Background: COVID-19 affects people with cystic fibrosis (PwCF) and CF carriers very differently.PwCF with two defective CF transmembrane conductance regulator (CFTR) genes and low CFTR function generally appear to suffer only mild COVID-19 disease, but recently published data from the University of Siena and the GEN-COVID study show that CF carriers, with one normal and one defective CFTR gene, and only approximately 50% CFTR function, have significantly more severe abnormal inflammatory responses and significantly earlier death by day 14 than did control COVID-19 patients with two normal CFTR genes.These comparisons between normal and CF-carrier patients suggested that low CFTR function may contribute to COVID-19 severity.How then could mild disease in PwCF be explained?We speculate that some PwCF might be less severely affected by COVID-19 disease because of compensating bystander genes that they inherited to survive with low CFTR function.We therefore hypothesized that inflammation in the normal COVID-19 airway might be due to inhibition of CFTR signaling by SARS-CoV-2.Methods: To test this hypothesis, we differentiated hTERT-transformed BCi-NS1.1 basal stem cells into epithelia on an air-liquid-interface (ALI) and exposed the differentiated epithelia to the SARS-CoV-2 Spike protein (S1S2) and measured cyclic adenosine monophosphate (cAMP)-activated CFTR chloride channels and CFTR protein and CFTR survival in endosomal recycling.We also measured nuclear factor kappa B (NHkB)-activated cytokine expression and proteolytic epithelial sodium channel (ENaC) activation.Results: Exposure of normal differentiated airway epithelia to SARS-CoV-2 Spike protein resulted in loss of normal CFTR protein expression and simultaneous loss of cAMP-activated CFTR chloride channel activity (Figure 1).Spike protein from the highly infectious b-1.315 strain,

Background/Aim: Circulating cell-free DNA (cfDNA) isolated from serum by noninvasive procedures can serve as a potential biomarker for the early detection of many cancers. The aim of this study was to implement a simple, yet effective quantitative method for measuring the cfDNA in serum and to investigate the relationship between cfDNA and the occurrence of recurrence in breast cancer (BrCa) patients. Patients and Methods: A total of 240 cases were selected, which comprised different subtypes of BrCa patients and control individuals. We selected 20 serum samples from patients which showed recurrence after 4-7 years of disease-free survival. SYBR green was used as a reporter molecule to estimate the amount of cfDNA in these serum samples. Results: A global Wilcoxon analysis was performed to compare the cfDNA abundance between non-recurrent and recurrent patients. The amount of cfDNA was higher in recurrent patients (recurrent vs. non-recurrent ratio=1.3; p=0.03; AUC=0.76) compared to non-recurrent patients. The data between normal/healthy controls and non-recurrent patients indicated no significant differences (n=20 in each group, healthy to non-recurrent ratio=1.03; p=0.20; AUC=0.61). Conclusion: We implemented a straightforward one-step technique to measure the amount of cfDNA in serum, which can translate into a clinical diagnostic tool in the near future. The high levels of cfDNA in the serum of recurrent BrCa patients compared to non-recurrent BrCa patients indicates a possible uncovered role for circulating genetic information, which either contributes to the cancer recurrence phenomenon or at the very least, serves as an identifier for the potential of recurrence.

Abstract Background: Quantitative estimation of circulating cell-free DNA (cfDNA) isolated from serum by noninvasive procedures can serve as a potential biomarker for the early detection of many cancers. However, a simple, straightforward technique is unavailable to estimate the cfDNA in clinical labs. Moreover, the prognostic value of cfDNA in patients with breast cancer (BrCa) is currently under debate. The aim of this study was to develop a simple yet effective quantitative method for measuring the cfDNA in serum and to eventually investigate the relationship between cfDNA and the occurrence of recurrence in BrCa patients. Methods: A total of 240 patient cases (n=240) were selected and are comprised of different subtypes of breast cancer patients and control individuals. We selected 21 serum samples from patients which showed recurrence after 4-7 years of disease-free survival. For the compare studies, each of the recurrent and non-recurrent serum samples was incubated with the SYBR Green I (2 μM). A standard graph was also made with known DNA concentration to calculate the amount of cfDNA in these recurrent and non-recurrent serum samples. Additionally, a comparative study was also performed with the serum of patients with non-recurrent BrCa versus healthy patients. Results: We develop a simple fluorescent based measuring technique which can easily estimate the cfDNA in one step. SYBR Green binds to DNA, and as a result, the fluorescence of SYBR Green increases substantially. Global Wilcoxon analyses were performed to compare the cfDNA amount between non-recurrent and recurrent patients. There is a significant difference in fluorescent intensities between recurrent patients' samples versus non-recurrent patients which are directly proportional to the cfDNA levels. The amount of cfDNA is higher in recurrent patient (ratio is 1.3 up; p= 0.03; AUC=0.76) compared to similar non-recurrent patients. While we compared the fluorescence data between normal/healthy patients versus non-recurrent is turned out as non-significant (healthy to non-recurrent ratio = 1.03; p= 0.20, AUC=0.61). Conclusion: In this current study, we developed a straightforward one-step technique to measure the amount of cfDNA in serum, which can easily translate into a clinical diagnostic tool. To the best of our knowledge, this is the first report which demonstrates serum cfDNA as an early detection marker for recurrent breast cancer patients. The relatively high level of cfDNA in the serum of recurrent breast cancer patients compared to non-recurrent breast cancer patients indicates an uncovered circulating genetic information which triggers the cancer recurrence pathway to relapse cancer in the near future. Citation Format: Bera A, Eidelman O, Russ E, Landa A, Karaian J, Eklund M, Hu H, Pollard HB, Shriver CD, Srivastava M. Circulating cell-free DNA in serum as a marker for the early detection of tumor recurrence in breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-26.

