Tick-borne encephalitis virus (TBEV) infection can lead to inflammation of the central nervous system. The disease can be effectively prevented by whole inactivated virus vaccines. Here, we investigated the innate immune profile induced in vitro by the antigen component of the vaccines, inactivated TBEV (I-TBEV), to gain insights into the mechanism of action of the TBE vaccine as compared to the live virus. To this end, we exposed human peripheral blood mononuclear cells (PBMCs) to inactivated and live TBEV and assessed cellular responses by RNA sequencing. Both inactivated and live TBEV significantly induced an interferon-dominated gene signature and an increased RIG-I-like receptor (RLR) expression. Using pathway-specific inhibitors, we assessed the involvement of pattern recognition receptors in the sensing of inactivated or live TBEV. Only RLR pathway inhibition significantly suppressed the downstream cascade induced by I-TBEV, while responses to the replicating virus were impacted by the inhibition of RIG-I-like, as well as Toll-like, receptors. Our results show that inactivated and live TBEV predominantly engaged an interferon response in our in vitro PBMC platform, and indicate RLRs as the main pattern recognition receptors involved in I-TBEV sensing. Tick-borne encephalitis is an inflammation of the central nervous system caused by the tick-borne encephalitis virus (TBEV), a flavivirus endemic in parts of Europe and Asia . The vir+ T cells ..17].c and e) or downregulated (cluster b) in both treatment groups, while 2 include DEGs that are downregulated in I-TBEV-treated cells and upregulated in live TBEV-treated cells (cluster a), or vice versa (cluster d). After discarding unexpressed and non-differentially expressed genes, 337 DEGs were obtained from the analysis of the control group, I-TBEV- and TBEV-treated cells. The PCA for differentially expressed genes shows distinctive clustering of the three treatment groups A. StimulTo validate these findings, and confirm that the differential profile induced by the live virus was not resulting from the extended incubation time, the expression of selected genes within each cluster was validated by RT-qPCR in PBMCs  treated with I-TBEV or live TBEV for 24 h. The changes in gene expression, averaged for the 3 donors, are shown and compared to the results from the RNA sequencing D. The exAgain, we evaluated the cytokine production in PBMCs from 4 donors in response to I-TBEV and live TBEV stimulation . On avera, which included genes downregulated following I-TBEV treatment and upregulated upon incubation with the live virus, comprises functions related to lipid metabolism, a process already identified in Analysis 1. Cluster b, including DEGs downregulated in both sample groups, shows enrichment of terms involved with RNA metabolism. Clusters c and e, both including genes upregulated in I-TBEV- and live virus-treated cells, show an over-representation of pathways related to the antiviral and IFN response and RLR signaling. Cluster d, comprising DEGs upregulated upon treatment with I-TBEV and downregulated by the live virus incubation, includes functions involved in the immune response.Functional enrichment analysis of the clusters was performed as previously described, and the top 7 hits for each cluster are shown in In summary, the analysis of DEGs identified in inactivated and live virus-treated PBMCs showed that the transcriptional profiles induced by the two treatments were mostly similar, with the exception of selected immune pathways (induced by the inactivated virus only) and of functions involved in the lipid metabolism (upregulated only upon incubation with the live virus). These differences may be ascribed to the intrinsic nature of the replicating virus, which acts to suppress certain immune responses  and to iTo gain a further insight into the molecular cascade engaged by I-TBEV, we assessed the cellular responses in the presence of inhibitors of downstream molecules in selected PRR signaling pathways.ISG56 in PBMCs from 3 healthy donors upon treatment with the positive control Poly I:C, we established 50 μg/mL to be the optimal concentration of ALX and 2 μM that of BX in terms of inhibitory capacity of the compounds  and BX795 (BX), two specific inhibitors of the noncanonical IkB kinases IKKε and TANK-binding kinase 1 (TBK1) ,31 downsompounds A. ALX anomarkers , indicat [CXCL10 C and, al0 TBEV, we turned to HEK Blue cells, human cells engineered to express a reporter construct upon ligand binding to various PRRs . The cells were incubated for 48 h with increasing concentrations of I-TBEV or its matrix control, and their level of stimulation is reported as a percentage of activation of the cells compared to the response to a fixed amount of TNF-α (set as 100% activation).While the different HEK Blue cell lines responded to their specific TLR and NOD2 ligands, none of the receptors were triggered specifically by I-TBEV A. The prIn this study, we assessed the cellular responses and pathways induced by I-TBEV, the main component of the TBE vaccine, and by the replicating virus in human primary cells and reporter cell lines. Our first aim was to extensively characterize the innate immune signature induced by I-TBEV in our human PBMC platform in comparison to untreated cells, as well as to cells treated with low-quality I-TBEV or live virus. The second aim was to identify the pattern recognition receptors responsible for (I-)TBEV sensing.Addressing the first aim, we found that I-TBEV induced an interferon-dominated immune profile and the upregulation of selected inflammatory genes. The low-quality formulation, HT I-TBEV, triggered a qualitatively similar expression signature; however, it had much lower response magnitudes. While the sequencing results were obtained in cells derived from only one donor, the same expression pattern emerged in the validation of the results with 2 additional donors, and the findings are in line with previous results on selected I-TBEV-induced responses in primary human immune cells from several donors ,33. IndeComparing the immune profiles in cells incubated with live or inactivated TBEV, we observed that the live virus induced a transcriptional signature overlapping in large parts with that induced by I-TBEV, with the exception of replication-related genes distinctly regulated in live virus-treated cells. Similarities in immune responses to live and whole inactivated viruses were previously observed in other studies ,40, but ISG56 and CXCL10, while inhibition of TBK1/IKKε—factors downstream of RLRs—halted the signaling cascade.After having established the distinctive transcriptional profile of (I-)TBEV-stimulated cells, we sought to determine which pattern recognition receptors, once triggered, led to the identified responses. Using inhibitors of downstream factors of PRRs, we demonstrated the involvement of RLRs in I-TBEV sensing in cryopreserved PBMCs. Inhibition of MyD88—an adapter protein downstream of TLRs—did not affect the expression of I-TBEV-induced genes such as Activation of RIG-I-like receptors is a predominant mechanism for cellular recognition of flaviviruses ,53; exprThe role of the viral genome in the immunogenicity of I-TBEV was previously assessed in plasmacytoid dendritic cells (pDCs) derived from freshly isolated PBMCs, which were found to sense I-TBEV through TLR7/8 . The disOur study provides new insights into how I-TBEV activates the innate immune system in vitro. Understanding the mechanism of action through which a virus particle interacts with the immune system can offer several benefits. Firstly, comparison between the live and the inactivated virus could, for example, indicate whether critical viral components are retained during the manufacturing process of inactivated virus vaccines. Secondly, the identification of key pathways might allow the selection or design of cellular platforms which express the relevant components. Such platforms would be particularly suited for assessing the potential of the vaccine in vitro. Thirdly, given that the TBE vaccine is highly effective, knowledge of the innate pathways engaged by this vaccine provides a lead for the design of effective vaccines for other pathogens.However, it has to be realized that the TBE vaccine contains aluminum hydroxide as an adjuvant, which by itself also affects the innate immune system ,68,69,70As the scientific community strives to bring safe and effective SARS-CoV-2 vaccines to the market, the process of vaccine development and assessment needs global attention now more than ever. While vaccine potency and effectiveness can be assessed relatively easily, knowledge of which host pathways should be activated for mounting a sufficient (but not excessive) immune response is not yet solid. This is especially the case with ‘difficult’ vaccines , where traditional “isolate, inactivate and inject” strategies might be inappropriate for vaccine development. Next generation sequencing techniques have been proposed as tools eventually enabling rational and directed vaccine design ,75,76. A


Patients with endogenous or exogenous glucocorticoid (GC) excess exhibit a range of side effects, including an increased risk of infections. Via both mechanism, immune impairments and cardiometabolic concomitant diseases, patients with GC excess could be at increased risk for COVID-19. The impact on incidence and outcome of a SARS-CoV-2 infection in this population are not yet completely clear. This review aims to compile the data available to date and to discuss the existing literature on this topic. Further we highlight potential effects of SARS-CoV-2 on the hypothalamic-pituitary-adrenal axis as well as the influence of endogenous or exogenous GC excess on SARS-CoV-2 mRNA vaccination. There is growing evidence suggesting an increased risk of infection and severe outcome in patients with high-dose GC therapy after contracting SARS-CoV-2. The few data and case reports on patients with endogenous GC excess and SARS-CoV-2 infection point in a similar direction: chronic GC excess seems to be associated with an unfavorable course of COVID-19. Whether this is mainly a primary immune-mediated effect, or also triggered by the many GC-associated comorbidities in this population, is not yet fully understood. Patients with endogenous or exogenous GC excess should be considered as a vulnerable group during the SARS-CoV-2 pandemic. Regardless of the cause, vaccination and consistent surveillance and control of associated comorbidities are recommended. The novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in January 2020. Since the initial reports of a cluster of pneumonia cases in Wuhan, China, in December 2019, acute infection by SARS-CoV-2, officially named as coronavirus disease-19 (COVID-19), has spread throughout the world . The symSince their discovery in the 1940s and the recognition of the immunosuppressive actions, GCs have been considered one of the most widely used and effective therapies to control inflammatory and autoimmune diseases . Patientper se already has many unfavorable side effects, and patients with underlying rheumatic diseases are more likely to have comorbidities, this should be considered and the data interpreted carefully.The risk for hospitalization due to COVID-19 was analyzed by Gianfrancesco and colleagues in a publication from the COVID-19 Global Rheumatology Alliance that analyzed factors associated with hospitalization in 600 cases of COVID-19 in patients with rheumatic diseases . They foThe risk for a severe course of disease or death due to COVID-19 was as well analyzed in studies of patients with chronic inflammatory diseases  vaccine response is not known. Systemic GC therapy affects both the innate and adaptive immunity. Because of the immunosuppressive actions and the known glucocorticoid receptor expression on all immune cells, the primary concern regarding SARS-CoV-2 vaccines in the setting of GC excess is efficacy. GCs are known to suppress the ability of antigen presenting cells to process antigen and to impair T cell activation . In animThe use of intraarticular corticosteroids was previously associated with an increased risk for developing influenza despite vaccination . Due to Patients with chronic immunosuppressive therapies have been excluded from the large studies of SARS-CoV-2 mRNA vaccines to date. In a recently published prospective study with 436 transplant recipients and immunosuppressive therapy including tacrolimus (83%), corticosteroids (54%), mycophenolate (66%), azathioprine (9%), sirolimus (4%), and everolimus (2%), antibodies were detectable in only 76 of 436 participants  at a median of 20 days after the first dose of SARS-CoV-2 mRNA vaccine . This isNoteworthy, all vaccines approved or currently in late-stage development, including vaccines based on mRNA, are considered inactivated vaccines. With regard to live vaccination, there are safety concern with systemic GC doses equivalent to 2 mg/kg or a dose of 20 mg per day of prednisone equivalents for 2 or more weeks , 111. HoA potentially decreased response to vaccination should not lead to a decision not to vaccinate, and vaccines should not be deferred in these vulnerable patients. As patients with endogenous or exogenous GC excess might be at higher risk for a severe course of COVID-19, immunization is recommended. Thus, it can be assumed that in patients with a higher risk, vaccination even with the result of a lower titer of antibodies is better than no vaccination. Depending on further studies on the immune response, repeated immunization after the period of altered immunocompetence, such as florid endogenous CS or high-dose GC treatment, may be necessary after achieving remission.There is growing evidence that patients under especially high doses of exogenous GC or during endogenous GC excess exhibit an increased risk for SARS-CoV-2 infection and for a severe course of COVID-19. Therefore, in patients with higher doses of exogenous GCs, the lowest possible dose is recommended to control the underlying inflammatory disease. In patients with endogenous CS, adequate control of hypercortisolism is recommended, as increased risk has been associated with the level of cortisol excess. Moreover, good monitoring of metabolic and cardiovascular comorbidities seems particularly important to ameliorate the risk profile of patients with GC excess.Many questions about the relation between SARS-CoV-2 and endogenous or exogenous GC excess remain unanswered. Further studies are required to understand the risk of these patients as well as the long-term effects of COVID-19 in this population. The immediate collection of data in the SARS-CoV-2 pandemic is important to answer key pressing questions. International collaboration between specialists appears to be of great importance for certain patient groups in order to obtain valid data . However


