Herpes simplex virus type 1 (HSV-1) is the only FDA- and EMA- approved oncolytic virus, and accordingly, many potential oncolytic HSVs (oHSV) are in clinical development. The utilized oHSV parental strains are, however, mostly based on laboratory reference strains, which may possess a compromised cytolytic capacity in contrast to circulating strains of HSV-1. Here, we assess the phenotype of thirty-six circulating HSV-1 strains from Finland to uncover their potential as oHSV backbones. First, we determined their capacity for cell-to-cell versus extracellular spread, to find strains with replication profiles favorable for each application. Second, to unfold the differences, we studied the genetic diversity of two relevant viral glycoproteins . Third, we examined the oncolytic potential of the strains in cells representing glioma, lymphoma, and colorectal adenocarcinoma. Our results suggest that the phenotype of a circulating isolate, including the oncolytic potential, is highly related to the host cell type. Nevertheless, we identified isolates with increased oncolytic potential in comparison with the reference viruses across many or all of the studied cancer cell types. Our research emphasizes the need for careful selection of the backbone virus in early vector design, and it highlights the potential of clinical isolates as backbones in oHSV development. Genetic variation exists between herpes simplex virus type 1 (HSV-1) clinical isolates, with differences ranging between 2–4% on a nucleotide level . Based o134.5, coding for ICP34.5 neurovirulence factor. The underlying mechanism is reversion of protein kinase R activity, which, in the absence of ICP34.5, blocks virus replication in noncancerous cells [To date, HSV-1 represents the only approved modality for oncolytic virotherapy in the US and in Europe. The European Medicines Agency (EMA) and the Food and Drug Administration (FDA) have approved Imlygic, a replication competent oncolytic HSV (oHSV) used as a repetitive inoculation directly to the tumor to treat melanoma. Like the other oHSVs in the current clinical pipeline , Imlygicus cells ,10.In addition to the aforementioned possibility of gaining clinical safety with the attenuation of one gene, HSV also boasts other features that separate this delivery modality from other approved viral platforms. In short, (1) the genome does not integrate, (2) it can harbor large transgenes, (3) it is neurotropic, (4) it has built-in small molecule drug sensitivity, (5) HSV is relatively easy to produce at an industrial scale as a replication competent vector, and (6) pre-existing immunity does not prevent repetitive local delivery . InspireWhen designing an oHSV, many aspects must be taken into account, including the safety or efficacy resulting from the gene deletions, the possible transgene(s), the potential for combinatorial treatment, and additional delivery or targeting methods (reviewed in ). HoweveThe early design of the thus far only approved oHSV, T-Vec (Imlygic), began with the comparison of two clinical strains for their oncolytic potential . Both ofIn this study, we compare 36 clinical HSV-1 isolates from Finland with respect to their potential as parental strains for future oHSV or HSV gene delivery vector development. Based on our results, we can conclude that the spread and the replication profiles as well as the oncolytic potential of the circulating isolates are highly dependent on the cell type. However, we were able to identify circulating strains with increased oncolytic properties in contrast to all studied reference viruses  across many or all of the studied cancerous cell types. This observation highlights the potential of clinical isolates as oHSV backbones as opposed to reference strains, and it emphasizes the need for careful selection of the backbone virus in early vector design, depending on the future implementation of the HSV construct.Five different cell lines were used in this experiment. U373MG cells, currently reclassified as U251  but here referred to with their original name for continuity with earlier publications from the group, were maintained in DMEM  with 5% heat inactivated fetal bovine serum (FBS), 1% GlutaMAX , and gentamycin. HCE cells  and SW480 cells  were maintained in the same medium as the U373MG cell line. Vero cells  were maintained in M199 medium (Gibco), 5% heat inactivated FBS, and gentamycin. Raji cells  were maintained in RPMI 1640 (Lonza) with 10% heat inactivated FBS, 1% GlutaMAX, and gentamycin.In this study, we used 36 anonymous archival HSV isolates from the Department of Virology, University of Turku. The clinical isolates were of early passage (passage 2–4) and had previously been confirmed as HSV-1 with an immunoperoxidase rapid culture assay . Additio50) value of 1.9 μg/mL.ACV sensitivity was studied with a plaque reduction assay on Vero cells, as before ,7. The aTo determine the viral yield of each viral strain, Vero cells were infected with 5 plaque forming units (pfu) per cell of each virus on a 96-well plate. Samples were taken from the culture supernatant at 6, 24, 48, and 72 hpi. A plaque assay was used to determine the titers at each time point.For each cell line, the concentrations of both the extracellular and the cell-associated virus were determined in Vero, U373MG, and HCE cells, as previously described [Viral genes UL27 and US7, encoding the glycoproteins B (gB) and I (gI), respectively, were sequenced for areas with maximal variability between 17+ (JN555585.1) and the recently full-genome sequenced Finnish clinical isolates . The pri® viability assay . The assay was conducted on a 96-well plate format for U373MG, SW480, and Raji cells with 2 pfu/cell infections. At 96 hpi, the cellular viability was quantified with the CellTiter-Glo® viability assay according to the manufacturer’s instructions. The resulting cellular viability representing the oncolytic effect for each strain was determined against untreated samples.The oncolytic potential of the strains was determined with a test for cytotoxicity, CellTiter-Glop-value of < 0.05. The sigmoidal dose–response curves and their associated EC50 values were fitted and calculated with Origin 2016 v.b9.2.3.303 (Academic) . The sequences were aligned with ClustalW-algorithm and the phylogenetic trees were created in ML-tree format, utilizing the Bootstrap method with 1000 replicates.Statistical analyses were performed with SPSS statistics v.25.0.0.1 . The statistical significances were calculated with Mann–Whitney’s non-parametric U-test by comparing two individual groups at a time, with the threshold of significance set as a To determine the virus yield from the clinical isolates in Vero cells, the cells were infected with 5 pfu/cell and a supernatant sample was collected at 24, 48, and 72 h post infection (hpi) to quantify the released virus. The results are presented in a stacked graph . All of The spread of the clinical isolates was studied in human corneal epithelial (HCE), astrocytoma (U373MG) and Vero cells by determination of cell-associated and extracellular titers separately at 24 h after a 0.1 pfu/cell infection. The titers are presented in overlaid columns in In Vero cells A strainsIn U373MG cells B, no isoIn HCE cells C, strainIsolates V4, V5, and V12 all yielded a significantly higher titer for released and cell-associated virus in both HCE and U373MG cells, whereas V7 and V36 yielded significantly higher titers for cell-associated virus amount in both HCE and U373MG cells . The onlThe reference viruses KOS and F were rather similar to the reference virus 17+ used as a point of comparison, with the exception that in HCE cells they yielded lower titers of released virus than 17+ . The rec50) of 1.90 µg/mL, as before [50 value of 3.7 µg/mL. As the IC50 values for the clinical strains ranged from 0.14 µg/mL to 1.13 µg/mL, none of the isolates were resistant to acyclovir , and, similar to H1211 [TCACCCCCTCAACCACCA, SNPs underlined). The reference strains KOS, F, and 17+ had 2, 3, or 2 repeats, respectively. In 17+, a sequence following the repeat (TCCCCGCTCCCTCGACCACCA), which was detected before in 17+ [The differences in US7 were more abundant than those of UL27 . In the to H1211 , the fire in 17+ , was obsThere is no obvious grouping in either the US7 or the UL27 genotype in the sequenced regions, but there is some evident grouping with the reference strains in both genes . AltogetTo assess whether the strains can be divided by geographical genotypes, the Finnish strains with the known geographic associations were included in the analysis , adenocarcinoma (SW480), and B-cell lymphoma (Raji) . After aThe cytolytic effect of the clinical isolates was most pronounced in Raji and SW480 cells, where cellular viability decreased approximately by 65–95% and by 55–85%, respectively. For U373MG cells, the viability decrease was 10–40%. In U373MG and Raji cells, the reference strains were within the range of the clinical isolates. However, in SW480 cells, all reference strains had a weaker effect on viability than those by any of the clinical isolates . There wIn U373MG cells A, three The yield of extracellular virus was studied in Vero cells , the mosAll clinical isolates (V1-V36) replicated in Vero cells with relatively identical profiles, displaying the highest extracellular shedding at 2 days post infection and plateauing at 3 days post infection . In contIn all cell types and with all studied strains, the cell-associated virus titers were higher than the extracellular virus titers , demonstIn the neuroglioma cell line (U373MG), KOS and F were similar to 17+ , but theIn HCE cells, the reference viruses KOS and F were different from 17+, as they both yielded significantly lower titers of released virus C. HoweveWe expected that the clinical isolates, being more adapted to human epithelium, would have altogether demonstrated a more abundant infection in HCE cells. Therefore, it was surprising that so few clinical isolates displayed a more abundant replication in HCE cells in contrast to the reference strains. However, HCE cells represent the epithelium of the cornea and not the oral epithelium from where a majority of the strains are likely collected. Furthermore, the reference strains have been accustomed to the epithelial cell type, such as Vero cells, for a long time in vitro. Nevertheless, in Vero cells, hardly any clinical isolates were more efficient in replication than the reference strain 17+, whereas many strains with an elevated capability for replication and spread  were ideAltogether, the replication characteristics of the isolates were dependent on the cell type , Table 3All clinical isolates were sensitive to acyclovir . The acyN = 2–5) of a previously described US7 tandem repeat in the studied clinical isolates [Two genes contributing to cell-to-cell spread, UL27 and US7, encoding for gB and gI, respectively, were partially sequenced. The aim of the sequencing was to potentially explain some differences uncovered in the phenotypes of the clinical isolates . The cliisolates , the onlisolates  nor has isolates . Therefoisolates ,8, the gThe oncolytic potential of the strains was studied in cancerous cell types representing neuroglioma (U373MG), adenocarcinoma (SW480), and B-cell lymphoma (Raji) . After aThe reference strains had some differences in their oncolytic potentials in cell lines representing B-cell lymphoma and adenocarcinoma, but they were similar to each other in the neuroglioma cell line . In shorBased on the replication profiles of the isolates in U373MG cells and HCE cells, potentially highly oncolytic strains could be at least V4, V5, and V12, as in both cell types, these strains had a phenotype with abundant replication . V4 and In U373MG cells, the significantly more oncolytic strains were V13, V27, and V35, of which V27 was more oncolytic than all three reference strains A. V35, wIn this study we characterized 36 clinical isolates from Finland with the aim of finding potential oncolytic backbones for oHSV development. The 36 isolates had varying characteristics for replication and spread , Table 3As a conclusion, clinical isolates appear to have obvious advantages over reference viruses as oHSV backbones. From a selection of 36 isolates, we were able to identify isolates with increased oncolytic potential in many types of cancer. All of the uncovered phenotypes were unrelated to the studied genotypes, and they were rather associated with the cell type. The future steps for this research are to perform full genome sequence of these clinical isolates and to continue research with selected oncolytic isolates by engineering their attenuated counterparts to confirm whether the observed increased oncolytic potential remains, as is suggested by previous literature.


