Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2–infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.

We evaluated the quantification strategies label-free (LF), stable isotope labeling by amino acids in cell culture (SILAC), and tandem mass tags (TMT) and their performance in quantification of proteins and phosphosites (p-sites) to identify the most powerful approach for monitoring cellular signaling. We analyzed the epidermal growth factor receptor (EGFR) signaling network, which plays an essential role in colorectal cancer, and studied its dynamics within 24 h upon treatment with the EGFR-blocking antibody cetuximab, representing the first cellular adaption toward therapy. LF achieved superior coverage but was outperformed by label-based approaches regarding technical variability, especially for quantification of p-sites. TMT showed the lowest coverage and most missing values. We found that its performance considerably decreases when experimental replicates are distributed over several TMT plexes. SILAC showed the highest precision and outstanding performance for quantification of p-sites, rendering it the method of choice for analyzing cellular signaling in cell culture models. On the protein level, we observed only little regulation upon cetuximab treatment, whereas a great fraction of p-sites was significantly regulated. These dynamics represented an initial downregulation of the MAPK pathway, which was partially rescued as early as 24 h after treatment. We identified upregulation and signaling via ERBB3 as well as calcium and cAMP signaling as possible mechanisms bypassing the blockage of EGFR.

Histopathological differentiation between severe urocystitis with reactive urothelial atypia and carcinoma in situ (CIS) can be difficult, particularly after a treatment that deliberately induces an inflammatory reaction, such as intravesical instillation of Bacillus Calmette-Guèrin. However, precise grading in bladder cancer is critical for therapeutic decision making and thus requires reliable immunohistochemical biomarkers. Herein, an exemplary potential biomarker in bladder cancer was identified by the novel approach of Fourier transform infrared imaging for label-free tissue annotation of tissue thin sections. Identified regions of interest are collected by laser microdissection to provide homogeneous samples for liquid chromatographyetandem mass spectrometryebased proteomic analysis. This approach afforded label-free spatial classification with a high accuracy and without interobserver variability, along with the molecular resolution of the proteomic analysis. Cystitis and invasive highgrade urothelial carcinoma samples were analyzed. Three candidate biomarkers were identified and verified by immunohistochemistry in a small cohort, including low-grade urothelial carcinoma samples. The best-performing candidate AHNAK2 was further evaluated in a much larger independent verification cohort that also included CIS samples. Reactive urothelial atypia and CIS were distinguishable on the basis of the expression of this newly identified and verified immunohistochemical biomarker, with a sensitivity of 97% and a specificity of 69%. AHNAK2 can differentiate between reactive urothelial atypia in the setting of an acute or chronic cystitis and nonmuscle invasive-type CIS. (Am J Pathol 2019, 189: 619e631; https://doi.org/10. 1016/j.ajpath.2018.11.018) Bladder cancer is the second most common urogenital malignancy, with approximately 430,000 new cases diagnosed worldwide in 2012. 1,2 Approximately 70% to 75% of patients are newly diagnosed with nonmuscle invasive, mostly low-grade bladder cancer, whereas 25% to 30% of de novo diagnoses are high-grade bladder cancer at the stage of infiltration of the muscularis, with metastases observed in 10% of cases. 3e5 A significant percentage (50% to 70%) of patients diagnosed with nonmuscle invasive cancer will have multiple recurrences, and 10% to 15% will even progress to invasion. 3,6,7 A special case of bladder cancer is carcinoma in situ (CIS), which is a flat, noninvasive tumor with a rate of progression to an invasive state of approximately 54%. 8

Cerebrospinal fluid is investigated in biomarker studies for various neurological disorders of the central nervous system due to its proximity to the brain. Currently, only a limited number of biomarkers have been validated in independent studies. The high variability in the protein composition and protein abundance of cerebrospinal fluid between as well as within individuals might be an important reason for this phenomenon. To evaluate this possibility, we investigated the inter- and intraindividual variability in the cerebrospinal fluid proteome globally, with a specific focus on disease biomarkers described in the literature. Cerebrospinal fluid from a longitudinal study group including 12 healthy control subjects was analyzed by label-free quantification (LFQ) via LC-MS/MS. Data were quantified via MaxQuant. Then, the intra- and interindividual variability and the reference change value were calculated for every protein. We identified and quantified 791 proteins, and 216 of these proteins were abundant in all samples and were selected for further analysis. For these proteins, we found an interindividual coefficient of variation of up to 101.5% and an intraindividual coefficient of variation of up to 29.3%. Remarkably, these values were comparably high for both proteins that were published as disease biomarkers and other proteins. Our results support the hypothesis that natural variability greatly impacts cerebrospinal fluid protein biomarkers because high variability can lead to unreliable results. Thus, we suggest controlling the variability of each protein to distinguish between good and bad biomarker candidates, e.g., by utilizing reference change values to improve the process of evaluating potential biomarkers in future studies.

Objective To investigate the effect of therapeutic suggestions played to patients through earphones during surgery on postoperative pain and opioid use. Design Blinded randomised controlled study. Setting Five tertiary care hospitals in Germany. Participants 385 of 400 patients consecutively recruited from January to December 2018 who were to undergo surgery for 1-3 hours under general anaesthesia. In the per protocol analysis 191 patients were included in the intervention group and 194 patients in the control group. Intervention The intervention comprised an audiotape of background music and positive suggestions based on hypnotherapeutic principles, which was played repeatedly for 20 minutes followed by 10 minutes of silence to patients through earphones during general anaesthesia. Patients in the control group were assigned to a blank tape. Main outcome measures The main outcome was dose of opioid administered by patient controlled analgesia or nurse controlled analgesia within the first postoperative 24 hours, based on regular evaluation of pain intensity on a numerical rating scale (range 0-10, with higher scores representing more severe pain). Results Compared with the control group, the intervention group required a significantly (P=0.002) lower opioid dose within 24 hours after surgery, with a median of 4.0 mg (interquartile range 0-8) morphine equivalents versus 5.3 (2-12), and an effect size (Cohen’s d) of 0.36 (95% confidence interval 0.16 to 0.56). The number of patients who needed opioids postoperatively was significantly (P=0.001) reduced in the intervention group: 121 of 191 (63%, 95% confidence interval 45% to 70%) patients in the intervention group versus 155 of 194 (80%, 74% to 85%) in the control group. The number needed to treat to avoid postoperative opioids was 6. Pain scores were consistently and significantly lower in the intervention group within 24 hours after surgery, with an average reduction of 25%. No adverse events were reported. Conclusions Therapeutic suggestions played through earphones during general anaesthesia could provide a safe, feasible, inexpensive, and non-drug technique to reduce postoperative pain and opioid use, with the potential for more general use. Based on the finding of intraoperative perception by a considerable number of patients, surgeons and anaesthetists should be careful about background noise and conversations during surgery. Trial registration German Clinical Trial Register DRKS00013800.