our recent study of M. tuberculosis infection within nonhuman primate (NHP) granulomas. Various mechanisms could limit T cell function, and one hypothesis is T cell exhaustion. T cell exhaustion is proposed to result from continual antigen stimulation, inducing them to enter a state characterized by low cytokine production, low proliferation, and expression of a series of inhibitory receptors, the most common being PD-1, LAG-3, and CTLA-4. In this work, we characterized the expression of inhibitory receptors on T cells and the functionality of these cells in tuberculosis (TB) lung granulomas. We then used these experimental data to calibrate and inform an agent-based computational model that captures environmental, cellular, and bacterial dynamics within granulomas in lungs during M. tuberculosis infection. Together, the results of the modeling and the experimental work suggest that T cell exhaustion alone is not responsible for the low quantity of M. tuberculosis-responsive T cells observed within TB granulomas and that the lack of exhaustion is likely an intrinsic property of granuloma structure.

Characterisation of asthma subgroups associated with circulating YKL-40 levels.

The chitinase-like protein YKL-40 mediates airway inflammation and serum levels are associated with asthma severity. However, asthma phenotypes associated with YKL-40 levels have not been precisely defined.We conducted an unsupervised cluster analysis of asthma patients treated at the Yale Center for Asthma and Airways Disease (n=156) to identify subgroups according to YKL-40 level. The resulting YKL-40 clusters were cross-validated in cohorts from the Severe Asthma Research Programme (n=167) and the New York University/Bellevue Asthma Repository (n=341). A sputum transcriptome analysis revealed molecular pathways associated with YKL-40 subgroups.Four YKL-40 clusters (C1-C4) were identified. C3 and C4 had high serum YKL-40 levels compared with C1 and C2. C3 was associated with earlier onset and longer duration of disease, severe airflow obstruction, and near-fatal asthma exacerbations. C4 had the highest serum YKL-40 levels, adult onset and less airflow obstruction, but frequent exacerbations. An airway transcriptome analysis in C3 and C4 showed activation of non-type 2 inflammatory pathways.Elevated serum YKL-40 levels were associated with two distinct clinical asthma phenotypes: one with irreversible airway obstruction and another with severe exacerbations. The YKL-40 clusters are potentially useful for identification of individuals with severe or exacerbation-prone asthma.

Evaluation of Autism-Related Health Information on the Web.

BACKGROUND: The Internet is a frequently accessed source of information for parents of a child with autism. To help parents make informed decisions about treatment options, websites should contain accurate information. This study aimed to evaluate the quality of information in a sample of autism-relevant websites. MATERIALS AND METHODS: Autism-related keywords were entered into three widely used search engines in April 2013 and the 20 most frequently appearing sites identified. Website quality was rated, by two independent raters, using the DISCERN tool. Websites were also coded according to the type of references/sources provided to support the intervention content presented. RESULTS: The mean DISCERN score was 46.5 (range 23-67.5), of a possible 80. Information about treatment risks and no treatment as an option was rarely described. Only six (30%) websites provided research references when describing intervention options. CONCLUSIONS: Many websites did not meet criteria for quality health information and failed to cite evidence supporting described interventions. Implications of these findings are discussed.

Systematic review of existing literature regarding the prevalence of pediatric atopic dermatitis in Honduras.

Background: Atopic dermatitis (AD) is an inflammatory skin condition, often multifactorial in origin, and most commonly manifests during childhood. Although there remains a deficit in literature, current data suggest Honduras may have the highest prevalence and severity of AD among all Latin American countries. Objective: To assess the current prevalence of pediatric AD in Honduras and evaluate existing gaps in available literature to monitor disease burden. Methods: A comprehensive literature search was performed in March 2023. Articles were removed if they were published before 2007, were of the incorrect study design, or were focused on countries outside of Honduras. The articles were independently reviewed by 2 authors. Results: The initial literature search yielded 174 studies, of which 7 met inclusion criteria. AD prevalence rates in children in Honduras ranged from 0.7% to 40.0%. Limitations: Limitations include elements of study design, analytic methods, study populations, and limited articles. Conclusion: There appears to be a disproportionately higher prevalence and disease burden of pediatric AD in Honduras. Future research should acquire accurate data to further understand the prevalence, incidence, and severity of AD in Honduras.