Aortoesophageal fistula (AEF) caused by esophageal cancer (EC) is a rare but life-threatening complication. However, the optimal management strategy remains undetermined. Previous cases have demonstrated that thoracic endovascular aortic repair (TEVAR) is effective for prophylactic management. In our study, we evaluated the management of AEF with elective TEVAR over salvage TEVAR. In our single-center retrospective cohort study, forty-seven patients with cT4M0 EC were included in this study, and we divided them into salvage (Group S) and elective (Group E) groups based on whether TEVAR was performed before the hemorrhagic AEF occurred. Our study outcomes included survival and complication rate after TEVAR. Group E showed better overall 90-day survival and aortic-event-free survival in 90-day and 180-day over Group S. More patients in Group E could receive subsequent chemoradiotherapy or esophagectomy. Significantly fewer AEF-related complications, including recurrent hemorrhagic events after TEVAR, hypoperfusion-related organ injury, and bloodstream infection, were noted in Group E. In patients with advanced EC-invading aorta, elective TEVAR offered an early overall and aortic-event-free survival benefit compared to salvage TEVAR. By reducing the AEF-related complications, elective TEVAR could provide more patients receiving subsequent curative-intent treatment. Aorto-esophageal fistula (AEF) is a rare and lethal entity, and the difficulty of making a diagnosis of AEF is well-known due to its representative symptom from sentinel hematemesis to massive hematemesis, and the symptom-free interval is unpredictable. Advanced esophageal cancer (EC) with aortic invasion is the third common cause of AEF, after thoracic aortic aneurysm and ingestion of a foreign body ,2, owingGiven the established advantage of endovascular thoracic aortic grafting (TEVAR) over the open aortic replacement for thoracic aortic aneurysm, several case series of esophagectomy after elective TEVAR have been reported. These findings indicated that the clinical benefit of TEVAR before esophagectomy provided better progression-free survival in patients with EC in terms of the intraoperative aortic hemorrhage and the achievement of R0 resection ,8,10,11.Our study aimed to compare the feasibility, safety, and clinical outcomes of elective TEVAR versus salvage TEVAR after the acute development of AEF in patients with advanced EC with T4b disease, stage IVA without distant metastasis. To our knowledge, this was currently the largest single-center retrospective cohort study on this subject.This retrospective single-centered cohort study was approved by the Institutional Review Board of the Ethics Committee in National Taiwan University Hospital, and the requirement of informed consent was waived because of the retrospective nature of the study. This study was registered in ResearchRegistry, and the study has been reported according to the STROCSS guideline .By reviewing the electronic medical records from March 2011 to March 2021 in National Taiwan University Hospital, patients with T4b EC of squamous cell type consulted for TEVAR were included in this study. Exclusion criteria included death due to hemorrhagic events before salvage TEVAR and distant metastatic EC. Data regarding independent prognostic factors for EC were collected ,19. The Information including the death registration was collected through the Integrated Medical Database in National Taiwan University Hospital and the National Health Insurance Research Database. The follow-up data were truncated on 30 June 2021. Patients were considered lost to follow-up if no information was available in any of the databases during follow-up.We divided patients into two groups: the salvage group (Group S), who received TEVAR after AEF-induced hemorrhagic events, and the elective group (Group E), who received TEVAR before any AEF-related hemorrhagic event was noted. A patient was considered the surgical candidate for elective TEVAR only if the staging computed tomography (CT) showed a T4b EC lesion without distant metastasis. The indication of TEVAR for patients in the elective group was either EC contacting greater than 90° of the descending thoracic aorta or obliteration of the triangular fat space between the esophagus and thoracic aorta on computed tomography (CT) or Positron emission tomography (PET), as indicated in All procedures were conducted under general anesthesia by using either femoral cutdown or percutaneous access in a hybrid suite . In Group E, the treatment goal was to cover the site of aortic invasion adjacent to the EC to prevent the aortic bleeding, while in Group S, this TEVAR procedure was performed to stop the acute hemorrhage from the AEF. The location of the invasive lesion or acute hemorrhage was determined by preoperative CT and PET scan, measuring proximally from the left subclavian artery (LSCA) or distally to the celiac artery. If the patient’s condition was stable before TEVAR, the oral edge of the tumor was marked with a radiopaque metallic clip by esophagoscopy to mark the optimal site for the stent graft. Both proximal and distal landing zones were selected between Zones 2 to 5 depending on the location of EC; at least 2 cm of the healthy aorta was required. The selected diameter size of the stent graft was 10–20% larger than the aortic diameter at the proximal landing zone. Tapered devices or telescopes with smaller devices were selectively used in patients with smaller distal aortic landing zones. A chimney procedure or physician-modified fenestration was performed as needed in some patients for LSCA revascularization to achieve an adequate proximal landing, but it was rarely performed for the distal landing zone. Routine spinal drainage was not required due to the short segment of aortic coverage or the emergent situation. Blood pressure was strictly controlled at 140/80 mmHg postoperatively to prevent spinal cord injury after TEVAR.After the procedure, patients in the elective group were admitted to the intensive care unit (ICU) for 1 day to monitor hemodynamics and neurological status. The prophylactic antibiotic with intravenous first-generation cephalosporin or vancomycin was given. In salvage cases, due to the unstable perioperative hemodynamics, patients were sent to the ICU for further monitoring of hemodynamics until stabilization. Considering the AEF, broad-spectrum prophylactic antibiotics with third or fourth generation cephalosporin with or without metronidazole was given for coverage of enteric aerobic and anaerobic flora.For elective patients, chemotherapy or radiotherapy generally starts 2–4 weeks after receiving TEVAR. In the salvage patients, due to the hemodynamic instability, the time of receiving CRT depends on the patient’s clinical condition.In our study, the primary outcomes included the freedom from all-cause mortality and aorta-related event-free survival analysis after TEVAR. The postoperative TEVAR secondary outcomes were the complications including recurrent or new aortic hemorrhagic events, EGI evidenced by the bloodstream infection with enteric commensals, reversible or irreversible hypoperfusion-related organ dysfunction, and neurological complications. The subsequent definitive CRT or salvage esophagectomy after TEVAR and the index length of hospital stay were also recorded.p-value less than 0.05 was considered significant. All statistical analyses were performed using SPSS, version 25.0 .For statistical analysis, continuous variables between Groups S and P were compared using the Mann–Whitney U-test. The chi-square and Fisher’s exact tests were used to compare categorical variables. The log-rank test was used to analyze overall survival and aortic-related event-free survival between groups. Multiple regression analysis and the Cox regression model were applied to establish the independence between two groups. A A total of 71 patients were identified. Among these patients, 24 of them were excluded from our study because of death from massive aortic bleeding at the emergency department before the salvage TEVAR (n = 1) or because they were diagnosed with EC stage IVb with distant metastasis (n = 23). Consequently, the data of the 47 remaining patients were included in our analyses, with 17 patients in Group S and 30 patients in Group E. The flow chart of patient selection was presented in 2. Before TEVAR, 12 (71%) of the patients received CRT, while two (12%) of the patients received esophagectomy in Group S, and six (20%) of the patients received CRT but none of the patients received esophagectomy in Group E.The results of the overall survival analysis were shown in Among all factors in multiple regression analysis of survival time, pre-TEVAR esophagectomy and alcohol statistically significantly predicted the length of survival time. The results of the statistical significance of each independent variable were listed in p < 0.001). Patients in Group E had a statistically higher chance of receiving subsequent definitive CRT and salvage esophagectomy than Group S.The perioperative details and secondary outcomes were summarized in In this study, elective TEVAR could prevent EC-associated AEF and more patients could undergo subsequent definitive CRT or salvage esophagectomy. In salvage TEVAR, though patients could achieve temporary hemostasis from the life-threatening AEF, it was associated with a poor 90-day overall survival, and 90-day to 180-day aorta-related event-free survival compared to elective TEVAR because of the increased periprocedural complications.AEF was a life-threatening complication of advanced EC. It was more commonly seen in patients with advanced EC invading the aorta after concurrent CRT with a reported incidence of 10–29% . In acutTraditionally, esophagectomy in EC patients with aortic invasion is considered contraindicated. According to the National Comprehensive Cancer Network guideline, the recommended treatment is definitive CRT without surgery. However, disease progression is frequently encountered after such treatment with a median survival of 10.6 months, and 14.6% of patients end up with AEF during or after treatment . InsteadIn this study, elective TEVAR did not provide a long-term survival benefit in patients with T4b EC compared to salvage TEVAR. Elective TEVAR could improve short-term survival by preventing aortic-related mortality. However, the long-term survival still depended considerably on tumor progression, which was difficult to be dealt with elective TEVAR. In the previous literature, the combined use of TEVAR and esophagectomy could improve local tumor control and progression-free survival but not the overall survival ,24, inclWe acknowledged the limitation of statistical power under such a single-center retrospective study with a small patient population, although it was currently the largest cohort in the literature to compare elective and salvage TEVAR in T4b EC patients. Our study focused explicitly on squamous cell carcinoma of the esophagus; a few patients with other histopathological subtypes of EC, including adenocarcinoma, small-cell carcinoma, and undifferentiated carcinoma, were excluded. Thus, our results might not apply to other types of EC. The generalizability of our study might be limited to Southeast Asian cohorts. Further multi-center prospective studies to investigate whether the intervention with elective TEVAR could make a difference to survival for such patients were required.In this study, elective TEVAR could offer an early 90-day survival benefit and 90- to 180-day aorta-related event-free survival over salvage TEVAR by reducing the AEF-related complications. It could also prevent EC-associated AEF and enable patients to receive subsequent definitive CRT or salvage esophagectomy for the curative-intent treatment. elective TEVAR appeared to be safe and effective and might become part of the routine approach before CRT or esophagectomy in patients with advanced EC with aortic invasion.


