Background: Oral squamous cell carcinoma (OSCC) is a devastating disease that is usually associated with a dense associated inflammatory infiltrate. Characterizing tumor-associated inflammation is critical to understand the pathogenies of tumor development and progression.Methods: We have tested a protocol to analyze tissue and salivary immune cells and mediators of 37 patients with OSCC at different stages and compared to eight chronic periodontitis patients and 24 healthy controls. Tissue analysis was based on fluorescent immunohistochemistry (FIHC) and inflammatory mediators were analyzed using a Luminex-based 30-Plex panel. Immune cells were analyzed using multichannel flow cytometry including CD45, CD66b, CD3, CD4, CD8, CD25, CD56, CD68, CD138, PD-1, and PD-L1.Results: We show an increase in OSCC-associated inflammation characterized by increased pro-inflammatory cytokines including IL-6, IL-8, TNFα, and GMCSF and increased salivary immune cells.Conclusion: We described a new method to analyze salivary inflammatory markers that can be used in future studies to monitor disease progression and prognosis. Oral squamous cell carcinoma (OSCC) is a neoplasm with squamous differentiation arising from the mucosal epithelium of the oral cavity  and accoPart of the challenges in understanding oral squamous cell carcinoma progression is the unique environment of the oral cavity which has well-developed innate and adaptive immune responses, frequent contact with the external environment, and exposure to carcinogenic factors along with a diverse microbiome . In thisSeveral studies have evaluated blood and saliva cytokines in head and neck squamous cell carcinomas. Recent studies found different salivary levels of some cytokines among oral cancer, OPMD, and healthy patients, suggesting these cytokines as potential diagnostic biomarkers for oral cancer and OPMD . Rhodus Cancer patients often have defective antitumor immunological responses ; despiteIn this study, we established a protocol for comprehensive analysis of the inflammatory response to OSCC in saliva and tissue samples, using both cytokines and immune cells compared to saliva of patients with a chronic oral inflammatory disease and healthy controls. We used the protocol to provide evidence that OSCC is associated with distinctive inflammatory response that could be observed in tissue and salivary cytokines.This was a prospective, case-controlled pilot study to investigate inflammatory changes in saliva in patients with OSCC. The study has been approved by the Research Ethics Board (REB) of Sunnybrook Health Sciences Center (project identification number 223-2015) and University of Toronto (protocol number 32724).The study population consisted of all patients referred for treatment of biopsy-proven OSCC at the Odette Cancer Center, Sunnybrook Health Sciences Center, Toronto, between September 2016 and October 2017. A total of 37 patients consented to participate in this study. A control group of 24 healthy patients with no history of oral cancer, as well as eight patients from the University of Toronto Faculty of Dentistry with moderate to severe periodontal disease and no history of oral cancer were included. The control patients were matched in age and sex with the OSCC patients. OSCC group: Inclusion criteria: biopsy proven OSCC referred for treatment at the Odette Cancer Center. Exclusion criteria: tumor location outside the oral cavity, non-squamous cell carcinoma, history of previous malignant disease, chemotherapy or head and neck radiation within the previous 5 years, presence of a second primary malignancy, immune disorders (such as Sjogren's syndrome and HIV infection), history of hepatitis, and severe systemic disease (ASA > 4). Patients with a biopsy confirmed OSCC that presented for surgery at the Odette cancer center but were found to have only premalignant lesions (dysplasia) and no invasive OSCC were included in the dysplasia group.The patients were recruited into four groups: (1) control patients without oral or systemic diseases, (2) Patients with a diagnosis of OSCC, (3) Patients with periodontal disease, and (4) Patients with dysplasia at resection. Control groups: healthy control patients with no reported oral disease or ongoing dental/periodontal treatment were included. Periodontal disease group: Periodontal disease patients from the Periodontal Disease Unit at the University of Toronto Faculty of Dentistry were only included if they had treatment appointments scheduled for diagnosis of moderate to severe periodontal disease, as recorded in their dental chart. Exclusion criteria included: history of previous malignant disease, chemotherapy or head and neck radiation within the previous 5 years, presence of a second primary malignancy, immune disorders (such as Sjogren's syndrome and HIV infection), history of hepatitis, and severe systemic disease (ASA > 4).As a pilot project, our first objective was to test a protocol for collection and analysis of saliva samples. The collection protocol using 3 ml of a saline rinse was well-tolerated by all patients and all patients consented to the collection of saliva after the study and protocols were explained. The collection was carried during the pre-surgical consult and did not cause any disruption to standard treatment protocols. The filtered saliva supernatant was adequate for cytokine analysis as all samples passed initial quality control and were within the range of the Luminex test. The establishment of FMO and single stain controls required the use of a control saliva sample as blood controls showed different staining patterns compared to the saliva samples. To overcome this issue, a single donor collected multiple saliva samples that were combined and used to generate all FMO and single marker controls.All patients were recruited during a pre-treatment oncology appointment at the Odette Cancer Center, Sunnybrook Hospital, Toronto. After informed consent was obtained, a short medical questionnaire and intraoral examination were performed. Saliva samples were obtained by rinsing the mouth for 30 s with 3 ml of saline. The samples were kept on ice and stabilized with 100 μl of protease inhibitor  until filtration using a 40 μm membrane filter. Time interval between collection and filtration of samples ranged from 20 to 150 min. The saliva samples after filtration were centrifuged  in the Sunnybrook Hospital Clinical Pathology lab to obtain cell pellets and supernatants. The latter were frozen at −80°C until use. Cell pellets were re-suspended and fixed in 200 μl paraformaldehyde 4% and PBS and kept at 4°C until use for a duration ranging from 1 to 12 months . Control6 cells/ml with 300 μl ice cold FACS buffer. Two Hundred microliters of Fc receptor blocking solution (5 μl of TruStain FcXTM per million cells) was added to each sample (Fc blocking solution 1% BSA and 1% FBS diluted in PBS buffer) and incubated on ice for 10 min. Samples were centrifuged at 1,500 rpm for 5 min at 4°C. Supernatant were discarded. Primary labeled antibodies were added and incubated for 20 min at room temperature in the dark. Cells were washed three times by centrifugation at 1,500 rpm for 5 min and resuspended in 1,000 μl of ice cold FACS buffer and kept in the dark at 4°C until analysis. Vortex and a final filtration (10 μm filter) were done immediately before FCM to ensure even distribution of the cells in the samples and to minimize cells and particles aggregates.A 12-antibody panel  was optiFluorescent-labeled saliva samples from the study and control patients  were analyzed in a flow cytometer (BD Fortessa x20 5 laser) in three sessions. For each FCM session, the instrument was calibrated with rainbow beads (BD Bioscience), to ensure that the voltage applied to lasers/detectors always returned the same median fluorescence. Compensation beads stained with the 12 fluorescent antibodies were used to set voltage and adjust compensation matrix. Full minus one controls (FMOs) for each of the 12 stains, as well as one fully stained control and one unstained control were prepared, using saliva provided by a single healthy donor. The instruments were calibrated with the same parameters for each session. Assays were conducted in 96-well plates according to the immunoassay protocol for the Millipore Human Cytokine/Chemokine Magnetic Bead Assay Panel   and Luminex? detection. The samples were prepared, and the data acquired and analyzed at the Princess Margaret Genomic Center  according to the manufacturer's instructions. Briefly, 25 μl magnetic beads, 25 μl assay buffer, and 25 μl of sample (1:2 diluted sample) were incubated overnight at 4°C with shaking. Beads were subsequently washed 2 X times and incubated with 25 μl of detection antibody for 1 h. Twenty-five microliters streptavidin phycoerythrin was added to the assay mixture for 30 min at room temperature. Beads were washed 2X and resuspended in 150 μl of Sheath Fluid. Assays were read with Luminex 100 Reader and data was analyzed using Bio plex Manager 6.0. Analyte concentrations are indicated in pg/ml units. From the 69 patients included in this cytokine analysis, 30 samples (17 cancer patients and 13 controls) were used as part of a previous publication [A total of 25 cancer cases were available for analysis and 10–15 tissue slides were prepared from the resection specimens. Fluorescent immunohistochemistry (FIHC) was performed as described previously . In brieDescriptive statistics, including means and standard deviations, were calculated for the outcome variables. These statistics were reported for the whole sample as well as separately by the four groups of interest. The Shapiro test of normality was run on the outcome variables and following this, non-parametric Kruskal-Wallis tests were used to examine whether there are significant bivariate associations between group membership and potential predictor variables. To explore differences among groups, multivariable logistic regression was applied to examine the relationship between group membership and predictors of interest. In the logistic regression model, group membership was dichotomized as Cancer vs. the others. Prior to modeling, multicollinearity among the predictors was examined using tolerance statistics . If multicollinearity was found to exist, then only one member of a correlated set of variables was retained for the final model. The model results were reported as odds ratios and their associated 95% confidence intervals. All analyses were carried out using SAS 9.4 . Correlation analyses and graphs were generated using GraphPad Prism 7.0 and IBM SPSS version 25. All figures were prepared using Adobe Illustrator CC 2019.th ed. based on the pathology report of the tumor resection; clinical staging was only used for two patients treated with primary chemoradiation and two patients did not have definitive treatment at the Odette Cancer Center (P = 0.07). There were more females in all groups except periodontal disease group (P = 0.049). Further analysis of dysplasia and cancer patients reveal that the most commonly involved site in all groups was tongue  followed by gingiva . 