Acinetobacter infections in the south of Sweden.The aim of this study was to provide a descriptive account of carbapenem resistance and risk factors for mortality from invasive Acinetobacter species between 2010 and 2019 in Skåne county were subtyped using MALDI-TOF and subjected to susceptibility testing against clinically relevant antibiotics. Association between risk factors and 30-day mortality were analysed in univariate and multivariate logistic regression models.Blood isolates with growth of Acinetobacter strains were carbapenem resistant. Independent risk factors associated with 30-day mortality in the multivariate regression model were Acinetobacter growth in all blood cultures drawn at the day of bacteraemia onset , baseline functional capacity  and correct empiric antibiotics at time of culture .There were 179 bacteraemia episodes in 176 patients included in the study. The 30-day all-cause mortality was 16%. In all, two percent of Acinetobacter bacteraemia in South Sweden found low 30-day mortality and low carbapenem-resistance rates compared to previous international studies which may be due to a higher rate of contaminant findings.This study on  Acinetobacter is a diverse group comprising gram-negative, strictly aerobic coccobacilli of more than 60 different species [Acinetobacter is primarily known as an opportunistic pathogen causing nosocomial infections, particularly in intensive care units (ICUs). Risk factors for infection include longer duration of hospitalization, exposure to broad-spectrum antibiotics, mechanical ventilation, venous and urinary catheterization and other impairments of natural barriers such as wounds and surgical procedures [Acinetobacter infections occur, albeit less frequent compared to nosocomial infections [A. baumannii is the most common species to cause infection[The bacterial genus of  species ,2. Acineocedures ,4. Pneumocedures ,4. Commufections ,4. A. bainfection.Acinetobacter isolates in Europe in 2019, with Greece, Croatia and Romania reporting rates of 92%, 92% and 88%, respectively[A. baumannii strains resistant to all available antibiotics have been reported[The southern and eastern parts of Europe saw the highest rates of carbapenem resistance amongst invasive pectively. A. baum reported.Acinetobacter is a pathogen of increasing importance globally, data on Acinetobacter infections in Sweden are scarce. Contrary to other bacteria with large potential for antibiotic resistance, Acinetobacter is not under national surveillance. We aimed to investigate invasive Acinetobacter infections in our clinical setting in the south of Sweden. The study objective was to provide a detailed descriptive account of invasive Acinetobacter infections with regard to epidemiology, antibiotic resistance, clinical characteristics, treatment and outcome.Even though Acinetobacter species (spp.) between the years of 2010–2019, including personal identification numbers, were obtained from the Department of Clinical Microbiology at Skåne University Hospital. This department handles and analyses all microbiological cultures from primary, secondary and tertiary care in Skåne. All blood cultures positive for an Acinetobacter-species during the study period were included. Exclusion criteria was complete absence of data in medical records and clinical microbiology records. Medical records were reviewed using the software Melior . Laboratory Registers at the Department of Clinical Microbiology, Region Skåne, were accessed through LIMS-systems ww-lab  and Bactius .We performed a population-based observational study in Skåne County in southern Sweden, with a population of 1.3 million. Data onAcinetobacter during the same hospitalization were considered one single event of bacteraemia. The bacteraemia was registered as carbapenem resistant if at least one of the blood cultures with Acinetobacter growth were carbapenem resistant. Patients with repeated bacteraemia at different episodes of in-patient care could be included multiple times. Cases were classified as primary bacteraemia if no other infectious foci were found and secondary if another focus was present . Bacteraemia were defined as nosocomial if the first blood cultures positive for Acinetobacter was drawn after more than 48 hours of hospitalization, otherwise the infection was defined as community acquired. The variable ‘abroad recently’ was defined as abroad stay within 30 days prior to the bacteraemia onset. Immunosuppression was defined as a daily corticosteroid dose equivalent to ≥15 mg of prednisolone, chemotherapy or other medication with significant immunosuppressive effect, such as TNF-alpha inhibitors, or innate immune deficiencies. Charlson comorbidity index was calculated according to the model developed and validated by Charlson et al [Multiple blood cultures positive for on et al . A modifon et al . Baselinon et al .Acinetobacter spp. were re-cultured. Isolates were plated on Mueller-Hinton-agars  and incubated for 18 hours. Isolates were tested for susceptibility to cefiderocol, doripenem, imipenem, meropenem, imipenem-relebactam, ciprofloxacin, levofloxacin, amikacin, gentamicin, tobramycin, colistin and trimethoprim-sulfamethoxazole by disc diffusion using antibiotic-impregnated discs  and E-tests . Inhibition zone diameters were interpreted using the NordicAST  breakpoints (www.nordicast.org). For strains not preserved (n = 72), the original susceptibility testing (performed at time when the Acinetobacter strain was uncovered) was used. Acinetobacter strains were identified to species level using Matrix Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) Mass Spectrometry (MS) and analysed with software MALDI Biotyper (MBT) Compass .All blood isolates drawn between 2010 and 2019 and preserved at the Department of Clinical Microbiology at Skåne University Hospital Lund with growth of p-values for all variables together with measures of discrimination (area under receiver operating curve) and model fit (pseudo R2) for the full model. We checked model assumptions using variable inflation factor <4 for multicollinearity and Hosmer-Lemeshow goodness-of-fit test for calibration. All results are reported with 95% confidence intervals and p-values below 0.05 were regarded as statistically significant. Statistical analyses were performed using Stata MP version 16.1 .We used multivariable logistic regression as our main statistical method for investigating an association between putative risk factors and the outcome. We limited the number of investigated risk factor to six due to a limited sample size and few events. Variables were chosen in two steps, the first part was based on a known or suspected association with the outcome to enable a well calibrated survival model . The second part was more exploratory, and variables were chosen to investigate putative associations with the outcome . We first fit univariate logistic regression models using all variables on their original scale and using complete case analysis respectively. We investigated patterns of missingness and concluded there to be a missing at random mechanism with >5% missingness in at least one variable. We performed chained multiple imputation using predictive mean matching and 10 imputations including all variables in the final model, the outcome variable, and additional data on nosocomial infection, immunosuppression and ICU admission. Non-linearity between the included variables and the outcome was tested with likelihood ratio tests using 4 knot restricted cubic splines for continuous variables and categorization for factor variables. We then fit a multivariable logistic regression model using original and imputed data and reported odds ratios, 95% confidence intervals and The study received ethical approval. This committee waived the need for written consent from the patients of the study due to the retrospective nature of the study and the fact that the patients are unidentifiable. The study was performed in accordance with the Declaration of Helsinki.Acinetobacter species in Skåne. When comparing multiple positive blood cultures against hospitalization episodes, 189 cases of bacteraemia were identified. Of these, 10 cases were excluded from the study due to absence of data. Thus, 179 cases of bacteraemia in 176 patients were included in the study; three patients had two episodes of bacteraemia each.Between 2010 and 2019, there were 233 blood cultures positive for Acinetobacter bacteraemia in Skåne remained fairly constant during the study period, ranging between 0.75 and 1.79 cases per 100,000 citizens and year (mean and median 1.3). Yearly incidence of cases 2010–2019 are displayed in The yearly incidence of Out of the 179 included cases of bacteraemia, 92 (51.4%) occurred in male patients, and the median age was 67 years (range 0–94). The median Charlson comorbidity index score was four (range 0–15), and 44 patients (25%) were considered immunosuppressed. The median length of hospitalization was 10 days (range 0–320), and 26 patients (15%) were admitted to the intensive care unit (ICU). The baseline characteristics of the study population are presented in Out of the 179 episodes of bacteraemia included in this study, 139 (78%) were community-acquired . Of the Acinetobacter strains out of 169 tested (3%) were carbapenem-resistant, of which four were found in patients hospitalized abroad immediately prior to the Acinetobacter bacteraemia. The result of the susceptibility testing is displayed in Acinetobacter were found in 81 (46%) of the bacteraemia episodes. Positive Acinetobacter cultures from locations other than blood were reported in 20 (12%) patients. Primary Acinetobacter bacteraemia, ie with no other foci of infection identified, was seen in 82 (46%) of cases while 97 (54%) were classified as secondary bacteraemia. In all, 107 isolates were preserved at the Department of Clinical Microbiology and available for MALDI-TOF analysis. A. lwoffii was most frequently identified  followed by A. pittii  and A. baumannii . Other than these, 11 different Acinetobacter species were identified  received antibiotics with proper antibacterial activity against Acinetobacter during the course of bacteraemia. The median treatment duration was 10 days (range 1–28) for those effectively treated, either empirically or after adjusting treatment according to the antibiogram. Out of the total 179 cases, 33 patients (18%) received source control treatment, including catheter removal, nephrostomy and wound revision. In 53 cases of bacteraemia (30%), effective antimicrobial treatment targeting Acinetobacter were discontinued or not at all initiated, either due to poor prognosis of the patients  or the Acinetobacter found being considered a contamination.As presented in n = 28), 20% (n = 36) and 30% (n = 54) at 30-days, 90-days and at 1-year, respectively.For all 179 episodes of bacteraemia, all-cause mortality rates were 16%  were significantly associated with 30-day mortality (Table S1). In the full multivariate model including six variables and multiple imputation for missing data (n = 176), all blood cultures positive had the strongest association with 30-day mortality , baseline functional capacity the second strongest  and finally, correct empiric antibiotics (when the culture was obtained) was also associated with an increased risk for death . The final full model showed good discriminatory abilities with an area under the receiver operating curve (AUROC) of 0.86 (95% CI: 0.79 to 0.93) but quite low model fit with an R2 of 23.2%. A complete case (CC) analysis (n = 148) shifted the effect estimates for blood culture positivity and baseline functional capacity somewhat, but the main difference was that correct empiric antibiotics and Charlson comorbidity index switched places for which variable had a p value below 0.05. The result of the multivariate regression analysis is detailed in In an initial univariate complete case analysis, both Charlson comorbidity index, baseline functional capacity, age, all blood cultures positive for Acinetobacter bacteraemia over 10 years in South Sweden. We found a low 30-day all-cause mortality of 16%, a high rate of community-acquired infections and only 3% of the Acinetobacter strains were carbapenem-resistant.The aim of this observational study was to report a detailed account of the epidemiology and outcome of Acinetobacter in our study remained relatively stable throughout the 10 years studied. This is encouraging given the increasingly problematic role of Acinetobacter globally and the continuous increase in international travel seen during the last decade [Acinetobacter infections can be kept at a constant level with the use of proper surveillance, infection prevention and control and restrictive carbapenem usage.The incidence of invasive t decade ,13. Our Acinetobacter isolates in Europe 2018 [Acinetobacter species. We found a predominance of A. lwoffii and A. pittii, and only a minority of strains were A. baumannii. Previous studies found appropriate antibiotic treatment associated with survival in infections caused by A. baumannii, but not by the less