Observational studies from China suggest that Kangbingdu oral liquid (KBD) may be effective in treating the common cold.  Reevaluation of efficacy and safety of Kangbingdu oral liquid after marketing and expanding population. Setting. Eleven hospitals from 3 provinces in China. Patients were recruited through 11 centers, including 7 teaching hospitals, 2 University health services, one military clinic, and one community hospital. Patients. 2647 persons aged 18 to 75 years with Common cold. Intervention. Patients were randomly allocated to 2 groups: the treatment group Kangbingdu oral liquid  and the placebo group were divided into a standard-dose group of 10 ml every time, a middle dose group of 20 ml every time, high dose group of 30 ml every time, 3 times daily. Interventions and control were given for 5 days. Measurements. The primary outcome is the mean amount of total scores measured by the 11-primary symptoms: to observe the change of main symptoms from severe to disappear and to calculate and compare the mean amount of total scores after the periods of observation. Secondary outcomes are the disappearance rate of each symptom and the median time of body temperature returned to normal.  Prospective, Pragmatic randomized controlled trial (Chictr.org.cn registration number: chiCTR-TRC-12002399). P < 0.0001). Significant reductions in time of body temperature to return to normal in the Kangbingdu liquid group . 13 (0.7%) participants in the Kangbingdu liquid group and 1(0.2%) participants in the placebo group (P > 0.05) had treatment-related AEs, which mainly include diarrhea and dyspepsia in the Kangbingdu liquid group and constipation in the placebo group.  On day 5, the Kangbingdu liquid group had significant reductions in the mean amount of total scores measured by the 11-primary symptoms (7.39 [95% CI 7.26 to 7.51] compared to the placebo group (6.43 [95%: CI 6.24 to 6.62]). The Kangbingdu liquid can improve the remission rate of accompanying symptoms on day 5 including aversion to wind, aversion to cold, fever, cough, stuffy, runny nose, sore throat, muscular aches, headache, fatigue, and sweat  Kangbingdu oral liquid can effectively improve the comprehensive clinical symptoms of common adult cold, also improved main symptoms, including sore throat, muscle aches, headache, and so on. (2) Kangbingdu oral liquid effectively shortens the time of body temperature to return to normal. Colds with a longer duration, worsening, and frequency will prolong the presence of eosinophilic neutrophils in the nose, making asthma challenging to control and exacerbating COPD severity [A common cold is associated with significant morbidity and economic consequences. The common cold is the most common respiratory illness and medical condition . Rhinoviseverity –9.Kangbingdu (KBD) oral liquid, a classic traditional Chinese medicinal formula that is revised based on the traditional Chinese medicine (TCM) formulations of “BaiHuTang” and “QingWenBaiDuYin,” is widely used for the clinical treatment of influenza and common cold ,11.We conducted a prospective, randomized, double-blind, placebo-controlled trial at 11 medical sites in three provinces in China. The plan was included in the sample of 2647 cases in this test, using the stratified-block randomization method, with fever and no fever. Each subgroup met 100 samples, and the central randomization system was used to screen subjects, randomize subjects, and dispense drugs. The institutional review board of the Institute of Basic Research in Clinical Medicine (IBRCM) reviewed and approved the protocol and consent forms before the start of the study. All participants signed written informed consent forms before enrollment.A Consensus Recommendation from an Expert Panel for Primary Care Clinicians . They us∗109/L; had antibiotics or other anticold medicines continuously for 48 hours; had other lesions or feelings that reduced the possibility of complicating.Exclusion criteria were as follows:(1) Patients with pneumonia, bronchitis, otitis media, pharyngeal isthmus, viral myocarditis, acute nephritis, rheumatic joint disease, and other diseases.(2) Patients had hepatic and renal insufficiency; (3) Patients with allergies to clear antivirals in KBD components . (4) Patients were pregnant or lactating women; had intellectual or behavioral dysfunction and were unable to cooperate with the completion of the clinical observer. (5) During a month, patients had taken part in other clinical studies. (6) Blood routine detection indicated a white blood cell count above 12Isatis indigotica Fortune ex Lindl. of family Brassicaceae), Rhizoma phragmitis , Radix Rehmanniae  DC of family Scrophulariaceae), Radix Curcumae , Rhizoma Anemarrhenae , Rhizoma acori tatarinowii , Herba pogostemonis  Benth. of family Lamiaceae), Fructus Forsythiae  Vahl. of family Oleaceae), and Gypsum fibrosum  in a dry weight of 129 g, 61 g, 32 g, 25 g, 25 g, 25 g, 29 g, 46 g and 57 g, respectively. The criteria for the quality of the herbs we used were by the 2005 Chinese pharmacopeia. And placebo keeps the color and taste the same as KBD.KBD and placebo that we used in our study were manufactured by Guangzhou Xiangxue Pharmaceutical Co., Ltd. KBD is composed of 9 herbs Radix isatidis  None : no symptoms of the condition. (2)Slightly: the symptom is present but occurs infrequently or very mildly. (3) More clearly: the symptom is present but not severe, between mild and severe, and generally tolerable. (4) Severe: the frequency or/and intensity of the symptom is very severe and significantly affects work and life. None, slightly, more clearly, and severe were assigned according to 0, 1, 2, and 3 points, with a maximum total score of 44 points for the 11 major symptoms.Patients and researchers conducted the evaluations. Before the study begins, all 11 centers' researchers must complete standard operating procedures (SOP) training, and records must be kept in accordance with SOP. Patients in this study were given patient-administered diaries, and coordinators instructed them on how to fill the diaries according to the trial's protocols. A case report form(CRF) must be filled out by research.The treatment period from the first day to the fifth day, as well as the interview, were conducted over the phone by the coordinator from the second to the fourth day, with the first and last days requiring face-to-face visits, with the last visit being automatically postponed to Monday in the event of a weekend. Patients were given test drugs on the first visit, and researchers should remind them to return all of the leftover test medications and packing boxes on the last visit. Clearly document the amount of distributing medicines that patients used during the experiment if drug recovery was challenging.The sample size is not based entirely on statistical considerations. The clinical trial included 2800 cases and ensured that 2600 cases were effective. The trial will be over once the situation is satisfying.All analyses were based on the intention-to-treat (ITT) principle. For the primary outcome, we used a mixed-effect model with baseline value as a covariate, therapy, and site as fixed effects to examine the change in total symptom score from baseline on day 5. For missing data on the primary outcome, we employed the last observation carried forward (LOCF) approach.We used the Chi-square test or Fisher exact test to compare the disappearance rate of single symptoms at baseline and the log-rank test to compare the time it took for body temperature to return to normal for fever cases at baseline. Secondary outcomes were performed in the observed cases without imputation of missing data.P value of less than 0.05 considered significant. No adjustment was made for multiple comparisons and interim analysis.All analyses were performed with SAS version 9.4 , with a 2-sided n = 1986) or the placebo (n = 661) group. Among the randomized participants, 1913 (96.3%) in the group and 638 (96.4%) in the placebo group completed the study  (P=0.0022)  at baseline and 1.1  on day 5 in the Kangbingdu liquid group; the mean number of total scores measured by the 11-symptoms was 8.4  at baseline and 2.0  at day 5 in the placebo group. The reduction in total scores was greater in the Kangbingdu liquid group  than in the placebo group , with a mean difference of 0.96  . When co 0.0001) . The med=0.0022) .P > 0.05) had treatment-related AEs, which mainly included diarrhea and dyspepsia in the Kangbingdu liquid group and constipation in the placebo group. Two participants, all from the Kangbingdu liquid group, withdrew from the study because of adverse events   participants in the Kangbingdu liquid group and 1 (0.2%) participant in the placebo group (zziness) .To our knowledge, this trial is one of the few prospective, randomized, double-blind, placebo-controlled trials investigating the efficacy and safety of TCM clinical treatment for influenza and the common cold in China. We found that the improvement in common cold-related symptoms was signiThe common cold mainly affects the upper respiratory tract and is typically characterized by nasopharyngeal catarrhal symptoms. Sore throat, stuffy nose, runny nose, cough, and fatigue usually peak in 1–3 days and last 7–10 days but can last for several weeks ,15. CougTreatment focuses on symptom improvement. While nonsteroidal anti-inflammatory drugs do not reduce the total symptom score of cold patients, they also increase the score of some symptoms, such as sneezing . The nasInvestigation and analysis of medication for common colds among students in school revealed that 97.12% of medical students and 85.96% of nonmedical students chose the self-purchase medicine types containing “proprietary Chinese medicine” or “traditional Chinese medicine decoction,” indicating that the use of Traditional Chinese medicine in the treatment of colds is relatively popular among students in our school, and most students recognize the benefits. . StudentIn our trial, the Kangbingdu Liquid group signiﬁcantly performed better than the placebo group  in terms of total symptom score reduction on day five compared to baseline with a mean difference of 0.96 .The mechanism of TCM in the treatment of influenza is complex. A pharmacodynamic study of Kangbingdu oral liquid shows that Kangbingdu oral liquid has sound therapeutic and preventive effects on influenza A (H1N1) virus infection in vivo and in vitro . AdminisThere were 14 adverse events in our trial (13 in the Kangbingdu liquid group and one in the placebo group). The adverse events observed in the KBD group were also reported in other KBD studies or reports ,26, and There are certain limitations to our research. Our study participants were mostly young and had been clinically diagnosed with the common cold. They went to the hospital to get relief from their symptoms and prevent aggravating their symptoms, which could lead to the development of other ailments. Following that, we intend to conduct real-world research to determine the appropriate population of KBD and, based on that, utilize positive control medications to evaluate the antiviral effect of KBD in clinical trials.Through this trial, Kangbingdu oral liquid effectively improved the comprehensive clinical symptoms of an adult common cold, also improving the main symptoms including sore throat, muscle aches, headache, and so on. Kangbingdu oral liquid effectively shortens the time it takes for the body temperature to return to normal.