Mycobacterium tuberculosis-Specific CD4 T Cells Expressing Transcription Factors T-Bet or RORγT Associate with Bacterial Control in Granulomas.

Despite the extensive research on CD4 T cells within the context of Mycobacterium tuberculosis (Mtb) infections, few studies have focused on identifying and investigating the profile of Mtb-specific T cells within lung granulomas. To facilitate the identification of Mtb-specific CD4 T cells, we identified immunodominant epitopes for two Mtb proteins, namely, Rv1196 and Rv0125, using a Mauritian cynomolgus macaque model of Mtb infection, thereby providing data for the synthesis of MHC class II tetramers. Using tetramers, we identified Mtb-specific cells within different immune compartments, postinfection. We found that granulomas were enriched sites for Mtb-specific cells and that tetramer+ cells had increased frequencies of the activation marker CD69 as well as the transcription factors T-bet and RORγT, compared to tetramer negative cells within the same sample. Our data revealed that while the frequency of Rv1196 tetramer+ cells was positively correlated with the granuloma bacterial burden, the frequency of RORγT or T-bet within tetramer+ cells was inversely correlated with the granuloma bacterial burden, thereby highlighting the importance of having activated, polarized, Mtb-specific cells for the control of Mtb in lung granulomas. IMPORTANCE Tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, kills 1.5 million people each year, despite the existence of effective drugs and a vaccine that is given to infants in most countries. Clearly, we need better vaccines against this disease. However, our understanding of the immune responses that are necessary to prevent tuberculosis is incomplete. This study seeks to understand the functions of T cells that are specific for M. tuberculosis at the site of the disease in the lungs. For this, we developed specialized tools called MHC class II tetramers to identify those T cells that can recognize M. tuberculosis and applied the tools to the study of this infection in nonhuman primate models that mimic human tuberculosis. We demonstrate that M. tuberculosis-specific T cells in lung lesions are associated with control of the bacteria only when those T cells are expressing certain functions, thereby highlighting the importance of combining the identification of specific T cells with functional analyses. Thus, we surmise that these functions of specific T cells are critical to the control of infection and should be considered as a part of the development of vaccines against tuberculosis.

The role of tumor histogenesis, FDG-PET, and short-course EPOCH with dose-dense rituximab (SC-EPOCH-RR) in HIV-associated diffuse large B-cell lymphoma.

This is a phase 2 study to assess the role of tumor histogenesis (subtype), fluorodeoxyglucose positron emission tomography (FDG-PET), and short-course etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin with dose-dense rituximab (SC-EPOCH-RR) in newly diagnosed HIV-associated CD20(+) diffuse large B-cell lymphoma. Patients received a minimum of 3 and a maximum of 6 cycles with 1 cycle beyond stable radiographic and FDG-PET scans. Overall, 79% of patients received 3 cycles. Combination antiretroviral therapy was suspended before and resumed after therapy. Thirty-three enrolled patients had a median age of 42 years (range, 9-61 years), and 76% had a high-intermediate or high age-adjusted international prognostic index. At 5 years median follow-up, progression-free and overall survival were 84% and 68%, respectively. There were no treatment-related deaths or new opportunistic infections during treatment, and patients had sustained CD4 cell count recovery and HIV viral control after treatment. FDG-PET after 2 cycles had an excellent negative but poor positive predictive value. Tumor histogenesis was the only characteristic associated with lymphoma-specific outcome with 95% of germinal center B-cell (GCB) versus 44% of non-GCB diffuse large B-cell lymphoma (DLBCL) progression-free at 5 years. SC-EPOCH-RR is highly effective and less immunosuppressive with shorter duration therapy compared with standard strategies. However, new therapeutic advances are needed for non-GCB DLBCL, which remains the important cause of lymphoma-specific death. This trial was registered at www.clinicaltrials.gov as NCT000019253.