virulent A. nosocomialis [The 30-day all-cause mortality of 16% found in this study is low compared to rates of 24–84% reported in previous studies . The reaope 2018 . Multiplcomialis .Acinetobacter, in some cases, might constitute contamination of blood cultures. A mere 21% of patients received empiric antibiotic treatment effective against Acinetobacter, and 26% of patients never received adequate antibiotics during the course of bacteraemia. In spite of this, and in spite of other studies recognizing early administration of active antibiotics as a particularly important factor determining clinical outcome in Acinetobacter infections, the survival rate was high [Acinetobacter might contaminate blood cultures [Acinetobacter growth in all blood cultures drawn at the day of bacteraemia onset had the strongest association with 30-day mortality, indicating that several positive blood cultures may be a marker of actual infection. Furthermore, almost half (46%) of the patients in this study had additional species of bacteria in their blood cultures beside Acinetobacter, which could indicate contamination. Many clinicians refrained from antibiotic treatment on the suspicion of contamination rather than infection. Acinetobacter being a contamination could also explain the low rates of ICU-treated patients, as well as the high rate of community acquired infection (78%); other studies primarily regard Acinetobacter as a nosocomial pathogen [Staphylococcus aureus, this would not indicate contamination but rather a mixed infection. To what extent Acinetobacter bacteraemia constitute a contamination should preferably be studied in future prospective studies.The low mortality also raises the question whether was high ,15,16,20cultures . In our pathogen . HoweverA strength of this study is the long inclusion time, the population-based nature and few exclusion criteria with few excluded patients and contemporary statistical methods. Limitations include the retrospective nature of the study, resulting in a risk for introducing selection bias. We used putative risk factors in our regression analyses believed to be most clinically relevant. This introduces the risk that other important variables associated with 30-day mortality were overseen. The sample size in our study is relatively small which limits the statistical power of the study. Therefore, regression modelling results should be interpreted with caution, including the association between empirical antibiotic treatment and mortality. Furthermore, since many of the standardized variables were collected from unstandardized written medical record, there is a risk of information bias. In addition, some variables have rather large proportions of missing data, although this was handled using multiple imputation. Furthermore, susceptibility testing and characterization to subspecies could only be performed in 107 of 179 strains, as the remaining were lost over time. In addition, we used Etest for testing colistin susceptibility, rather than the broth microdilution method recommended by EUCAST. This could have rendered a falsely high rate of isolates exhibiting colistin resistance and susceptibility, increased exposure.Acinetobacter bacteraemia in the south of Sweden, we found a high degree of community-acquired infection, a low rate of carbapenem resistance and low 30-day mortality, compared to previous international studies. This may reflect a high rate of microbiological contamination rather than clinical infection.In this retrospective cohort study of a decade of Click here for additional data file.


Global epidemic crises, such as the coronavirus (COVID-19), usually expose small and medium enterprises (SMEs) to various kinds of challenges and may put their lives at risk. This study aims to develop a theoretical model to provide insights about the association between innovation practices and the SMEs’ performance and survival while underlining the auxiliary role of external support in such a relationship. Online questionnaire has been used to collect the data from 259 randomly selected SME managers in Saudi Arabia, and the data was analyzed using the SmartPLS3 software. The structural equation modeling results showed that the innovation practices adopted by SMEs to face the repercussions of COVID-19 had a positive impact on the performance and likelihood of business survival. PLS-SEM bootstrap results indicated that external support aids strengthen the positive impact of SMEs’ innovation practices on business survival rather than its performance. The study has several significant practical implications for SME managers, governments, and policy makers that have been stated. Since its emergence in late 2019, the COVID-19 epidemic has caused negative effects on the economies of countries and has had a disastrous impact on human health. The conditions and restrictions imposed in most countries to limit the virus’s spread among people, such as social distancing and quarantines, have led to distortions in the system of supply and demand for goods and slowed many countries’ economies. The repercussions of the COVID-19 pandemic have been felt across all economic sectors and institutions, including small and medium enterprises (SMEs)  have provided various forms of support to SMEs. Governments have adopted several policies that will mitigate the negative effects of this crisis . Then, the direct association between SME innovation practices and business survival is considered (H2). An analysis of the effect of external support on the strength of the relationship between SMEs’ innovation practices and their business performance is performed (H3). Finally, the role of external support in the link between innovation practice constitutions and SME survival is scrutinized (H4).In the next section, the hypothetical relationships of the independent variable and dependent variables in the model are discussed.The current study is limited to SMEs in Saudi Arabia that employ a number of employees, ranging between six and 250, with revenue less than 200 million Saudi Riyals. The online questionnaire prepared through SurveyMonkey was used to collect the data from SME managers. The online questionnaire is less expensive and helps obtain large responses in a short period of time . At the end of the survey period, 259 participants completed the questionnaire, resulting in a response rate of 52%. The total number of questionnaires was sufficient to represent the SMEs in Saudi Arabia, and they were analyzed using PLS-SEM . The questionnaire survey was used to examine the impact of SMEs’ innovative responses to the COVID-19 pandemic and external support received during COVID-19 on SMEs’ business performance and survival. The questionnaire consisted of five sections. The first section addressed the personal profile of the enterprise manager and the enterprise characteristics. The second section focused on the innovation practices that have been adopted in the enterprise since the emergence of COVID-19. The third section dealt with enterprise performance after the outbreak of the epidemic. The fourth section addressed project survival indicators, and the fifth section focused on the usage of external support packages provided by the government and NGOs.Our dependent variables are SME performance and business survival. SME performance was measured using a subjective scale adapted from Bouchikhi , Miller Business survival was measured using two set indicators  derived from Barbosa . The finInnovation practices are presented as the main independent variable in the research model. It comprises five sub-constructs adapted from Crossan and Apaydin . InnovatExternal knowledge is indicated by knowledge and information obtained as a result of existing within social business-related networks in addition to other types of knowledge required to develop enterprise innovation capabilities .The research hypotheses were tested through the partial least squares structural equation modeling (PLS-SEM) using the SmartPLS 3.2.9 software , average variance extracted (AVE), and discriminant validity , and model goodness of fit (GoF) . R2 indicates the degree to which exogenous latent variables explain the variation in the endogenous variables , moderate explanation , and weak explanation . Accordingly, the endogenous variables business survival and business performance have achieved sufficient variance explained values whereas endogenous variable business performance has scored moderate explanation. Therefore, we can conclude that our proposed structural model has sufficient predictive power.Structural model examination results in Table ile Chin  classifiR2 and outer construct average AVE  was estimated by calculating the square root of product of inner construct average t-value. Results indicate that hypothesis (H1) is confirmed, and SME innovation practices have significant positive influence on business performance . Similarly, hypothesis (H2), which is concerned with the strength of the link between SMEs innovation practices and business survival, is supported .PLS-SEM bootstrapping was used to evaluate the hypothesized relationship among research structural model constructs. The analysis results in Table t= 1.029, p>0.10). Hypothesis H4 is concerned with the strengthening impact of “external support” in the relationship between “innovations practices” and “business survival”. Results exhibited that “external support” has a significant moderation effect in the relationship between “innovation practices” and “business survival”; the t-value was found to be 1.698 which is greater than the threshold 1.96, p<0.10. Therefore, hypothesis H4 was accepted on a statistical basis.For analyzing the moderating effects, the variable “external support” was added to the original structural model as a proposed assistant for strengthening the relationship between the independent variable (innovation practices) and the dependent variables  in Fig. . PLS-SEMThe SME sector has been significantly affected by the COVID-19 pandemic. During this crisis, SMEs faced difficulties in performing their operational activities and severe financial risks . These results indicate that the new management practices  that have been implemented in SMEs after the COVID-19 outbreak may result in improved performance and increased chances of survival for these enterprises. In other words, SME managers’ intensive communication with others to obtain business information and assistance, including using social media to market their products, spending reductions through workplace sharing and performing tasks online, worker participation in thinking about the business’s future, and active involvement in SME social networks, may positively reflect on the business’s financial performance. These results partially verified the findings of Gerald et al. . Indeed, the research findings indicated that SME survival indicators were positively affected by innovative practices in the fields of external knowledge, structures, leadership, and renewal of employee activities. These findings indicated that SME managers’ intensive communication with others to obtain business information and assistance may increase the likelihood of the business’s survival. The results of the current study supported the findings of Omar et al.  outweighs their impact on enterprise survival (0.054). This indicates that managerial innovation practices have a greater impact on an enterprise’s short-term compared to long-term performance. These findings agreed with Freeman , who poiThe results also indicated that external support provided to SMEs has a significant role in tempering the relationship between innovation practices and enterprise survival. These results indicate that a moderating role of external support is mainly limited to the relationship between innovation practices and enterprise survival, rather than the enterprise short-term performance. The external support provided to SMEs during the COVID-19 pandemic, whether in the form of training, consultancy, or finance, supports the continuity and survival of these enterprises. The findings partially support the arguments of Song et al.  who callThis study has implications for SME managers, governments, and policymakers. This study has four implications that may help SME managers mitigate the repercussions of this crisis. First, SME managers should continue to develop creative practices in relation to all enterprise activities to adapt to the challenges imposed by the pandemic. Second, SME managers should keep abreast of necessary business information solutions (whether through networking with other entrepreneurs or consultations and training) to help them make rational decisions to overcome the ordeal. Third, SME managers should constantly update their plans and strategies to achieve the flexibility required to respond to the ramifications of COVID-19. Fourth, because the situation of SMEs after the pandemic will largely differ from their pre-pandemic status, SME managers should develop a strategic business plan to address the negative effects of the crisis on their