The Patient-Focused Drug Development initiative of the U.S. Food and Drug Administration (FDA) aims to ensure that the patient experience of disease and treatment is an integral component of the drug development process. The 21st Century Cures Act and Prescription Drug User Fee Act (PDUFA) VI require the FDA to publicly report the type of patient-experience data reviewed in a new drug application (NDA) to inform regulatory decision-making. This report describes a recent approach adopted at Janssen of integrating patient-experience data into the NDA for esketamine (SPRAVATO®) nasal spray with a newly initiated oral antidepressant (esketamine + AD) for treatment-resistant depression. During the development of esketamine + AD, patient-experience data were collected using several patient-reported outcomes, including the Sheehan Disability Scale and 9-item Patient Health Questionnaire (PHQ-9). Additionally, a patient-preference study assessed the relative importance of benefits and harms that patients allocated to different attributes of treatment. Preferences were collected from patients enrolled in phase 3 esketamine trials and from an online panel of primarily ketamine-naive patients. Patient-experience data were integrated into the esketamine NDA, the FDA advisory committee meeting briefing document, and the Sponsor’s presentation. The FDA acknowledged reviewing the patient-experience data and determined that they supported esketamine + AD for treatment-resistant depression. This report highlights the importance of integrating patient-experience methods early in drug development, their impact on assessing patient-relevant benefits and risks, and how they can help improve clinical program design. The Patient-Focused Drug Development (PFDD) initiative of the U.S. Food and Drug Administration (FDA) has evolved to ensure that the patient-experience of disease and treatment becomes an integral component of drug development and regulatory review . The FDAThe FDA considers patient-experience data to include information from patients about (1) the signs and symptoms of their condition and how they affect patients’ day-to-day functioning and quality of life, (2) the natural history of their condition and the changes in symptoms over time, (3) their experience with the symptoms and burdens related to treatment, (4) their views on potential disease or treatment outcomes and how they weigh the importance of different possible outcomes, and 5) how they view the impact of the disease, treatment, and outcomes, and potential tradeoffs between disease outcomes and treatment benefits and risks  how they, 7.Esketamine (SPRAVATO®), the S-enantiomer of ketamine, was developed by Janssen and is now approved in the US  , Europe,This report summarizes patient-experience activities assessed in the esketamine clinical development program and the corresponding comments from the FDA. Additionally, it presents a recent approach adopted at Janssen to generating and integrating patient-experience data into the esketamine NDA, as well as the FDA regulatory review and decision-making process.The efficacy and safety of esketamine + AD was assessed in international, phase 3 TRANSFORM and SUSTAIN trials in adults with TRD. The TRANSFORM trials were randomized, double-blind, multicenter, active-controlled , phase 3 studies that assessed the efficacy, safety, and tolerability of esketamine + AD. TRANSFORM-1 NCT02417064) and TRANSFORM-2 NCT02418585) assessed fixed and flexible doses, respectively, of esketamine + AD , 13. SUS064 and T585 assesThe PRO strategy included research to understand which symptoms and impacts of depression are important to patients and how they perceive effects of depression and its treatment on health and functioning (literature review not available in the public domain). Specific PROs were carefully selected, focusing on key concepts of the patients experience of depression, with the consideration that patient “feelings and function” are adequately captured. Evidence supporting the content validity and measurement properties of the PROs was reviewed to assess whether the instruments were fit for purpose in the context of the phase 3 trials. When gaps in the supportive evidence were identified, additional data  were generated to support selected PROs for use in the phase 3 TRD trials. Phase 2 clinical trials included limited PRO assessments that assisted with selection of PROs, and in addition, patients receiving investigational drugs for TRD in two phase 2 trials were interviewed to assess their experience of treatment, including positive and negative health effects . ResultsThe 9-item Patient Health Questionnaire (PHQ-9), a PRO that includes items addressing the Diagnostic and Statistical Manual of Mental Disorders (5th edition) criteria for depression, was selected to assess patient perceptions of symptoms of depression. Changes in social, family, and work functioning were identified as important functional impacts of depression that are not adequately captured by the primary endpoint measure for the clinical trials, ie, change in the Montgomery-Åsberg Depression Rating Scale (MADRS) from baseline to day 28 , 13. TheThe SDS and PHQ-9 PROs, which are validated instruments that are widely used to assess experience of symptoms and disability in patients with depression –28, wereThe same PROs were also included as secondary endpoints in SUSTAIN-1 and SUSTAIN-2. All 4 trials included GAD-7 and EQ-5D-5L as secondary endpoints. The FDA’s PRO guidance was followed regarding appropriate implementation of PROs in clinical trials .For the PRO data from the TRANSFORM clinical trials, change from baseline in instrument scores was assessed at day 28 during the double-blind induction phase. The statistical plan in these trials required the PHQ-9 and SDS scores to be assessed in a hierarchical manner after another secondary endpoint of clinical response at day 2 that was maintained through day 28. Additional, PRO analyses were performed in the clinical trials but are not reported here.In both TRANSFORM-1 and TRANSFORM-2, the PRO results were consistent with and supportive of the primary endpoint  , 13. TheA discrete-choice survey was conducted to evaluate relative importance of benefits and harms associated with treatment and to quantify the maximum acceptable risk patients would accept for treatments that relieved depression. Specifically, degree of depression relief (efficacy), time to response, transitory postdose issues—a composite attribute of postdose short-term altered sensations and the inconvenience associated with logistics of drug administration—and putative long-term extreme safety risks associated with ketamine abuse  were included in the preference survey. Planning for the preference study began prior to the start of the phase 3 trials. Development, implementation, and analysis of the preference study spanned more than 1.5 years. The comprehensive and detailed patient-preference study design along with the complete statistical information  and preference study have been published elsewhere . The comn = 161) who had direct experience with esketamine, including its efficacy, postdose symptoms, and treatment burden. The second set was a sample of patients from a national online panel (n = 301), most of whom had no experience with off-label ketamine; this population represented patients who would consider esketamine + AD, once approved, for the treatment of TRD. Patients in the second set relied solely on textual description of treatments in the survey. Patient preferences between these two sets were compared to inform the degree to which prior experience with esketamine influenced willingness to accept tolerability issues and risks. The survey was completed on a computer or tablet.Because patients with TRD have failed several previous antidepressant therapies, it was unclear whether patients would believe textual descriptions of rapid onset of efficacy without direct experience of novel therapies that provide such efficacy. To address this, the survey was administered to two sets of patients with TRD. The first set was a sample of patients enrolled in SUSTAIN-2 and SUSTAIN-3 at English-speaking sites  . Th. Th32]. Patient-experience data are considered a valuable part of the totality of evidence evaluated in the regulatory review process and are becoming increasingly important, particularly in the US , 7. ThesThe FDA encourages sponsors to discuss patient engagement methods and approaches early in development and often. This is, in part, to ensure that any PRO measure proposed for collecting patient-experience data are clinically relevant and fit for purpose, especially if the PRO data are under consideration for inclusion in the product label. These early discussions may necessitate generation of additional validation data, which could further strengthen the NDA. Importantly, clinical trial sponsors should consider providing a comprehensive package of patient-experience data in the NDA submission so that the regulatory authorities can assess important elements from the patients’ perspective. This could include summary information on PRO findings and data to support validation and interpretation of PROs (where available), as well as patient preferences or other relevant patient-experience data in supporting benefit-risk assessment and overall findings of clinical trial(s). Finally, clinical trial sponsors should consider including information on the PROs in NDA summary documents such as Summary of Clinical Efficacy and Clinical Overview.This was a single example where patient-experience data were integrated into the NDA. More examples are needed to more fully understand the effectiveness of the FDA’s PFDD initiative. A recent study that systematically assessed 59 new drugs approved by the FDA in 2018 found that among 48 approvals that reported whether or not patient-experience data were included in the review, 34 (71%) had included these data . AdditioThe patient-experience data collected by Janssen and its integration into the NDA for esketamine for the treatment of patients with TRD assisted the FDA in its regulatory evaluation and decision-making. The FDA’s publicly available clinical review of esketamine + AD highlighted its focus on patient-experience data. The FDA acknowledged that functional outcomes were reviewed and provided interpretation on how they supported the primary efficacy endpoint results. Additionally, both the FDA and its advisory committee used the patient-preference data as part of their assessment of patient acceptance of the benefit-risk profile of esketamine + AD. The example of esketamine NDA demonstrates the importance of early planning for and integrating patient-experience methods early in drug development, which can help identify the patient-relevant risks and benefits and ultimately benefit patients and clinical program designs. The data presented here support the FDA’s PFDD initiative and underscore its goal of including patient-experience in its decision-making, especially for conditions such as depression, where objective scales are lacking.