86.4% of lesions were painful and 40% of patients had abundant visible dental plaque. The presence of visible plaque and daily tooth brushing was significantly increased in periodontal disease patients compared to cancer patients (P = 0.0498 and P = 0.0333). There were no significant differences in alcohol consumption or smoking between groups and between cancer stages. Most dysplasia and cancer patients had a normal appearing remaining oral mucosa  while 18.9% (7/37) had other areas of leukoplakia , IL-6 , Rantes/CCL5  and MIP1α (CCL3)  in OSCC patients compared to control and periodontal disease patients. TNFα (p = 0.0038), MCP1 (CCL2) (p = 0.0493), IL-1β (P = 0.0043), and IL-8 (p = 0.0035) were increased in OSCC compared to control only while MIP1β (CCL4) (p < 0.0365) was increased in OSCC patients compared to periodontal disease patients. IL-10 (p = 0.0364), IL-12p40 (p < 0.0364) were statistically significant but multiple comparison analysis did not show significant differences between groups. There were no significant differences between the dysplasia patients and OSCC patients.Thirty cytokines/chemokine were evaluated in the saliva samples and the results are reported as the fold change of the specific cytokine over control in a log2 scale . There wp = 0.0040, odds ratio 3.991). Similar analysis including IL-1β, Rantes/CCL5, MCP1, GMCSF, TNFα, and IL-6 showed similar results with elevated IL-6 increasing the probability of having a cancer diagnosis (p < 0.0046)  was performed. The first logistic regression model including IL-6, IL-8, and TNFα showed that elevated IL-6 significantly increased the likelihood of having a diagnosis of cancer ( 0.0046) .r > 0.8 and significance of p < 0.01. The selected cytokine correlations were analyzed using Cytoscape to create an interaction map in control  but not periodontal disease patients . We also calculated the total number of identified cells  and OSCC patients showed significantly more cells  compared to periodontal disease  and control patients . Cancer patients also had more neutrophils (p = 0.04) and lymphocytes (p = 0.05) compared to control patients. There were very few immune cells other than neutrophils and T lymphocytes in saliva, preventing further characterization of the cells  followed by lymphocytes (23.98% ± 29.59). The cancer group had the highest number of immune cells (CD45+) in saliva  and was significantly higher than control patients .P = 0.0262) in high Stage (III–IV) tumors compared to low stage (0-II) tumors (P = 0.038) (P = 0.038) (P = 0.219) and CD3+CD8+ (p = 0.33) T cells (We investigated neutrophils (CD45+ CD66b+), CD8+ T cells (CD3+CD8+), and CD4+ T cells (CD3+CD4+) in addition to PD-1 and PD-L1 expression in 25 tissue samples that were available from the cancer cohort . To norm) tumors . There w= 0.038)  and CD45= 0.038)  cells in T cells .p = 0.309) and presence of metastasis .The patient outcomes are summarized on Understanding the characteristics of the inflammatory infiltrate in oral cancer is essential to determine pathogenesis and new strategies to predict outcomes and control the disease. In this pilot study, we have created and tested a protocol to collect saliva samples for a comprehensive analysis of inflammatory cells and mediators and used it to study the inflammatory signature of oral cancer patients compared to control and periodontal disease patients. Our protocol is based on a single collection that can identify 30 cytokines and was also able to identify cell surface markers in salivary immune cells. This pilot study allowed us to optimize a non-invasive collection and analysis and the results will be used to create a large clinical study to characterize the salivary inflammatory signature of oral cancer patients. The protocol also allows for repeated sampling.The analysis of inflammatory cell profile in saliva by FCM is challenging, and we believe this is the first report of its kind. Saliva inherently contains debris and proteases that contribute to sample degradation and formation of aggregates. To minimize this, we used multiple filtrations prior to processing the samples. The use of formalin has the potential to alter antigen availability, therefore, compensation was done using fixed saliva from the same donor and the antibodies were tested in both fixed and fresh saliva. To determine the different cell populations and exclude debris, gating was carefully done using forward and side scatter and by applying multiple markers/gates for each cell. An important limitation of studying inflammatory cell profile in saliva by FCM is the number of cells available in each sample, particularly in control patients. By vigorously rinsing with saline, we were able to collect at least 100,000 inflammatory cells per sample which made FCM analysis feasible. To achieve reproducible results, we had to have access to filters, fixatives, protease inhibitors, ice, and a centrifuge adjacent to the collection area. This allowed us to minimize the time between collection, processing, and storage which was critical to achieve consistency.Our results show an increased number of inflammatory cells and a distinct change in inflammatory mediators in the saliva of cancer and periodontal disease patients compared to control. The levels of salivary inflammatory mediators GMCSF, IL-6, Rantes/CCL5, MIP1α (CCL3), and MIP1β (CCL4) were elevated in OSCC compared to periodontal disease which supports that the inflammatory response in OSCC is specific and different from other causes of chronic inflammation such as periodontitis in the oral cavity. The changes in immune cells in saliva of OSCC patients were primarily due to an increase in total number of cells and not from specific shifts in different cell populations. This is potentially due to the fact the highly motile neutrophils would extravasate to saliva while most other inflammatory cells would stay attached to the stroma or connective tissue, therefore the changes in immune cells are expected to be seen in the tissue and not in saliva. The findings also raise the possibility that neutrophils in saliva are primarily caused by the presence of ulceration rather than a specific recruitment mechanism. Several findings challenge this hypothesis. First, there was no difference in the number of salivary neutrophils in lesions that were primarily ulcerated vs. non-homogeneous hyperkeratotic lesions or exophytic lesion. Second, there were no significant differences between the number of salivary neutrophils in different cancer Stages, suggesting that tumor size alone does not explain the observed neutrophilia. Finally, some patients with non-ulcerated lesions in periodontal disease showed similar levels of salivary neutrophils compared to cancer patients, suggesting that ulceration alone also does not explain the increase recruitment of neutrophils. Our data supports the conclusion that the increased number of immune cells in saliva of OSCC patients is due to the recruitment of inflammatory cells to the tumor microenvironment  which thin vitro [Our results suggest a markedly pro-inflammatory immune environment in OSCC. Signaling pathways downstream of growth factors and cytokines such as interleukin IL-1β, IL-6, TNFα, vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) , 25, 26 in vitro . Previouin vitro –31. Specin vitro . Our resThis pilot study shows that salivary and tissue analysis of a broad panel of inflammatory mediators and cells is feasible and can be used to characterize in detail the immune responses that are unique to OSCC. Despite the small sample size, our results indicate that OSCC patients show an increase in immune cells in the tumor microenvironment and a unique salivary cytokine profile. These findings can be used to design clinical tests that could be used as part of treatment decision making; such assays can help to identify patients at higher risk of developing disease adverse events and recurrence. For example, OSCC patients with elevated salivary pro-inflammatory chemokines/cytokines after definitive treatment may indicate higher risk of recurrence or persistent disease activity. This could be incorporated as part of the regular follow up visits. Regarding the development of unique cancer therapies, there are numerous ongoing studies in different cancers targeting chemokine networks and immunomodulatory pathways in cancer treatment as recently reviewed in , 33. TheSaliva is easy and quick to collect as per our protocol, making it a potential non-invasive, easy to perform, saliva-based test. Using our collection protocol, saliva collection can be performed in a variety of clinical settings by various health professionals, regardless of clinical expertise for assessing presence of oral cancer. Lastly, in the era of immunotherapy, finding biomarkers to identify cancer patients eligible for immunotherapy is urgent. In this study, we set the basis on which we can build to further explore the characteristics of inflammatory mediators in the saliva and their value as predictive markers.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 Research Ethics Board (REB) of Sunnybrook Health Sciences Center (project identification number 223-2015) and the University of Toronto (protocol number 32724). The patients/participants provided their written informed consent to participate in this study.CL completed all patient recruitment and data collection and helped in the data analysis. MM has conceptualized the project, analyzed data, and wrote the manuscript. NN has completed all the tissue analysis experiments. AK completed all statistical analyses. KH has provided clinical support to recruit patients.GB participated in the creation of the project and manuscript review. DE and AA have provided technical support and development. All authors contributed to the article and approved the submitted version.This study was funded by the University of Toronto, Faculty of Dentistry, Oral Pathology Department research fund.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.