T cell transcription factor expression evolves over time in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a global health concern, yearly resulting in 10 million new cases of active TB. Immunologic investigation of lung granulomas is essential for understanding host control of bacterial replication. Here, we identify and compare the pathological, cellular, and functional differences in granulomas at 4, 12, and 20 weeks post-infection in Chinese cynomolgus macaques. Original granulomas differ in transcription-factor expression within adaptive lymphocytes, with those at 12 weeks showing higher frequencies of CD8+T-bet+ T cells, while CD4+T-bet+ T cells increase at 20 weeks post-infection. The appearance of T-bet+ adaptive T cells at 12 and 20 weeks is coincident with a reduction in bacterial burden, suggesting their critical role in Mtb control. This study highlights the evolution of T cell responses within lung granulomas, suggesting that vaccines promoting the development and migration of T-bet+ T cells would enhance mycobacterial control.

Complete Genome Sequences of Microbacterium paraoxydans Phages Cassita and Fransoyer.

Phages Cassita and Fransoyer were isolated from soil in northwestern Wisconsin using Microbacterium paraoxydans as the host. The genomes of Cassita and Fransoyer are 61,868 bp and 62,277 bp, respectively, with direct terminal repeats. Both phages exhibit siphoviral morphology and are predicted to have lytic life cycles.

In the heat of the night--alternative pathway respiration drives thermogenesis in Philodendron bipinnatifidum.

• Philodendron bipinnatifidum inflorescences heat up to 42 °C and thermoregulate. We investigated whether they generate heat via the cytochrome oxidase pathway uncoupled by uncoupling proteins (pUCPs), or the alternative oxidase (AOX). • Contribution of AOX and pUCPs to heating in fertile (FM) and sterile (SM) male florets was determined using a combination of oxygen isotope discrimination, protein and substrate analyses. • Both FM and SM florets thermoregulated independently for up to 30 h ex planta. In both floret types, AOX contributed > 90% of respiratory flux during peak heating. The AOX protein increased fivefold with the onset of thermogenesis in both floret types, whereas pUCP remained low throughout development. These data indicate that AOX is primarily responsible for heating, despite FM and SM florets potentially using different substrates, carbohydrates or lipids, respectively. Measurements of discrimination between O2 isotopes in strongly respiring SM florets were affected by diffusion; however, this diffusional limitation was largely overcome using elevated O2. • The first in vivo respiratory flux measurements in an arum show AOX contributes the bulk of heating in P. bipinnatifidum. Fine-scale regulation of AOX activity is post-translational. We also demonstrate that elevated O2 can aid measurement of respiratory pathway fluxes in dense tissues.

Differential efficacy of bortezomib plus chemotherapy within molecular subtypes of diffuse large B-cell lymphoma.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed distinct molecular subtypes that include germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. ABC DLBCL has a worse survival after upfront chemotherapy and is characterized by constitutive activation of the antiapoptotic nuclear factor-kappa B (NF-kappaB) pathway, which can inhibit chemotherapy. We hypothesized that inhibition of NF-kappaB might sensitize ABC but not GCB DLBCL to chemotherapy and improve outcome. As the proteasome inhibitor bortezomib can inhibit NF-kappaB through blocking IkappaBalpha degradation, we investigated bortezomib alone followed by bortezomib and doxorubicin-based chemotherapy in recurrent DLBCL. Tumor tissue was analyzed by gene expression profiling and/or immunohistochemistry to identify molecular DLBCL subtypes. As a control, we showed that relapsed/refractory ABC and GCB DLBCL have equally poor survivals after upfront chemotherapy. Bortezomib alone had no activity in DLBCL, but when combined with chemotherapy, it demonstrated a significantly higher response (83% vs 13%; P < .001) and median overall survival (10.8 vs 3.4 months; P = .003) in ABC compared with GCB DLBCL, respectively. These results suggest bortezomib enhances the activity of chemotherapy in ABC but not GCB DLBCL, and provide a rational therapeutic approach based on genetically distinct DLBCL subtypes. This trial is registered with http://www.ClinicalTrials.gov under identifier NCT00057902.

Nature and outcomes of sanctioned medical misconduct in six international jurisdictions: a case series.