The method 3D polarised light imaging (3D-PLI) measures the birefringence of histological brain sections to determine the spatial course of nerve fibres (myelinated axons). While the in-plane fibre directions can be determined with high accuracy, the computation of the out-of-plane fibre inclinations is more challenging because they are derived from the amplitude of the birefringence signals, which depends e.g. on the amount of nerve fibres. One possibility to improve the accuracy is to consider the average transmitted light intensity (transmittance weighting). The current procedure requires effortful manual adjustment of parameters and anatomical knowledge. Here, we introduce an automated, optimised computation of the fibre inclinations, allowing for a much faster, reproducible determination of fibre orientations in 3D-PLI. Depending on the degree of myelination, the algorithm uses different models , allowing to account for regionally specific behaviour. As the algorithm is parallelised and GPU optimised, it can be applied to large data sets. Moreover, it only uses images from standard 3D-PLI measurements without tilting, and can therefore be applied to existing data sets from previous measurements. The functionality is demonstrated on unstained coronal and sagittal histological sections of vervet monkey and rat brains. Microscopy techniques like optical coherence tomography6, light-sheet microscopy8, or two-photon fluorescence microscopy10 allow an in-depth scan of the sample and provide microscopic resolution in all three dimensions. However, they can only be applied to smaller brain volumes and need advanced image processing to follow the course of nerve fibres, which is especially challenging in regions with densely packed fibres. The microscopy technique 3D polarised light imaging (3D-PLI)12 analyses the spatial course of nerve fibre pathways by transmitting polarised light through unstained histological brain sections and measuring their birefringence . In contrast to the other techniques, 3D-PLI requires sectioning of the brain and advanced registration algorithms to recover the original brain volume. But it allows to retrieve the three-dimensional orientations of the nerve fibres also in densely grown regions and for large brain sections, making 3D-PLI a powerful approach for analysing the brain’s nerve fibre network in microscopic detail.To better understand the function of the brain and to treat neurological diseases, a detailed reconstruction of the intricate and densely grown nerve fibre network is needed. Diffusion magnetic resonance imaging allows to study the course of nerve fibre pathways in vivo and for whole brain volumesmyelin—a stack of cell membranes with highly ordered molecular structure that surrounds most axons in the white matter13.  The orientation of the nerve fibres within the section plane can be determined with high degree of accuracy, provided the fibre bundles have a well-defined orientation14. The computation of the out-of-plane fibre orientation (inclination) requires a more sophisticated analysis: The fibre inclination is directly related to the amplitude of the birefringence signal, which also depends on other factors such as the amount of nerve fibres15. In order to determine the fibre inclinations independent of the tissue composition, a tiltable specimen stage can be used: By repeating the 3D-PLI measurement for different tilting angles, it is possible to change the perspective onto the nerve fibres in a predictable manner and reconstruct the fibre inclinations. Tilting has successfully been employed to improve the determined inclination angles for 3D-PLI measurements with in-plane resolutions comparable to the brain section thickness16. However, analysing images obtained from tilting microscopes with higher in-plane resolution remains a challenge. Moreover, many high-resolution polarising microscopes are not equipped with a tilting stage.The three-dimensional nerve fibre orientations are derived from the measured birefringence signals, which are mainly caused by transmittance weighting)17: Due to its highly layered structure, myelin has a large scattering coefficient18 so that regions with a high density of nerve fibres (myelinated axons) appear dark in the transmittance image.An alternative way to adjust the determined inclination angles is to use the transmittance (polarisation-independent transmitted light intensity) as a measure of the amount of nerve fibres in the brain section  if the attenuation of myelin is dominant compared to the attenuation of other tissue components. Apart from myelinated axons, brain tissue contains unmyelinated axons, neuronal cell bodies, dendrites, synapses, glial cells, and blood capillaries, which are all differently distributed and have locally variable attenuation properties20. Therefore, the transmittance cannot serve as a measure of myelination in regions with a low amount of myelinated axons.Transmittance weighting has the advantage that it can be applied to (existing) data sets from standard 3D-PLI measurements without tilting. However, using the transmittance to adjust the computed fibre inclinations poses several challenges: Previous work of our groupUp to now, the nerve fibre inclinations were computed using a transmittance-weighted model for the whole brain section, requiring manual adjustment of the resulting parameters and advanced anatomical knowledge to account for different tissue compositions. Based on transmittance and retardation histograms, an expert needed to iteratively adjust various threshold parameters to identify the optimum set of parameters that yields particular fibre inclinations in anatomically known brain regions while reducing the number of regions with under/over-estimated fibre inclinations.21. However, instead of using a binary classification, we introduce the important concept of transition zones, allowing to consider subtle changes in the tissue composition and to compute more accurate fibre inclinations at boundaries. As the automated computation does not require any manual adjustment of the parameters, it allows for a much faster and reproducible computation of the three-dimensional nerve fibre orientations for 3D-PLI measurements of brain tissue.Here, we introduce a fully automated computation of the nerve fibre inclinations, taking the inclination-dependence of the transmittance and different degrees of myelination into account. Instead of applying the transmittance-weighted model to the whole brain section, we use a regionally specific computation of the fibre inclinations: The transmittance-weighted model is only used in regions with a high degree of myelination. In regions with a low degree of myelination, an unweighted model is used. The separation of regions with low and high degrees of myelination is based on an algorithm proposed by Benning et al.We demonstrate the functionality of our algorithm on coronal and sagittal sections of primate and rodent brains. To validate the automatically computed nerve fibre inclinations, we compared our results from 3D-PLI to in-depth-tissue scans from two-photon fluorescence microscopy.The brain sections were obtained from three healthy rats  and two healthy vervet monkeys . All animal procedures were approved by the institutional animal welfare committee at Forschungszentrum Jlülich GmbH, Germany, and were in accordance with the European Union and National Institutes of Health guidelines for the use and care of laboratory animals and in compliance with the ARRIVE guidelines. Euthanasia of rats was carried out under controlled isoflurane inhalation followed by decapitation. The monkey brains were obtained in accordance with the Wake Forest Institutional Animal Care and Use Committee (IACUC #A11-219). Euthanasia procedures conformed to the AVMA Guidelines for the Euthanasia of Animals, using ketamine/pentobarbital anesthesia followed by perfusion with phosphate buffered saline and fixation with 4% paraformaldehyde.The brains were removed from the skull within 24 h after death, fixed with 4% buffered formaldehyde for several weeks, cryo-protected with 20% glycerine and 2% dimethyl sulfoxide, deeply frozen, and coronally or sagittally cut with a cryostat microtome  into sections of 60 µm. A coronal vervet monkey brain section (no. 512), a sagittal vervet monkey brain section (no. 374), a coronal rat brain section (no. 185), and a sagittal rat brain section (no. 176) were selected for evaluation. The sections were mounted on glass slides, embedded in 20% glycerine solution, cover-slipped, sealed with lacquer, and measured with 3D-PLI afterwards.19: Incoherent light with a wavelength of about 550 nm was transmitted through a linear polariser, the brain section, and a circular analyser (quarter-wave retarder and linear polariser), see Fig. 12:transmittancedirectionretardationinclinationThe 3D-PLI measurements were performed with the polarising microscope , as described in Menzel et al.19, with a custom-made two-photon fluorescence microscope23 consisting of a model-locked titanium-sapphire laser with a wavelength of 800 nm and a water-immersion 3. The fluorescence signals were collected by two photomultiplier tubes, detecting red and green fluorescence, respectively. TPFM allows a full in-depth scan of the 60 µm thin brain section, yielding one image every 2, with an overlap of 10%, and the resulting images were stitched together using the software TeraStitcher24. Due to the slightly different autofluorescence signal of different tissue components, the nerve fibre bundles can be manually traced across different slices of a TPFM image stack. To determine the inclination angle of a fibre bundle, the cross-sections of the fibre bundle were determined in the first and last slices of the image stack that still show the fibre bundle. The inclination angle To validate the automatically computed nerve fibre inclinations, we compared the results from 3D-PLI to two-photon fluorescence microscopy (TPFM) measurements of the same brain section. The TPFM measurements were performed on a coronal rat brain section (R) via25:11, the fibre inclinations can only be computed based on certain assumptions.The out-of-plane inclination angle Assuming that d and attenuation coefficient T is attenuated according to the Lambert–Beer law:17:Usually, the amount of nerve fibres , the attenuation can be considered to be mostly caused by the scattering of myelin so that the fibre inclinations can be computed with the transmittance-weighted model. However, in regions with a low degree of myelination (LM-regions), the transmittance is dominated by other tissue components such as cell bodies so that the unweighted model should be used instead to compute the fibre inclinations.As mentioned in the Introduction, the transmittance is only a good measure of the amount of nerve fibres (myelinated axons) if the attenuation of myelin is dominant compared to the attenuation of other tissue components. In regions with a high degree of myelination . Note that the authors used the terms “grey matter” and “white matter”, while we use the terms LM- and HM-regions to avoid confusion with the anatomical definitions. The separation of regions was performed by analysing the transmittance (T) and retardation (R) images from 3D-PLI.We followed the classification introduced in Benning et al.19, the transmittance in a region with many parallel in-plane fibres can be used as a reference (HM-regions (high myelination): LM-regions (low myelination): BG-regions (no tissue): All three threshold parameters  that has the highest retardation values.The threshold parameter entclass1pt{minimatransition zones between HM- and LM-regions.When the inclination angles are computed separately for HM- and LM-regions, artificially sharp borders might occur, e.g. in regions where nerve fibres from the white matter spread out into the cortex. To account for regions that do not clearly belong to one or the other tissue type, we defined bootstrapping (random sampling with replacement): Pixels were randomly drawn from the retardation and transmittance images; the same pixel could be selected multiple times. To identify the best compromise between computing time and accuracy, the algorithm was run for different sample sizes  for 500 iterations. From the resulting values for positive/negative means , there is a smooth transition