Premature ventricular contractions (PVCs) may increase during pregnancy, however, few studies have evaluated the relationship between PVCs and the pregnant outcomes.PVCs may increase the adverse fetal/neonatal outcomes in pregnant women.Six thousand one hundred and forty‐eight pregnant women were prospectively enrolled in our center between 2017 and 2019 in the study. The average PVC burden was determined by calculating the number of PVCs in total beats. Those who had a PVC burden >0.5% were divided into two groups based on the presence or absence of adverse fetal or neonatal events. The adverse outcomes were compared between the groups to assess the impact of PVCs on pregnancy.A total of 103 (1.68%) women with a PVC burden >0.5% were recorded. Among them, 17 adverse events (12 cases) were documented, which was significantly higher than that among women without PVCs . The median PVC burden among pregnant women with PVCs was 2.84% (1.02%–6.1%). Furthermore, compared with that of the women without adverse events, the median PVC burden of women with adverse fetal or neonatal outcomes was significantly higher . Multivariate logistic regression analysis demonstrated that not the LVEF, heart rate and bigeminy, but only the PVC burden was associated with adverse fetal or neonatal outcomes among pregnant women with PVCs .Frequent PVCs have adverse effects on pregnancy, and the PVC burden might be an important factor associated with adverse fetal and neonatal outcomes among pregnant women with PVCs. An estimated prevalence of 1%–4% is found in the general population on standard 12‐lead electrocardiography (ECG).22.1This is a single‐center prospective cohort study of consecutive pregnancies referred to The First Affiliated Hospital of Nanjing Medical University from July 2017 to July 2019. The inclusion criteria were pregnant women with normal cardiac structure and function based on echocardiographic examination. Pregnant women with PVC burdens greater than 0.5% on Holter examination each time during the whole pregnancy period were classified as the PVC group. The exclusion criteria were as follows: Pregnant women with (1) hyperthyroidism, (2) hypothyroidism, (3) gestational hypertension or pregnancy with hypertension, (4) Type I or II diabetes, (5) chronic nephritis or impaired renal function , (6) autoimmune disease, (7) congenital heart diseases, (8) pulmonary artery hypertension, (9) cardiomyopathy, (10) alcohol abuse, (11) anemia (Hgb <110 g/L), (12) smoking, and (13) a family history of sudden cardiac death. The study protocol was performed in accordance with the ethical standards established in the 1964 Declaration of Helsinki and its later amendments and was approved by the institutional ethics committee of The First Affiliated Hospital of Nanjing Medical University (2019SR‐503). Informed consent was obtained from all pregnant women enrolled in the study.2.2Clinical and 12‐lead ECG data were collected at the time of the initial clinic visit or for routine obstetric examinations during the first trimester of pregnancy. If the standard 12‐lead ECG recorded the PVC events, Holter monitoring was then performed and reexamined in each subsequent trimester to assess the burden of ventricular premature contractions and analyze the total number of QRS complexes, total number of PVCs and total runs of nonsustained ventricular tachycardia (NSVT) (run with >3 consecutive PVCs but <30 s). Furthermore, if the women had self‐reported symptoms such as palpitation during pregnancy, the 12‐lead ECG examination was then performed. If no arrhythmia was recorded, a wearable single‐lead ECG recorder  was then weared for three continuous days to record the possible PVC events in those pregnant women. The single‐lead wearable ECG recorder was designed to recognize the arrhythmia based on the convolutional neural network technology. It can easily identify the PVCs when it happens. And if the PVCs was documented by 12‐lead ECG or the wearable single‐lead ECG recorder and confirmed by the ECG experts in our center (Lin J & Chen QS), the Holter was examined thereafter once a trimester for the remainder of the pregnancy period. The average PVC burden was determined by calculating the total number of PVCs in the total QRS complexes. PVC morphological patterns were analyzed to determine the PVC origin. PVCs with inferior axis and left bundle branch block (LBBB) patterns were determined to most likely originate from the right ventricular outflow tract (RVOT). Specific diagnostic procedures were carried out with reference to published diagnostic criteria.Echocardiography was performed in the first and third trimesters in all pregnant women. Experienced echocardiographers performed echocardiography to ensure that there was no evidence of SHD in the pregnant women and to ensure that the cardiac function of the enrolled pregnant women was within the normal range (left ventricular ejection function [LVEF] >60%).All pregnant women were followed, and the data on characteristics including age, comorbid medical conditions and family history were recorded. All women were followed from preconception to 1 week after delivery. If the exclusion criteria were met, they were excluded from the study.2.3Two physicians blinded to the women's baseline characteristics independently verified adverse events. Adverse fetal and neonatal events were defined as previously described2.4t‐test or the Wilcoxon rank‐sum test was used to compare continuous variables between the women with and without adverse fetal/neonatal events who had PVCs >0.5%. Possible risk factors of adverse events were analyzed using univariable logistic regression, followed by multivariable logistic regression. A p value <.05 was considered statistically significant.Statistical analysis was performed using the SPSS (Version 22.0). Data are presented as the means ± SDs, medians (25th–75th percentiles), or proportions. Student's 33.1Six thousand one hundred and forty‐eight pregnant women who fulfilled the inclusion criteria were enrolled consecutively, of which 103 (1.68%) had symptomatic or asymptomatic PVCs. A total of 17 adverse events occurred in 103 pregnant women with PVCs, including five cases of respiratory distress syndrome, five preterm births, and seven small‐for‐gestational‐age births. Finally, only 12 women with adverse events were counted, as multiple events occurred in some of them simultaneously. The remaining defined adverse events did not occur. The other 91 pregnant women with PVCs delivered safely without adverse events. A total of 177 fetal and neonatal adverse cases were counted in the cohort of 6045 pregnant women without PVCs. The incidence of adverse events was significantly higher in PVC cases (11.65%) than in those without PVC (2.93%)  and inferior axis, frequently associated with the left ventricular outflow tract (LVOT), accounted for approximately 11.65%, far less than PVCs associated with the RVOT. According to the special morphology of PVCs reported in the previous literature,3.3The baseline characteristics of the study cohort suggested that the average burden of PVCs was related to adverse outcomes, so the burden of PVCs was further divided into low (<33rd percentile), middle (33rd–67th percentile) and high groups (>67th percentile) according to the percentile of the average PVC burden (Figure Univariable logistic regression analysis demonstrated that the LVEF, heart rate, bigeminy and burden of PVCs were associated with adverse fetal or neonatal outcomes among pregnant women with PVCs. Variables which were statistically significant in univariate analysis were further analyzed in multivariate logistic regression analysis; however, statistical significance was only evident for PVC burden in the multivariate logistic regression analysis (OR: 1.34, 95% CI [1.11–1.61], p < .05, Table 4It is widely recognized that idiopathic PVCs are relatively benign in structurally normal hearts;In our study, 1.68% pregnancies with structurally normal heart were found to have high PVC burden in 6148 consecutive enrolled pregnant women. And the rate of adverse fetal/neonatal events in women with PVCs was 11.65%. Similar results were reported in Tong C et al.,Idiopathic PVCs are distributed mainly to specific cardiac structural sites, of which the RVOT accounts for approximately 67%, and the rest are mainly followed by the LVOT around the annulus, papillary muscles and other special sites.4.1Although this study is a prospective, single‐center study, the number of participants included is still relatively small; therefore, a large‐scale and multicenter study should be performed in the future. Second, PVC origin was classified by a standard 12‐lead ECG pattern in the study. Undeniably, changes in diaphragm position during pregnancy and cardiac rotation may cause misjudgment of the origin of PVCs. Third, because the PVC burden may fluctuate throughout the day and throughout stages of pregnancy, we applied the average PVC burden screened by Holter in each trimester to represent the PVC burden during pregnancy. It is therefore suggested to use long‐term wearable monitoring equipment to screen the PVC burden in the future. Finally, all patients underwent only two‐dimensional echocardiography in the study, and cardiac magnetic resonance might be suitable for further study to eliminate the possibility of latent cardiomyopathy.In conclusion, our study revealed that the prevalence of PVCs in pregnancy is higher than that in the normal population. Frequent PVCs have adverse effects on pregnancy, and the PVC burden might be the main factor associated with adverse pregnancy outcomes.The authors declare no potential conflict of interest.Junhong Wang, Xin Wu: Designed, supervised the study and prepared the manuscript. Jing Lin, Yanxia Qian, Qiushi Chen: Data collection and analysis, writing original draft. Yaoxi Chen, Ruijie Xu, Jingxian Chen, Yukang Shi, Shunxin Yang, and Xinyi Luo: Data collection and analysis. Mingming Zhang: Writing ‐ Review and Editing. Qiang Ding: Project administration and data analysis.Figure S1 The origin of PVC in pregnant women. LVOT, left ventricular outflow tract; RVOT, right ventricular outflow tract; TV, tricuspid valve; LP, left posterior; LA, left anterior; others indicate 10 unidentified classifications, 1 from right ventricular inflow tract, 1 from right ventricular apex, 1 from His bundle and 1 from right ventricular mid‐septum.Click here for additional data file.


Bluefin tuna (BFT) is an apex predatory, long-lived, migratory pelagic fish that is widely distributed throughout the world's oceans. These fish have very high concentrations of neurotoxic methylmercury (MeHg) in their tissues, which increase with age. Our study shows that Hg accumulation rates (MARs) in BFT as a global pollution index can reveal global patterns of Hg pollution and bioavailability in the oceans and further reflect both natural and anthropogenic emissions and regional environmental features. Overall, MARs provide a means to compare Hg bioavailability among geographically distinct populations of upper trophic level marine fish across ocean subbasins, to investigate trophic dynamics of Hg in marine food webs, and furthermore, to improve public health risk assessments of Hg exposure from seafood. Bluefin tuna (BFT), highly prized among consumers, accumulate high levels of mercury (Hg) as neurotoxic methylmercury (MeHg). However, how Hg bioaccumulation varies among globally distributed BFT populations is not understood. Here, we show mercury accumulation rates (MARs) in BFT are highest in the Mediterranean Sea and decrease as North Pacific Ocean > Indian Ocean > North Atlantic Ocean. Moreover, MARs increase in proportion to the concentrations of MeHg in regional seawater and zooplankton, linking MeHg accumulation in BFT to MeHg bioavailability at the base of each subbasin's food web. Observed global patterns correspond to levels of Hg in each ocean subbasin; the Mediterranean, North Pacific, and Indian Oceans are subject to geogenic enrichment and anthropogenic contamination, while the North Atlantic Ocean is less so. MAR in BFT as a global pollution index reflects natural and human sources and global thermohaline circulation. SI Appendix, Table S1) (8 times higher than those in seawater (3−1 wet weight (w.w.) set by the US Food and Drug Administration and the World Health Organization (As the world’s largest tuna (up to 300 cm and 600 kg), bluefin tuna (BFT) is an apex predatory and long-lived (>30 y) fish (able S1) . At the able S1) –6. Efficeawater 3. As a reThunnus maccoyii), Pacific BFT , and Atlantic BFT (Thunnus thynnus), whose migratory routes are confined to southern hemisphere oceans , the North Pacific Ocean (NPO), and the North Atlantic Ocean (NAO)/Mediterranean Sea (MS), respectively . Although they have slightly different biological and ecological characteristics , being closely related, the three