Protein activity state, rather than protein or mRNA abundance, is a biologically regulated and relevant input to many processes in signaling, differentiation, development, and diseases such as cancer. While there are numerous methods to detect and quantify mRNA and protein abundance in biological samples, there are no general approaches to detect and quantify endogenous protein activity with single-cell resolution. Here, we report the development of a chemoproteomic platform, single-cell activity-dependent proximity ligation, which uses automated, microfluidics-based single-cell capture and nanoliter volume manipulations to convert the interactions of family-wide chemical activity probes with native protein targets into multiplexed, amplifiable oligonucleotide barcodes. We demonstrate accurate, reproducible, and multiplexed quantitation of a six-enzyme (Ag-6) panel with known ties to cancer cell aggressiveness directly in single cells. We further identified increased Ag-6 enzyme activity across breast cancer cell lines of increasing metastatic potential, as well as in primary patient-derived tumor cells and organoids from patients with breast cancer.

Abstract Objective Airborne spread of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) remains a significant risk for healthcare workers. Understanding transmission of SARS‐CoV‐2 in the hospital could help minimize nosocomial infection. The objective of this pilot study was to measure aerosolization of SARS‐CoV‐2 in the hospital rooms of COVID‐19 patients. Methods Two air samplers (Inspirotec) were placed 1 and 4 m away from adults with SARS‐CoV‐2 infection hospitalized at an urban, academic tertiary care center from June to October 2020. Airborne SARS‐CoV‐2 concentration was measured by quantitative reverse transcription polymerase chain reaction and analyzed by clinical parameters and patient demographics. Results Thirteen patients with COVID‐19 (eight females [61.5%], median age: 57 years old, range 25–82) presented with shortness of breath (100%), cough (38.5%) and fever (15.4%). Respiratory therapy during air sampling varied: mechanical ventilation via endotracheal tube (n = 3), high flow nasal cannula (n = 4), nasal cannula (n = 4), respiratory helmet (n = 1), and room air (n = 1). SARS‐CoV‐2 RNA was identified in rooms of three out of three intubated patients compared with one out of 10 of the non‐intubated patients (p = .014). Airborne SARS‐CoV‐2 tended to decrease with distance (1 vs. 4 m) in rooms of intubated patients. Conclusions Hospital rooms of intubated patients had higher levels of aerosolized SARS‐CoV‐2, consistent with increased aerosolization of virus in patients with severe disease or treatment with positive pressure ventilation through an endotracheal tube. While preliminary, these data have safety implications for health care workers and design of protective measures in the hospital. Level of Evidence 2

Digital proximity ligation assay (PLA) detects single protein molecules with a pair of oligonucleotide-conjugated antibodies and digital PCR (dPCR) readout, which allows absolute quantitation of proteins in single cells with high sensitivity. The pipeline also allows simultaneous measurement of protein and mRNA from the same single cell. The sensitivity of the assay has been further improved with implementation of the assay on a microfluidic system, which enables quantitation of rare protein species, with expression level as low as ~3000 protein molecules per cell.

RATIONALE: Survivors of critical illness are at risk for long-term physical disability and poor quality of life. Changes in health care delivery, such as visitor restrictions and limited availability of therapy services coupled with economic hardship due to the COVID-19 pandemic may exacerbate these long-term consequences. Methods: Patients with respiratory failure requiring advanced respiratory support (endotracheal tube, noninvasive ventilation, or high-flow nasal cannula) and/or shock admitted to the intensive care unit (ICU) with COVID-19 were eligible for enrollment in this prospective observational study. We assessed quality of life (using short form-36 [SF-36]), work status, and independence in activities of daily living (ADLs) in survivors at hospital discharge and 6 months. Quality of life scores were compared to US population norms. An SF-36 score ≥50 is the population norm for both physical and mental scores;scores <45 define significant physical or mental health impairment. Patients underwent assessment for ICU-acquired weakness (ICU-AW) and functional impairment (using Functional Status Score for the ICU (FSS-ICU)) by physical and occupational therapists upon hospital discharge. An FSS-ICU score ≥28 is associated with discharge to home. Analysis of changes over time for the quality of life assessment were performed using paired t-test and tests of proportions. Results: 100 patients were enrolled from April to November 2020. On hospital discharge, 39% of patients had ICU-AW. The median FSS-ICU score on discharge was 24 [20-29], consistent with functional impairment. Fifty-six patients completed the 6-month evaluation with 74.7% follow-up (56/75 alive patients). Mental health impairment was reported by 33% of patients on hospital discharge and declined to 13% at 6 months (p=0.007). Physical health impairment was reported by 70% at discharge and persisted in 46% of patients at 6 months (p=0.007). Patients were independent in ADLs at both hospital admission and 6 months post discharge (90 vs 91%). Only 56% of patients who had been working prior to hospitalization had returned to work at 6 months. Conclusions: Impairments in quality of life and neuromuscular weakness were present at hospital discharge in a significant number of ICU survivors of COVID-19. Similar to the trajectory described in survivors of acute respiratory distress syndrome, despite relatively normal mental health quality of life, persistent physical health impairment was noted in almost half of the survivors at 6 months. Further investigation of the long-term effects of COVID-19 related critical illness and its distinction from post-ICU syndromes from non-COVID-19 critical illness is warranted.