for fibres spreading from the white matter into the cortex (magenta arrow). The slightly lower ons see “”.T need to be adjusted before computing the fibre inclinations.Before Eq.  can be uAs the transmittance values are used to correct the fibre inclinations in the HM-regions cf. Eq. , cellulaTo avoid the saturation of image pixels (19), all (median-filtered) transmittance values smaller than As out-of-plane nerve fibres have lower transmittance values than in-plane nerve fibres due to an increased amount of scattering . Depending on the available graphics memory, the images were processed in a different number of chunks to avoid overflow. The coronal and sagittal rat brain sections  were computed using an NVIDIA RTX 3070 and 8 GB of RAM. The coronal and sagittal vervet brain sections (6 GB and 6.5 GB) were processed on the supercomputer JURECA27. Using 512 GB RAM, one GPU (NVIDIA A100 40GB) and 128 CPU cores on one node, the computing time for the coronal vervet brain section  was 15 min for the HM-probability map and 5 min for the rest.The HM-probability maps and the nerve fibre inclinations were computed with the specifically developed open-source software To illustrate the difference between the unweighted model and the transmittance-weighted model for computing the fibre inclination Eqs.  and 7 asWhen applying the unweighted model to the whole image, using the highest retardation of the brain section as reference  as described in the Methods.As TPFM allows an in-depth scan of the sample, it allows to directly assess the three-dimensional course of nerve fibre bundles in the caudate putamen cf. Fig. c, so thaWhile the direction angles computed from 3D-PLI correspond mostly to those obtained from TPFM see Fig. d, the coFigure The HM-probability maps reliably separate regions with low myelination (black) from the rest. Individual nerve fibres are visible in the putamen (Pu) of the coronal vervet brain section, and in the caudate putamen (CPu) of the coronal and sagittal rat brain sections. The HM-probability values in the white matter are slightly reduced (light green) for in-plane crossing fibres (cr) and steep out-of-plane fibres , yielding slightly lower estimations of the inclination angles cf. Fig. e. The inWhen analysing measurement data from 3D-polarised light imaging (3D-PLI) of histological brain sections, the determination of the out-of-plane nerve fibre inclinations has been a major challenge, requiring anatomical knowledge and effortful manual adjustment of parameters. Here, we introduced an automatic computation of the fibre inclinations, by evaluating the retardation and transmittance images . As these images are generated during standard 3D-PLI measurements, past measurements can be easily evaluated to improve the interpretation of fibre orientations in 3D-PLI data. In particular, no special equipment like a tilting stage is required to compute the nerve fibre inclinations. In contrast to the manual adjustment, no expert knowledge is required, the results for each measured brain section are reproducible, and large data sets can be analysed. In this way, we were able to provide a reliable estimate of the nerve fibre inclinations, which can still be adjusted by an expert in a second step.21 proposed a binary separation of regions with low and high myelination. We improved the existing algorithm (taking different features/plateaus of the histograms into account and excluding background tissue when determining the threshold parameters). Furthermore, we introduced the important concept of transition zones. The classification of brain regions into regions with high myelination (HM-regions), low myelination (LM-regions), and transition zones is not only relevant when applying different inclination formulas. Another possible application might be Independent Component Analysis (ICA), which has been used for noise and artefact removal in 3D-PLI images28, especially in regions with low myelination 21. The HM-probability map allows to determine different degrees of myelination, helping to improve the ICA in those regions and enabling enhanced tissue segmentation.In addition to the fibre inclinations, we provided an HM-probability map, indicating the probability that a region is highly myelinated. Benning et al.The HM-probability maps and fibre inclinations were computed with a specifically developed open-source software, which is parallelised and uses GPU acceleration to efficiently process large images. While the computation of the fibre inclinations takes less than 5 min for the coronal vervet brain section  or when the transmittance image shows irregularities (caused e.g. by an inhomogeneous distribution of the embedding glycerine solution or tile/stitching artefacts), this leads to artefacts in the resulting inclination map, which would not be visible when solely using the birefringence signal (retardation) for computing the inclination, as in the LM-regions. Hence, the software should only be used when the transmittance image contrast is free of artefacts. Another point to take into account is that the algorithm uses the transmittance and retardation histograms to compute the parameters in the inclination formula. A reliable computation of parameters only works when the histograms include a broad variety of tissue components as well as various nerve fibre structures (in-plane and out-of-plane fibres).15. As shown in Menzel et al.19, the transmittance image can serve as an indicator whether large inclination angles belong to out-of-plane nerve fibres or are caused by in-plane crossing fibres or a low fibre density. Regions with high inclination values can be considered as reliable if the transmittance in these regions is notably lower than in regions with in-plane nerve fibres. When comparing the transmittance image . So far, these nerve fibre inclinations were computed by applying a transmittance-weighted model to the whole brain section, requiring anatomical knowledge and effortful manual adjustment of parameters. By distinguishing areas with different degrees of myelination and defining transition zones, we were able to apply a regionally specific computation of fibre inclinations that accounts for subtle changes in the tissue composition. The developed algorithm is parallelised and uses GPU acceleration, allowing for a fast and reproducible computation of nerve fibre inclinations in large brain sections, thus greatly improving the analysis and interpretation of 3D-PLI data also for past measurements.


Lipids are indispensable in the SARS-CoV-2 infection process. The clinical significance of plasma lipid profile during COVID-19 has not been rigorously evaluated. We aim to ascertain the association of the plasma lipid profile with SARS-CoV-2 infection clinical evolution. Observational cross-sectional study including 1411 hospitalized patients with COVID-19 and an available standard lipid profile prior (n: 1305) or during hospitalization (n: 297). The usefulness of serum total, LDL, non-HDL and HDL cholesterol to predict the COVID-19 prognosis (severe vs mild) was analysed. Patients with severe COVID-19 evolution had lower HDL cholesterol and higher triglyceride levels before the infection. The lipid profile measured during hospitalization also showed that a severe outcome was associated with lower HDL cholesterol levels and higher triglycerides. HDL cholesterol and triglyceride concentrations were correlated with ferritin and D-dimer levels but not with CRP levels. The presence of atherogenic dyslipidaemia during the infection was strongly and independently associated with a worse COVID-19 infection prognosis. The low HDL cholesterol and high triglyceride concentrations measured before or during hospitalization are strong predictors of a severe course of the disease. The lipid profile should be considered as a sensitive marker of inflammation and should be measured in patients with COVID-19. High blood pressure and dysglycaemia are associated with a worse prognosis; however, although dyslipidaemia is one of the main cardiovascular risk factors, the association between baseline lipid levels and the prognosis has been less frequently studied. In this respect, a recent umbrella review of systematic reviews suggested that dyslipidaemia may play a role in the severity of SARS-CoV-2 infection4.The COVID-19 pandemic has rapidly spread worldwide, and it appears to be far from being controlled. While the main pathophysiological mechanisms are being revealed, only some of the factors determining whether some patients have an asymptomatic course while others die have been identified. Given the limited treatment options, the identification of risk factors related to severe COVID-19 is of paramount importance to improve the prognosis. Age, sex, background therapies and comorbidities are the main clinical determinants of severity5. The membrane lipid composition and particularly the lipid rafts influence this process6. Once inside the cell, the virus replicates using the metabolic machinery of the invaded cell7. The newly synthesized viral particles exit the cells again by crossing the lipid-rich cell membrane. Based on studies of animal models, intracellular cholesterol may increase cellular SARS-CoV-2 infectivity8. Recently, the SREBP-2 C-terminal fragment discovered in blood samples from patients with COVID-19 was identified as an indicator of the severity of the diagnosis and a therapeutic target for preventing the cytokine storm and organ damage9. In the host cell, the virus modifies cellular metabolism by altering energy production pathways for its own benefit. Both cellular glucose and lipid metabolism are extensively modified6, and a recent metabolomic study of patients infected with SARS-CoV-2 has described a specific lipidomic fingerprint10.Lipids are crucial in the infection process, as they are important structural components of cellular and subcellular organellar membranes. Membrane lipid components participate in the regulation of transmembrane molecular trafficking, including infectious materials such as viruses. Viral internalization requires the attachment of the virus to the host cell membrane, activating an endocytosis mechanism5.In addition to structural and energy supply functions, intracellular lipids also act as intracellular signalling molecules or transcription factors. Viral particles also interfere with these pathways, altering cell physiology and leading to apoptosis and cell death11. Low HDL-C levels have been proposed as a risk biomarker for different infections12. Regarding the SARS-CoV-2 infection, low LDL-C, HDL-C and triglyceride (TG) levels have been described to be associated with an increasing infection severity6, and a role for these lipids in immune mechanisms has been suggested13. Low lipid levels during the infection have been associated with the severity of COVID-1914. Meanwhile, low plasma lipid concentrations are regarded as a consequence of the hypermetabolic state and undernutrition in the infected patient; however, many metabolic pathways associated with the immune response and infection itself participate in these alterations11. Cytokines, inflammatory mediators, modified lipids and intermediate lipid classes generated during the infection interfere with several steps of lipid metabolism by reducing cholesterol synthesis and absorption, decreasing triglyceride-rich lipoprotein clearance or reducing apolipoprotein (apo) A1 synthesis15. Taken together, we hypothesise that an analysis of lipid metabolism should be contemplated as another clinical tool to evaluate the SARS-CoV-2 infection state and prognosis. Thus, the present study aimed to determine the variation in the lipid profile due to COVID-19 and its association with disease severity.The standard clinical approach to assessing alterations in lipid metabolism is based on the measurement of cholesterol in several lipoprotein particles and triglyceride levels. Researchers have not determined how these parameters will improve our understanding of the important role of cellular lipids in viral infections. Infectious diseases are usually associated with low HDL cholesterol (HDL-C) concentrations and sometimes with low LDL cholesterol (LDL-C) concentrations, while triglyceride levels are typically maintained or even increasedOf the 2159 patients included in the STACOV database, 1305 had an available