BFT species have similar pelagic migration and feeding behaviors. BFT are oceanic epipelagic fish that commonly spend most of their time (>90%) in waters shallower than 200 m, although they can dive to depths of several hundred meters . While their spawning is confined to specific locations and seasons, BFT engage in opportunistic feeding across vast areas of the ocean subbasin each inhabits . Since Hg levels in marine fish reflect concentrations in their prey and ultimately local seawater . Furthermore, observed differences in MARs in BFT were examined in relation to the variation of MeHg concentrations in water and plankton, obtained from previous studies, in surface (0 to 150 m) and thermocline layers  and in water column inventories  across four ocean subbasins. Finally, based on a detailed compilation of the global patterns of Hg cycling and fluxes in the ocean, we examine relationships between Hg accumulation in BFT and human sources and global thermohaline circulation.In view of the above, the primary goal of this study was to evaluate how Hg bioaccumulation in BFT varies on a global scale and whether such variation corresponds with levels of Hg pollution across ocean basins. To examine recent global patterns of Hg pollution in the world's oceans (including the 22-y period from 1998 to 2019), we first calculate the apparent MAR as the slope of the relationship between muscle THg concentrations and age based on new measurements of Pacific BFT from the western NPO caught in 2017 to 2018 and Southern BFT from the IO caught in 2018 to 2019, as well as previously available results for fish from the NAO and MS collected from 1998 to 2017 , and based on fork length (FL) (SI Appendix), varied in age from 5 to 27 y old (∼85% in age groups of 5 to 15 y). THg levels in the muscle tissue of these Pacific bluefin tuna (Pacific BFT) ranged from 0.49 to 5.65 µg ⋅ g−1 w.w. , ∼94% of which exceeded the safe consumption guideline of 1 µg ⋅ g−1 w.w. The Southern bluefin tuna (Southern BFT) from the IO we analyzed were 2 to 15 y old (ref. SI Appendix) and had muscle tissue THg concentrations of 0.20 to 1.85 μg ⋅ g−1 w.w. .Our results include THg concentrations in adult Pacific BFT from the NPO (≥5 y old) and in >4 y old Southern BFT from the IO. The adult Pacific BFT from the NPO we analyzed spanned wide ranges of size and body mass (gth (FL)  . Indeed, among all four populations examined, mean concentrations of THg in BFT declined in the order NPO > MS > NAO > IO. This differs from previously reported trends, including those suggesting that BFT from NAO have higher average Hg concentrations than BFT from all other oceans  consistent with expected allometric relationships (16−1) were at the upper limit of the FL–age model and therefore of uncertain age . To account for differences in fish age and size across studies and geographical domains, and to eliminate the influence of very old/large fish, we used age-based MARs estimated for ≤15-y-old fish to scale Hg accumulation among BFT subpopulations from different ocean subbasins.Based on these and previously published results, and as shown for other tuna species , mean THr oceans . Howeverionships , 16–18. nships 16 and the nships 16. AtlantiSI Appendix, Tables S3 and S4). Overall, the highest age-based MAR for BFT was observed in the MS, followed by the NPO, the IO, and lastly the NAO . The same global trend in MARs was found when BFT of all ages were included, although the inclusion of older fish resulted in somewhat higher MARs for the NAO and lower MARs for the NPO . Please note that MARs for two samples of Atlantic BFT from the MS, one collected in 2000 to 2005 and the other in 2019 (P > 0.1) and were both the highest among the four ocean basins we examined . Thus, while Hg concentrations in BFT within the same region might vary slightly over two decades, such variation does not affect the global patterns of MARs we observed.Our analysis shows that age-based MARs estimated for ≤15-y-old BFT binned into 1-y age classes varied significantly among BFT populations from four ocean subbasins  integrated from the surface to 1,000 m (P < 0.001). The relationship between MeHg inventories from the surface to 1,000 m and MARs in BFT may be particularly strong (R2 = 0.97) because the inventories integrate possible differences in vertical foraging habits of BFT that may influence their Hg concentrations. Furthermore, MARs in BFT from the MS, NPO, and NAO were also positively correlated with concentrations and bioaccumulation factors of MeHg in phytoplankton and zooplankton from each ocean basin . Such correlations provide evidence linking dissolved MeHg to its accumulation in apex predators at the top of oceanic food webs, which is expected but rarely observed directly due to inadequate sample sizes. This further implies that Hg concentrations in BFT prey also vary with MeHg concentrations in seawater . Those factors are mostly controlled by water temperature in different geographic locations. Dell'Apa et al. (P = 0.7) with Hg accumulation rates in the four BFT populations examined. However, Hg accumulation rates in BFT populations are strongly positively correlated with the concentration of MeHg in the water column and in planktonic organisms at the base of each basin's food web . This is consistent with recent model results, which indicate that most (60 to 75%) of the variation in MeHg concentrations in Atlantic BFT is due to variation in the concentration of MeHg in seawater . The ocean basin with the highest MAR for BFT is the MS. This indicates that the effects of regional contamination and/or the production and food web bioavailability of MeHg are greater in the MS compared to other ocean subbasins. Hg contamination of the MS ecosystem has been a concern for decades . Relatively high fluxes of Hg to this nearly landlocked marginal basin, which experiences little exchange with the open North Atlantic results in lower variability of THg concentrations in the surface waters and thermocline of the MS than in other ocean basins . In addition, as a result of high seawater temperatures and metabolic (oxygen consumption) rates in strongly stratified and primarily oligotrophic surface waters  and higher levels of Hg in Mediterranean BFT . The NPO is downwind of East Asia, which is the world's largest emitter of Hg to the atmosphere  . Since the majority of the mercury in contemporary atmospheric and riverine inputs is anthropogenic  . AdditioFig. S5) , 38. Ignopogenic , the higopogenic  and modeopogenic . Given topogenic  Fig. 1)SI Appendopogenic , 38.Tissue Hg concentrations are available for Pacific BFT from the eastern NPO, but these include only juvenile fish with ages under 5 y , 41. JuvSI Appendix, Table S8). As a result of lower atmospheric deposition and riverine inputs from North America and Europe, net inputs of Hg to the NAO (∼10 nmol ⋅ m−2 ⋅ y−1) are also lower than those in the other subbasins . Lee et al. , but other factors related to ocean circulation and biogeochemical processes may also contribute to these differences. For instance, little upwelled or anthropogenic MeHg from the NPO and the IO is expected to return to the NAO via global thermohaline circulation .The MARs for the North Atlantic population of Atlantic BFT that we estimated from the results of Lee et al.  were cone et al.  suggestee et al.  and in se et al. , were tie et al. . Lower i−2 ⋅ y−1) is much lower than that to the NPO (55 nmol ⋅ m−2 ⋅ y−1), it is nearly double that to the NAO (10 nmol ⋅ m−2 ⋅ y−1) . While MeHg concentrations are relatively low in the northern IO, including the oxygen minimum zones of the Arabian Sea .Our observation that MARs for Southern BFT from the IO were higher than those for Atlantic BFT from the NAO is somewhat surprising given that there are fewer terrestrial sources and anthropogenic emissions of Hg to oceans in the southern hemisphere, where open ocean accounts for ∼81% of the surface area, than to the NPO and NAO . Howeverbian Sea , surfaceSI Appendix, Fig. S5). This variation may well reflect different levels of Hg pollution or natural sources and different mechanisms of MeHg production and transport associated with contrasting ecological structures and circulation patterns across ocean subbasins. MARs in BFT and potentially other large marine predators can therefore be used as global indices of Hg concentrations and bioavailability in the world's oceans. Additionally, the MAR index presents a possible refinement of safe consumption guidelines, which currently treat all fish of a certain species the same. For example, the MAR along with information on the fish size  and the ocean basin of capture could be used to estimate whether or not Hg concentrations in an individual fish exceed food safety limits and safe consumption rates (SI Appendix). Thus, variations in Hg accumulation among populations of marine fish from distant ocean basins can be used to study global Hg contamination, manage marine fisheries, and safeguard human health.We found significant variation in MARs among four geographically distinct populations of BFT (n = 261) captured by Taiwan longline fishing vessels in the western NPO  from April to July in 2017 and 2018 during the spawning period and from Southern BFT (n = 83) in the southern IO  from March to July in 2018 and 2019 . Samples were taken from whole fish landed at the fishing ports in Taiwan, and the FLs of the individual Pacific BFT and Southern BFT  were measured by the observer. Muscle tissue was taken from the hypaxial dorsal musculature and immediately frozen at −5 °C and then stored at −20 °C once returned to the laboratory. Muscle tissue was subsequently freeze-dried for 96 h and then homogenized before Hg analysis.Muscle tissue was sampled from Pacific BFT  and 113 to 392 kg (212 ± 61) and of the 83 SBFT from 85 to 166 cm (120 ± 16) and 13 to 96 kg (28 ± 13), respectively. The age of the captured Pacific BFT was estimated from FL by an empirical age-length equation that was directly developed from otoliths of the Pacific BFT population in the southern spawning ground near Taiwan . Estimat3 (4 mL) and HCl  (2), two-stage gold (Au) amalgamation, and thermal desorption together with cold vapor atomic fluorescence spectrometry (SI Appendix). THg concentrations in BFT were expressed in μg ⋅ g−1 w.w. through the correction of water content  from the dry weight (d.w.) concentration. Detailed information regarding the analytical performance and data quality with recoveries of Hg from the two certified biological reference materials -436, tuna fish, 4.19 ± 0.36 μg ⋅ g−1 d.w.) was described in SI Appendix.In this study, THg analysis was carried out after quantitative recoveries of all Hg from lyophilized muscle tissues (0.02 to 0.05 g) by microwave-assisted acid bomb digestion with a 4:1 (volume:volume) mixture of HNO4, 1 mL) , 49. Tuntrometry . THg conhttps://automeris.io/WebPlotDigitizer) was used to extract the values of Hg, fish size, age, etc. All data were expressed in μg ⋅ g−1 w.w. basis for analyses.A focus of this work is a comparison of relationships between Hg and fish age or size in a variety of locations worldwide. Our preferred metric for this comparison was the MAR for each fish population. We thus collected published total Hg concentrations in muscle tissue of BFT and accompanying metadata, including capture date and location, FL, fish weight, and age. Mean concentrations of Hg in size/weight groups of BFT were included if raw individual data were not reported. In some cases, data were reported graphically, and Web Plot Digitizer  , 41, 51,16SI Appendix, Table S3 by group and individual). Within each population, MARs for age-binned and unbinned values were nearly the same. MAR values were hence retrieved as the slopes for age-binned values as described below. Since the maximum age of BFT from the four populations studied varied from 9 y (MS) to 29 y (NAO), MARs were estimated for three groups of BFT, those ≤10 y old, those ≤15, and those of all ages In this study, data for BFT collected from 1998 to 2019 were evaluated, a time interval that is within the life span of BFT and which represents contemporary bioaccumulation status and exposure risk. MARs for BFT from each ocean basin were obtained as the slope of THg concentration versus age relationships for fish binned into 1-y age classes . Regression slope values  of Hg versus age and average Hg concentrations of BFT across ocean regions were compared using a t test for their significant differences. This was finally followed by the one-way ANOVA to test differences of significance for regional effects. Relationships between MARs and the concentrations and column inventories of MeHg in seawater  from the four ocean subbasins were shown in In order to eliminate the variabilities of fish body and Hg levels in certain age ranges, we grouped values into age groups in increments of 1 y . The varSupplementary File