Citrus flavonoids are well-known for their beneficial effects at the cardiovascular and cardio-metabolic level, but often the encouraging in vitro results are not confirmed by in vivo approaches; in addition, the clinical trials are also inconsistent. Their limited bioavailability can be, at least in part, the reason for these discrepancies. Therefore, many efforts have been made towards the improvement of their bioavailability. Hydrodynamic cavitation methods were successfully applied to the extraction of byproducts of the Citrus fruits industry, showing high process yields and affording stable phytocomplexes, known as IntegroPectin, endowed with great amounts of bioactive compounds and high water solubility. The cardioprotective effects of grapefruit IntegroPectin were evaluated by an ex vivo ischemia/reperfusion protocol. Further pharmacological characterization was carried out to assess the involvement of mitochondrial potassium channels. Grapefruit IntegroPectin, where naringin represented 98% of the flavonoids, showed anti-ischemic cardioprotective activity, which was better than pure naringenin (the bioactive aglycone of naringin). On cardiac-isolated mitochondria, this extract confirmed that naringenin/naringin were involved in the activation of mitochondrial potassium channels. The hydrodynamic cavitation-based extraction confirmed a valuable opportunity for the exploitation of Citrus fruits waste, with the end product presenting high levels of Citrus flavonoids and improved bioaccessibility that enhances its nutraceutical and economic value. Citrus flavonoids are well-known for their beneficial effects at the cardiovascular and cardio-metabolic level [Citrus flavonoids naringin and naringenin, the latter being the aglycone of naringin, are promising nutraceuticals in the strategy regarding the management of cardiovascular complications, improving the systolic pressure levels and the metabolic profile [Citrus flavonoids, hesperetin and naringenin, were shown to be endowed with cardioprotective effects. For example, hesperetin protects the heart against the toxicity induced by doxorubicin treatment [ic level . Recentlic level . Likewis profile . In vitr profile ,5,6. How profile ,8,9,10. reatment , while nreatment ,12. Recereatment ,14.Citrus flavonoids’ nutraceutical value in the maintenance of cardiovascular and metabolic homeostasis, often clinical trials show inconsistencies, probably due to the poor systemic bioavailability of Citrus flavonoids. Many efforts have been made in order to improve their pharmacokinetic profile, mainly attempting to make secondary metabolites more soluble and more accessible at the intestinal level, via structural transformation  and pharmacological technologies [Despite the fact that numerous pre-clinical studies suggest nologies . Citrus fruits industry [Citrus fruits, complying with the principles of circular economy, as well as with the principles of green extraction of natural products [In the last few years, hydrodynamic cavitation (HC) technology has been successfully applied to the extraction of waste streams of the industry . This teindustry ,18. The products . Citrus pectins [Citrus pectins showed lower protective efficacy, if compared to IntegroPectin. The authors hypothesized that terpenes and flavonoids may be adsorbed on the pectic surface rich in RG-I “hairy” regions, containing galactose and arabinose units, making the biologically active secondary metabolites more bio-accessible [Grapefruit IntegroPectin showed remarkable water solubility, which was directly related to the cavitation-based extraction process, as well as in vitro bactericidal activity against both Gram-negative and Gram-positive bacteria, that was much higher than commercial  pectins . Even mocessible .In this study, we investigated, for the first time, the in vivo efficacy of grapefruit IntegroPectin in a model of myocardial ischemia/reperfusion injury.4× g rpm in 80 mL Falcon probes and freeze drying of the resulting surnatant. The newly obtained grapefruit IntegroPectin is a low pectin rich in adsorbed naringin and hesperidin [The grapefruit IntegroPectin was isolated via freeze-drying of the water phase extract upon hydrodynamic cavitation of waste peels of organic pink grapefruit, kindly donated by OPAC Campisi . The details of the hydrodynamic cavitation-based extractor, comprising a closed hydraulic circuit with a centrifugal pump and a Venturi-shaped reactor, with electricity as the only energy source, were described in a previous study about the extraction of waste orange peel . The detsperidin , as wellsperidin .Male Wistar rats of about 12–15 weeks and weight between 300 and 400 g were used. The animals were housed in cages, with freedom of movement, supplied with water and food, and exposed to light/dark cycles of 12 h each, at 22 °C. The study was carried out in line with EU legislation (EEC Directive 63/2010) and Italian legislation (Legislative Decree No. 26/2014) on the protection of animals used for scientific purposes. Formal approval to conduct the described experiments was obtained from the Animal Subjects Review Board of the University of Pisa (protocol number 491/2018-PR). With regards to the ex vivo approach, we discussed it and obtained the following protocol number: 45975/2016.The animals were treated with intraperitoneal injection (i.p.) of naringenin (100 mg/Kg), vehicle  or grapefruit IntegroPectin , 2 h before heart removal. 3 25.0 mM, Glucose 11.7 mM, NaCl 117.9 mM, KCl 4.8 mM, MgSO4 2.5 mM, CaCl2 2.2 mM, KH2PO4 1.2 mM; clioxicarb: 95% O2 and 5% CO2; pH 7.4; 4 °C). Then, the heart was set up on a Langendorff apparatus and perfused with Krebs saline solution at 37 °C and constant pressure of 70–80 mmHg, through a peristaltic pump . A latex balloon filled with bidistilled water at a pressure of 5–10 mmHg was placed inside the left ventricle through the mitral valve to monitor the functional parameters of the heart. The ischemia/reperfusion protocol consisted of 30 min stabilization time, 30 min of global ischemia and 120 min of reperfusion. At the end of reperfusion time, the heart was removed, dried, weighed, then the left ventricle was isolated. This was sliced into cross sections and immersed in a 1% w/v solution of 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich) dissolved in PBS (pH 7.4) (Sigma-Aldrich) for 20 min at 37 °C, in the dark. Finally, the slices were fixed in a 10% v/v aqueous formaldehyde solution. As a result, it gave the vital areas a red color. The animals were subjected to an i.p. injection of heparin (2500 I.U.) , and to a subsequent one of pentothal Sodium (100 mg/kg)  to induce a deep anesthesia. Each animal was sacrificed, and the explanted heart was immersed in a Krebs solution  and a data acquisition system . Recorded parameters were left ventricular pressure (LVDP), heart rate (HR), and contraction/relaxation time (dp/dt). The rate-pressure product (RPP) was calculated as the product of the first two parameters. The effects of the different doses of grapefruit IntegroPectin on cardiac functional parameters  were expressed in % of the basal values measured at the end of the stabilization period.The cardiac areas involved in ischemia/reperfusion damage, necrotic or apoptotic, remained white or pale pink. Ischemic area was calculated as % of left ventricle total area (Ai/Avs %) using the software GIMP .t-test, with p < 0.05 considered as an indicator of significant difference.All data were obtained from 6 different animals and graphed using the GraphPad Prism 8.0 program and expressed as mean ± standard error of mean (SEM). Statistical analyzes were performed with the Student’s The animals were sacrificed after isoflurane anesthesia . Then, the hearts were removed and immediately placed in isolation buffer  constantly kept ice cold.The heart was cleaned and finely chopped. Heart fragments were suspended in 10 mL of STE and homogenized using Ultra-Turrax homogenizer . The suspension obtained was subjected to centrifugations to isolate the mitochondrial component, taking care to keep it refrigerated during each step to preserve mitochondrial integrity and functionality ,24. Mito+) and a selective electrode coupled with a reference one , connected to data acquisition software . The electrodes were calibrated with known concentrations of TPP+Cl− before each experiment.The membrane potential (ΔΨ) of the isolated mitochondria was determined by a potentiometric method using the liposoluble cation tetraphenylphosphonium , with the addition of TPP+Cl− (30 μM). Grapefruit IntegroPectin was tested at concentration levels between 0.01 and 0.3 mg/mL and naringenin between 1 and 30 µM. The effects of the corresponding vehicle (DMSO 0.1%) were also evaluated. ΔΨ was calculated using the following fitted Nernst equation, as previously described [t-test using the GraphPad Prism 8.0 program , with p < 0.05 considered as an indicator of significant difference.The isolated mitochondria (1 mg of protein/mL) were kept on suspension with gentle and constant shaking in the incubation medium Δψ=602+ sensitive mini electrode  coupled to a reference one , using data acquisition software . Calibration curves were generated before each experiment using known concentrations of CaCl2. The electrodes were placed in IM with the addition of CaCl2 solution (100 μM) and grapefruit IntegroPectin at the concentration levels of 0.03, 0.1 and 0.3 mg/mL or naringenin 30 and 100 μM or vehicle (1% DMSO). Then, mitochondria (1 mg protein/mL) were added under gentle stirring and the changes in calcium uptake were evaluated by recording mV variations. Mitochondrial calcium uptake was measured with a Cap < 0.05 considered as an indicator of significant difference.Decreasing mV levels in the medium concentration of calcium was linked to its accumulation in the mitochondrial matrix. Each result was obtained with mitochondria isolated from the heart of 6 different animals. All data were expressed as mean ± SEM and were analyzed by GraphPad Prism 8.0. The data were statistically analyzed using the Student’s t-test, with An ischemic/reperfusion episode produced marked damage to the isolated hearts of vehicle-treated rats. In this regard, a decrease in the functional parameters of myocardial contractile function (RPP) and myocardial performance (dP/dt) was observed during the reperfusion time. At the end, the levels of RPP and dP/dt were 14 ± 5% and 21 ± 9%, respectively, of the levels observed in the pre-ischemic period, as shown in Based on the concentration level of the flavonoids in