businesses after the pandemic to ensure continuity and survival.Additionally, this study provided empirical evidence of the importance of external support  for the survival of SMEs in times of crisis. As such, this study has important implications for governments and policymakers, who should develop policies to provide more stimulus packages for SMEs that include financing facilities, advisory services, and training. Moreover, governments should encourage NGOs to provide different kinds of support to SMEs in the form of consultations, training, advice, guidance, and psychological support to help them cope with the difficulties caused by COVID-19. Additionally, because the COVID-19 crisis has greatly affected SMEs’ financial position, governments must encourage finance providers to adopt more flexible policies when financing SMEs, such as low-interest loans and the consideration of the enterprise’s financial position for loan installments.This research also has significant theoretical implications because it developed a comprehensive model to examine the role of external support received by SMEs during the COVID-19 pandemic in moderating the relationship between innovation practices adopted by SMEs and their performance and survival. Thus, this study added to the literature by arguing that in times of crisis similar to COVID-19, external support can help an enterprise obtain more positive results from innovation practices in the form of performance improvements and strengthened survival indicators. Crises usually weaken the performance of SMEs and their ability to survive (Michael & Robbins, The current research proposes a theoretical model for studying the moderating effect of external support, provided during the COVID-19 epidemic crisis, in strengthening the link between innovation practices and the performance and survival of SMEs using the PLS-SEM algorithm. The study based on four basic hypotheses in relation to the association between these variables. The main findings of the study suggest that the innovation practices of SMEs have a significant impact on the performance and survival of SMEs. Additionally, the study results confirmed the significant and moderating role of external support provided to SMEs during the COVID-19 epidemic crisis and the survival of the business. Results of the study showed that the policies adopted by Saudi’s government to reduce the repercussions of the COVID-19 epidemic crisis on SMEs, which represented numerous financial support packages and encouraged the support of nongovernmental organizations, was expected to contribute to the resilience of these enterprises in facing such a crisis.Although the current study has achieved findings that have significant implications for SME managers and policy makers, it has some limitations. Because of the wide range of innovation practices, the study focused only on administrative innovation practices and excluded other fields, such as technological innovations. Another limitation of this study is the measurement of the performance of SMEs using financial and marketing indicators and ignoring other indicators, such as administrative, social, and psychological elements.Future research could expand upon these conclusions by addressing the shortcomings of the current study. Because of the diversity of the sectors to which small enterprises belong, it would be beneficial to conduct a sector-based examination of their practices. Furthermore, to obtain comprehensive and in-depth insight into the nature of the relationship between SMEs’ innovation practices, external support, and business performance and survival, all indicators for measuring enterprise performance should be considered, and the types of innovation must be addressed.


Hantaan virus (HTNV), the causative pathogen of hemorrhagic fever with renal syndrome (HFRS), is a negative RNA virus belonging to the Orthohantaviridae family. HTNV envelope glycoprotein (GP), encoded by the genomic medium segment, is immunogenic and is therefore a promising vaccine candidate. Major histocompatibility complex class I (MHC-I) epitopes derived from HTNV has been extensively studied, but little is known of MHC-II epitopes. In silico predictions based on four databases indicated that the full-length HTNV GP has 1121 15-mer epitopes, of which 289 had a high score for binding to the human and murine MHC-II superfamily. It found that epitope ILTVLKFIANIFHTS could potentially bind most MHC-II molecules covering human and murine haplotypes. Dominant epitopes were validated by enzyme-linked immunospot assay of splenocytes from immunized mice; 6 of 10 epitopes supported the predictions including TATYSIVGPANAKVP, TKTLVIGQCIYTITS, FSLLPGVAHSIAVEL, CETYKELKAHGVSCP, CGLYLDRLKPVGSAY, and NLGENPCKIGLQTSS. Conservation analysis of dominant epitopes revealed host–virus interactions without geographic stratification, thus meeting the requirements of candidate vaccines for large-population prophylaxis. These findings provide insight into hantavirus antigenicity and suggest that vaccines targeting MHC-II could provide immune protection in large population to complement symptomatic therapies for the treatment of HFRS. Orthohantavirus genus , and viral RNA polymerase, respectively . The patMHC-II proteins on antigen-presenting cells (APCs), such as dendritic cells, macrophages, and B cells, bind and display peptides from self and foreign proteins at the cell surface that interact with CD4+ T cells. Most peptides bound by MHC-II proteins are 13 to 25 amino acid (a.a.) residues, with an average length of 15 residues . MHC-II The glycoprotein  of Hantaan virus 76-118 were obtained from NCBI GenBank and used as an input for various bioinformatics tools for epitope prediction, conservation analysis, and the experiment. For calculating the conservation rank of predicted epitopes overlapping HTNV GP among Hantaan virus strains, the protein sequences of different isolates (148 envelope glycoproteins as shown in http://tools.iedb.org/mhcii/), NetMHCIIpan 3.2 Server (http://www.cbs.dtu.dk/services/NetMHCIIpan/) (http://imed.med.ucm.es/Tools/rankpep.html) (http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm) were used to predict. Predicted epitopes are chosen, which are highlighted in red for Rankpep or ranking top 2 percentile for IEDB and NetMHCIIpan and top 2% of number for SYFPEITHI. Finally, the epitopes predicted by any tools are regarded the dominant epitope.For H2-A and H2-E epitope prediction, Web-based tools like IEDB recommended method (CIIpan/) , Rankpepep.html) , and SYFThe epitopes of alleles of major human leukocyte antigen (HLA)-II supertypes like DRB1 , DRB3/4/5 , DQA1/DQB1 , DPB1  were predicted using four bioinformatics tools include IEDB recommended, NetMHCIIpan, Rankpep, and SYFPEITHI. It is estimated that these alleles cover more than 98% in all major ethnicities worldwide . Eventua®) which was produced by the Changchun Institute of Biological Products Co., Ltd., Changchun, China was used in this study. The vaccine was made of inactivated and purified HTNV and SEOV in Hamster Kidney Cells .HTNV 76-118 strains were propagated in BHK-21 cells . The bivEight-week-old specific-pathogen-free female mice of four kinds were purchased from the Laboratory Animal Centre of the Fourth Military Medical University. Three types of inbred mice included c57, BALB/c, and C3H within MHC-II haplotypes of H2b, H2d, and H2k, respectively, and the other one was closed colony mice of Kunming. They were randomly divided into two groups of each kind and 10 mice in each group. The first groups of mice were inoculated with 50 μl 1:5 diluted bivalent inactivated vaccine (referring to the instructions of the manufacturer to determine the immune dose). The vaccine is injected every 3 weeks, twice in total. The mice in other groups were injected with 50 μl 100 TCID50 diluted HTNV strain 76-118 only once. Five weeks after the first immunization, the immunized mice were sacrificed. ELISpot assay was used for cellular evaluation. All animals were kept in a pathogen-free animal laboratory with quiet raising condition. The animals were fed with standard feeds and filtered water and bedding was changed every week. More than 10 h of continuous night time were ensured to maintain circadian rhythm. Sterile gloves were taken in all experimental operations. The sacrifice of animals was inhaled carbon dioxide. The whole procedures for care and use of animals were approved by the Ethics Committee of the Fourth Military Medical University’s Animal Center and all applicable institutional and governmental regulations concerning the ethical use of animals were followed.6 splenocytes were added to each pore and stimulated with a final concentration of 20 μg/ml synthetic GP peptides. The plates were cultured in a 5% CO2 incubator of 37°C for 24 h. Completed medium was used as the negative control. Con A (10 μg/ml) was used as the positive control. After incubation, the culture plates were washed with H2O and PBST, and then incubated with 2 mg/ml relevant biotinylated rat anti-mouse IFN-γ antibody at room temperature for 2 h. After washing with PBST, the plates were incubated with streptavidin-HRP 1:100 for 1 h, and then 3-amino-9-ethylcarbazole  was added to the HRP substrate, and the reaction was stopped by washing with water. After air-drying, use the AID ELISpot Reader Classic–ELR06  to count IFN-spots generated by AEC substrate (BD Pharmingen). Each experiment was performed in triplicate, and all the results were shown as the average value of spot forming cells (SFC) per 106 splenocytes.Full-length HTNV GP yielded 147 peptides with an 8-amino acid overlapping 15-mer design strategy, including 83 peptides of Gn and 64 peptides of Gc  because of the inherent difficulties in their production. As mentioned previously, single peptides were diluted with 20 μg/ml in PBS for INF-γ ELISpot assay . Brieflyhttps://bioinfogp.cnb.csic.es/tools/venny/index.html).The criterion for dominant epitopes verification corresponded to the first quarter of values from high to low by ELISpot assay. To compare the experimental and predicted epitopes, predicted epitopes were clustered to eight-amino acid overlapping 15-mer strategies parallel to experimental peptides and the predicted epitopes with common part more than 12 amino acids were clustered. The three groups of epitopes in forecast, inactivated and live virus were drawn into Venn diagram with VENNY 2.1  was used for the bidirectional hierarchical clustering, and the expression values were presented by heatmaps .http://www.allelefrequencies.net/hla6006a.asp) was used to obtain the geographical distribution of different HLA-II subtypes and the results of heatmaps were determined whether there were bias in virus immunity among individuals caused by geographical regions and ethnic groups.The web  and alleles of major HLA-II supertypes. in silico analysis and effectiveness of ELISpot validation of the predicted epitopes.Injection of vaccine or live virus induced cellular responses specific to HTNV. In C57, BALB/c, C3H, and Kunming mice, 38, 34, 35, and 33 epitopes, respectively, were obtained by vaccine injection and 43, 39, 35, and 34, respectively, were obtained by live virus injection . These rTo evaluate the conservation of qualified epitopes among HTNV GPs, we performed multiple sequence alignment of 148 HTNV strains. The dominant HTNV epitopes showed high intraspecific conservation and similar peptides location within GP protein sequences . The distribution of different alleles in same supertype is highly expressed in different regions and races. In Allele frequencies were searched in the website  not only recognize antigenic peptide but also polymorphisms of MHC during antigen presentation by APCs or MHC-expressing cells, which is known as MHC restriction . EpitopePrevious studies have evaluated the efficacy of vaccines that cross-react with heterologous hantaviruses and yielded antibodies that were shown to neutralize 3 HFRS hantaviruses  or 7 HPSThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by experimental animal ethics committee of Fourth Military Medical University.All authors have made substantial contributions to the manuscript. KY, DJ, and HS contributed to the original conception and design of the study. HS, YaL, and JZ predicted epitopes. HS, GX, YF, TW, SX, YuL, BS, and DJ carried out wet-lab experiments. HS, LC, and DJ evaluated experimental results. HS, ZL, and DJ conducted conservative and interactive analysis. LC performed viral challenge assay and relative experiments under BSL-3 conditions. HS, ZL, ML, JX, and DJ presented the tables and figures. HS drafted the manuscript and DJ revised it critically. KY and DJ supervised the whole project. XZ, YS, SY, YunZ, and YusZ assisted the experiments. All authors contributed to the article and approved the submitted versionThis study was supported by the National Natural Science Foundation of China (No. 81772763 and No. 82073154 to KY) and Key Research and Development Program of Shaanxi Province (No. 2020SF-200 to KY).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.