High-Performance Thin-Layer Chromatography (HPTLC) was used in a chemometric investigation of the derived sugar and organic extract profiles of two different honeys (Manuka and Jarrah) with adulterants. Each honey was adulterated with one of six different sugar syrups  in five different concentrations . The chemometric analysis was based on the combined sugar and organic extract profiles’ datasets. To obtain the respective sugar profiles, the amount of fructose, glucose, maltose, and sucrose present in the honey was quantified and for the organic extract profile, the honey’s dichloromethane extract was investigated at 254 and 366 nm, as well as at T (Transmittance) white light and at 366 nm after derivatisation. The presence of sugar syrups, even at a concentration of only 10%, significantly influenced the honeys’ sugar and organic extract profiles and multivariate data analysis of these profiles, in particular cluster analysis (CA), principal component analysis (PCA), principal component regression (PCR), partial least-squares regression (PLSR) and Machine Learning using an artificial neural network (ANN), were able to detect post-harvest syrup adulterations and to discriminate between neat and adulterated honey samples. Cluster analysis and principal component analysis, for instance, could easily differentiate between neat and adulterated honeys through the use of CA or PCA plots. In particular the presence of excess amounts of maltose and sucrose allowed for the detection of sugar adulterants and adulterated honeys by HPTLC-multivariate data analysis. Partial least-squares regression and artificial neural networking were employed, with augmented datasets, to develop optimal calibration for the adulterated honeys and to predict those accurately, which suggests a good predictive capacity of the developed model. Honey has been regarded as a nutritional and medicinal food for thousands of years . About 713C/12C Stable Carbon Isotope Ratio Analysis   . HPTLC iication) . Multivaication) .The aim of this study was to determine the feasibility of HPTLC-based profiling in combination with MVDA for the quality control of Manuka and Jarrah honeys, specifically the detection of post-harvest adulterations with a range of sugar syrups. It was found that the combination of HPTLC and multivariate data analysis can not only assist in confirming and quantifying these adulterations, but also in identifying some of the adulterants used.All chemicals and reagents were of analytical grade. Commercial sugar syrups and honeys  were obt4 followed by filtration, the solvent was evaporated at ambient temperature and the resulting extracts stored at 4 °C. Prior to HPTLC analysis they were reconstituted in 100 µL of dichloromethane. A solution of 4,5,7-trihydroxyflavanone (0.5 mg/mL) in methanol was prepared as a reference solution.Adulterated honey samples were prepared by mixing the respective syrups  with honey (Manuka and Jarrah) in a final concentration of 10%, 20%, 30%, 40% and 50% (w/w) respectively. The samples were warmed in a water bath at 36 °C for about 30 min to assist with their mixing into homogenous blends. As described in The HPTLC sugar and organic honey extract profiles of all samples were obtained using a CAMAG HPTLC instrumentation  following previously established methods .254 plates) using a semi-automated HPTLC application device . Track distances were 11.4 mm with 15 tracks on each 20 × 10 HPTLC plate, which represents the default plate setting. The development chamber was saturated for 60 min (33% relative humidity) and the plates developed to a migration distance of 85 mm in 1-butanol: 2-propanol: aqueous boric acid . After drying for 5 min, the plates were derivatised with two ml of aniline-diphenylamine-phosphoric acid reagent  and heated for 10 min at 115 °C (CAMAG TLC Plate Heater III). After cooling to room temperature for two min, the plates were analysed at white light with the HPTLC imaging device .In brief, for sugar analysis, three µL of each sample were applied as eight mm wide bands eight mm from the lower edge and 20 mm from the side edge of the HPTLC plate (20 cm × 10 cm glass-backed silica gel 60 F254 plates) with a semi-automated HPTLC application device . Track distances were 11.4 mm with 15 tracks on each 20 × 10 HPTLC plate, which represents the default plate setting. The development chamber was saturated for 20 min (33% relative humidity) and the plates developed to a final distance of 70 mm in toluene: ethyl acetate: formic acid (6:5:1 v/v/v). After drying for 5 min, the chromatographic results were documented at 254 and 366 nm using the HPTLC imaging device  before being derivatised with three ml vanillin with sulfuric acid reagent  and being heated for 3 min at 115 °C (CAMAG TLC Plate Heater III). After cooling to room temperature for 2 min, the obtained images were again recorded at white light and 366 nm. The chromatographic analysis was performed using VisionCATS software , which was also used to control the individual instrumentation modules.To obtain the organic extract profile, the reference solution (four µL) and the reconstituted extracts (five µL) were applied as eight mm bands at eight mm from the lower edge and 20 mm from the side edge of the HPTLC plates , the six syrup adulterants  and the resulting 60 adulterated honeys. Sugar data included quantities of fructose, glucose, sucrose and maltose. From the organic honey extract HPTLC profiles, four sets of images  were converted into their respective chromatograms. The intensity (AU) of bands expressed as absorption peak height were extracted for data calculation along with their corresponding Rf values. To reduce complexity, only bands between Rf 0.05 and Rf 0.60 were considered, as this captured the majority of all detected bands . The obtMultivariate data analysis included non-supervised techniques, like cluster analysis and principal component analysis, as well as supervised techniques, including principal component regression, partial least-squares regression and machine learning .k groups of samples, called folds of equal sizes (if possible). The prediction function is learned using k-1 folds, and the fold left out is used for testing. Each fold is used for testing once. This process was applied to supervised PCR, PLSR and ANN chemometric techniques. For PCR and PLSR Root Mean Square Error of Cross Validation (RMSECV), Root Mean Square Error (RMSE) and Root Mean Square Error of Prediction (RMSEP) were deemed useful to predict the validation parameters.The sugar contents data had a dynamic range of 0 to ~2,000. All the methods chosen for the analysis prefer standardised data in the number range −1 to 1 or 0 to 1; so, the data was standardised. With the ANN, the AU values of the organic extracts naturally have a range from 0 to 1 and thus did not need standardising. To ensure the supervised models would work with unseen data, and not just repeat the labels of the samples that they had just seen, cross validation and k-fold cross validation was used. This involved holding back 30% of the data as a test set to validate the training of the models. With the basic raw data only 68 samples were available. This meant standard cross validation was inappropriate for PCR, PLSR and ANN, and k-fold cross validation was required. k-Fold cross validation divides all the samples into For the bands obtained in four different analysis conditions -a drift of ±0.0173 Rf and adding Gaussian noise with a standard deviation of 1.25 times the standard deviation of the original AU value. The data was separated into the four light bands and the standard deviation of each band was calculated.For the sugar content values–Gaussian noise with a standard deviation of 5% of the value was add to the samples.This study aimed to predict the honey/syrup type and the adulteration level of all samples, so all 68 classes. The original data set was too small to be used directly as input to train an ANN, PCR, PLSR, and potentially, a PCA. To address this, the fact that an analysis run using HPTLC is not deterministic was used. As different runs will produce small various in AU and the Rf value can slightly drift, this was used to augment the original dataset by repeating the following process 50 times:This increased the dataset from 68 to 71,468  as shown in The obtained HPTLC sugars profiles of honey were very simple as they only captured glucose (Rf 0.30) and fructose (Rf 0.14) within the limit of quantification, as can be seen in Cluster analysis was applied in this study in order to identify interrelationships between different honey groups. Clustering allows the grouping of similar data points in such a way that points in the same group, known as cluster, are more similar to each other than points in other groups. There are several types of cluster analysis, in this paper Hierarchical Clustering, K-Means Clustering and Density Based Clustering were investigated.Hierarchical clustering (HC), an unsupervised clustering algorithm, is one of the most popular and easy to understand clustering techniques. It starts with placing each observation in its own cluster and then stepwise merging clusters by analysing the distances between adjacent points. HC gives an indication not only to what extent individual points are similar to each other but also how similar different clusters are to each other. The number of significant clusters found and displayed in the respective dendrogram is based on the Euclidian distance of the normalised data . The hieThe dendrogram derived from the study’s dataset displays three main clusters. The first contains sugar syrups (with the exception of TRE), the other two contain the pure and adulterated honeys. Within the other clusters two major sub-cluster can be distinguished; one contains pure Manuka and syrup adulterated Manuka honeys, the other contains pure Jarrah and syrup adulterated Jarrah honeys. These findings demonstrate the usefulness of hierarchical clustering in distinguishing syrups from pure honeys and syrup adulterated honeys.vs Sucrose + Maltose are shown in K-means clustering is also a very simple and popular unsupervised algorithm. Typically, unsupervised algorithms use information from datasets as input vectors. A target number k is set first, which refers to the number of centroids needed in the dataset . A centrepsilon or ϵ-neighbourhood) value, the radius of neighbourhood around a point  and Principal Component 2 (PC2) had the greatest variability, PC2, PC3 and PC4 had marginal variability. Little variability could be detected for PC5 and beyond .Although similar chromatographic profiles were obtained for all honey samples –5, PCA cA clear differentiation between two clusters can be noted. Cluster 1 contains Manuka and Manuka-syrup adulterated honeys and Cluster 2 contains Jarrah and Jarrah-syrup adulterated honeys. Further exploring the PCA data reveals that in Cluster 1 pure Manuka honey is clearly separated from the other data points within this cluster, the same can be noted for Cluster 2, where pure Jarrah honey is also clearly separated from the adulterated samples . Five ofvs PC2 was retained, but the importance of PC1 and PC2 was significantly reduced . Generally, the number of PLS factors and the characteristic variables of the data can affect the performance of the PLS model. Too few PLS factors tend to decrease the reliability of the model, too many in turn might increase the model’s complexity and weaken its stability . In thisThe main practical difference between PCR and PLSR is that PCR often needs more components than PLSR to achieve the same prediction error . On thisAn ANN is a supervised mathematical model inspired by biological systems. It simulates the way mammalian brains work in processing information and learning from data. The ANN consists of a number of layers that perform different functions, such as multiplying weights, randomly dropping out nodes and performing non-linear activations. Training is performed iteratively, so that as it progresses, the ANN learns by comparing the ground truth to the predictions, and back propagating the losses to adjust the weights of the varies layers. Upon completion the model will have established (linear or non-linear) relationships between input and output data  through The augmented data was split into six groups to perform K-fold learning with six folds. For each of the six runs a different fold of the data was retained for testing as discussed in “Chemometric validation”. HPTLC derived organic extract profiles allow a reliable and reproducible authentication of a honey’s floral source , but any10.7717/peerj.12186/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12186/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12186/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.12186/supp-4Supplemental Information 4Click here for additional data file.