RationaleThe novel coronavirus, COVID-19, can cause critical illness in up to 5% of infected patients. Although the sequelae of surviving critical illness are known, limited data exist regarding the psychological and cognitive outcomes specifically in survivors of COVID-19. Given the social isolation, scarcity of multidisciplinary staff, and socio-economic impact of the pandemic, understanding the cognitive and psychological impact of surviving critical illness due to COVID-19 is of utmost importance. MethodsA prospective observational cohort study at an academic medical center enrolled critically ill patients with confirmed COVID-19 infection. Upon hospital discharge, cognitive and psychological sequelae were measured using the following validated assessments: Montreal Cognitive Assessment (MoCA), Impact of Event Score (IES), and the Hospital Anxiety and Depression scale (HADS). Cognitive impairment was defined as a MoCA score 32. Significant symptoms of anxiety and depression were defined as a HADS subscale score of ≥8. ResultsFrom April 10, 2020 through November 17, 2020, 100 adult critically ill patients were enrolled, of which 27 were invasively mechanically ventilated. Eighty-nine patients underwent cognitive and psychological evaluation upon hospital discharge. Of the 11 patients who did not complete the evaluation, 2 died prior to discharge, 4 had significant cognitive impairment, 2 were discharged prior to survey completion, and 3 refused. Cognitive impairment was present in 93% of patients (n=83) with a median MoCA score of 17 [13-22]. Patients with cognitive impairment tended to be older (62 years old [53-71] vs 50 [41-58];p=0.17). Ten patients (11%) had probable PTSD with a median IES score of 4 [0-17] in the overall cohort. Seventeen patients (19%) reported significant depressive symptoms and twenty patients (22%) noted significant symptoms of anxiety. ConclusionPrior coronavirus outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) were associated with anxiety, depression, impaired memory and confusion occurring in approximately one-third of patients at hospital discharge. In contrast, cognitive impairment in ICU survivors of COVID-19 infection was nearly universal on hospital discharge. This impairment is not completely explained by coexistent psychiatric symptoms of anxiety or depression, which were present in only one-fifth of the ICU survivors.

Rationale: Intensive care unit (ICU) survivorship has well documented physical disability1,2 psychological sequelae, and cognitive dysfunction3,4 all combined under the umbrella of Post-Intensive Care Syndrome (PICS). The COVID-19 pandemic is concerning for overwhelming health care resources in the short-term, but the longterm consequences of this large cohort of patients surviving critical illness remains ill-defined. It also remains unclear if the unique context of health care delivery such as lack of visitors and cohorting patients during the pandemic exacerbates the psychological and cognitive impairments of PICS. Methods: All patients admitted to the ICU with COVID-19 were eligible for enrollment in this prospective observational study. We performed a global assessment of cognitive function (using the Montreal Cognitive Assessment tool (T-MoCA)), posttraumatic stress disorder (using the Impact event scale (IES-Revised)), and depression/anxiety (using Hospital Anxiety and Depression scale (HADS)) in ICU survivors at 6 months after hospital discharge. Interviews were conducted via telephone or in-person when possible. Results: From April 10, 2020 thru November 17th, 2020 one hundred patients were enrolled upon hospital discharge. Eighty-two patients reached the 6-month milestone after hospital discharge and 22 (26.8%) of these patients required invasive mechanical ventilation. Of this eligible cohort, seven patients died, two were cognitively unable to complete the evaluations, four refused to participate, and thirteen were lost to follow-up. Fifty-six patients completed the evaluation with 74.7% follow-up (56/75 alive patients). Symptoms of probable post-traumatic stress disorder were reported in 2 patients (3.6%). Depression and Anxiety was reported in 5 patients (8.9%) respectively. Cognitive impairment was present in 47.3% of patients with a median T-MoCA score of 18 [15.5-19]. There was no difference in T-MoCA scores based on whether patients required invasive mechanical ventilation (IMV) (IMV 17 [15-20.5] vs not intubated 18 [16-19];p=0.89). Conclusions: Our preliminary long-term follow-up data suggest that symptoms of post-traumatic stress after COVID-19 infection are rare. In addition, less than 10% of patients reported anxiety and depression six months after hospital discharge. Interestingly, cognitive impairment as measured by T-MOCA was present in almost half of the patients. Further follow-up on the long term effects of COVID-19 related critical illness is warranted as we adapt during this pandemic.

Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.

Cells receive a wide range of dynamic signaling inputs during immune regulation, but how gene regulatory networks measure such dynamic inputs is not well understood. Here, we used microfluidic single-cell analysis and mathematical modeling to study how the NF-κB pathway responds to immune inputs that vary over time such as increasing, decreasing, or fluctuating cytokine signals. We found that NF-κB activity responded to the absolute difference in cytokine concentration and not to the concentration itself. Our analyses revealed that negative feedback by the regulatory proteins A20 and IκBα enabled differential responses to changes in cytokine dose by providing a short-term memory of previous cytokine concentrations and by continuously resetting kinase cycling and receptor abundance. Investigation of NF-κB target gene expression showed that cells exhibited distinct transcriptional responses under different dynamic cytokine profiles. Our results demonstrate how cells use simple network motifs and transcription factor dynamics to efficiently extract information from complex signaling environments.