The 5-year survival rate of non-small cell lung cancer (NSCLC) patients has not significantly improved despite advancements in the currently applied treatments. Thus, efforts are put forth in developing novel immunotherapeutic agents targeting cancer-testis antigens (CTA) in NSCLC. This work utilized reverse vaccinology approach in designing a novel multi-epitope vaccine targeting melanoma-associated antigen 3 (MAGEA3), MAGEA4, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), and Kita-Kyushu lung cancer antigen 1 (KK-LC1), being the most frequently expressed CTAs in NSCLC. Epitopes were mapped from the sequences of CTAs. The population coverage (PC) of identified CD4+ and CD8+ epitopes were estimated. Candidate linear B cell (BL), CD4+, and CD8+ epitopes were adjoined in a multi-epitope construct (Mvax) with flagellin domain as an adjuvant. Antigenicity, and cross-reactivity of Mvax were examined. The tertiary structure of Mvax was modelled, and validated. All epitopes included in the vaccine were docked with their human leukocyte antigen (HLA) binders. The immunogenicity of epitopes in Mvax was validated through molecular dynamics analysis. Mvax contains 22 epitopes from MAGEA3, MAGEA4, NY-ESO-1, and KK-LC1. It is classified as antigenic, non-allergen, non-toxic, and possesses physicochemical stability. Epitopes have no significant hits with other human proteins, except for 2 other CTAs frequently expressed in NSCLC. The stretch of BL epitopes in Mvax confers flexibility, and accessibility emphasizing its antigenicity. The tertiary structure analysis showed that Mvax model has good structural quality. All epitopes included in the vaccine are highly immunogenic as indicated by favorable binding affinity, low binding energy, and acceptable root-mean-square deviation (RMSD). CD4+ and CD8+ epitopes have global PC of 81.81%, and 84.15%, respectively. Overall, in silico evaluations show that Mvax is a potential immunotherapeutic agent against NSCLC.  Lung cancer is the primary cause of cancer-related deaths in the world with non-small cell lung cancer (NSCLC) making up 84% to 87% of the lung cancer cases . Current treatments include radiation therapy, surgery, chemotherapy, monoclonal antibodies, or the combination of such. However, these methods are often accompanied by adverse reactions, metastasis, and tumor regression. Despite the advancements made in the last decades, the 5-year survival rate of lung cancer patients remains poor . MAGEA genes has been strongly associated with lung cancer . MAGEA3 is one of the most frequently expressed CTAs in NSCLC . Similar to MAGEA3, MAGEA4 is also frequently expressed in NSCLC patients . Another commonly expressed CTA is the New York esophageal squamous cell carcinoma-1 (NY-ESO-1), also known as cancer/testis antigen 1. It is encoded by gene cancer/testis antigen A1 (CTAGA1) which is expressed in testis, ovary, and various cancer tissues such as NSCLC. NY-ESO-1 is currently known as the most immunogenic CTA . Kita-Kyushu lung cancer antigen 1 (KK-LC1), encoded by cancer/testis antigen 83 (CT83), is a single-pass membrane protein normally expressed in testis, and other cancer types including lung cancer . Strong immunogenicity, tumor-restricted, and biased expression of MAGEA3, MAGEA4, NY-ESO-1, and KK-LC1 can offer vast drug-development opportunities, including immunotherapeutic agents and vaccines. Therefore, the primary goal of this work is to apply reverse vaccinology to efficiently design a novel multi-epitope vaccine that can potentially induce immune response against multiple CTAs expressed in NSCLC. Multi-epitope vaccine can be more advantageous than whole recombinant proteins, or even single peptides. It may cover wider population coverage, and exclude cross-reactive sequences to prevent adverse reactions. With the aid of immunoinformatics, the cost, effort, and time required to develop vaccines, can be minimized .One of the alternative treatments used for lung cancer is immunotherapy. This approach takes advantage of the patient’s immune system by training it to recognize, and respond to antigens expressed by cancer cells, resulting to tumor-mechanism inhibition or direct cytotoxicity to cancer cells. To develop effective immunotherapeutic agents, cancer antigens should be identified and strategically targeted. Tumor antigens can be overexpressed proteins, mutated gene products, or preferentially expressed in cancer cells other than normal cells such as the cancer-testis antigens (CTA) . The expression of CTAs in normal tissues is restricted to the testis, ovaries , and are widely expressed in several human cancer tissues. CTAs have been shown to induce humoral or cellular antitumor responses in cancer patients . Nevertheless, autoimmunity is prevented due to the downregulation of human leukocyte antigen (HLA), and the expression of immunosuppressors  in placenta, ovary, and testis. Well-studied CTAs include the members of melanoma-associated antigen (MAGE) family. The expression of Identification of linear B cell and T cell epitopes50 ≤ 150nM) in NN-align-2.3, and with rank ≤ 10 in NetMHCIIPan4.0E, were selected as final CD4+ epitopes. CD8+ epitopes and their MHC I binders were identified using NetMHCcons method in the Proteasomal cleavage/TAP transport/MHC class I combined predictor which combines three MHC-peptide binding prediction methods to give more reliable result . A list containing the most frequent MHC I binders was uploaded . Epitopes with 8-11 residues, binding to at least 5 MHC I, with TAP and proteasome scores > 1.0, and with IC50 ≤ 150nM, were selected as final CD8+ epitopes. Peptides with IC50<500nM are classified as good binders . A multi-epitope vaccine can offer larger population coverage (PC). To estimate global PC, the set of final CD4+ and CD8+ epitopes were queried separately, using the Population Coverage tool in IEDB. The amino acid sequences of Homo sapiens MAGEA3 (P43357), MAGEA4 (P43358), NY-ESO-1 (P78358), and KK-LC1 (Q5H943) were retrieved from UniProt Database. Extracellular sequence of KK-LC1 (22-113) was mapped to identify linear B cell (BL) epitopes. ABCPred, and Emini Surface Accessibility (ESA), BepiPred Linear Epitope (BLE), and Kolaskar and Tongaonkar Antigenicity (KTA) tools in the Immune Epitope Database (IEDB) were utilized. Epitopes from ABCPred with overlapping sequences from at least 2 tools in IEDB, were chosen as final BL epitopes. Among the four tumor antigens, only KK-LC1 has an extracellular sequence; thus, it was the only antigen used to identify BL epitopes. The full-length amino acid sequences of MAGEA3, MAGEA4, NY-ESO-1, and KK-LC1 were mapped for T cell epitopes using default thresholds. CD4+ epitopes, and their corresponding major histocompatibility complex (MHC) II binders were identified using NN-align-2.3 (netMHCII-2.3), and NetMHCPanII4.0E in IEDB. A reference list for the most common MHC II binders was utilized . Consensus CD4+ epitopes (15 residues) with good binding affinity to at least 5 MHC II  sequence was retrieved from Uniprot database (P06179), and used as an adjuvant. Its N-terminal domain 1 (ND1) region (46-175) was linked to the cluster of CD8+ and CD4+ epitopes, via flexible EAAAK linker. Then, the C-terminus of the construct was fused with the C-terminal D1 (CD1) region of fliC (398-455) using EAAAK. The series of BL epitopes were connected to CD1 of fliC. Lastly, a valine residue was added at the N-terminus of the construct (Mvax) with the aim of increasing its half-life .Evaluation of antigenicity, allergenicity, toxicity, cross-reactivity, and physicochemical properties of MvaxThe whole sequence of Mvax construct was assessed for antigencity in VaxiJen2.0 using ≥0.5 threshold in tumor model. It utilizes an alignment-independent method predicting an antigen with 70%-89% accuracy . The sequence was further evaluated in ANTIGENpro, with estimated accuracy of 76% using cross-validated experiments . AllergenFPv1.0 was used to identify potential allergen sequence in Mvax. This tool generates the highest Tanimoto score to the nearest allergenic sequence in the database . Sequences with exact match to human proteome, other than the target CTAs, may result to cross-reactivity; thus, the sequence of Mvax was queried against human proteome databases using default settings in the protein basic local alignment search tool proteins (BLASTp). The physicochemical properties of Mvax were estimated in sillico in ExPASy ProtParam tool (https://web.expasy.org/protparam/). Secondary structure analysis, and tertiary structure modelling with validationThe series of B cell epitopes in Mvax must be flexible, and exposed enough for B cell receptors (BCR) to effectively bind to it. In this work, Mvax sequence was evaluated for its secondary structure composition, disordered, accessible, and hydrophilic regions. The position of B cell epitopes were particularly investigated. GOR4 web tool (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html) was utilized to evaluate the