Post-traumatic stress disorder (PTSD) is psychiatric disease, which can occur following exposure to traumatic events. PTSD may be acute or chronic, and can have a waxing and waning course of symptoms. It has been hypothesized that proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) or plasma might be mediators of the psychophysiological mechanisms relating a history of trauma exposure to changes in behavior and mental health disorders, and medical morbidity. Here we test the cytokine/chemokine hypothesis for PTSD by examining levels of 17 classical cytokines and chemokines in CSF, sampled at 0900 hours, and in plasma sampled hourly for 24 h. The PTSD and healthy control patients are from the NIMH Chronic PTSD and healthy control cohort, initially described by Bonne et al. (2011), in which the PTSD patients have relatively low comorbidity for major depressive disorder (MDD), drug or alcohol use. We find that in plasma, but not CSF, the bivariate MCP4 (CCL13)/ MCP1(CCL2) ratio is ca. twofold elevated in PTSD patients compared with healthy controls. The MCP-4/MCP-1 ratio is invariant over circadian time, and is independent of gender, body mass index or the age at which the trauma was suffered. By contrast, MIP-1β is a candidate biomarker for PTSD only in females, whereas TARC is a candidate biomarker for PTSD only in males. It remains to be discovered whether these disease-specific differences in circadian expression for these specific immune signaling molecules are biomarkers, surrogates, or drivers for PTSD, or whether any of these analytes could contribute to therapy.

Devising molecular strategies that overcome ΔF508 folding and trafficking defects comprise a central objective of CF therapeutic development. An understanding of disease mechanisms can be improved by new method(s) and/or compounds that redirect ΔF508-CFTR to the plasma membrane. RNA interference (siRNA) mediated knock-down of gene expression has proven to be a powerful tool for investigating protein function(s) and advancing drug discovery. BioFocus (a Galapagos company) has developed adenoviral vectors expressing small hairpin RNAs (shRNAs) for genomewide functional screening that allow robust transduction and durable gene repression. In this project, sixty-eight adenoviral-shRNA constructs (targeting 28 high priority genes) were provided by BioFocus to five collaborating laboratories in the United States and Canada. Putative gene targets were identified by a consortium-based review of the existing CF literature. Each research group established independent protocols to investigate effect(s) of gene knock-down (via Ad-shRNA) on ΔF508-CFTR maturation. Protocols included 1) A study of CF bronchial epithelial cells expressing the halide sensitive variant of eYFP and measurement of ΔF508-CFTR activity at the cell surface. 2) Short circuit current in primary human bronchial epithelial cells (ΔF508/ΔF508), 3) Appearance of rescued ΔF508 CFTR at the plasma membrane in CF bronchial epithelial cells monitored biochemically, 4) Effects on CFTR-dependent release of inflammatory markers (chemokines and cytokines) from IB3 cells (ΔF508/W1282X), and 5) Short circuit current and Western blotting in CFBE cells transduced with lentivirus encoding ΔF508-CFTR. Preliminary results indicate significant activity of BioFocus shRNAs in several of the independent protocols and laboratories, particularly against gene products such as AHSA1 and 2 (activators of HSP 90) and HDAC7A (a member of the histone deacetylase family). A profile of shRNAs found to improve ΔF508 processing, including spectrum of activity data and gene-network annotation of the relevant pathways, will be presented. Identifying the most robust molecular targets for ΔF508 CFTR correction (from among hundreds of candidates in the CFTR “interactome”) has been limited by the complexity of the relevant cellular pathways. The studies described here provide a means by which chaperones and other contributors to CFTR misprocessing can be evaluated, prioritized, and better understood in the future for development of new therapeutic approaches and delineating genetic modifiers that contribute to variation in severity of CF onset and disease progression. The project represents a collaboration among members of the CFTR Folding Consortium. Supported by the CFF and NIH.

Antibody microarrays are a new proteomic technology, which we have developed as a platform for identifying a cystic fibrosis (CF)-specific serum proteomic signature. Serum samples from CF patients have been pooled and compared with equivalent pools of control sera in order to identify patterns of protein expression unique to CF. We find that the set of significantly differentially expressed proteins is enriched in protein mediators of inflammation from the NFκB signaling pathway, and in proteins that may be selectively expressed in CF-affected tissues such as lung and intestine. In several instances, we validate the data from the antibody microarrays by quantitative analysis with Reverse Capture Protein Microarrays. We conclude that antibody microarray technology is sensitive, quantitative, and robust, and can be useful as a proteomic platform to discriminate between sera from CF and control patients.