Introduction: The severity of injury from motor vehicle crashes (MVCs) depends on complex biomechanical factors, and the bodily features of the injured person account for some of these factors. By assuming that vulnerable road users (VRUs) have limited protection resulting from vehicles and safety equipment, the current study analyzed the characteristics of fat distribution measured by computed tomography (CT) imaging and investigated the existence of a “cushion effect” in VRUs. Materials and Methods: This retrospective study enrolled 592 VRUs involved in MVCs who underwent CT scans. Visceral fat area and subcutaneous fat cross-sectional area were measured and adjusted according to total body area (TBA) and are presented as the visceral fat ratio and the subQ fat ratio (subcutaneous fat ratio). Risk factors for serious abdominal injury ) resulting from MVCs were determined by univariate and multivariate analysis. Results: MAISabd ≥ 3 was observed in 104 (17.6%) of the patients. The subQ fat ratio at the L4 vertebral level was significantly lower in the MAISabd ≥ 3 group than in the MAISabd < 3 group . A decreased L4 subQ fat ratio was associated with a higher risk for MAISabd ≥ 3 in multivariate analysis . Conclusion: The current study supported the “cushion effect” theory, and protection was apparently provided by subcutaneous fat tissue. This concept may further improve vehicle and safety designation in the future. Motor vehicle crashes (MVCs) are a leading cause of death in the young population . Injury 2 by adopting National Institutes of Health (NIH) and World Health Organization (WHO) guidelines [Body mass index (BMI) is the most discussed assessment of an individual’s body size. It is a simple but also an indirect measurement of physical status. One of the most popular bodily features, obesity, is characterized as excess fat accumulation. It is defined as a BMI ≥ 30 kg/midelines . It has idelines ,17,18,19Although a greater body mass carries higher momentum, the injury pattern of an obese occupant diverges from those with normal weight or those who are underweight in the crash. Arbabi et al. introduced the “cushion effect” in 2003, suggesting that increased abdominal adiposity provides a “cushion” for abdominal trauma during the injury event ,22. A coHowever, the ease of use of BMI does not overcome its inability to distinguish between bone, muscle mass, and fat tissue. Regardless of the fact that the development of modern computed tomography (CT) imaging and software enables the precise measurement of body composition, the exact mechanism and interaction between the human body and collision is still poorly understood. To further investigate the impact of bodily characteristics during an MVC, the current study was performed by assessing VRUs with limited abdominal protection during the collision. We analyzed the characteristics of body fat distribution in VRUs and hypothesized the existence of a “cushion effect” in blunt abdominal trauma.The data from May 2008 to December 2016 were collected from the trauma registry of our institution. Individuals who were involved in MVCs were included if they were older than 16 years old, had an abdominal multidetector helical computed tomography (MDCT) scan performed primarily for trauma indications, and were admitted to the ward or intensive care unit (ICU). Indications for an MDCT scan included a positive Focus Assessment with Sonography in Trauma (FAST), an abnormal finding in physical examination of the abdomen or pelvis, an abnormal chest or pelvic X-ray, an unconscious occupant with blunt torso injury, or clinical judgment. All occupants underwent MDCT scans if they were hemodynamically stable either with or without resuscitation. Occupants with incomplete medical records or missing body height or weight measurements were excluded.Data on the following variables were collected: age, sex, height, weight, BMI, Glasgow coma scale (GCS), and vital signs at the emergency department. Vehicle data, including vehicle type and safety equipment, were collected. The VRUs are defined as road users that do not have shell protection during an MVC, who consist of pedestrians, bicyclists, and motorcyclists . Injury Hemodynamic instability was defined according to at least one episode of systolic pressure less than 90 mmHg upon emergency admission. A MAIS of 3–6 (MAIS ≥ 3) was defined as serious injury in each body region.2) .Subcutaneous fat area: the cross-sectional area between skin and fascia with fat density thresholds between −205 and −51 HU.Visceral fat ratio (%) = SubQ fat ratio (%) = CT images were assessed with analytic morphomics, which has been previously described ,30. The 2) . Becauset test or the Mann–Whitney U test . A multivariable logistic regression model was performed to examine the relationship between serious abdominal injury and the subcutaneous fat ratio, adjusting for patient and crash characteristics.Descriptive statistics were calculated for the cohort. Categorical data are presented as numbers and percentages and were compared using the chi-square test. Continuous variables are presented as the mean with standard deviation (SD) or median with interquartile range . Continuous variables were compared using Student’s A significance level of α = 0.05 was used. All analyses were performed using IBM SPSS statistics 25.0 . Microsoft Excel (V16.19) was used for data entry and to create associated figures.2, respectively. Of these patients, 59 (10.0%) were pedestrians, and 533 (90.0%) patients were bicyclists or motorcyclists involved in an MVC. A total of 98 patients did not use safety equipment, while the other 494 wore helmets. Upon arrival at our emergency department (ED), 73 individuals (12.3%) had unstable hemodynamics. The median GCS was 15, and the median ISS was 18. Among these individuals, 156 (26.4%) had serious (MAIS ≥ 3) head injuries, 269 (45.4%) had serious thoracic injuries, 104 (17.6%) had serious abdominal injuries, and 175 (29.6%) had serious limb injuries. Overall mortality in the cohort was 5.4%, with a mean length of hospital stay of 16.3 ± 15.7 days and a mean length of ICU stay of 5.1 ± 7.9 days. The distribution of BMI and BMI classifications of the enrolled VRUs is shown in Between May 2008 and December 2016, 592 VRUs involved in MVCs underwent abdominal CT scans primarily for trauma evaluation. The demographics of these studied patients are summarized in abd ≥ 3, the differences were only significant in subQ fat ratios at the L3, L4, and L5 vertebral levels . There were no significant differences in age, BMI, sex, vehicle, or safety equipment usage. VRUs with serious abdominal injury did not have a longer length of stay (LOS), and they did not have a higher mortality rate according to the analysis. However, the MAISabd ≥ 3 group had a longer ICU LOS . Notably, VRU with a lower mean L4 subQ fat ratio was more likely to have serious abdominal trauma , although the BMI was similar.p = 0.009). BMI and L2 visceral fat ratio were not independent predictors of serious abdominal injury. The correlations between the L4 subQ ratio and MAISabd are shown in p = 0.028).The prevalence of obesity is increasing worldwide and has been well studied in terms of the associated cardiovascular and metabolic risks ,32,33. WAdipose tissue can be stored beneath the skin, around organs, and within bone marrow or muscle. In addition to its role in energy storage and endocrine function , it alsoThe utility of CT-measured visceral and subcutaneous fat tissue in the trauma population grew because of the development of CT imaging and software. A prospective study by Shashaty et al. reported that increased L4-L5 visceral fat increased prevalence of acute kidney injury in 327 trauma patients , though As a growing issue worldwide , obesityViano et al. found that obese patients had a 40% higher risk of serious injury than normal weight patients. However, the abbreviated injury scales of each body region were not discussed . In the The current cohort was from a level I trauma center in Taiwan, with a median ISS of 17.5 and an overall in-hospital mortality rate of 5%, which was in line with a previous report ,14,41,42Finally, it is important to realize that the current study was limited due to its retrospective nature. A lower rate of obesity and morbid obesity in Asia also limited the ability to demonstrate a full picture of the “cushion effect”. Furthermore, vital organs such as the liver, spleen, and kidneys are located between the T11 and L2 vertebral levels. An analysis of fat area at a single vertebral level may not be conclusive.The advancement of CT imaging provides a chance to inspect the complex interaction between the human body and injury. The results of the current study supported the existence of a fat cushion effect. Increased subcutaneous fat in VRU involved in MVCs is associated with lower prevalence of serious abdominal trauma. The concept may further improve the development of vehicles and safety features in the future.