Objective The aim of this study was to determine the types of medical misconduct, the practitioner, specialities and jurisdictions at risk, patient outcomes and the sanctions imposed. Methods This study was a retrospective case series of 822 adverse medical tribunal determinations in Australia, New Zealand, Canada (Ontario, Alberta), Pennsylvania (USA), Singapore and Hong Kong in 2013-17. Results Inappropriate medical care and illegal or unethical prescribing were the most common types of misconduct. Misconduct varied with practitioner sex, international medical graduate status, speciality and jurisdiction (P<0.05). Cases of inappropriate medical care were more common in Singapore (46.7% of all Singapore cases; 95% confidence interval (CI) 31.9-62.0) and among surgeons (47.6% of all surgeon cases; 95% CI 36.5-58.8). Illegal or unethical prescribing was more common in Australia (31.1%; 95% CI 24.8-38.2) and among general or family practitioners (26.9%; 95% CI 20.0-35.0). Misconduct not related to patients was more common in Pennsylvania (30.3%; 95% CI 25.2-36.0) and among local graduates (20.5%; 95% CI 17.1-24.5). Sexual misconduct was more common in Australia (29.6%; 95% CI 23.4-36.6) and among males (19.6%, 95% CI 16.7-22.8). Healthcare dishonesty was more common in Hong Kong (21.8%; 95% CI 14.0-32.2) and among surgeons (13.4%; 95% CI 7.2-23.2). The most common patient outcomes were patient risk (40.6%; 95% CI 36.1-45.4) and death and actual physical harm combined (31.2%; 95% CI 26.9-35.7). Sanctions were most commonly suspension or deregistration. Deregistration was most common in cases of sexual misconduct. Conclusion Medical misconduct varies widely. Risk factors for particular misconduct types are apparent among jurisdictions and practitioner characteristics. The nature of patient harm varied by type of misconduct, with illegal unethical prescribing commonly leading to drug dependency and sexual misconduct leading to psychiatric injury. What is known about the topic? Medical misconduct is a continuing problem. Tribunals and medical boards sanction misconduct to protect patient safety and public trust. What does this paper add? Tribunals and boards differ in misconduct reporting and permitting public access to determinations. Types of misconduct vary between international jurisdictions, practitioner sex, international graduate status and speciality. Risk and physical injury (including death) are the most common patient outcomes. The nature of patient harm varied by type of misconduct, with illegal unethical prescribing commonly leading to drug dependency and sexual misconduct leading to psychiatric injury. What are the implications for practitioners? Medical colleges should tailor trainee programs to address the common types of misconduct within their specialities. Standardisation of misconduct reporting, and report access, across jurisdictions would facilitate ongoing surveillance and intervention evaluation.

Discordance between patient-reported and actual emergency department pain management.

OBJECTIVE: To determine patient knowledge of the nature of their pain management in the ED. METHODS: This was a planned sub-study of data collected during a randomised, controlled trial of the nature of the informed consent process in a single ED. Patients aged ≥18 years, with a triage pain score of ≥4, were enrolled. Forty-eight hours post-ED discharge, patients were asked if they had declined analgesia or if a range of pain management options had been administered. The primary outcome was discordance between the patient report and the ED report (proportion of cases where these reports differed). RESULTS: Outcome data were collected on 655 patients. There was significant discordance for all variables examined (P < 0.001). Discordance for patients declining analgesia was lowest at 8.9% (95% confidence interval [CI] 6.8-11.4). Discordance for administration of pain management 'other' than analgesia was highest at 32.6% (95% CI 29.0-36.4). Discordance for the administration of oral analgesia or 'any' analgesia was 17.1% (95% CI 14.3-20.3) and 14.4% (95% CI 11.8-17.3), respectively. For both of these outcomes, patients with chest pain and lower triage pain scores were more likely to report discordant responses. With the exception of 'other' pain management, smaller proportions of patients incorrectly reported not receiving management than incorrectly reporting that they did receive it. CONCLUSION: Patients are often unaware of the nature of their pain management. They are most often unaware of management other than analgesia. Patients with chest pain and lower triage pain scores had the least knowledge of their pain management.