standard lipid profile  before the infection and 297 had a lipid profile that was measured during hospitalization. One hundred ninety-one patients had values for profiles measured at both time points  or during n = 297) hospitalization sorted by the COVID-19 severity according the aforementioned criteria are shown in supplementary material Table . Regardi305 or du97 hospitTotal cholesterol (TC), LDL-C, non-HDL-C and HDL-C concentrations obtained before COVID-19 were significantly higher than the levels measured during hospitalization. In contrast, higher TG levels were measured during the infection , showing statistically significant differences in all lipid parameters Fig. . InteresAs shown in Fig. Triglyceride concentrations showed a weak but significant direct correlation with ferritin levels , while HDL-C levels correlated inversely with ferritin  and D-dimer  concentrations. CRP levels were not significantly correlated with any lipid parameter. We also performed a multivariate analysis as outlined in the statistical methods section to assess the effectiveness of lipid parameters as COVID-19 biomarkers. The highest performing model was a random forest model, with an accuracy of 76% for the test set and an AUC of 0.84 (95% CI 0.79–0.88). The most important variables in this model were Ferritin, CRP and D-dimer, followed closely by the lipid profile, specifically TG, HDL-cholesterol and LDL-cholesterol levels, which showed the same importance level as age and body mass index  included in the database had a lipid profile that was determined during hospitalization, which was lower than other inflammatory markers, such as CRP or ferritin. Lipids are usually associated with the nutritional status of patients, but not as markers of infection or inflammation. Our data support the relevance of lipids, even the standard lipid profile, as markers of the global inflammatory status and helpful risk markers.4. Therefore, a major gap may exist between the presence of dyslipidaemia prior to viral infection and the time at which lipid disturbances were detected at the time of hospitalization. In the present study, infected patients presented lower TC, LDL-C, HDL-C and non-HDL-C levels compared to the levels measured before the disease; however, TG levels were significantly higher . In the presence of inflammation, several mediators interfere with glucose and lipid metabolism22. Several cytokines and inflammatory mediators that are overexpressed during COVID-19 may interact directly with LPL or its regulatory proteins, such as apo CII23. Among these proteins, several products delivered by activated macrophages, such as tumour necrosis factor (TNF), interleukin (IL)-1, IL-11, and Interferon-γ25, as well as products derived from bacteria, such as lipopolysaccharide (LPS) associated with septicaemia, and modified lipoproteins such as oxidized LDL or lysophosphatidylcholine have been shown to inhibit LPL activity26.Strong scientific evidence supports the hypothesis that both inflammatory states and infectious diseases are associated with striking changes in lipid metabolism28. Although some of these pathways may partially explain the low HDL levels, other factors, including the inhibition of apo A1 synthesis or increased HDL clearance due to the uptake of inflammatory mediators such as serum amyloid A (SAA) by HDL, must be taken into consideration11.Lower LPL activity results in a decreased conversion of triglyceride-rich lipoproteins (TRL) to LDL, leading to high TG and low HDL levels. Moreover, altered activity of the cholesteryl ester transfer protein (CETP) leading to smaller LDL particles and lower HDL concentrations has also been described and associated with a poor infection prognosis10. Interestingly, clinical lipid parameters were differentially correlated with lipid families. In general, cholesterol levels in both apo B and apo A1 lipoproteins tend to display direct correlations with plasma phosphatidylcholine levels and extracellular vesicle sphingomyelin levels, while triglyceride concentrations were more strongly associated with phosphatidylethanolamine levels10. Although the clinical implications of these observations remain to be completely elucidated, they provide evidence for a relationship between standard lipids measured at the clinical level and lipid derangements throughout COVID-19.Recent plasma lipidomic data from patients infected with SARS-CoV-2 have revealed substantial modifications of lipid families during the infection. Plasma monosialodihexosyl ganglioside concentrations were negatively correlated with CD4 + T cells and positively correlated with disease severity26 due to their capacity to bind toxins. Furthermore, low HDL cholesterol levels may cause dysfunction in innate immunity, a first-line defence mechanism against COVID-1929. HDL is an inflammatory mediator that buffers toxic molecules by absorbing them and transporting them to the liver for clearance11. Therefore, the structural and functional changes observed during COVID-19 might influence the disease evolution.Lipoprotein modifications observed during COVID-19, as in other infectious situations, should not be considered as only a marker of severity or a surrogate marker of inflammation, as they may have a pathogenic role. Triglyceride-rich lipoproteins have been associated with innate immunityThe present study has some limitations. First, it was a retrospective study, precluding any analysis of the causal relationship in our results. Second, the lipid concentrations measured before the infection were obtained at any moment during the previous year; therefore, we were unable to ascertain the proximity of these concentrations to the clinical event. Similarly, we were unable to precisely determine when the lipid profile was measured during hospitalization for COVID-19, although it is usually included in the first standard blood test performed after admission; therefore, we were only able to deduce that the lipid profile was measured within the first 48 h. Finally, only 191 patients had both measurements before and during the infection, limiting our ability to obtain robust conclusions about the effect of lipid variations during the infection.Standard lipid concentrations are not only a marker of nutritional status but also a biomarker of infection and the degree of inflammation that may play a pathophysiological role. A high triglyceride level associated with a low HDL-C concentration must be considered as a marker of a poor prognosis. Our data strongly support the need for lipid profile measurements during the course of COVID-19, since the lipid profile appears to be a potent risk biomarker.18. Briefly, members of the Lipids and Arteriosclerosis Units Net (XULA) of Catalonia (Spain) retrieved clinical data collected from patients admitted to their hospitals because of a SARS-CoV-2 infection up to May 31, 2020. Consecutive patients aged at least 18 years who were hospitalized for at least 24 h were eligible. Only data from patients with a definite COVID-19 diagnosis obtained by reverse transcription-polymerase chain reaction (PCR) whose infections were acquired in the community were included. A total of 2159 patients were included in the final database.This study employed a observational cross-sectional design. Data from patients included in the STACOV cohort (Cohort registration code NCT04407273-STACOV) with an available lipid profile were included in the analysis. Clinical characteristics of the STACOV cohort have been described previouslyAll collected data were anonymized, preventing reidentification. All procedures were conducted in accordance with the legal provisions of the protection of personal data in Spain and according the European Union Regulations (EU) 2016/6799 on the physical protection of the treatment of personal data. The study was compliant with the Declaration of Helsinki. It was approved by the Ethics Committee of the University Research Institute “Pere Virgili” (Reus), including the exemption of the requirement for informed consent.Only those patients from the STACOV database with an available lipid profile  that was measured within the previous year or during hospitalization were included to study the effect of circulating lipid and lipoprotein levels on patients who were admitted to hospital for COVID-19.The main objective of the study was to assess the relationship between standard serum lipid concentrations measured before or during hospitalization for COVID-19 with the infection evolution. Infection severity was categorized as mild  or severe .Lipid variations associated with COVID-19 and their correlations to clinical variables and effects on clinical severity were also analysed.Continuous variables were tested for normality using the Shapiro–Wilk test. Data are presented as medians and 25th and 75th percentiles for continuous variables with a non-normal distribution or as the means and standard deviations (SDs) for variables with a normal distribution. Unless indicated otherwise, categorical variables are reported as percentages. Differences between groups were analysed using the non-parametric Mann–Whitney test or Student’s parametric t test for continuous variables and the chi-square test or Fisher’s exact test for categorical variables. All continuous variables were standardized and normalized when necessary.Regarding the multivariate test, we constructed a series of predictive models to assess the relationship between clinical and lipid variables and the COVID-19 prognosis (mild vs. severe). The methodology is as follows: first, we divided our dataset into a training set, consisting of 80% of the observations (patients), in which models were constructed, and a test set, with the other 20%, where we evaluated the model performance. Two types of models were used: logistic regression models regularized via an elastic net and random forest models. For each scenario, we trained several versions of each type of model and adjusted their parameters via fivefold cross-validation and chose the model with better performance. This model was then evaluated using the test set, and its performance is the one that we report.30. Furthermore, they allow us to assess the relative importance and p-value of each variable against all others by determining the out-of-bag accuracy and importance before and after variable permutation31. However, these models do not provide information as to the protective or harmful effect of each variable on the prognosis of patients with COVID-19. Since this assessment is clearly a very important measure in our clinical setting, we also computed the regularized logistic regression analyses, through which we evaluated the direction of the effect based on the sign of the coefficient. Since this effect is the only parameter in which we are interested and variable importance will be inferred from the non-linear models, confidence intervals and p-values were not reported. As we shall see, both models were slightly different in terms of variable selection, but sufficiently similar to be able to combine them to draw meaningful conclusions.The system under study proved to be rather complex, especially due to the non-linearity of the relationships between predictor and predicted variables and also because of the relations between predictor variables. It's for this reason that we resorted to Random Forests models, which are able to capture non-linearity as well as accommodate relations between predictor variables and are much better suited for these complex scenarioshttps://www.R-project.org/).Statistical analyses were performed using the R software package version 3.5. (R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing. 2020. Vienna, Austria. URL: https://osf.io/mwc8n/?view_only=82bb2153fbb04aad890a3fb266fc7ba4 previous requirement to authors.The dataset supporting the conclusions of this article is available in Supplementary Information