OBJECTIVETo investigate the down-regulation effect of let-7b-5p on the expression of FTO in acute myeloid leukemia cell line THP-1 and inhibitory effect on THP-1 proliferation via m6A/MYC signaling pathway.METHODSThe acute myeloid leukemia cell line THP-1 and the normal human peripheral blood mononuclear cells (PBMNC) were selected as subjects. The expression of let-7b-5p and FTO mRNA in those cells was detected by qPCR, further the expression of FTO protein in those cells was detected by Western blot. And, the luciferase reporter gene assay was used to verify the targeting effect of let-7b-5p on FTO. Finally, THP-1 cells were transfected respectively with let-7b-5p mimic, and PBMNC with let-7b-5p inhibitor, there after the C-MYC mRNA m6A enrichment level in transfected cells was analyzed by dot blot, the expression levels of let-7b-5p, FTO and c-MYC were assayed by RT-PCR, the expressions of FTO and c-MYC protein were verified by Western blot, and the proliferation level of cells after transfection was detected by MTT assay.RESULTSCompared with PBMNC, the expression of let-7b-5p in THP-1 significantly decreased, while the expression of FTO was significantly increased (P<0.05). After transfection with let-7b-5p mimic combined with FTO 3'-UTR, the luciferase activity of transfected THP-1 cells significantly decreased, but the luciferase activity significantly increased after transfection with mutant 3'-UTR, which was significantly different from the negative control group(blank vector) (P<0.05). Let-7b-5p inhibitor down-regulated c-MYC mRNA m6A enrichment, and then up-regulated the expression of FTO in transfected PBMNC cells, the effects of which were significant (P<0.05). However, let-7b-5p mimic up-regulated c-MYC mRNA m6A enrichment level and down-regulated the expression of FTO in the transfected THP-1 cells, and the cell proliferation rate was significantly lower than that in the negative control group (blank vector) (P<0.05).CONCLUSIONHuman acute myeloid leukemia cell line THP-1 low expresses the let-7b-5p, which regulates c-MYC expression through let-7b-5p-/FTO-/m6A axis and promotes the proliferation of leukemia cell line THP-1.

Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases. Here, we demonstrate sensitive and rapid quantification of bacterial load and cytokines from human biological samples to generate actionable hypotheses. Our digital assay measures IL-6 and TNF-α proteins, gram-negative (GN) and gram-positive (GP) bacterial DNA, and the antibiotic-resistance gene blaTEM with femtomolar sensitivity. We use our method to characterize bronchoalveolar lavage fluid from patients with asthma and find elevated GN bacteria and IL-6 levels compared to healthy subjects. We then analyze plasma from patients with septic shock and find that increasing levels of IL-6 and blaTEM are associated with mortality, while decreasing IL-6 levels are associated with recovery. Surprisingly, lower GN bacteria levels are associated with higher probability of death. Applying decision-tree analysis to our measurements, we are able to predict mortality and rate of recovery from septic shock with over 90% accuracy.

Cells receive a wide range of dynamic signaling inputs during immune regulation, but how gene regulatory networks measure and interpret such dynamic inputs is not understood. Here, we used microfluidic live-cell analysis and mathematical modeling to study how NF-κB pathway in single-cells responds to time-varying immune inputs such as increasing, decreasing or fluctuating cytokine signals. Surprisingly, we found that NF-κB acts as a differentiator, responding strictly to the absolute difference in cytokine concentration, and not to the concentration itself. Our analyses revealed that negative feedbacks by the regulatory proteins A20 and IκBα enable dose differentiation by providing short-term memory of prior cytokine level and continuously resetting kinase cycling and receptor levels. Investigation of NF-κB target gene expression showed that cells create unique transcriptional responses under different dynamic cytokine profiles. Our results demonstrate how cells use simple network motifs and transcription factor dynamics to efficiently extract information from complex signaling environments.

Simultaneous measurement of proteins and mRNA in single cells enables quantitative understanding and modeling of cellular functions. Here, we present an automated micro-fluidic system for multi-parameter and ultra-sensitive protein/mRNA measurements in single cells. Our technology improves the sensitivity of digital proximity ligation assay by up to 55-fold, with a detection limit of 2277 proteins per cell and with detection efficiency of as few as 29 protein molecules. Our measurements using this system reveal higher mRNA/protein correlation in single mammalian cells than previous estimates. Furthermore, time-lapse imaging of herpes simplex virus 1 infected epithelial cells enabled by our device shows that expression of ICP4 -a major transcription factor regulating hundreds of viral genes- is only partially correlated with viral protein counts, suggesting that many cells go through abortive infection. These results highlight the importance of high-sensitivity protein/mRNA quantification for understanding fundamental molecular mechanisms in individual cells.