secondary structure composition of Mvax. Disordered, and solvent-accessible regions were identified in RaptorX (http://raptorx.uchicago.edu/StructurePropertyPred/predict/). GalaxyTBM tool  was used in generating tertiary structure model for Mvax. GalaxyRefine server  was employed to improve resulting tertiary structure model. Different validation tools were employed to check the quality of refined structures. PROCHECK generated Ramachandran plot to show percentage of residues lying within the favoured, and disallowed regions. ProSa-web calculates the z-score of a structure by estimating its deviation from validated x-ray crystallography and NMR structures of native proteins . Qualitative model energy analysis (QMEAN) score provides an estimate of the degree of nativeness in the structural model by comparing it to experimental structures of similar size. Scores ≤-4.0 indicate a low-quality model . Finally, the best structure model for Mvax was chosen and viewed in Pymol. Structural B cell epitopesThe BL epitopes incorporated in Mvax must be protruded enough so BCRs can bind to it. This work utilized Ellipro to identify structural epitopes on the tertiary structure model of Mvax. As the best structure-based algorithm amongst the others, Ellipro predicts the conformational and the linear epitopes based from protrusion index (PI) of a residue, and provides PI score for each protruded sequence . Molecular docking and simulation50 = 143.49). Most CD4+ epitopes were docked with their most common HLA-DRB1*01:01, while epitopes VLHHMVKISGGPHIS, ERVIKNYKRCFPVIF, and ALIVFWKYRRFQRNT were docked with HLA-DRB1*11:01 as their common binder. PDB structures of HLA-B*15:01 (PDB ID: 6uzs), HLA-B*35:01 (PDB ID: 4lnr), HLA-DRB1*01:01 (PDB ID: 1aqd), and HLA-DRB1*11:01 (PDB ID: 6cpl) were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). Epitopes were docked to their respective binders using GalaxyPepDock which performance has been proven superior versus other web servers . Then, GalaxyRefineComplex tool was used to refine the structures of docked complexes, and viewed in iCn3D server (https://www.ncbi.nlm.nih.gov/Structure/icn3d/full.html). The binding energies, and dissociation constants of docked complexes at 37oC were calculated in PRODIGY web server which makes use of both intermolecular interactions, and non-interface surface properties . Molecular dynamics simulation was performed to evaluate the stability of interaction within the complex using plot of root-mean-square deviation (RMSD) per residue. For this process, C-alpha Brownian dynamics was set in 100 ps time, time change 0.01 ps, 3.8Ǻ distance between alpha carbon atoms, output frequency of 10 steps, and force constant of 40 kcal/mol Ǻ2. It was performed in MDWeb server (http://mmb.irbbarcelona.org/MDWeb/) which employs force-field Amber-99sb in GROMACS MD setup with solvation .All CD8+ epitopes were docked with their most common HLA I binder(HLA-B*15:01), except for GSVVGNWQYFF which was docked with HLA-B*35:01 having the lowest binding affinity (ICB cell and T cell epitopesBL epitopes with residues 1-20 were mapped from the extracellular sequence of KK-LC1 (22-113). BL epitopes (16 residues) from ABCPred tool which overlapped with sequences from at least 2 tools in IEDB are KK-LC1 sequences 22-37, 77-92, and 89-113. Finally, sequences were merged to 22-37, and 77-113, and were included in the vaccine construct. Mvax constructThe schematic presentation of the whole Mvax construct  shows thAntigenicity, allergenicity, toxicity, cross-reactivity, and physicochemical properties of MvaxE. coli, in vivo). Its instability index is 31.55, aliphatic index (thermostability) is 78.08, and its grand average hydropathicity (GRAVY) is -0.156 which classifies it as slightly hydrophilic. Mvax is validated antigenic in Vaxijen server (0.5956), and in ANTIGENpro (0.903634). It is classified as non-allergen, having the highest Tanimoto similarity index of 0.86 with Q96PE2. Potentially toxic sequences were not mapped in the vaccine. Results showed that four epitopes have exact sequence match with other known CTAs—MAGEA6 and CTAG2. These include MAGEA3 CD8+ 137-147, 142-151, 176-186 with MAGEA6; and NY-ESO-1 CD4+ 143-157 with CTAG2. More importantly, none of the epitopes has significant match with other human proteins in the databases. Mvax construct has 540 residues with molecular weight of 57,839.58 Da, and 9.23 theoretical pI. The estimated half-life is 100 hours , >20 hours , and >10 hours  involved in its tumor-progression mechanisms. These require more extensive, yet more specific antigen-targeting approach to increase its efficacy, and to cover the majority of its cases. A clinical trial which utilized engineered T cell receptor targeting MAGEA3, lead to severe adverse events which might have been due to the cross-reaction of the peptide with MAGEA12. MAGEA12, A1, A8, and A9 were also assayed to be positively expressed in the brain in low levels . A recently concluded phase III clinical trial utilized MAGEA3 as an adjuvant immunotherapeutic agent for NSCLC patients, but the disease-free survival rate did not improve . Thus, this work targeted more than 1 type of antigen. The most frequently associated CTAs to NSCLC were utilized with the objective of designing a multi-epitope vaccine that can potentially induce immune responses against NSCLC expressing MAGEA3, MAGEA4, NY-ESO-1, and KK-LC1. Epitopes were cautiously evaluated to prevent potential cross-reaction with MAGEA12, A1, A8, and A9. Sequences with significant match to these antigens were excluded. Three BL epitopes identified from the extracellular sequence of KK-LC1 are highly antigenic, and can potentially induce humoral immune response against KK-LC1. Because this response is induced with the aid of T helper cells, CD4+ epitopes were also mapped in KK-LC1. CD4+ epitopes were identified from MAGEA3, MAGEA4, and NY-ESO-1. To be able to induce cytotoxic immune responses, CD8+ epitopes were also identified. Consensus approach was employed and at least 2 tools were utilized in the identification of all BL, CD4+, and CD8+ epitopes, increasing the accuracy of prediction. Besides good binding affinity, proteosomal cleavage, and TAP transport scores were also considered. In addition, Mvax possess large global population coverage with CD4+ (81.81%) and CD8+ (84.15%) epitopes. Some MHC binders identified in this study are not currently available in the IEDB tool, and were not included by the tool in its calculations; thus, it must be noted that the estimated %PC in this work can be larger in reality. The choice of adjuvant is another crucial step in vaccine design. Herein, the type of pathogen recognition receptor (PRR) activated by the adjuvant was carefully considered, as the activation of some toll-like receptors (TLRs) is associated to lung cancer progression . On the contrary, TLR5 activation was found to have antitumor effects in NSCLC cells . Flagellin is a well-studied ligand of TLR5. Due to its reported efficacy and safety, fliC was incorporated as adjuvant in Mvax. Among all 4 domains of fliC, D1 is known to be highly conserved, and has the essential interaction binding-sites to activate TLR5 . Only D1 sequence was included to avoid possible adverse reactions. Overall, the inclusion of flagellin in Mvax may offer antitumor effects while enhancing the immunogenicity of epitopes. EAAAK linkers were used to preserve the bioactivity of fliC D1 . Total of 17 peptides, merged from 22 epitopes, were adjoined using AAY and GPGPG linkers known to effectively present epitopes in vivo . Besides efficacy, vaccines must possess safety and stability. Mvax is classified as non-allergen, and non-toxic. In addition, no sequence in the human proteome databases was found to significantly match the epitopes in Mvax, except for the 4 epitopes that match exactly with MAGEA6 and CTAG2. MAGEA6 and CTAG2 are expressed in testis, and in very low levels in placenta but are observed to be highly expressed in NSCLC and various cancer types . Thus, the potential cross-reactivity of Mvax with MAGEA6 and CTAG2 may offer additional antitumor benefits. The vaccine is classified as stable (instability index <40), and can also be utilized in areas with warmer climates as it possess thermostability indicated by higher aliphatic index. In addition, the valine residue linked to the N-terminus of Mvax has notably lengthen its half-life. Mvax is classified as antigenic in tumor models. It has large percentage of random coils, disordered or flexible regions, and exposed sequences, more importantly within the series of B cell epitopes , which pIn conclusion, this is the first work to use reverse vaccinology approach in designing a multi-epitope vaccine targeting MAGEA3, MAGEA4, NY-ESO-1, and KK-LC1 in NSCLC. In silico assessments showed that Mvax confers antigenicity, immunogenicity, stability, and safety. In vitro and in vivo studies are anticipated. The author solely conducted all the requirements for the accomplishment of this work.