grapefruit IntegroPectin , we deciHearts of the animals treated with naringenin showed, at the 120th minute of reperfusion, an RPP level of 42 ± 9%, dP/dt level of 38 ± 15% and Ai/ALV level of 22 ± 2%, again meant as percentages of the respective levels observed in the pre-ischemic period, as shown in Citrus flavonoid naringenin, as shown in The addition of grapefruit IntegroPectin in isolated cardiac mitochondria, at the concentration levels of 0.01, 0.03, 0.1 and 0.3 mg/mL, corresponding to about 1, 3, 10 and 30 micromolar of naringin (the glycoside-derivative of naringenin), respectively, showed concentration-dependent depolarization, similar to the Citrus flavonoids can be, at least in part, the reason for these discrepancies. Indeed, Citrus flavonoids show poor water solubility, their bioavailability is low and they undergo extensive metabolism [Citrus fruits industry is represented by the amount of waste [Citrus flavonoids, such as naringin [Naringin and its aglycone naringenin have shown several beneficial health effects, including cardiovascular, metabolic and, recently, anti-SARS-CoV-2 effects , but thetabolism . These ptabolism . Anotherof waste . Indeed,naringin ; therefoCitrus fruits industry, complying with the principles of bioeconomy [Citrus fruits IntegroPectins) with higher water solubility, in which a great amount of bioactive compounds are adsorbed at the surface of pectin. Indeed, high levels of naringin (about 73 mg/g of extract) and other characteristic flavanones, as well as volatile compounds , have emerged from the phytochemical analyses [In this context, HC may be considered as an innovative green technique that allows the convenient exploitation of waste streams from the oeconomy . This teanalyses ,30. Citrus pectins have been characterized from the phytochemical and pharmacological point of view; however, they have not been found to contain relevant levels of polyphenols and their interest is mainly linked to the polysaccharide portion [Other commercial  portion ,31. Conv portion . Indeed, portion . This enIn order to explore this new product from a pharmacodynamic and pharmacokinetic point of view, we carried out an in vivo treatment through intraperitoneal injection and, 2 h later, the heart was explanted and set up on Langendorff apparatus to proceed with the ischemia/reperfusion protocol. Through this type of administration, bioactive compounds do not need to be adsorbed at the intestinal level; however, they must pass in the blood and be distributed among the organs and tissues, in order to carry out pharmacological processes; among these organs, the liver can be recognized as the main district responsible for the metabolism of flavonoids. Therefore, we can discuss, even if not exhaustively, the putative improvement of bioavailability. Moreover, we chose to evaluate the anti-ischemic cardioprotection that has been previously demonstrated for naringin and naringenin ,34,35,36A deeper insight into the mechanism of action in cardiac-isolated mitochondria confirmed that the flavonoid portion, most likely naringenin/naringin, is the main actor of this promising pharmacological profile, being involved in the activation of mitoK channels. Indeed, usually an activator of mitoK channels is able to promote mild depolarization of mitochondrial membrane potential to contain the accumulation of calcium ions in the matrix, thus reducing apoptosis and preserving the cell viability. Indeed, grapefruit IntegroPectin was shown to be able to induce moderate depolarization, in addition to a reduction in intramitochondrial calcium uptake.Citrus pectin may be due to the more favorable pharmacokinetics of naringin, at a systemic level, vehiculated by the highly soluble IntegroPectin acting as a drug carrier. Further studies will be aimed at verifying this hypothesis.In conclusion, the results of this in vivo investigation demonstrate that grapefruit IntegroPectin displays anti-ischemic cardioprotective activity that exceeds that of the pure bioactive flavanone naringenin on a dose-dependent basis. Investigation of the mechanism of action in cardiac-isolated mitochondria confirms that the flavonoid portion, most likely naringenin/naringin, is the main actor of this promising pharmacological profile, being involved in the activation of mitoK channels. We hypothesize that the enhanced anti-ischemic cardioprotective activity of this whole Citrus fruits, in particular from grapefruit, with the end product presenting high concentration levels of Citrus flavonoids and improved bioaccessibility that enhances its nutraceutical and, consequently, economic value. This study further confirms that the emerging extraction technique based on HC processes offers a valuable opportunity for the convenient exploitation of waste


Finally, soft-NMS is introduced in the prediction layer to optimize the final prediction results of overlapping pests. We evaluated the performance of our method on a large scale multi pest image dataset containing 24 classes and 25k images. Experimental results show that AgriPest-YOLO achieves end-to-end real-time pest detection with high accuracy, obtaining 71.3% mAP on the test dataset, outperforming the classical detection models  and lightweight detection models , meanwhile our method demonstrates better balanced performance in terms of model size, detection speed and accuracy. The method has good accuracy and efficiency in detecting multi-class pests from light-trap images which is a key component of pest forecasting and intelligent pest monitoring technology.Light traps have been widely used for automatic monitoring of pests in the field as an alternative to time-consuming and labor-intensive manual investigations. However, the scale variation, complex background and dense distribution of pests in light-trap images bring challenges to the rapid and accurate detection when utilizing vision technology. To overcome these challenges, in this paper, we put forward a lightweight pest detection model, AgriPest-YOLO, for achieving a well-balanced between efficiency, accuracy and model size for pest detection. Firstly, we propose a coordination and local attention (CLA) mechanism for obtaining richer and smoother pest features as well as reducing the interference of noise, especially for pests with complex backgrounds. Secondly, a novel grouping spatial pyramid pooling fast (GSPPF) is designed, which enriches the multi-scale representation of pest features  Crop pest control has always been a priority issue for agricultural producers, and plays an important role in guaranteeing world food security and normal economic development. Chemical pesticides have been an important weapon in the fight against pests for a long time, and agricultural workers need to configure pesticide species and doses according to the population dynamics of pests in real-world scenarios , to prevFortunately, with the development of information science, new problem-solving ideas are offered , namely via attention mechanism and contextual information to detect pest regions in maize with different growth cycles. In addition to targeting specific agricultural pests, some studies have reported advances in detection methods for multi-class agricultural pests. With the upgrading of hardware and software, the rapid development of deep learning has been driven. Compared with traditional machine learning, deep learning techniques based on Convolutional Neural Network (CNN) have the potential to become an effective approach to solve challenging tasks in intelligent pest monitoring because of their efficient feature self-learning and self-organizing strategy power . Object YOLOv5  is a staThe remainder of this paper is organized as follows. Section 2 contains a brief introduction of the material used in this study. Section 3 elaborates on the proposed pest detection method. Section 4 reports the comparison experiment results and analysis. Finally, we conclude our work and discuss future work in section 5.2We evaluate our methods on a pest dataset called Pest24 . Pest24 Data has a substantial impact on deep learning. In order to enrich the diversity of training samples in the Pest24 dataset, improve model robustness and avoid overfitting. As shown in 33.1S×S grids, and each grid is responsible for detecting targets in which the central point falls within this grid, and then generating prediction bounding box information and confidence scores through regression. The prediction parameters consist of the prediction bounding box information , the confidence score (Conf) and the probability of different classes (Pred). The confidence score is calculated as follows:YOLOv5 network is continuously optimized from YOLO series algorithm , which iPr(Object) is 1 when the predicted bounding box contains pest and 0 vice versa. where The main components of YOLOv5 network include backbone network, SPPF, neck, and head. There are five versions of YOLOv5 depending on the depth and width of the network, including YOLOv5n, YOLOv5s, YOLOv5m, YOLOv5l, and YOLOv5x. The lightweight model YOLOv5s has the better overall performance among the five versions and meets the demand of real-time detection. However, it ignores the particularity of pest images, so we propose an improved YOLOv5s network, AgriPest-YOLO, for real-time pest detection. The overall structure is shown in 3.1.1Backbone network mainly consists of Focus and CSP. Focus module is mainly used to perform slicing operation on the input image to assure adequate feature extraction. In the latest version of YOLOv5s, Focus module has been replaced by a 6×6 convolution operation  and Path Aggregation Network (PAN). PAN adds a new bottom-up pathway to the feature pyramid network to improve the utilization of feature information.3.1.4Same as YOLOv4, YOLOv5s has inherited the head structure of YOLOv3. In the training phase, the GIoU loss function  as regre3.2Attention mechanism can guide the model to pay attention to the region of interest instead of the whole image. The pest datasets generally have problems such as small size and complex background. Based on the idea of coordinate attention  and ParsX∈RC×H×W as input, where the feature map height and width are H,W, respectively, and the channel dimension is C. Firstly, one-dimensional pooling operations with kernel sizes  and  are used to encode along the horizontal and vertical directions of the input feature map, respectively, then the output of the c-th channel in height h and width w respectively can be denoted as follows:Given an intermediate feature map xc denotes