Outcomes of laceration suture repair in the emergency department.

OBJECTIVE: To assess patient satisfaction with laceration management, post-ED care, cosmesis and complication rates. METHODS: We undertook a prospective observational study of adult patients with lacerations sutured in two EDs over a 4-month period. ED data included participant demographics, laceration characteristics and management. A telephone survey was undertaken approximately 14 days post-ED discharge. Patient satisfaction with post-ED pain management, advice on wound care and follow up, overall management and wound cosmesis were evaluated using a six-item satisfaction scale (very dissatisfied to very satisfied). Details of wound infection, dehiscence and suture failure were recorded. RESULTS: Eighty-nine patients participated. The number (% [95% confidence interval]) of patients very satisfied with their laceration management were: post-ED pain management 55 (62.5% [51.5-72.4]), wound care advice 51 (57.3% [46.4-67.6]), follow-up advice 39 (43.8% [33.5-54.7]), overall management 61 (68.5% [57.7-77.7]) and cosmetic appearance 46 (51.7% [40.9-62.3]). Infection, dehiscence and suture failure occurred in 5 (5.6%), 8 (9.0%) and 8 (9.0%) cases, respectively. These complications were not associated with being very satisfied overall (P = 0.96). Patients very satisfied with post-ED pain management, wound care advice, follow-up advice or wound cosmesis were much more likely to be very satisfied overall (P < 0.001). CONCLUSIONS: Most patients are very satisfied with their laceration management. However, there is scope for improvement, especially for follow-up and wound care advice. Complications are infrequent and not associated with overall satisfaction.

Corrigendum: Spatial Organization and Recruitment of Non-Specific T Cells May Limit T Cell-Macrophage Interactions Within Mycobacterium tuberculosis Granulomas.

[This corrects the article DOI: 10.3389/fimmu.2020.613638.].

Approaches for integrating heterogeneous RNA-seq data reveal cross-talk between microbes and genes in asthmatic patients.

Sputum induction is a non-invasive method to evaluate the airway environment, particularly for asthma. RNA sequencing (RNA-seq) of sputum samples can be challenging to interpret due to the complex and heterogeneous mixtures of human cells and exogenous (microbial) material. In this study, we develop a pipeline that integrates dimensionality reduction and statistical modeling to grapple with the heterogeneity. LDA(Latent Dirichlet allocation)-link connects microbes to genes using reduced-dimensionality LDA topics. We validate our method with single-cell RNA-seq and microscopy and then apply it to the sputum of asthmatic patients to find known and novel relationships between microbes and genes.

Synchronicity of thermogenic activity, alternative pathway respiratory flux, AOX protein content, and carbohydrates in receptacle tissues of sacred lotus during floral development.

The relationships between heat production, alternative oxidase (AOX) pathway flux, AOX protein, and carbohydrates during floral development in Nelumbo nucifera (Gaertn.) were investigated. Three distinct physiological phases were identified: pre-thermogenic, thermogenic, and post-thermogenic. The shift to thermogenic activity was associated with a rapid, 10-fold increase in AOX protein. Similarly, a rapid decrease in AOX protein occurred post-thermogenesis. This synchronicity between AOX protein and thermogenic activity contrasts with other thermogenic plants where AOX protein increases some days prior to heating. AOX protein in thermogenic receptacles was significantly higher than in post-thermogenic and leaf tissues. Stable oxygen isotope measurements confirmed that the increased respiratory flux supporting thermogenesis was largely via the AOX, with little or no contribution from the cytochrome oxidase pathway. During the thermogenic phase, no significant relationship was found between AOX protein content and either heating or AOX flux, suggesting that regulation is likely to be post-translational. Further, no evidence of substrate limitation was found; starch accumulated during the early stages of floral development, peaking in thermogenic receptacles, before declining by 89% in post-thermogenic receptacles. Whilst coarse regulation of AOX flux occurs via protein synthesis, the ability to thermoregulate probably involves precise regulation of AOX protein, most probably by effectors such as alpha-keto acids.