Novel vaccines are required to effectively combat the epidemic spread of tuberculosis. Using in silico approaches, this study focuses on prediction of potential B cell and T cell binding immunogenic epitopes for 30 putative outer membrane proteins of Mtb. Among these, certain immunodominant epitopes of Rv0172, Rv0295c, Rv1006, Rv2264c, and Rv2525c were found, which are capable of binding B-cell and a maximum number of MHC alleles. The selected immunodominant epitopes were screened for their allergenic and antigenic properties, their percentage identity against the human proteome and their structural properties. Further, the binding efficacy of the immunodominant epitopes of Rv0295c and Rv1006 with HLA-DRB1*04:01 was analyzed using molecular docking and molecular dynamics studies. Hence, the in silico-derived immunogenic peptides (epitopes) could potentially be used for the design of subunit vaccines against tuberculosis.The online version contains supplementary material available at 10.1186/s43141-021-00148-9. Mycobacterium tuberculosis (Mtb) and is a deadly disease that affects millions of people worldwide. In accordance with the WHO Global tuberculosis report 2018, TB is one of the top ten causes for human deaths and estimated around 1.3 million deaths in HIV-negative people. Moreover, 10.0 million people developed TB disease in 2017. The emergence of multi-drug and extensively drug-resistant strains of Mtb increases the burden of the drug treatment regimen for TB. Currently, Bacille-Calmette-Guerin (BCG) is the only available vaccine for treating TB. In infants, it is shown to have a protective effect against tuberculous meningitis and miliary tuberculosis [Tuberculosis (TB) is caused by pathogenic bacillus rculosis . HoweverImmunoinformatics involves the use of computational tools to predict the immunogenic epitopes or peptides which could be used to design ideal subunit vaccine candidates. These tools simply use the organism’s genetic information, and it reduces the cost and time taken for the development of vaccines . SubunitVibrio anguillarum. Similarly, Rauta et al., in [Vibrio cholerae. Song et al., in [Outer membrane proteins (OMPs) play an important role in the host-pathogen interactions and in maintaining the integrity and permeability of the cell membranes. Due to their localization on the mycobacterial surfaces, they can be easily targeted by the host immune system and hence they are ideal candidates for vaccine design . Recentl al., in , have pr al., in , have idThe overall methodology adopted in this study to determine potential vaccine candidates of the putative OMPs of Mtb is depicted in Fig. FASTA Sequences of 143 putative OMPs of Mtb were retrieved from UniProtKB protein database and subjected for epitope prediction. UniprotKB Ids of the retrieved sequence is given in Table B cell epitope prediction for the retrieved FASTA sequences of the OMP’s of Mtb was performed using IEDB tools. BepiPred Linear Epitope prediction method  was emplAntigenic and allergenic values of the predicted B cell epitopes were calculated using VaxiJen (antigenic proteins should possess a score above 0.4)  and AlgPThe B cell binding epitopes were further screened for their similarity against humans, in order to avoid cross-reactivity. BLASTp program  was usedThe predicted B cell epitopes were further subjected for T cell binding prediction. The MHC-I binding predictions were made using the IEDB analysis resource Consensus tool  which coThe MHCII binding predictions were made using the IEDB analysis resource Consensus tool , 31. TheS. typhi.Immunodominant epitopes (IDEs) are regions which can bind B cell as well as maximum number of MHC-I and MHC-II alleles. The identification of IDEs has immense potential as it can lead to strong immune response and it can be effectively used to design peptide based vaccines. This method of finding IDEs was successfully employed by Verma et al. in  for the Structural properties of the IDEs such as solvent accessibility, transmembrane topology, and solubility upon overexpression were predicted using ACCpro, ABTMpro, and SOLpro tools respectively, found in the SCRATCH protein prediction server . SolventThe selected IDEs of Rv0295c and Rv1006 were modeled using PEPFOLD 3 server  and wereSong et al., in , have idThe list of B cell epitopes predicted from the 30 putative OMPs of Mtb, along with their Vaxijen and AlgPred scores and the number of MHC alleles capable of binding these epitopes is given in Table 382ASTASTLPKE IAYSEPRLQPPNGYKDTTV PGIWVPDTPLSHRNTQPGWVVA432 of Rv0172 is predicted to be a B cell binding epitope and is predicted to bind all the 27 reference alleles of MHC Class I and Class II, respectively. Moreover, it is predicted to be antigenic and non-allergenic, cannot find significant similarity against the human proteome. This IE is a non-transmembrane protein; solvent exposed and is predicted to be soluble when over-expressed. Additionally, Rv0172 belongs to Mce  family of proteins which are crucial for the virulence of Mtb [e of Mtb .228AIGQDPKLAPA PMLERQANQRSDEW VDRYRAEAPRLG264B-cell binding epitope of Rv0295c is non-allergenic and antigenic and binds 26 alleles of MHC Class I and 27 alleles of MHC Class II. It has no sequence similarity with the human proteome, solvent-exposed, non-transmembrane protein and is predicted to be soluble when over-expressed. In fact, Rv0295c is a Trehalose 2-sulfotransferaseand it involves in catalyzing the transfer of a sulfuryl group from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to trehalose, which leads to the synthesis of trehalose-2-sulfate (T2S) [Similarly, we found a potential IE. te (T2S) .24LNGCSSSASHRG PLNAMGSPAI PSTAQEIPNPLRGQ59” from Rv1006 is predicted to be a B-cell binding epitope which also binds 26 and 27 alleles of MHC Class I and Class II alleles, respectively. It is antigenic, non-allergenic and has least similarity to the human host. Additionally, it is solvent exposed, non-transmembrane and soluble upon overexpression. Rv1006 is believed to be a conserved hypothetical protein.The next IDE is “383AWSEADEDSHI GPAPGYTAARPSL SFDHDAHAEPEPKSPPIPW425” is predicted to be a B cell binding epitope from Rv2264c, it also binds all of the 27 reference alleles of Class I and Class II, respectively. It is also predicted to be antigenic, non-allergenic and has least similarity to the human proteome. It is solvent exposed non-transmembrane and soluble upon overexpression. Rv2264c is a conserved hypothetical protein.“102YGKGSTADWLGGA SAGVQHARRGSELHA AAGGPTSAPIYA SIDDNPSYEQYK153” is predicted to be a B cell binding epitope from Rv2525c, it also binds all of the 27 reference alleles of Class I and Class II, respectively. It is also predicted to be antigenic, non-allergenic and has least similarity to the human proteome. It is solvent exposed non-transmembrane and soluble upon overexpression. Rv2525c is a tat secreted protein and it functions as a putative peptidoglycan hydrolase [“ydrolase .Five selected IDEs are discussed below:The epitopes of Rv0295c and Rv1006 was modeled using PEPFOLD 3 server and was subjected to molecular docking studies with the 3D structure of HLA-DRB1*04:01 using Cluspro server. The other epitopes could not be modeled by the PEPFOLD 3 server as the length of the epitope was greater than 50 amino acids. The epitope of Rv2264c was having 52.17% similarity  . The antGenerally, bacterial OMPs serve as potential vaccine candidates, as their exposed epitopes on the bacterial cell surface could be easily recognized by the host immune system , 22. In Therefore, from our in silico study of the OMP’s of Mtb, we have retrieved five IDEs Table  which caIn this study, through immunoinformatics approach, potentially immunogenic epitopes for 30 putative OMPs of Mtb have been identified. Immuno dominant epitopes designed for Rv0172, Rv0295c, Rv1006, Rv2264c, and Rv2525c were predicted to be non-allergenic, antigenic and capable of binding B cells and a maximum number of MHC alleles. These epitopes also show lesser or no sequence similarity with the human proteome, solvent-exposed, non-transmembrane and soluble upon overexpression. Molecular docking and molecular dynamics analysis of Rv0295c and Rv1006 epitopes-HLA-DRB1*04:01 complex further enhance our study. Thus, we suggest that these in silico-derived epitopes could be useful in developing peptide-based subunit vaccines against tuberculosis.Additional file 1: Table S1. UniprotKB IDs of the putative OMPs of Mtb. Table S2. List of MHC-I and MHC-II alleles employed in the study. Figure S3. Ramachandran plot of IDE of Rv0295c. Figure 4. Ramachandran plot of IDE of Rv1006. Figure S5. Ramachandran plot of IDE of Rv2265.