the c-th channel feature map of the intermediate feature map X . xc , and contain the awareness information of xc in horizontal and vertical directions, respectively. Then both of the above generated feature maps are aggregated by transpose and concatenate operations, and a feature map where δ is the nonlinear activation function Leaky ReLu, Conv is the convolution operation with convolution kernel parameter of F is split into two different feature tensors, gh∈RC×H×W and gw∈RC×H×W , which are formulated as follows:where σ is the sigmoid activation function, Conv_h and Conv_w are the convolution kernel parameters as Where Next, local attention information is extracted along the channel dimension by local attention consisting of 1×1 convolutional blocks in the CLA module, which helps the network to detect locally distributed tiny pest targets. The local context X′ of CLA can be represented as:where C1, C2 are the convolution operations with convolution kernel parameters CLA attention module captures long-term dependencies with precise location information and different scale channel attention through the aforementioned behaviors, gains weight coefficients of effective pest features and filters background noise, which can effectively improve the recognition accuracy of pests in complex backgrounds.3.3Convs block, the input feature map is uniformly divided into four groups, each of which is a subset of the deep feature map i∈{1,2,3,4} . SPPF works on the latter three feature subsets with the purpose of fusing multiple receptive fields, improving the representational power of the feature subsets. The output yi−1 of the previous SPPFi−1 is added to the current subset vi and then fed into the SPPFi  to get the output yi of the feature subset. Hence, yi and the output Y of GSPPF can be written as:Spatial Pyramid Pooling (SPP) is a common module for YOLOv3,YOLOv4, constructed by four pooling operations with different kernel sizes for extracting salient feature information and improving the classification ability of the model. The spatial pyramid pooling fast (SPPF) in YOLOv5s is an improved version of SPP, which ensures feature information extraction as well as significantly improves computational efficiency, with 277.8% growth compared to SPP. To further intensify the network for multi-scale pest feature extraction, based on the concept of scale dimensionality , we desiGSPPF has the same position as SPPF in the network, and functions on the deep feature map that contains more semantic information but has lost part of the fine-grained information. The module intensifies the multi-scale representation at a finer-grained level. Through grouping the deep feature maps, each feature subset is treated as an independent feature map with SPPF to extract feature information and expand the receptive fields to achieve feature information reuse and multiple receptive field fusion of features of different scales, which enriches the feature map representation and facilitates the detection of complex multi-target and scale-variant scenarios in the pest dataset.3.4In the testing phase, the detector often generates multiple prediction bounding boxes for the same pest object, and the redundant prediction bounding boxes are filtered out in the post-processing phase of YOLOv5s with the NMS approach : selectiSi is the score of the i-th prediction bounding box, Bboxmax is the prediction bounding box with the highest score, bboxi is the adjacent bounding box, and the hyperparameter α is set to 0.5.where 3.5In the object detection task, the metrics commonly employed to evaluate the accuracy of the model are Precision, Recall, Average Precision (AP) and mean Average Precision (mAP). The specific calculation formula is as follows:C is the number of pest categories, which in this paper is 24. For each pest category in the detection, AP is the area under the Precision-Recall curve, APi is the AP of the i-th category, mAP0.5 is the average of the AP of all pest categories. AP50, AP55,…,AP90,AP95 are the mean of Precision under different Recall when taking different IoU thresholds (thresholds from 0.5 to 0.95 with a step size of 0.05), respectively. AP50:95 is the average of the ten values of AP50, AP55,…,AP90,AP95 . mAP@[0.5:0.95] is the average mAP under different IoU thresholds. In agricultural pest detection tasks, for more comprehensive and fairer measurement of pest detection model performance, AP and mAP are usually adopted as the main evaluation metrics. Thus, in this paper, we mainly discuss AP, AP50:95, mAP0.5and mAP@[0.5:0.95] as reference metrics.Where TP (true positive) represents the number of correctly detected pest targets, FP  represents the number of incorrectly detected pest targets, and FN  represents the number of missed pest targets. 44.14.1.1All experiments in this section were performed on one NVIDIA Tesla V100 GPU with 32G of memory. The software environment is Ubuntu 18.04, Python 3.8, and Pytorch. To accelerate the training process, NVIDIA CUDA10.2 and CUDNN7.6.5 neural network packages were used.4.1.2The default hyperparameters were set as follows: the initial learning rate was 0.01, the weight decay was 0.0005, and the momentum was set to 0.937. The remaining parameters were adjusted as follows: the batch-size was set to 16, the iteration period (epoch) was set to 300, and the input image size was set to 640×640 . The online data augmentation technique mentioned in Section 2 was used to preprocess the input images during the network training. Furthermore, this paper used transfer learning, based on the pre-training weights obtained from training YOLOv5s on the COCO dataset were used to initialize the model parameters with the aim of equipping the network with fast learning capabilities and generalization . Finally4.2@0.5, 3.5% improvement compared to YOLOv5s, 10.2% improvement compared to Cascade RCNN, the best performing model in the two-stage models. Moreover, to further investigate the localization performance of AgriPest-YOLO in pest detection, mAP@[0.5:0.95] of each model calculated at different thresholds are given in To assess the overall performance of the proposed pest detection method, we compared it with several advanced detectors, including classical object detection models: Faster RCNN, Cascade RCNN , DynamicThe goal of this paper is proposed a real-time lightweight detection model for achieving a sound balance between efficiency, accuracy and model size for agricultural pest detection. Hence, not only the accuracy of detection but also its size and detection speed should be discussed when evaluating the pest detection model. We compare GFLOPs, parameters, model size, and inference time of AgriPest-YOLO with other detectors  were present in the images, as shown in In this section, we visualize part of the pest detection results to directly observe the strengths of our proposed pest detection method, as shown in 4.64.6.1@[0.5:0.95] computed at higher thresholds, the proposed methods contribute to the generation of high-quality bounding boxes.Our proposed pest detector based on YOLOv5s model contributes four elements, including the attention and GSPPF modules developed, the introduction of Soft-NMS as a post-processing method, and online data augmentation methods to enrich the samples. To further investigate the impact of each element on pest detection, the results of the ablation study are given in 4.6.2Considering the special properties of pest detection tasks such as small targets, dense distribution, and complex backgrounds, it is necessary for the attention module to select the suitable activation functions for optimal detection performance. We selected several state-of-the-art activation functions including ReLu6, Mish, SiLu, and Leaky ReLu, and conducted multiple control experiments to investigate their impact on the pest detection task. Experimental results are reported in 4.6.3To enrich the diversity in the training samples in the Pest24 dataset and to improve the robustness of the detector, five online data augmentation methods were adopted, including HSV, Filp, Translate, Scale, and Mosaic. We performed multiple comparison experiments using the control variables method, adding one data augmentation method to the training model at a time, to validate the impact of different augmentation methods for the multi-category pest detection task. The results are shown in 4.7To further investigate the quality of AgriPest-YOLO, we have added different levels of Salt & Pepper noise and Gaussian noise in all images of the test set and analyzed the detection results to evaluate the robustness of AgriPest-YOLO. The Salt & Pepper noise will generate random white or black dots on the images. The noise level ranges from 0.005 to 0.05 in steps of 0.005. In addition, the mean value of Gaussian noise is 0.1 with standard deviation of 0.05. Experimental results are shown in 5To address the limitations of multi-class agricultural pest detection from light-trap images, this work proposes a lightweight pest detection method called Agripest-YOLO. As part of our proposal, a new attention mechanism was designed that can improve pest feature extraction, filter out worthless features, which can augment the detection performance of pests in complex backgrounds. Then, GSPPF was developed to represent multi-scale features of pests at a finer granularity level, achieve feature reuse and multiple fusion of pest features of different scales, enrich multi-scale representation of pest features, and thereby obtain better multiple scales pest detection performance. Experimental results show that AgriPest-YOLO outperforms other advanced methods in several aspects, improving the detection accuracy while considering lightweight and detection speed. Furthermore, AgriPest-YOLO has the advantage of recognizing densely distributed and complex background pests. And it has broad application prospects due to its lightweight design. However, AgriPest-YOLO still has limitations, such as for the extreme small pest, Rice planthopper (index 1) and Plutella xylostella (index 14), although it has been optimized, the detection accuracy remains poor. In the future work, we will try to further address the problem of extreme small pest identification and localization.The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.WZ: methodology, conceptualization, software, investigation, and writing draft. HH: supervision, writing and revising. YS: supervision, writing and revising. XW: validation. All authors contributed to the article and approved the submitted version.


Classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is a rare autosomal recessive condition characterized by cortisol deficiency and excess androgen production. The current standard of care is glucocorticoid (GC) therapy, and sometimes mineralocorticoids, to replace endogenous cortisol deficiency; however, supraphysiologic GC doses are usually needed to reduce excess androgen production. Monitoring/titrating GC treatment remains a major challenge, and there is no agreement on assessment of treatment adequacy. This study surveyed expert opinions on current treatment practices and unmet needs in adults with classic CAH.via online questionnaire. Survey questions were organized into three categories: practice characteristics/CAH experience, GC management, and unmet needs/complications. Anonymized aggregate data from Round 1 were provided as feedback for Round 2. Responses from both rounds were analyzed using descriptive statistics. Consensus was defined a priori as: full consensus ; near consensus ; no consensus .A modified two-round Delphi process with adult endocrinologists was conducted The same nine panelists participated in both survey rounds; five (56%) were based in North America and four (44%) in Europe. Most panelists (78%) used hydrocortisone in the majority of patients, but two (22%) preferred prednisone/prednisolone. Panelists agreed (89%) that adequate control is best evaluated using a balance of clinical presentation and androgen/precursor laboratory values; no consensus was reached on optimal timing of collecting samples for androgen testing or laboratory values indicating good control. Despite lack of consensus on many aspects of CAH management, panelists agreed on the importance of many disease- and GC-related complications, and that there is a large unmet need for new treatments. With currently available treatments, panelists reported that 46% of classic CAH patients did not have optimized androgen levels, regardless of GC dose.The limited areas of consensus obtained in this study reflect the variability in treatment practices for adults with classic CAH, even among clinicians with expertise in treating this population. However, all panelists agreed on the need for new treatments for classic CAH and the importance of many disease- and GC-related complications, which are difficult to manage with currently available treatments. Approl cortex . Severe The “classic” form of CAH is associated with severe 21-hydroxylase deficiency and occurs in approximately 1:14,000 to 1:18,000 births , 8.Androgen excess during childhood and adolescence raises the risk for precocious puberty or pseudopuberty, as well as accelerated somatic growth with advanced bone age, which results in below-predicted adult height , 7. DuriManagement of classic CAH presents unique challenges distinct from other forms of adrenal insufficiency –5, 8. ThThe purpose of this study was to survey expert opinions on current GC treatment practices and unmet needs in adult patients with classic CAH. The Delphi method, a systematic group communication process, was developed by the RAND Corporation in the 1950s to forecast the impact of technology on warfare and is well suited to assist in decision-making when evidence is incomplete, unclear, or unavailable –28. The 22.1Survey panelists from the US, Canada, and Europe were recruited by Evidera upon recommendation by Neurocrine Biosciences, Inc., the study sponsor. Participation was voluntary, but respondents were compensated for their time in completing the survey. Recruitment efforts for the survey panel focused on adult endocrinologists who frequently managed patients with classic CAH . Additional criteria for recruitment included involvement in publications on CAH, participation in CAH clinical trials, or participation in the development of CAH guidelines. Of the 21 panelists invited to participate in the survey panel, nine agreed to participate Figure 12.2Two internet-based survey rounds were conducted from September 2020 to April 2021 Figure 1The Delphi process traditionally begins with an open-ended (free-response) questionnaire in Round 1, but a common and acceptable modification is to use more structured questions if basic information on the target issue is available . In thisFor Round 2, panelists were provided anonymous aggregate data from Round 1  as feedback. Questions for Round 2 were refined as follows: 1) If there was general agreement in Round 1 responses, panelists were asked if they agreed or disagreed with the conclusion to establish consensus; 2) If there was variability in Round 1 responses, the question was re-circulated  for a second round of input from panelists.2.3a priori as follows: full consensus ; near consensus ; no consensus .Quantitative responses from both survey rounds were analyzed using descriptive statistics, including mean, standard deviation, range, and frequency. Qualitative data from the free-response questions were analyzed using key codes developed with clinical input from Neurocrine. For Round 2 survey results, consensus was defined 33.1Of the nine total panel members, five (56%) were based in North America, and four (44%) were based in Europe ; ✘ indicates no consensus . 17-OHP, 17-hydroxyprogesterone; A4, androstenedione; CAH, congenital adrenal hyperplasia; GC, glucocorticoid; ULN, upper limit of normal.Key Findings: Glucocorticoid Management in Adults with Classic CAH. ✔ indicates full consensus ; 3.2.1In Round 1, all nine panelists reported prescribing hydrocortisone in an average of 62% of patients, with individual responses ranging from 10% to 96% of patients Table 1.When asked in Round 2 to provide the average daily GC dose and/or average dose range used globally to treat patients with classic CAH, the mean hydrocortisone dose was 27.2 mg/day, and the mean hydrocortisone dose range was 14.2 to 40.8 mg/day Table 1.In Round 2, near consensus was reached that hydrocortisone is the most widely used GC globally (89%), and that dexamethasone should be prescribed at bedtime if given once daily (78%) Box 1.3.2.2When asked in Round 1 to provide what they considered to be the upper end of a physiologic GC dose with hydrocortisone, panelists reported a mean dose of 27.2 mg/day, with individual responses ranging from 15 to 40 mg/day – which was consistent with hydrocortisone dosing that the panelists reported using in clinical practice (see previous section). All but one panelist felt that the dose they indicated was reliable  or certain . In Round 2, consensus agreement was not reached, but six (67%) panelists agreed that 25-30 mg/day was the upper end for a physiologic hydrocortisone dose Box 1. T3.2.3In Round 1, panelists were asked to provide an appropriate hydrocortisone equivalency ratio to use when summarizing a data set . The mean  hydrocortisone equivalency ratios reported by panelists were as follows: dexamethasone, 43.1 ; prednisone, 4.4 ; prednisolone, 4.4 ; methylprednisolone, 4.8 . In Round 2, all but one panelist (89%) agreed that the Round 1 equivalency ratios for prednisone, prednisolone, and methylprednisolone were appropriate to use when summarizing a dataset Box 1. C3.2.4For A4, consensus was not reached in any subgroup on an appropriate laboratory value to indicate good control, but six (67%) panelists agreed in Round 2 that within ULN was appropriate in males with TARTs Box 1. TIn Round 2, near consensus (89%) was reached that the leading indicator of control is the balance of clinical presentation and laboratory values Box 1. B3.2.5Based on their own definitions for what they considered to be “optimized” androgen levels and “physiologic” GCs, panelists reported that almost half of their adult patients with classic CAH (46%) did not have optimized androgen levels, regardless of GC doses  panelists reporting making changes at least twice a year in these patients ; ✘ indicates no consensus . CAH, congenital adrenal hyperplasia; GC, glucocorticoid; TARTs, testicular adrenal rest tumors.Key Findings: Unmet Needs and Complications in Adults with Classic CAH. ✔ indicates full consensus ; 3.3.1In Round 1, the panelists were asked free-response questions to provide the most important short- and long-term unmet needs, as well as the most important unmet needs for classic CAH patients categorized by sex, age, and GC treatment optimization. Based on responses in Round 1, the unmet needs were categorized into three groups for Round 2 questioning:o Good androgen control/avoidance of hyperandrogenism o Managing/reducing supraphysiologic GC doses o Treatment-related needs (including simplified dosing and affordability)In Round 2, the panel ranked the importance of the three unmet needs categories from 1 “not at all important” to 5 “very important”. Near consensus (78%) was reached that good androgen control was very important for short- and long-term treatment, as well as for females, younger patients (age 18 to ≤55 years), and patients whose androgens were not optimized Box 2. C3.3.2In Round 1, panelists were asked to rank the importance of several disease- or GC-related complications from 1 “not at all important” to 5 “very important”. Based on the mean response from Round 1, panelists were asked in Round 2 if they agreed (yes/no) that the complication was “important/very important”  or “moderately important” . In Round 2, consensus (100%) or near consensus (78%) was reached that all of the complications related to cardiovascular and metabolic health, bone health, female health, and male health were important or very important, except dyslipidemia, which the panel agreed was moderately important (78%) Box 2. N4Despite the advances in the past several decades in the understanding of the genetics, pathophysiology, and treatment of classic CAH, many challenges remain in managing the condition , 8, 21. This study aimed to provide a view of expert opinions on current practices and unmet needs in the management of adult patients with classic CAH. The survey results showed some areas of agreement in GC management, including near consensus that hydrocortisone was the most widely used GC. Most panelists reported using hydrocortisone in the majority of their patients, but two panelists preferred the long-acting GCs, prednisone or prednisolone. These findings align with recent published reviews, which describe hydrocortisone split in two to three doses as the most common treatment option for adult patients due to its lower risk of adverse effects on metabolic, cardiovascular, and bone health , 3, 7. LA potential limitation of this study is the small number of panel participants, whose opinions might not reflect those of other endocrinologists who treat adults with CAH. In addition, the small number of panelists meant that consensus or near consensus would not be reached if only one or three panelists gave dissenting opinions, respectively, which could skew results. To ensure a representative sample of expert opinions, panelists from different institutional and clinical settings throughout the US and Europe were recruited who met academic and clinical criteria. However, classic CAH is a rare disorder; thus, there were few clinicians who met the study inclusion criteria of seeing at least 10-20 adults with classic CAH every quarter. Larger studies surveying a broader geographical range of expert opinions (beyond the US and Europe) may expand our findings and help to provide a more comprehensive, global view of adult CAH care. Financial support for the study was provided by Neurocrine, who is investigating crinecerfont, a corticotropin-releasing factor type 1 receptor (CRF1R) antagonist, for