The outbreak of coronavirus disease in Nepal led medical colleges to suspend inperson teaching-learning activities and ultimately online platform was introduced to deliver the contents of medical education. The objective of this study was to describe the perception of medical students towards online teaching-learning introduced during the COVID-19 outbreak in Nepal.An online survey using a descriptive cross-sectional study design was carried out among 515 undergraduate medical students currently enrolled in medical colleges in Nepal. A semi-structured questionnaire in Google form was utilized to collect data. The link of the Google form was sent to the potential respondents through email and social media. Descriptive statistics, including frequency, percentage, mean, and standard deviation were used to analyze data in SPSS vs20. Ethical approval was sought from Nepal Health Research Council to conduct this study, and digital informed consent was taken from study respondents.The overall score of perception of online teaching-learning was 17.61±7.19, which indicated many problems in this method of teaching-learning. The mean score of perception of online teaching-learning was found to be different across sex, location of enrolled medical colleges, having a personal electronic device, having an internet connection at residence, having separate room/space for attending online classes, and self-rated computer skills. Moreover, only 28 (5.4%) of respondents had perceived online teaching-learning as a better method of delivering content of medical curricula.Surveyed medical students in Nepal were found to perceive many problems in online teaching-learning. Moreover, management and faculty members need to take the necessary measures for enhancing the online teaching-learning quality. This suspension would lead to a loss of learning time, lower students' self-esteem, confidence, and loss of clinical skills.2 To reduce this loss, medical colleges in Nepal felt the need to use information technology to deliver medical curricula,4 and resumed undergraduate medical education through online teaching-learning platforms.With coronavirus disease (COVID-19) hitting Nepal, the Government enforced lockdown and travel restrictions,6 Online teaching provides both opportunities and challenges to students.8 The use of an online platform to deliver medical education is comparatively new in Nepal and its effectiveness from students' perspective is a must.The success of online learning hinges on accessibility, usage of appropriate methods, and course content.Hence, this study aimed to describe medical students' perception towards online teaching-learning introduced during the COVID-19 outbreak in Nepal.A web-based cross-sectional descriptive study was carried out among the Bachelor of Medicine and Bachelor of Surgery (MBBS) students currently enrolled from the first year to final year at medical colleges of different provinces in Nepal, who were 18 years or above, and who gave digital informed consent to participate. The data was collected via a web-based online survey by sending the link of the semi-structured questionnaire in the form of a Google document to the undergraduate medical students enrolled in medical colleges in Nepal by email and/or social media via purposive sampling. Medical students from 12 different medical colleges were approached for their participation in the survey. These 12 colleges were selected purposively in a way that we covered medical colleges from all provinces where medical colleges are located. The minimum sample size was estimated using the following formula:Where,Z = 1.96 for confidence interval at 95%p = prevalence 50%q = 1-pe = margin of error 5%With a non-response of 20%, the estimated minimum sample size was 461. However, with a larger response than expected, the total number of participants enrolled in the study was 515 within a span of one week of the online survey (12 July to 18 July 2020). Ethical approval was taken from the Ethical Review Board of Nepal Health Research Council (NHRC) to conduct the study (reference number 2810).11 and questions exploring perceived experiences of online teaching-learning which were adapted from data collection tool from a similar study.12 The adapted rating scale consists of 12 items with five points Likert scale (0 to 4). Each of the twelve items with a mean score of ≥3.5 is true positive points; ≤2 are problem areas; between ≥3.5 and ≤2 indicate aspects that could be enhanced.11 The maximum score from the rating scale that an individual can obtain is 48 and are interpreted as follows: 0-12 (≤25% score)= very poor, 13-24 (25-50% score)= many problems, 25-36 (50-75% score)= more positive than negative, 37-48 (≥75% score)= excellent.11 Moreover, the adapted questions assessing perceived experiences of online teaching-learning explored the preference of online teaching-learning method over in-person, attendant of the online class, enjoyment during online class and its reason, duration of active enjoyment during online class, perception of optimal mix of the online and in-person class, and recommended duration of online class per day.The questionnaire used in the online survey assessed background characteristics and perception towards online teaching-learning. The part of the questionnaire assessing perception towards online teaching-learning comprised of: a rating scale adapted from the subscale of students' perception of learning of Dundee Ready Educational Environment Measure,Data collected via the online survey were imported to an excel sheet, cleaned, and analyzed in SPSS  version 20.The age of the respondents ranged from 18 to 29 years, with a mean age of 21.09±1.96 years. Out of the total 515 respondents, 284 (55.1%) were female; 146 (28.3% ) were enrolled in the first year; 324 (62.9%) were enrolled in medical colleges at Bagmati Province. While most of them 488 (94.8%) had their personal electronic device for attending online classes, a quarter 128 (24.9%) did not have a separate internet connection at their residence, and around one-third 162 (31.5%) did not have a separate room/space where they could attend online classes. Regarding self-rated computer skills, 216 (41.9%) responded as having only basic skills.Individual item-wise analysis of the rating scale, none of the items on respondents' perception of online teaching-learning scored more than 3.5 . FurtherThere was a difference in the score of respondents' perceptions of online learning across sex, provincial location of enrolled medical colleges, having a personal electronic device, having an internet connection at residence, having separate room/space for attending online classes, and self-rated computer skills .Only 5.4% of the respondents perceived online teaching-learning as a better method in medical education . Regardi3 One of the reasons for this negative perception and ineffectiveness in online teaching-learning was the lack of skill-building and laboratory sessions in this method of delivering medical education. This is consistent with medical students' attitudes in studies in various nations, including Pakistan, China, Malaysia, and Singapore.15 Moreover, differences in the score of perception of online teaching-learning were found across characteristics of students, including computer skills, availability of the personal electronic device, and internet connection at residence. This is logical as online teaching-learning requires reliable internet connection and sound skills on software and hardware.16 However, there was a similar score of perception of online teaching-learning among respondents of different enrollment years. This may be because whatsoever be the year of enrolment, the medical students in Nepal are on the same page regarding exposure to online teaching-learning, as it is a relatively new pedagogy method adopted in medical education in Nepal.The overall mean score of students' perceptions towards online teaching-learning in the current study was 17.61, indicating that there were many problems in the online teaching-learning method. The finding is supported by a study conducted in Pakistan where most medical students had a negative perception of online learning.17 Similar responses were observed in a study conducted among Polish medical students where the majority reported to be less active during online learning.18 The reason for this inactivity may be a lack of an interactive approach while preparing and conducting online medical education sessions.18 Similarly, only 26.5% of the present study students perceived online teaching-learning classes to be positive and helpful. The Pakistani study showed similar results where 14% of the medical students considered online learning impact to be positive.3The mean score of the item regarding getting encouraged to participate in online classes in this study was 1.29, indicating a problem area that needs to be emphasized while delivering medical education contents via the online method. The lack of effective participation in an online class was found among students in the current study, with 61.3% feeling lonely due to inadequate discussion during online sessions. This result is congruent to the study done in Pakistani medical students, where 84% of students felt increased isolation due to low student-teacher interaction during online teaching-learning sessions.3 This finding is also consistent with other studies where a majority of the students preferred face-to-face learning.20 However, studies conducted in Korea and Oman suggested that medical and nursing students preferred and were satisfied with e-learning and digital lectures.22 The reason for this discrepancy may be that online learning introduced during the lockdown phase in Nepal is new for medical education in Nepal as compared to medical education in Korea and Oman. Furthermore, lack of interaction with patients in the clinical setting and lack of clinical skill learning sessions may be the reasons for fewer adherences to a perceived preference towards online teaching-learning among medical students.Furthermore, the problem area in online learning indicated by the score of perception of online teaching-learning was also evident from the proportion of students (only 5.4%) who perceived online teaching-learning as a better method of learning in medical education. This finding is similar to the study conducted in Pakistan, where 85% of the medical students preferred traditional in-person teaching over online teaching.23 and this, in turn, may help generate enthusiasm in online learning among the students during the crisis situations like COVID-19 outbreak where the in-person teaching-learning activity is a challenge. However, as depicted from the current study's findings, medical colleges' management and faculty members need to frame online classes of not more than two hours session a day. Nevertheless, each online session should not be more than an hour based on an active engagement during an online class.To some extent, use of virtual laboratory materials and virtual patients can be utilized to train medical students and prepare them before encountering a real-life patient,12 This discrepancy in the level of enjoyment of online learning may be due to prior exposure to virtual learning in medical schools in high-income countries.Moreover, the negative perception towards online teaching-learning in the present study is also reflected by the proportion of the respondents (73.5%) who did not enjoy online classes. This finding is in contrast with the study done in the United States, where 59% of the medical students enjoyed online learning.The limitations of the study include the design of the study because of which the findings need to be interpreted with caution as it does not establish causation. Furthermore, due to the nature of the study being a web-based online survey, potential respondents who were not in access to the internet during the data collection phase might have been left out whose perspective would have been important for the study.Surveyed medical students in Nepal perceived many problems in online teaching-learning in medical education. The majority of the medical students did not perceive online teaching-learning as a better method of online teaching-learning. In this regard, medical educators in Nepal should emphasize addressing the problem areas in online teaching-learning methods, thereby enhancing medical education quality in an interrupted way during emergencies. Further study is recommended to determine the perception of teaching medical education via online platforms among faculties as teachers' perception and experience of delivering education contents via virtual platform is an important aspect to be explored to enhance the quality of teaching-learning methodology medical education.