Background: The category of the “stromal tumors of the lower female genital tract” encompasses a wide spectrum of lesions with variable heterogeneity, which can be nosologically classified on the basis of their morphologic and immunohistochemical profiles as deep (aggressive) angiomyxoma (DAM), cellular angiofibroma (CAF), angiomyofibroblastoma (AMFB) or myofibroblastoma (MFB). Despite the differential diagnosis between these entities being usually straightforward, their increasingly recognized unusual morphological variants, along with the overlapping morphological and immunohistochemical features among these tumours, may raise serious differential diagnostic problems. Methods and Results: The data presented in the present paper have been retrieved from the entire published literature on the PubMed website about DAM, CAF, AFMB and MFB from 1984 to 2021. The selected articles are mainly represented by small-series, and, more rarely, single-case reports with unusual clinicopathologic features. The present review focuses on the diagnostic clues of the stromal tumours of the lower female genital tract to achieve a correct classification. The main clinicopathologic features of each single entity, emphasizing their differential diagnostic clues, are discussed and summarized in tables. Representative illustrations, including the unusual morphological variants, of each single tumour are also provided. Conclusion: Awareness by pathologists of the wide morphological and immunohistochemical spectrum exhibited by these tumours is crucial to achieve correct diagnoses and to avoid confusion with reactive conditions or other benign or malignant entities. MTG1–CYP2E1 fusion transcripts (AMFB and MFB), it is likely that they are histogenetically related, as previously suggested [The category of the “stromal tumours of the lower female genital tract” covers a wide spectrum of lesions with variable morphological and immunohistochemical heterogeneity. They arise from the specialized, hormonally responsive stroma of the lower female genital tract, and, based on morphological and immunohistochemical features, at least four tumour entities can be nosologically recognized: (i) deep (aggressive) angiomyxoma (DAM); (ii) cellular angiofibroma (CAF); (iii) angiomyofibroblastoma (AMFB) and (iv) myofibroblastoma (MFB) ,2. Amonguggested ,4,5. Notuggested .Based on these morphological, immunohistochemical and cytogenetic findings, the hypothesis that vulvovaginal CAF, MFB and AMFB are in the spectrum of a single entity, likely arising from a common precursor stromal cell of the lower female genital tract, has been postulated ,4,5,6,7.The data presented in the present paper have been retrieved by the entire published literature on the PubMed website about DAM, CAF, AFMB and MFB from 1984 to 2021. The selected articles were mainly represented by small-series (due to the relative rarity of these tumours) and, more rarely, by single-case reports with unusual clinicopathologic features. The histological illustrations have been retrieved from a personal consultation series of DAM, CAF, AMFB and MFB (63 cases) by Prof. G. Magro. The morphological and immunohistochemical diagnostic clues for each single tumour are provided. In addition, the unusual morphological features that can be diagnostically challenging are emphasized in the form of tables.DAM is a rare, locally infiltrative and non-metastasizing myofibroblastic stromal tumour first described by Steeper and Rosai in 1983 ,8,9. TheAlthough the diagnosis of DAM is usually straightforward if the classic-type morphology is encountered, in a recent paper some clinicians reported that several unusual morphological features do exist, causing diagnostic problems, especially in recurrent tumours .By immunohistochemistry, DAM is typically a desmin-positive myofibroblastic tumour F with vaatypical CAF”) to, frankly, areas of sarcomatous transformation [RB1 or FOXO1 at the 13q14 locus [CAF, originally described by Nucci et al. in 1997 , is a raormation ,23,24. Tormation ,23,24. Tormation ,22. The ormation ; variablormation . Nuclearormation . Notably14 locus . CAF is 14 locus ,25. NotaHMGA1 and HMGA2 rearrangements [MTG1–CYP2E1 fusion, has been reported in AMFB [AMFB is a benign, superficially located (subcutaneous) stromal tumour, firstly described by Fletcher et al. in 1992 , that mangements  and the ngements , the latngements , the neo in AMFB . AMFB is in AMFB . DistantMTG1–CYP2E1 fusion transcripts [MFB of the lower female genital tract is a benign neoplasm composed of spindled cells with myofibroblastic profile, that arises from the subepithelial stroma of the vagina and, less frequently, of the vulva and cervix  48,49,5,52,53,54nscripts ; howeverHMGA2 rearrangements, while the latter shows the absence of RB1 nuclear expression and the overexpression of CY2E1, respectively, as a surrogate of the 13q14 deletion and MTG1–CYP2E1 fusion transcripts [RB1 and FOXO1 at the 13q14 locus but MTG1–CYP2E1 fusion transcripts are commonly identified.Differential diagnosis of the vulvovaginal stromal tumours may be challenging, as they share several clinical, morphological , immunohnscripts . AMFB maHMGA2 rearrangements by means of immunohistochemistry or molecular biology is helpful for the diagnosis of DAM; (iv) if the pathologist is dealing with a tumour exhibiting overlapping morphological features among the different histotypes  it could be a trivial problem to try to subtype a specific tumour at any cost and the use of the generic term “benign stromal tumour of the lower female genital tract” seems to be appropriate.Based on our experience in approaching the diagnosis of the benign stromal tumours of the lower female genital tract, namely DAM, CAF, AMFB and MFB, we strongly suggest adopting the following recommendations: (i) the diagnosis should be mainly based on histological features in combination with clinical and macroscopic features; (ii) immunohistochemical analyses may be misleading for correct tumour classification due to the non-specific results in the different histotypes; however, a diffuse desmin immunoreactivity in a tumour with abundant myxoid stroma is highly suggestive of DAM; (iii) in cases with ambiguous morphological and immunohistochemical features, F.I.S.H. analysis showing a 13q14 deletion is helpful in ruling out the diagnosis of DAM and AMFB; similarly, the detection of


Micropterus salmoides). Four isonitrogenous and isolipidic diets were prepared: two basal diets containing low (9.0%) and high (14.4%) levels of starch (named as LS and HS), and 0.125% OE was supplemented to each basal diet (named LSOE and HSOE). The results show that high-starch diets had significant negative effects on growth performance, with lower FR, SGR and higher FCR, whereas OE significantly lowered FCR, determined by two-way ANOVA analysis. High-starch diets induced oxidative stress, inflammatory response and liver function injury, with significant increases in the content of plasmatic AKP, AST, ALT, hepatic SOD and MDA, and up-regulation of hepatic TNFα, IL1β, and TGFβ1 gene expression. In addition, a high-starch diet decreased the phosphorylation of AMPK and upregulated the expression of SREBP, together with higher hepatic liver lipid and HSI. The oxidative stress and lipid metabolism disorders indicate metabolic liver disease (MLD) of largemouth bass fed high-starch diets. Feeding on OE-supplemented diets increased the hepatic antioxidant capacity by decreasing the content of MDA and SOD. Fish fed the HSOE diet had an activated phosphorylation of JNK and decreased expression of pro-inflammatory IL1β compared with those fed the HS diet, which strongly indicated that the degree of inflammatory responses was reduced after OE supplementation. Interestingly, this study demonstrated that OE regulates hepatic lipid metabolism in fish by inhibiting the expression of hepatic lipogenesis genes (ACC1 and FASN) and promoting lipolysis (ATGL) and β-oxidation (CPT1α) to prevent TG accumulation. In conclusion, high-starch feed induced oxidative stress and lipid metabolic disorder of largemouth bass, while supplementation with OE improved its antioxidant capacity, anti-inflammatory responses and lipid metabolism. However, hepatic histopathological results suggested that OE supplementation could not completely repair the MLD caused by the high level of starch in largemouth bass.An 8-week feeding trial was conducted to investigate the effects of high-starch diets and the supplementation of an olive extract (OE) on the growth performance, liver health and lipid metabolism of largemouth bass ( Micropterus salmoides) is a North America-native carnivorous species, which has been one of the most widely cultured and consumed fish species in China because of its rapid growth, good flesh quality, and high market value. Its production in 2020 was up to 600 thousand tons in China. As a typical carnivorous fish, M. salmoides was traditionally fed on chilled fish; however, with the requirements for sustainable development of aquaculture, more and more formulated diets are used nowadays. Starch is necessary as a binder and sweller during extrusion processing of formulated, pelleted feeds [World aquaculture production has increased tremendously over the past years. However, due to intensive farming practices, metabolic diseases of fish have posed a great threat to fish production, causing heavy economic losses. Largemouth bass  [Cyprinus carpio) by activating digestive enzyme activity in the intestine and increasing the expression level of several genes (GH and IGF-I) related to growth in the brain, liver, head kidney and muscle [Acanthopagrus schlegelii) [To develop environmentally friendly aquaculture and ensure food safety, various non-antibiotic feed additives have been used to improve animal health . Differe mykiss) . Furtherd muscle . On the d muscle . Triterpd muscle ,12,13,14legelii) . In addilegelii) . TherefoThe objective of the present study was to investigate effects of high-starch diets and the supplementation of OE on the growth performance, hepatic antioxidant capacity and lipid metabolism of largemouth bass. The results of this study provide insights into the environmentally friendly prevention of MLD in carnivorous fish.Two isonitrogenous and isoenergetic experimental diets with 9.0%  and 14.4%  starch were prepared, and 0.125% OE  was added to these two basal diets, respectively (named as LSOE and HSOE). The OE product was prepared from the olive cake remaining from typical two-phase olive oil production, with a standardized composition (determined by HPLC) of minimum 7.5% triterpenes (maslinic acid and oleanolic acid). The product also contains > 2% protein, > 5% fat and > 30% fiber. The feed ingredients were ground into fine powder through a 247 μm mesh. Each diet was processed into 3 mm-diameter floating pellets under the following extrusion condition: feeding section (90 °C/5 s), compression section (130 °C/5 s), and metering section (150 °C/4 s), using a Twin-screwed extruder  according to our previous studies . All dieLargemouth bass were obtained from a commercial aquafarm . All fish were acclimated and fed the control experimental diet (LS diet) for 4 weeks before the formal feeding trial. After a 24 h fasting period, fish  were distributed into 12 cylindrical plastic tanks (capacity: 256 L) with three replicates per treatment and 20 fish per tank, and each diet was randomly assigned to 12 tanks. Fish were fed to apparent satiation twice daily at 08:00 h and 17:00 h. During the experiment, water temperature was maintained at 21–25 °C, pH at 7.2–8.0, dissolved oxygen > 6.0 mg/L, and ammonia-N < 0.3 mg/L.g, 10 min, 4 °C) to obtain plasma for the analysis of hematological parameters. Four liver samples from each tank were dissected and immediately frozen in liquid nitrogen, and kept at −80 °C for mRNA isolation and tissue homogenate analysis until used. Four liver samples near the bile duct in each tank were fixed in 4% paraformaldehyde  for