potential use in CAH. To mitigate potential bias introduced by the commercial sponsor, the survey questions were specifically designed to address a broad and comprehensive clinical approach to the management of classic CAH.www.i-cah.org) also revealed large variations in GC treatments and doses  and dexamethasone (1.5 vs 0.5 mg) , 7. Thesnd doses .2/day, or approximately 15-25 mg/day of hydrocortisone  might be an adequate physiologic dose , treatment goals, and side effects to guide shared decision making. This need for individualized treatment is also reflected in the relatively broad Endocrine Society treatment guidelines, which recommend the use of daily hydrocortisone and/or long-acting GCs plus mineralocorticoids for adults with classic CAH “as clinically indicated”, with limited guidance on treatment optimization or patient monitoring .Despite the lack of consensus on many aspects of CAH management, there was consensus agreement on the importance of many disease- and GC-related complications. In addition, all panelists agreed that there is a large unmet need for new treatments. With the currently available treatment options, panelists reported that almost half of their patients with classic CAH did not have optimized androgen levels, and another 29% had androgens optimized but were receiving supraphysiologic GC doses. Just 25% of patients were receiving physiologic GC doses and were perceived to have optimized androgen levels. These findings are in agreement with the previously mentioned cross-sectional studies in the UK and US, in which only 36% and 40% of adults with classic CAH, respectively, had normal serum A4 levels , 35.Newer therapies, such as modified release hydrocortisone preparations and alternative hydrocortisone delivery systems (continuous subcutaneous infusion), have been developed as alternatives to long-acting synthetic GCs –44. Stud5The limited areas of consensus obtained in this study reflect the variability in treatment practices for adults with classic CAH, even among clinicians with expertise in treating this population. The management of classic CAH is heterogeneous and varies widely by patient and provider; there is no single agreed-upon way to treat or manage classic CAH. However, this study found full consensus on the need for new treatments for classic CAH and the importance of many disease- and GC-related complications, which are difficult to manage with currently available therapeutic options.The original contributions presented in the study are included in the article/MF, CO’D, CY, and ML contributed to the conception and design of the Delphi methodology. RA, CC, AD, DE-M, HF, AL, and PT were expert panel members and survey participants. MA, KC, and NT collected and collated the survey responses. MA, KC, NT, and ML contributed to the analysis and interpretation of the results. All authors contributed to the drafting and critical revision of this manuscript and approved the submitted version.This study was supported by Neurocrine Biosciences, Inc., San Diego, CA.Survey responses were collected and collated by Evidera  and analyzed by Evidera and IQVIA , with support from Neurocrine Biosciences, Inc. . Medical writing and editorial services were provided by Prescott Medical Communications Group , with support from Neurocrine.MF, CO’D and CY were employed by Neurocrine Biosciences, Inc. MA, KC, and NT were employed by Evidera. ML, full-time employee of IQVIA. IQVIA received consulting fees for the advice of ML on this research. RA received research funding from Neurocrine Biosciences, Inc., Diurnal, LTD, and Spruce Biosciences; RA served as consultant for Neurocrine Biosciences, Inc., Crinetics Pharmaceuticals, OMass Therapeutics, H Lundbeck A/S, and Adrenas Therapeutics. HF served as consultant for Neurocrine Biosciences, Inc., Diurnal Ltd., Roche Diagnostics International Ltd., H Lundbeck A/S, and Adrenas Therapeutics. AL served as editor Adrenal Section, UpToDate.The authors declare that this study received funding from Neurocrine Biosciences, Inc. The funder had the following involvement in the study: Contracted with Evidera and IQVIA to conduct the Delphi surveys, collate data, perform statistical analyses, and assist with interpretation of data and preparation of the manuscript. Contracted with Prescott Medical Communications Group to assist with preparation of the manuscript.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


QEEG reflects neuronal activity directly rather than using indirect parameters, such as blood deoxygenation and glucose utilization, as in fMRI and PET. The correlation between QEEG spectral power density and Symptom Check List-90-R may help identify biomarkers pertaining to brain function, associated with affective disorder symptoms. This study aims at determining whether there is a relation between QEEG spectral power density and Symptom Check List-90-R symptom scores in affective disorders.This study evaluates 363 patients who were referred for the initial application and diagnosed with affective disorders according to DSM-V, with QEEG and Scl-90-R. Spectral power density was calculated for the 18 electrodes representing brain regions.Somatization scores were found to be correlated with Pz and O1 theta, O1 and O2 high beta. Whereas FP1 delta activities were correlated with anxiety, F3, F4, and Pz theta were correlated with obsession scores. Interpersonal sensitivity scores were found to be correlated with F4 delta, P3, T5, P4, T6 alpha and T5, and T6 theta activities. While depression scores were correlated with P3 and T4 delta, as well as T4 theta, there was a correlation between anger and F4, as well as T4 alpha and F8 high beta activities. Paranoia scores are correlated with FP1, F7, T6 and F8 theta, T5 and F8 delta, and O2 high beta activities.According to our results, anxiety, obsession, interpersonal sensitivity, depression, anger, and paranoia are related to some spectral powers of QEEG. Delta-beta coupling seems to be a neural biomarker for affective dysregulation. Kraepelin, who suggested the medical disease model for the first time in psychiatry a hundred years ago, included recurrent unipolar depression under the category of bipolar disorder and conceptualized the spectrum disorders . As currQEEG reflects neuronal activity directly rather than using indirect parameters, such as blood deoxygenation and glucose utilization, as in fMRI and PET. Additionally, QEEG has very high temporal resolution. Cortical discharges of 1.5 mV amplitude are amplified and decomposed with Fourier transformation. Spectral power density is the power EEG waves carry per unit frequency in a defined frequency range. QEEG eliminates the confusion originating from the need to take references. Therefore, this new approach is reported to yield more direct results .This study aims at determining the probable relation between QEEG spectral power density and Symptom Check List-90-R symptom scores of the newcomers diagnosed with affective disorders in psychiatry outpatient clinic.The sample of this cross-sectional study was composed of 816 patients who are drug-free but with psychiatric complaints, applying for the first time in the outpatient clinic of NPIstanbul Brain Hospital, between 2017 and 2018. Patients gave written informed consent in accordance with the Declaration of Helsinki. The Institutional Review Board of Uskudar University approved the study. All data and material archived at our institution according to Information and Consent Form on processing and protection of Personal Data.n = 35); presence of intellectual disability, dementia, delirium or other amnestic disorders (n = 8), and chronic medical diseases (n = 122).QEEG and Symptom Check List-90-R (Scl-90-R) are routine evaluation tools in our NPIstanbul Brain Hospital outpatient clinic. Scl-90-R is present in cross-sectional data with symptom profile. QEEG and Scl-90-R were applied to all consenting participants (n= 702). A total of 528 patients were diagnosed with affective disorders according to DSM-V criteria: depressive disorder, bipolar disorder, and no other specified (NOS). Exclusion criteria were as follows: being younger than 18 and older than 65 years of age  were used for reference. Recording time was 3 min. The data of each subject were averaged across the recording epochs for each electrode and absolute power was computed for the five bands. The article implements 18 electrodes by 128 frequency bins, ranging from 1 Hz to 30 Hz with a resolution of.0078 Hz. We implemented a normative database embedded in the Neuroscan software z-scores which calculates of spectral values. The Z-scores were used to correct individual variation. Spectral power density was calculated for the 18 electrodes representing brain regions .p-value of ≤ 0.05. Regression analysis is applied with ROC curve.QEEG data were analyzed using Neuroguide Deluxe v.2.5.1 . The statistical analyses were performed using the SPSS  20 software for 363 patients. The Kolmogorov-Smirnov test was used to check for normal distribution. The correlation analysis was performed with Pearson's correlation test according to normal distribution. The false discovery rate (FDR) was computed using methodology described by Benjamini and Hochberg. Significant results were determined based on an FDR-adjusted There are 363 cases, of which 207 are women and 156 are men, with mean age of 29.4 ± 5.1 .Somatization scores were found to be correlated with Pz and O1 theta, and O1 and O2 high beta ; whereasR2 = 0.79, p < 0.001),  are significant for dimension obsession, interpersonal sensitivity, depression, anger, and paranoia ( 0.001), .This study is the first one to investigate the symptomatology with spectral power density of QEEG in cases with affective disorders. Conventional EEG studies demonstrated EEG anomalies in 20–40% of cases diagnosed with major depressive disorder (MDD) .A slowed posterior dominant rhythm is characteristic for a major depressive disorder . SpecifiA correlation among anger scores, F4, and T4 alpha, as well as F8 high beta power, was found in this study. Alpha activity was interpreted in favor of bipolar depression when found together with beta activity, and in favor of mania, when it is correlated with beta power alone. As a matter of fact, frequency diversity is higher in the manic period than in remission and depressive period, while increased beta activity is frequently observed , 10.Paranoia scores were correlated with FP1, F7, T6, and F8 theta; T5 and F8 delta, O2 beta powers. No study on QEEG in affective disorders with psychotic features was found in the literature. Also, the frontal and occipitoparietal alpha activity that distinguished between psychotic and non-psychotic manic cases in our previous study was not found in consequential remission period in this study done on acutely presenting cases. In their hypothesis testing study, Buckley and Miller  reportedSuffin and Emory  studied The bodies of evidence summarized up to this point regarding affective disorders and QEEG are necessary but not adequate, especially for subgroups.Interpersonal sensitivity is a usual finding in affective disorders, especially in bipolar affective disorder type II . This stThere is a correlation between somatization scores and FP1 delta, F4 and F8 delta and theta, and T4, T6, and O2 theta activities. A predisposition defined as ‘dissociative mental state' in the literature is reported to coexist with posterior temporal and parietooccipital EEG abnormalities in 58% of the cases . The priObsession scores are found to be correlated with F3, F4, and Pz theta