Introduction: Gallbladder carcinosarcoma with osteoclast-like multinucleated giant cells is known to be most uncommon form of gallbladder cancer. Owing to its rarity, the pathogenesis of gallbladder carcinosarcoma with osteoclast-like multinucleated giant cells is largely unknown.Case Presentation: We present a case of carcinosarcoma with osteoclast-like multinucleated giant cells in the gallbladder. A 57-year-old woman visited our hospital due to jaundice. An examination revealed calculous cholecystitis and gallbladder carcinoma. After cholecystectomy, macroscopic examination disclosed one whitish mass and another distinct brown and pendulous mass in the body of the gallbladder. A pathological examination revealed that each mass had a different histological type: adenosquamous carcinoma and carcinosarcoma with osteoclast-like multinucleated giant cells. Immunohistochemistry revealed that these osteoclast-like multinucleated giant cells are CD68(+), CD163(−), and MIB-1(−). In addition, the osteoclast-like multinucleated giant cells showed the strong expression of RANK and sarcoma cells around the osteoclast-like multinucleated giant cells, were positive for RANKL. Furthermore, RUNX2 was positive for some sarcoma cells. The result indicated that osteoclastic and osteoblast-like differentiation occurred in our case.Conclusion: To our knowledge, this is the first case to show the interaction of RANK-RANKL signaling in gallbladder carcinosarcoma with osteoclast-like multinucleated giant cells. Gallbladder carcinoma is the most common malignant neoplasm of the biliary tract . The annGallbladder carcinosarcoma is a tumor that contains both epithelial and mesenchymal tumor cells. There are few report cases because carcinosarcoma is rare; however, our search of the PubMed database revealed more than 90 reports. Gallbladder carcinosarcomas have a poor prognosis and often infiltrate and metastasize to other organs. Zang et al.  reviewedGallbladder carcinosarcoma itself is rare, gallbladder carcinosarcoma with osteoclast-like multinucleated giant cells is even less common. Taskin et al.  reportedvia interaction with receptor activator of nuclear factor-κB (RANK) and RANK-Ligand (RANKL), which are known to be contained in the tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies. In bone tissue, osteoblasts secrete RANKL, and RANKL binds to RANK on monocyte/macrophage lineage progenitor cells for osteoclastic differentiation (It is known that the differentiation and activation of osteoclastic multinucleated giant cells are induced ntiation . Recentlntiation . In addintiation –10. In gRunt-related transcription factor 2 (RUNX2) is known to be a key transcription factor for differentiation and maturation process of osteoblasts from mesenchymal stem cells . It was We herein report a rare case of gallbladder carcinosarcoma and adenosquamous carcinoma. The former tumor consisted of spindle cell sarcoma with osteoclast-like multinucleated giant cells and adenocarcinoma. We further analyzed the expression of RANK, RANKL and RUNX2 by immunohistochemistry to investigate the role of RANK-RANKL signaling in this rare gallbladder neoplasm.A 57-year-old woman with a history of anemia and cataracts, noticed general malaise when taking Kampo medicine for approximately 3 weeks prior to her admission. Apart from this symptom, she had been healthy all her life and was a non-smoker with no history of alcohol consumption. She noticed yellow skin and her family doctor pointed out hyperbilirubinemia of her conjunctiva. She was then admitted to the Department of Gastroenterology of Gifu Municipal Hospital and was hospitalized on the same day in order to clarify the cause and treatment.Laboratory analyses on admission showed significantly elevated serum levels of CA19-9 , ALP , and γ-GT . Her serum total bilirubin level was also elevated . Abdominal contrast-enhanced computer tomography (CT) revealed swollen gallbladder with calculous cholecystitis and broadly rooted elevated lesions, suggesting carcinoma. After the removal of gallstones by endoscopic retrograde cholangiopancreatography, the patient underwent extended cholecystectomy.Two solid tumors were observed in the body region of the resected gallbladder . MacroscThe patient did not receive additional treatment such as chemotherapy after the operation, and no recurrence of the tumor was observed even at 6 months.A histopathological examination revealed that the two tumors have quite different microscopic features. The tumor 1  consisteIn the tumor 2 , atypicaThe intervening gallbladder mucosal epithelium between the two tumors was shedding partially and atrophic, and the gallbladder wall was thickened with mild inflammatory cell infiltration and fibrosis, indicating chronic cholecystitis. The dysplasia of gallbladder epithelium in the borders of the carcinosarcoma was observed, and it is a very localized area.Immunohistochemistry was performed to confirm our histopathological diagnosis. In the tumor 1, the squamous cell carcinoma component was positive for the expression of CK5/6 and p63, whereas the adenocarcinoma component showed negative for the expression of both antibodies. In the tumor 2, the adenocarcinoma component was positive for the expression of CKAE1/AE3 , EMA andFurther, to determine the property of the osteoclast-like multinucleated giant cells present in the carcinosarcoma, we performed an immunohistochemical analysis. The osteoclast-like multinucleated giant cells were CD68-positive  and CD16Additionally, to investigate the association of carcinosarcoma with osteoclast-like multinucleated giant cells and RANK-RANKL signaling, we performed immunohistochemistry with anti-RANK and anti-RANKL antibodies , as well as sarcomatoid hepatocellular carcinoma , 10, 25.To our knowledge, the expression of RANK and RANKL in gallbladder malignancy was analyzed for the first time, as described. Although the accumulation of further cases is necessary, our case gives new insight regarding RANK-RANKL signaling, which may contribute information about the nature and characteristics of the tumor.


An electron backscattered diffraction (EBSD) method provides information about the crystallographic structure of materials. However, a surface subjected to analysis needs to be well-prepared. This usually requires following a time-consuming procedure of mechanical polishing. The alternative methods of surface preparation for EBSD are performed via electropolishing or focus ion beam (FIB). In the present study, plasma etching using a glow discharge optical emission spectrometer (GD-OES) was applied for surface preparation for EBSD analysis. The obtained results revealed that plasma etching through GD-OES can be successfully used for surface preparation for EBSD analysis. However, it was also found that the plasma etching is sensitive for the alloy microstructure, i.e., the presence of intermetallic phases and precipitates such as carbides possess a different sputtering rate, resulting in non-uniform plasma etching. Preparation of the cross-section of oxidized CM247 revealed a similar problem with non-uniformity of plasma etching. The carbides and oxide scale possess a lower sputtering rate than the metallic matrix, which caused formation of relief. Based on obtained results, possible resolutions to suppress the effect of different sputtering rates are proposed. The2 layers , carbon 2 layers  or Ti-Alaminates . Usuallyaminates . The difaminates . The gloaminates ,21 or thaminates ,23. In tIn the present study, a glow discharge optical emission spectrometer (GD-OES) GD-Profiler fabricated by Horiba Jobin Yvon  was used as a tool for sample preparation via plasma etching for electron backscattered diffraction (EBSD) analysis. The used GD-OES device is a GD HR Profiler with RF electrodes. An anode fabricated of copper with the internal diameter of 4 mm was used. Thus, the sputtered area possesses a circle shape with the diameter of 4 mm. In this device argon pressure can easily be controlled and the available pressure range lies between 0 and 1100 Pa. However, it is known that the lowering of the Ar pressure significantly lowers the sputtering rate of metallic material due to the limited number of Ar ions in the plasma able to collide with surface of the material. Thus, an Ar pressure at the level of 300 Pa was adjusted. The studied materials included the steels: 1.4509 (AISI 441)  and 1.4521 (AISI 444)  and Ni-base superalloy CM247  with the chemical compositions observed in 2 of plasma-etched area enabled EBSD analysis. To study the potential of plasma etching using the GD-OES device as a fast method of surface preparation for EBSD analysis, flat, mechanically polished specimens of steel were investigated. For plasma etching, an anode with a 4 mm diameter of pure copper was chosen. Plasma etching was performed for 10, 20, 30 and 40 s. EBSD analyses revealed that the best results are obtained for plasma etching for 40 s. Thus, only the results obtained for 40 s plasma etching are shown. The image of the plasma-etched surface of steel 1.4521 obtained using the forward scatter detector (FSD) shown in 2O3 is present. In the middle part of the oxide scale TiTaO4 particles and Ni/Co/Cr-spinel formed, and at the oxide scale/alloy interface Al2O3 formed. The cross-section of the sample was subjected to preparation for EBSD analysis by GD-OES plasma sputtering. The obtained crater is shown in After validation of successful surface preparation by GD-OES on flat metallic surfaces, we attempted to prepare the cross-section of CM247 after oxidation. The microstructure of CM247 after air oxidation at 1050 °C for 20 h is shown in -Shortening of plasma etching time;-Lowering the plasma parameters, e.g., argon pressure;-Using so-called “pulse mode” to lower the sputtering rate.The results obtained on flat, mechanically polished, metallic samples clearly show that a surface etching by plasma sputtering using the GD-OES device can successfully be applied as a method for fast preparation of a surface for EBSD analysis. However, plasma etching was sensitive for the alloy microstructure and phase composition. As described by Niewolak et al. , steel 1All proposed solutions are the topic of ongoing work.Based on the results obtained in the present study, the following conclusions can be drawn:-Plasma etching by GD-OES is a very fast method for surface preparation for EBSD analysis of homogeneous metallic materials;-The plasma etching is sensitive to the presence of intermetallic phases as well as carbides due to a large difference in their sputtering rates;-Preparation of oxidized CM247 by plasma etching in GD-OES failed due to the difference in sputtering rate of the oxide scale and metallic matrix;Surface preparation by plasma etching using GD-OES is limited to the preparation of metallic materials without secondary phases such as oxides, carbides etc.