histology determination. The rest of the livers in each tank were pooled into zip-lock bags and then stored at −20 °C for the assay of crude lipids.Growth performance factors , survival rate (SR), specific growth rate (SGR), feed conversion ratio (FCR), feeding rate (FR)) were determined by batch weighing the fish at the end of the 8 weeks after starvation for 24 h. All the sampled fish were anesthetized with chlorobutanol (300 mg/mL). Individual body weight, body length, viscera, liver and visceral adipose tissue weight of four fish in each tank were recorded to calculate the condition factor (CF), viscerosomatic index (VSI), hepatosomatic index (HSI) and visceral adipose index (VAI). Blood was rapidly drawn from the caudal vein and centrifuged , total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total bile acids (TBA), alkaline phosphatase (AKP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hepatic TG, TC, TBA, HDL-C, LDL-C, malondialdehyde (MDA) and activities of total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) were determined by commercial assay kits  following the manufacturer’s protocols. Reactive oxygen species (ROS) was determined using a commercial kit . Liver samples were fixed, dehydrated, embedded, and stained for hematoxylin and eosin (H&E) as previously described ,16. The ® Green Supermix . Each sample was run in triplicate and analyzed using the 2−ΔΔCt method. EF1α was used as an endogenous reference gene. Primer sequences are shown in Total RNA extraction and cDNA synthesis were carried out as described previously ,16. The Protein extraction of the liver samples was carried out as described previously . Proteinp < 0.05 was considered significantly different. The graphics were drawn using GraphPad Prism 7.0 .All data were presented as the mean value ± standard error of the mean (S.E.M). Statistical analyses were performed using the SPSS software version 22.0 for Windows . All data means were analyzed after homogeneity of variances were tested. Two-way ANOVA was used to analyze the significant differences among treatment means based on starch levels (9.0 and 14.4%) and OE levels (0 and 0.125%). Meanwhile, one-way ANOVA was also performed for the data analysis of four groups in figures. p > 0.05). According to the two-way ANOVA analysis, high-starch diets significantly reduced the FBW, SGR, FR and FCR compared with low-starch diets (p < 0.05). There were no significant differences in FBW, SGR, and FR after adding 0.125% OE to the diets (p > 0.05). However, supplementation of OE significantly decreased the FCR (p < 0.05). Both a high starch level and OE supplementation significantly increased the HSI, while OE supplementation significantly decreased the CF and high starch significantly increased the VSI (p < 0.05). Both a high starch level and OE supplementation had no effect on the crude protein content of the whole body (p > 0.05). OE supplementation significantly increased the crude lipid level of the whole body (p < 0.05) (The results of growth performance and morphometric parameters are presented in  < 0.05) . p > 0.05). OE supplementation significantly increased plasma TC, which was mainly induced by the increase in HDL-C (p < 0.05), and plasma LDL-C/TC was decreased with 0.125% OE supplementation (p < 0.05). Moreover, high levels of dietary starch led to abnormal liver function with significantly higher AST, ALT and AKP, and supplementation with OE significantly decreased ALT in plasma, which indicated that OE may improve liver function.The hematological and liver function parameters of largemouth bass are presented in p < 0.05), which indicated that OE may improve hepatic antioxidant capacity . Simultaneously, the P-ERK/ERK ratio showed a tendency towards an increase in the high-starch groups. Supplementation with OE significantly increased ERK levels (p < 0.05), but had no significant effect on P-ERK and P-ERK/ERK levels (p > 0.05). Phosphorylated JNK (P-JNK(Thr183/Tyr185)) levels, and the P-JNK/JNK ratio in liver tissues, were significantly lower in the high-starch groups than in the low-starch groups (p < 0.05). Adding OE significantly decreased phosphorylated JNK (P-JNK) levels (p < 0.05), but had no significant effect on P-JNK(Thr183/Tyr185)/JNK levels (p > 0.05). Moreover, One-way ANOVA analysis showed that the ratio of P-JNK/JNK was significantly up-regulated in the HSOE group compared to the HS group (p < 0.05). We also observed a significant up-regulation of mRNA levels of pro-inflammatory cytokines  and anti-inflammatory cytokines (TGFβ1) in the high starch groups (p < 0.05), while supplementation with OE had no significant effect on the expression of inflammatory cytokines by two-way ANOVA (p > 0.05). However, one-way ANOVA analysis showed that the mRNA level of IL1β was significantly decreased in the HSOE group compared with the HS group (p < 0.05) (As shown in  < 0.05) B. The rep > 0.05). High-starch diets induced a higher content of lipid in the liver (p < 0.05). Supplementation of OE significantly decreased hepatic TG, LDL-C, LDL-C/TC and TBA (p < 0.05). Although the two-way ANOVA results showed that OE had no significant effect on the level of liver lipids (p > 0.05), there was a clear reduction in hepatic lipids after adding OE to the low starch diet.As shown in p < 0.05). In terms of mRNA levels, high-starch diets significantly increased SREBP1 compared with diets containing low starch (p < 0.05) (ACC1 and FASN) were significantly down-regulated, and TG hydrolysis (ATGL) and fatty acid β-oxidation (CPT1α)-related genes were significantly up-regulated by OE supplementation (p < 0.05) (As shown in  < 0.05) B. On the < 0.05) C.Oryzias latipes), which is based on feeding the fish a high-fat diet (HFD). Medaka that are fed a HFD exhibited macrovesicular fat deposition and liver dysfunction [Oreochromis niloticus) fed diets including extracts of ginseng (Ginsana® G115) [Paralichthys olivaceus) fed diets including extract of green tea [Cyprinus carpio) were enhanced after feeding diets with 0.10–0.25% OLE, but decreased after feeding diets with high levels of OLE (0.50–1.00%) [Starch is typically used as a technical ingredient (stabilizer and swelling agent) in the manufacture of aquatic feeds, and is sometimes used as a cheap energy source in diets for some fish species. However, carnivorous fish having a metabolism adapted to diets low in carbohydrates exhibit symptoms of glucose intolerance after the intake of high-starch diets ,18. Prevfunction . In aquafunction ,23,24. Afunction . For insfunction ,27. Simia® G115)  or limona® G115) , and in reen tea . Growth 0–1.00%) . In thisTo avoid metabolic stress, such as that potentially caused by high carbohydrate, organisms rely on an antioxidant protection system to prevent oxidative injury. SOD enzymes control the levels of a variety of ROS and reactive nitrogen, thus limiting the potential toxicity of these molecules and controlling broad aspects of cellular life that are regulated by their signaling functions . MDA is IL-1β in head kidney tissue and TNF-α in spleen tissue) and enhanced the survival rate of common carp juveniles by inhibiting the pathogenicity of E. tarda [TNFα, IL1-β and IL-8, although the expression levels of these genes decreased with higher doses of OLE, such as 0.25, 0.5, and 1.0% [Acanthopagrus schlegelii), repairing the hampered immune response induced by cadmium exposure [Penaeus vannamei) [IL1β, TNFα and TGFβ1 suggested that high starch contents activated the immune response of largemouth bass, while supplementation with OE in the diet containing high starch level reduced transcript levels of pro-inflammatory gene IL1β and expression of anti-inflammatory genes remained at a high level.At present, the most widely reported application of olive extracts in fish is related to their immunomodulatory effects. Dietary supplementation of 0.1% OLE increased the expression levels of immune-associated genes (E. tarda . Adding and 1.0% . Triterpexposure . Moreoveannamei) . In thisMegalobrama amblycephala and Rhynchocypris lagowskii Dybowski [IL1β. However, hepatic histopathological analysis did not show significant effects of OE supplementation on the repair of liver injury induced by the high starch diet, which may be a result of the short feeding time (eight weeks) in the study.Oxidative stress induced by oxidized fish oil activated NF-κB signaling and release of inflammatory cytokines in Dybowski ,38. In mDybowski ,40. In fDybowski . MAPK faDybowski . A high Dybowski . Indeed,Dybowski . Our resSREBP-1. OE showed a tendency to decrease SREBP-1 expression in the HS diet.Hepatic lipid content and HSI are important indicators of liver health in fish. In this study, we demonstrated that a high-starch diet led to an over-accumulation of lipid and enlarged liver in largemouth bass, with a higher content of hepatic lipid and HSI. In addition, our results suggested that both AMPK and SREBP were involved in the over-accumulation of lipid. Up-regulation of SREBP increases the uptake and absorption of glycerol by hepatocytes and enhances the synthesis of fatty acids and cholesterol . When thSREBP1 involved in fatty acid synthesis, including ACC1 and FASN. Surprisingly, no significant effects of starch level were observed in the expression of these genes. However, one-way ANOVA analysis showed that expression of ACC1 was the highest in the HS group, which was similar to the expression of SREBP1. A significantly lower expression of ACC1 and FASN was observed in the OE-supplemented diets, strongly indicating that OE decreased lipogenesis. In addition, OE supplementation increased the expression levels of the lipolysis gene ATGL and β-oxidation gene CPT1α. These results are consistent with a previous study showing that a Chinese olive fruit extract improved the metabolic abnormalities of mice associated with fatty liver under high fat challenge by increasing the protein expression of phosphorylated AMPK, ACC1, CPT-1, and PPARα, but downregulating the expression of mature SREBP-1c and FAS [In order to determine the functional consequences of activated SREBP1, we further examined the gene expression levels of several key target enzymes of  and FAS . In thisAt the level tested in this study (0.125%), supplementation with OE was associated with several beneficial effects. However, it is important to note that the concentration of OE supplemented into diets should be optimized, as hepatotoxic effects of some triterpenes have been reported in a dose-dependent manner. For instance, the anti-inflammatory pentacyclic triterpene oleanolic acid at about 20% caused body weight loss, inflammation, hepatocellular apoptosis, necrosis, and feathery degeneration (indicative of cholestasis) in mice . TherefoIn summary, the intake of high starch diets induced oxidative stress, inflammatory response, lipid metabolism disorder and even MLD, which significantly reduced the growth performance of largemouth bass. Supplementation with 0.125% of an olive extract obtained as a byproduct of olive oil production significantly reduced FCR, which would result in a lower farming feed cost of largemouth bass. In addition, our results demonstrate that supplementation with OE in the HS diet increased the hepatic antioxidant capacity, promoted the activation of JNK signaling pathway and decreased inflammatory responses. In this study, we present the first evidence that OE regulates hepatic lipid metabolism in fish. The protective mechanisms induced by the supplementation of OE mainly depend on inhibiting hepatic lipogenesis and promoting lipolysis and β-oxidation, leading to the prevention of hepatic TG over-accumulation in fish. A limited effect of OE was observed histologically on the repair of MLD induced by high starch in largemouth bass, but considering the short trial period and single doses tested, it would be worthwhile to further investigate the effects of different OE doses and feeding time on the growth, hepatoprotection and immune status of fish in future studies.