activities. Zielinska et al.  mentioneWe found correlations between anxiety scores and FP1 delta power. Studies comparing panic disorder cases with healthy controls , 24 poinTo summarize our findings up to this point, we suggest that beta power, which was associated with bipolar depression, mania, schizophrenia-like psychoses, OCD, phobia, and panic disorder with anxiety in the literature, which we accordingly found to be associated with depression, anger, paranoia, obsession, and phobia scores, is characterized with anxiety accompanied by hypervigilance. This mood reactivity was linked with irritability . On the Relationships between slow and fast wave frequency bands are considered to be of interest of dispositional affective traits as a continuity between depression and mania proposed first by Areteus, such as mania is a severer form of depression and anxiety,  accompanThese results are discussed with a focus on the correlations of spectral power density of brain regions and affective temperament in predicting risk, resistance, and resilience for bipolar disorder in our previous study . AffectiIn the mentioned study, all patients who are euthymic bipolar included in the study were on lithium prophylaxis. The aim of giving a uniform treatment was to standardize the prophylactic treatment. Whilst lithium normalizes beta, delta, and theta activities, treatment response is most closely associated with basal delta activity , 32. In Methodological differences, such as recording different brain regions, using different hardware configurations, complicate the replication of study findings and diminish the consistency of the normative database, in which an abnormal EEG rate of 12% is reported among healthy subjects . While tIn conclusion, a considerable number of authors have the opinion that QEEG findings are not clearly associated with specific diagnostic categories. At this point, the absence of evidence must be carefully distinguished from evidence of absence. However, a dimensional approach will yield more consistent results than categorical diagnosis. In this study we set forth relations with high internal and external consistencies about affective disorders. CFC delta-beta coupling seems to be a neural biomarker for affective dysregulation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board of Uskudar University. The patients/participants provided their written informed consent to participate in this study.SK conceived and designed the experiments, performed the experiments, and analyzed and interpreted the data with AM. SK wrote the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To describe clinical features and laboratory data of myocarditis after the mRNA COVID-19 vaccine in children.We reviewed patients younger than 18 years of age, who visited our hospital because of myocarditis within 1 week after BNT162b2 from June 2021 to January 2022.We identified five male patients aged 12–16 years who presented to our hospital with myocarditis within 2–3 days after the second dose of BNT162b2 COVID-19 vaccination between June 2021 and January 2022. All patients experienced chest pain, and fever, pain other than chest pain, and shortness of breath were present in two, three, and two patients, respectively. The serum troponin I level was increased in all patients except one, and electrocardiogram (ECG) showed ST elevation in all patients. Echocardiography revealed pericardial effusion and decreased ejection fraction in three and one patients, respectively. In accordance with the Japanese guidelines for myocarditis, the patients were treated with colchicine and aspirin. Chest pain improved within a few days with no hemodynamic instability. The patients were discharged with no sequelae.ST changes on ECG and elevated troponin I levels may aid the diagnosis of myocarditis after mRNA COVID-19 vaccination Vaccines are the mainstay intervention for controlling the SARS-CoV-2 pandemic. In Japan, the messenger RNA (mRNA) coronavirus disease 2019 (COVID-19) vaccine was approved for children aged ≥12 years in June 2021. Although the BNT162b2 vaccine is effective for children aged 12–15 years . In IsraIn Japan, the Ministry of Health, Labour and Welfare (MHLW) reported that myocarditis occurred in 3.7 per 1 million males aged 12–19 years after BNT162b2 vaccination . Some caOkazaki City Hospital is a tertiary emergency hospital that provides services to Okazaki City and Koda Town, which contain approximately 30,000 children aged 12–19 years. In this region, a COVID-19 vaccination program for children aged ≥12 years was initiated in June 2021 using the BNT162b2 vaccine. Children who developed myocarditis after vaccination would be expected to visit Okazaki City Hospital. Waiver of informed consent was approved by institutional review board of Okazaki City Hospital, because we retrospectively analyzed the existing data with no identifiable private information.This study is a retrospective case series including patients aged <17 years who presented to Okazaki City Hospital with myocarditis within 1 week after COVID-19 vaccination, between June 2021 and January 2022. We extracted clinical information from the medical records of hospitalized patients. In this study, myocarditis was diagnosed when a patient with one or more clinical symptoms including chest pain, pressure, discomfort, dyspnea, shortness of breath, pain during breathing, palpitations, and syncope had at least one or more of the following pathological findings: (1) elevated serum troponin level, (2) electrocardiogram (ECG) abnormalities consistent with myocarditis, (3) echocardiogram abnormalities such as reduced ejection fraction and wall motion abnormalities. Patients were excluded when there were other identifiable underlying conditions causing cardiac dysfunction. These conditions met are the criteria for probable myocarditis proposed by Gargano et al. . All patThe following information was collected from the patients' medical records: age, sex, presence or absence of chest pain, fever, shortness of breath, fatigue, pain other than chest pain, length of hospitalization, and treatment. We also recorded the serum C-reactive protein and troponin I levels (reference level: <28.6 pg/mL), and ECG and echocardiographic findings. The initial echocardiogram findings were recorded by the attending pediatrician and subsequently confirmed by a board-certified pediatric cardiologist within 2 days. In this study, ejection fraction of 50% or less was defined as reduced ejection fraction. Cardiac MRI with gadolinium enhancement was performed only in one patient, because abnormal findings were considered unlikely in most patients, judging from echocardiographic findings. Quantitative antigen test for SARS-CoV-2 was performed using nasopharyngeal swab in all patients. Systematic viral screening was not performed, although we asked the presence or absence of other prodromes such as viral infections.We identified five patients with myocarditis after mRNA COVID-19 vaccination during the study period . The medDuring the clinical course, an increased serum troponin I level was observed in all patients except one . In the At presentation, echocardiography showed small amount of pericardial effusion in three patients and slightly decreased ejection fraction in one patient. Cardiac MRI was performed in Patient 5 and showed no abnormal findings.According to Japanese guidelines for myocarditis, patients were treated with colchicine, aspirin, acetaminophen, loxoprofen, and famotidine. Chest pain improved within a few days, and none of the patients were hemodynamically unstable. The median duration of admission was 3 days (range: 0–8 days), and all patients were discharged with no sequelae.Patient 5 was a 12-year-old boy who presented with chest pain. His personal and family history were unremarkable. After the first dose of BNT162b2 vaccine, the patient experienced chest pain that lasted for several hours and resolved spontaneously. Two days before presentation, the patient received the second dose of BNT162b2 vaccine. One day after vaccination, he developed fever and fatigue, followed by chest pain on the day of presentation. At presentation, the patient's body temperature was 37.0°C, the heart rate was 82 beats/min, blood pressure was 102/57 mmHg, and the respiratory rate was 18 breaths/min. Physical examination was unremarkable. Laboratory tests revealed a white blood cell count of 8,700/μL, creatine kinase of 278 U/L (reference level: <248 U/L), troponin I of 1,369 pg/mL, and N-terminal pro-brain natriuretic peptide (NT-proBNP) of 261 pg/mL (reference level: <125 pg/mL). The ECG showed ST elevation in leads I, II, and V3–6 . EchocarIn the study region, a total of 22,557 individuals aged 12–19 years had received two doses of BNT162b2 by January 26, 2022 . AssuminMevorach et al.  reportedThe median interval from the second vaccination dose to the onset of myocarditis was 3 days in this study. Oster et al.  reportedIn the present study, chest pain was observed in all patients, and fever, shortness of breath, and pain other than chest pain were also common. Marshall et al.  reportedIn the present study, ST elevation was present in all patients with myocarditis. In the study of Marshall et al., all seven patients with myocarditis after mRNA COVID-19 vaccination had abnormal ECGs, and ST changes were present in 86% of patients. Oster et al. reported that 72% of patients with myocarditis after mRNA COVID-19 vaccination, including adolescent males, had abnormal ECGs. Although acute myocarditis may lead to sinus tachycardia with non-specific ST-segment and T wave abnormalities on ECG , the senThe serum troponin I level was elevated in all patients except one in this study. An increased serum troponin I level was observed at the initial presentation in all patients except Patient 3. Troponin I is a highly specific marker of myocardial damage, with high specificity but limited sensitivity for the diagnosis of myocarditis . Das et There were several limitations to this study. First, it was a retrospective, single-center case series. Thus, the results cannot be generalized. Second, the frequency of myocarditis may have been overestimated because our hospital is easily accessible for patients with myocarditis. In addition, none of the patients developed myocarditis after receiving vaccines other than BNT162b2; therefore, the frequency of myocarditis after other vaccine types could not be determined. Cardiac MRI was performed only in one patient in our study. Recent studies have suggested the usefulness of cardiac MRI in the diagnosis of myocarditis . Thus, wWe report five cases of myocarditis after mRNA COVID-19 vaccination. All patients were males and the median interval from vaccination to the onset of myocarditis was 3 days. All patients had chest pain, as well as ST and serum troponin I elevation, suggesting the usefulness of these parameters for diagnosing myocarditis after mRNA COVID-19 vaccination. Although myocarditis after mRNA COVID-19 vaccination appears to be mild, further studies are required to confirm this.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. Written informed consent was not obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.HMu contributed to the study design, interpretation of date, and was a major contributor in writing the manuscript. YZ, KS, HMi, NS, and NN contributed to the acquisition and analysis of date from the patients. HMo, HI, and AO revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript as submitted and agree to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.