To analyse the risk factors for intracranial infection after neuroendoscopic transnasal pituitary adenoma resection (NTPAR) to provide a reference for the prevention and treatment of postoperative intracranial infection.The clinical data of 387 patients who underwent NTPAR in the Department of Neurosurgery of the First People’s Hospital of Yichang from March 2013 to March 2021 were retrospectively analysed. The patients were divided into an infected group and a noninfected group according to the occurrence of intracranial infection. The detailed clinical data of the two groups were collected. Univariate and multivariate logistic regression was used to analyse the risk factors for intracranial infection after NTPAR.Among the 387 surgical patients, 32 patients (8.27%) were in the intracranially infected group and 355 patients (91.73%) were in the noninfected group. The results of the univariate analysis suggested that age > 45 years, tumour size > 1 cm, operation time > 240 min, blood loss > 400 ml, Kelly Grade of cerebrospinal fluid (CSF) leakage > Grade 2, postoperative CSF leakage, lumbar cistern drainage and blood transfusion were the influencing factors for postoperative intracranial infection, while the results of multivariate logistic regression analysis implied that intraoperative CSF leakage (Kelly Grade > 2) and postoperative CSF leakage were independent influencing factors for intracranial infection after NTPAR, and perioperative use of antibiotics was an independent protective factor for postoperative intracranial infection.There are a variety of risk factors for intracranial infection after NTPAR, which indicates that it is necessary to develop different repair strategies for CSF leakage according to the Kelly Grade, timely treatment of postoperative CSF leakage and perioperative use of antibiotics. These measures have been shown to effectively reduce the probability of intracranial infection after NTPAR. Pituitary adenomas are common benign tumours of the nervous system, second only to gliomas and meningiomas, accounting for approximately 15% of intracranial tumours . With thThe clinical data of 387 NTPAR patients in the Department of Neurosurgery of the First People’s Hospital of Yichang City from March 2013 to March 2021 were collected retrospectively. According to the occurrence of postoperative intracranial infection, the patients were divided into an infected group (n = 32) and a noninfected group (n = 355). This study is in line with the relevant ethical standards and was approved by the hospital ethics committee.Inclusion criteria: 1) NTPAR operation was performed for the first time; (2) the Diagnostic Standards for Hospital Infection  criteria for central nervous system infection, established by the Ministry of Health of the People’s Republic of China in 2001, were met ; (3) no  NTPAR opData from patients who met the inclusion criteria were retrospectively collected through the hospital electronic medical record system. The data collected included sex, age, body mass index (BMI), a history of hypertension, a history of diabetes, American Society of Anesthesiologists (ASA) score, tumour size, operation time, blood loss, CSF leakage during operation , decreased vision (n = 98), amenorrhea syndrome (n = 33), and acromegaly (n = 19); 54 patients were asymptomatic. Pituitary dynamic contrast-enhanced magnetic resonance imaging (MRI) was performed before the operation, and tumour measurements, three-dimensional computed tomography (CT) of the head and computed tomography angiography (CTA) examination of the head were performed. No significant differences in sex, age, BMI, hypertension history, diabetes history, ASA score or sinusitis were noted between the two groups  and postoperative CSF leakage were independent influencing factors for intracranial infection after NTPAR. Perioperative use of antibiotics was an independent protective factor for intracranial infection after NTPAR  Macroadenoma and tumour size > 1 cm were risk factors for intracranial infection . Macroadnfection . Therefo et al.  . Kingsle et al.   stated t et al.  , and NTPvery low .Intraoperative CSF leakage (Kelly Grade > 2) and postoperative CSF leakage were independent influencing factors for intracranial infection after NTPAR, and perioperative use of antibiotics was a protective factor against postoperative intracranial infection. During transnasal pituitary adenoma resection, the rate of intraoperative CSF leakage can reach 17.9%, and the rate of postoperative CSF leakage is 2.7% . As in tThis study has limitations. This study was a retrospective analysis of the data from patients from a single centre, but some of our findings have been confirmed in multicentre studies. In addition, the follow-up period of this study was short, with no long-term follow-up of patients; therefore, the long-term effect of intracranial infection on patients undergoing NTPAR is not clear.There are a variety of risk factors for intracranial infection after NTPAR. Age > 45 years, tumour size > 1 cm, operation time > 240 min, blood loss > 400 ml, Kelly Grade of CSF leakage > Grade 2, postoperative CSF leakage, lumbar cistern drainage and blood transfusion were the influencing factors for postoperative intracranial infection. Perioperative use of antibiotics was a protective factor against postoperative intracranial infection. Intraoperative CSF leakage (Kelly Grade > 2) and postoperative CSF leakage were independent influencing factors for intracranial infection after NTPAR. Perioperative use of antibiotics was an independent protective factor for postoperative intracranial infection. Finally, the analysis of the causes of different risk factors can be helpful for clinicians to formulate individualized and accurate prevention strategies, which can effectively reduce the probability of intracranial infection after NTPAR.


VHSV  is a globally important group of viruses, infecting a wide range of fish species, in both marine and freshwater. New initiatives are now being taken to re-establish Atlantic cod as an aquaculture species. Any susceptibility to diseases would be relevant for this industry. Atlantic cod was experimentally infected with a strain of VHSV originating from a disease outbreak of farmed rainbow trout in Norway. The fish infected by injection did develop the disease, as verified by investigations of tissue samples. However, no transmission of the virus to cohabitant fish  was found. This indicates that, despite the fact that the virus is capable of causing disease in Atlantic cod, the degree and ability to infect the fish is limited.The first known outbreak caused by a viral haemorrhagic septicaemia virus (VHSV) strain of genotype III in rainbow trout occurred in 2007 at a marine farm in Storfjorden, Norway. The source of the virus is unknown, and cod and other marine fish around the farms are suspected as a possible reservoir. The main objective of this study was to test the susceptibility of juvenile Atlantic cod to the VHSV isolate from Storfjorden. As the pathology of VHS in cod is sparsely described, an additional aim of the study was to give a histopathological description of the disease. Two separate challenge experiments were carried out, using both intra peritoneal (ip) injection and cohabitation as challenge methods. Mortality in the ip injection experiment leveled at approximately 50% three weeks post challenge. Both immunohistochemical and rRT-PCR analysis of organs sampled from diseased and surviving fish confirmed VHSV infection. No VHSV was detected in the cohabitants. The results indicate that Atlantic cod has a low natural susceptibility to this VHSV genotype III strain. One of the most extensive pathological changes was degeneration of cardiac myocytes. Immunohistochemistry confirmed that the lesions were related to VHSV. In some fish, the hematopoietic tissue of spleen and kidney showed degeneration and immunostaining, classical signs of VHS, as described in rainbow trout. Positive immunostaining of the capillaries of the gills, suggests this organ as a useful alternative when screening for VHSV. Rhabdoviridae family, genus Novirhabdovirus. Four genotypes, including several subtypes, have been identified. The different genotypes are mainly associated with geographical regions reviewed in [Viral haemorrhagic septicaemia virus (VHSV) causes viral haemorrhagic septicaemia (VHS) in a wide range of wild and farmed fish species in marine and fresh water, both in Europe, USA, and other parts of the northern hemisphere. VHSV is a member of the iewed in .Oncorhynchus mykiss) in a fish farm in Storfjorden, Møre og Romsdal County, on the west coast of Norway. The fish showed classical signs and pathology related to VHS [Scophthalmus maximus) reviewed in [Gadus morhua and turbot show, low susceptibility to rainbow trout [In 2007, the first confirmed VHS outbreak caused by a genotype III isolate NO-2007-50-385) occurred in rainbow trout .VHSV has been isolated from various wild-caught asymptomatic fish species, including Atlantic cod. The susceptibility of Atlantic cod to VHSV is of interest both in terms of a potential reservoir in marine fish populations, and as a risk factor affecting the Atlantic cod farming industry. A marine VHSV strain was first isolated from Atlantic cod suffering from ulcus syndrome . Low preThe experimental susceptibility of Atlantic cod to various VHSV strains has been found relatively low, except when using injection as the challenge method ,13,14. HThe experiments were approved by the Norwegian Food Safety Authority, as application No. (FOTS) 2011/141935. By this authorization, the experiments were judged, by the relevant national authority, to be in accordance with the WMA declaration of Helsinki.−1) [7.3 TCID50 mL−1 and 107.5 TCID50 mL−1, were used for the ip injection and cohabitant challenge, while 100 μL BF-2 cell growth medium, containing lysed cells, was used for the control fish.VHSV genotype III (NO-2007-50-385) isolated from rainbow trout during a disease outbreak in Norway in 2007 . Culture−1) . A 100 μ−1 (6.7 L min−1). The cod were fed a pellet commercial diet (Skretting). Fish groups and tanks are described in 7.3 TCID50 mL−1, and thereafter rinsed for 1 h. Control fish were ip injected with 100 μL cell growth medium. The fish were monitored daily, and dead fish were removed and recorded.Prior to challenge 15 fish were randomly sampled, using a landing net to verify that the fish were negative for VHSV. The juvenile cod, average size 24.1 g ± 5.3 standard deviation (STDV), were provided by the Institute of Marine Research (IMR) research station Parisvatnet. The fish were bath vaccinated against vibriosis, using the commercial vaccine Norwax Vibriose Marine (Europharma), 4 weeks prior to shipment to the challenge facilities at IMR, Bergen. When arriving at the challenge facilities, the cod was randomly placed, using a landing net in the respective eleven 250 L experiments tanks and acclimatized for 4 weeks until the start of the experiment. Full salinity sea water was used. The temperature was 9.4 °C at the start of the experiment, which had gradually decreased to 8.7 °C by the end of the experiment. Water flow was approximately 400 L hSampling times and selection of organs are described in −1 (8.3 L min−1). The experimental tanks and the diet were similar to experiment 1. Description of fish groups and tanks are given in −1, while the control fish (n = 20) were ip injected with 100 µL cell growth medium. These 20 fish were housed in two separate, greyish coloured 17 L baskets, within the 250 L tanks, to keep them separated from the unchallenged fish , were provided by IMR’s research station at Austevoll. The fish were bath vaccinated against vibriosis using the commercial vaccine Norwax Vibriose Marine (Europharma) at six and two weeks prior to shipment to the challenge facilities at IMR, Bergen. The cod was randomly placed in the respective eleven identical experiments tanks using a landing net and left to acclimatize for 3 weeks until the start of the experiment. Temperature throughout the experiment was 12 ± 0.2 °C and water flow approximately 500 L hged fish . The sidSampling times and selection of organs are provided in Samples used for PCR-detection were stored in RNAlater  at 4 °C for 24 h prior to freezing at −20 °C. RNA was extracted with the automated easyMAG protocol  and measured using NanoDrop ND-1000 . The rRT-PCR assay was performed in accordance with Johansen et al. , using p® universal ABC-AP kit , including both the secondary antibody and the ABC-AP, and the DAKO Fuchsin Substrate and Chromogen system  were used according to manufacturers’ recommendation. The tissue sections were counterstained with Shandon’s haematoxylin and mounted in aqueous mounting medium . Tissue samples from the control fish were used as negative control, while VHSV positive material  served as positive control. All incubations were performed at room temperature (20 °C) in a humidity chamber in a fume hood. A Leica DMBE microscope, equipped with a QImaging MicroPublisher 5 RTV camera, and QCapture software for Mac were used to capture image of the tissue sections. Tissue samples from two individuals with granulomas in kidney and one with vacuoles in brain were tested with IHC, using specific antibodies against Francisellosis [Tissue samples were fixed in Bouin’s fixative and treated according to Evensen & Olesen . Tissue sellosis  and Nodasellosis , respectSwabs from head kidney and ulcers were taken from clinically affected fish during both experiments and grown in pure culture on marine agar (MA) (Difco 2216) and blood agar (BA) (nutrient agar (Oxoid) according to . Bacterip value was corrected by a factor of 5 (p = 0.05/5 = 0.01). Experiment 2: We tested the mortality differences between the 6 fish tanks and the p value was corrected by a factor of 6 (p = 0.05/6 = 0.0083): see Rice (1989). Yates’ correction was used since there was only 1 degree of freedom (df).Since the survival and mortality data are not normally distributed, non-parametric tests were used. A 2 × 2 contingency table, performed in Statistica v 10.0 (StatSoft), was used to test for mortality differences among the treatment and control groups. As multiple independent tests were used to test differences in mortality rate among all challenged groups and control groups, a Bonferroni correction was applied to minimize the possibility of doing a type II error (Rice 1989). Experiment 1: We tested the mortality differences between the 5 fish tanks used for mortality registration and the Mortality in virus injected fish groups started at 5 dpc and increased, until it declined out around 21 dpc and ended between 75 and 88% . During Due to high mortality rate earlier than expected the number of sampled fish was reduced from 5 to 3 fish per tank from day 14 . Later iThe fish displayed normal swimming behavior until death occurred. At five dpc the first observations of petechiae around the fin bases, head and operculum was observed on ip injected fish. A few fish had haemorrhages around the eyes and inside the mouth cavity. Pale gills and clear ascites in addition to a pale and patched miscoloration of the spleen were frequent observations during necropsy of clinically affected and recently deceased fish. Most of the sampled fish had no stomach or intestinal content except for a yellow sticky intestinal substance. Fish that survived the acute stage of the disease (21 dpc) showed bleedings limited to the head and abdominal areas.All fish sampled prior to challenge (n = 15) and control fish sampled during the experiment (n = 15) tested negative for VHSV by rRT-PCR.Initially a selection of brain and heart samples from the first three sampling points at 7, 14, and 28 dpc (9–15 fish per sampling), were tested with two PCR assays to evaluate the diagnostic sensitivity. Assay 2 by Duesund et al.  showed aIn addition to the 92 fish sampled at the fixed sampling points, 16 clinically affected or recent mortalities were sampled and tested by rRT-PCR . All orgA total of 25 ip injected individuals were tested and compared in all organs by both IHC and PCR. All tissues with a Ct value below 33 were found positive by IHC. No positive staining with IHC was observed in tissue samples with Ct values above 33. The last verification of positively stained tissue was found in fish sampled 28 dpc.One control fish was examined at each sampling point. No histopathology or positive immunostaining was detected in tissue samples from these fish A.Positive VHSV-immunostaining was seen in all tissue samples showing histopathological changes. When examining heart tissue, a general observation was degeneration of the myocardial cells B and extThe histopathology, observed in the spleen, varied from no detection (n = 17) to various degrees of necrosis and degeneration of both white and red pulpa with immunostaining (n = 8) D. The poPositive staining of head kidney tissue samples was also variable. Extensive staining of most of the hematopoietic tissue was observed in samples taken at 6–7 dpc (n = 5) E. Later,Another trend was that in organs with less positive staining, such as liver, gills, and eye, the positive staining was associated with endothelial cells in blood vessels and capillaries F–I. PosiOn one occasion, the intestine was included in the organ material sampled for histological examination (16 dpc). Degeneration of muscle cells in muscularis circularis and muscularis transversum was observed D, and thPositive staining in the brain was weak and difficult to observe. Twenty-five brains were analysed with both IHC and PCR method. Of these, only four out of 17 PCR-positive brains were confirmed positive for VHSV with IHC . The positive staining was observed in the metencephalon, both in single neuron cells M and focThe majority of ip injected fish died between three and six dpc . By 14 dPost-mortem examinations of the virus injected fish revealed similar findings to the challenged fish in experiment 1. Cohabitant fish, sampled during the regular samplings, did not show any signs of pathology or disease. Post-mortem examination of diseased cohabitant fish showed petechiae on the abdomen, around the fin base, and head. Pale gills were found on a few individuals. In addition, ulcers on the caudal peduncle were seen in most individuals. Erosion of fins, especially the caudal fin, was also frequently observed.All fish sampled prior to challenge (n =12) and control fish sampled during the experiment (n = 22) tested negative for VHSV by rRT-PCR. The ip injected fish (n = 7) were tested and found positive for VHS-virus. None of the cohabitant fish (n = 48) tested positive for VHSV in any organ samples .The CodELF PCR assay gave stable Ct values within the range of 12–15.Fish injected with virus showed pathology and positive immunostaining with anti-VHSV serum similar to findings in experiment 1 (n = 6). None of the cohabitant fish tested positive with anti-VHSV serum or showed any signs of pathology related to VHS infection (n = 16).Vibrio spp. and Tenacibaculum spp. from ulcers, while Vibrio spp. and Photobacterium spp. were identified from the kidneys. Experiment 2: A mixed bacterial flora of Vibrio spp. (n = 8) was isolated from ulcers of two individuals. No bacterial growth was obtained from bacterial swabs taken from head kidney of four individuals. Bacterial sampling did not provide sufficient data to conclude whether or not some fish died of bacterial causes.Bacterial analysis was performed on diseased fish during both experiments to possibly explain mortality not related to VHS. Experiment 1: Bacterial isolates sampled from ulcers (n = 19) and head kidney (n = 12) of seven fish were sequenced. The 16S rRNA sequencing analysis revealed a mixed bacterial micro flora of Our results show that Atlantic cod are susceptible to VHSV genotype III isolate (NO-2007-50-385) when the virus is injected into the fish. Increased mortality and classical pathological signs, such as degeneration of the hematopoietic tissue, were observed, along with more unique changes in the heart. The virus was, however, not horizontally transferred in our cohabitation experiment, which indicates that Atlantic cod has a low susceptibility to this VHSV isolate at this study’s given conditions.Pimephales promelas, in which presence of VHSV was not detectable in tissue samples towards the end of the experiments (74 days post infection) [Paralichthys olivaceus testing negative at 50 dpc [Although experimental studies have shown that Atlantic cod is not highly susceptible to VHSV ,13,14, tfection)  and expet 50 dpc . This coScophthalmus maximus [Aplodinotus grunniens [Esox masquinongy [Paralichthys olivaceus [Our results show that when examining Atlantic cod challenged by ip injection the heart and/or brain were always among the positive organs in individuals testing positive for VHSV by rRT-PCR. No individuals tested positive in the eyes, gills, liver, spleen, or head kidney samples if the heart and/or brain tested negative. The highest frequency of PCR positive fish was found in heart samples . This co maximus , freshwarunniens , and musquinongy . The healivaceus . Iida etlivaceus  detectedlivaceus ,38,39,40In the present study the histopathological examinations of heart samples revealed activation of the RES system in the endocardium D. The REClupea palasii, after the virus has been cleared from other tissues.Brain samples frequently tested positive for VHSV with PCR assays; however, the IHC staining in brain tissue was scarce, weak, difficult to observe, and limited to single cells. In general, results from IHC studies may, to some extent, depend on the location of the tissue section and, in particular, tissues with few positive cells. Careful analyses of several serial sections of such tissue samples may limit this inaccuracy. Our results are in contrast to the histological findings by Dale et al. , which dClupea harengus [per se. Not only for their water filtering function, but also as an organ in which VHSV seems to persist post infection. In addition, gills are also a relatively large organ and easy to sample.Positive immunostaining of gill tissue was observed in the endothelial cells of capillaries G,H and nharengus . More reharengus  suggesteThe positive staining of circulatory cells and endothelia of blood vessels was a frequent and consistent observation in several tissues, including the gills, eye, and liver F–J. VascSalmo salar using the same genotype III isolate NO-2007-50-385 resulted in a cumulative mortality of 52% around day 20 post infection [5 TCID50 injected in each fish, was lower than in this study (107.3 TCID50 mL−1 and 107.5 TCID50 mL−1), suggesting that salmon is slightly more susceptible to this genotype III isolate than Atlantic cod.The ip injection experiment in this study with VHSV resulted in a cumulative mortality rate between 40–55% approximately 3 weeks post challenge. An ip injection experiment on salmon The cohabitation challenge in this study did not result in a VHS infection in cohabiting fish. This is comparable to previous studies with cod . The amoSalmo trutta, has been experimentally shown for the northern pike Esox lucius [VHSV is widely distributed on the Northern hemisphere and isolated from an increasing number of fish species. VHSV genotype IVb has caused disease and mortality to several wild fish species inhabiting in the Great Lakes of North America ,48,49,50x lucius  and rainx lucius . Cod werx lucius . It is pOur results demonstrate that Atlantic cod is susceptible to the VHSV genotype III isolate from rainbow trout in Norway 2007, following intraperitoneal injection, but no horizontal transfer of infection to cohabitants was found. This indicate that, despite findings of VHSV in wild cod and the positive findings in the i.p injected fish, VHSV genotype III is not highly pathogenic to the species. Atlantic cod can still be carriers of the virus and spread the disease to other fish species. Even so, it is important to keep in mind that VHSV is a virus with a great variety of genotypes and host specificity, and that other genotypes may have different patterns, regarding host range and virulence.The findings also show that the gills are affected by VHSV, and gills may be suitable for sampling and screening for the virus.