Fasciola hepatica in humans have been reported. Four animal species are the reservoir species for F. hepatica in this area, namely, sheep, cattle, pigs, and donkeys. Livestock for the Aymara inhabitants is crucial because vegetable cultures are not viable due to the inhospitality of the very high altitude of 3,820–4,100 m. A One Health initiative has been implemented in this area in recent years, as the first such control action in a human endemic area ever. Among the different control axes included, special focus is devoted to the two main reservoirs sheep and cattle. Egg embryonation, miracidial infectivity, intramolluscan development, cercarial production, infected snail survival, and metacercarial infectivity were experimentally studied in altiplanic sheep and cattle isolates. These laboratory studies were performed using altiplanic isolates of the lymnaeid species Galba truncatula, the only vector present in the hyperendemic area. Experiments were made at constant 12 h day/12 h night and varying 20/20°C and 22/5°C photoperiods. Infections were implemented using mono-, bi-, and trimiracidial doses. Results demonstrate that sheep and cattle have the capacity to assure F. hepatica transmission in this very high-altitude area. Field surveys included prevalence studies by coprology on fecal samples from 1,202 sheep and 2,690 cattle collected from different zones of the Northern Bolivian Altiplano. Prevalences were pronouncedly higher and more homogeneous in sheep  than in cattle  in each one of the different zones. Although similarities between the prevalences in sheep and cattle appeared in the zones of the highest and lowest infection rates, this disappeared in the other zones due to cattle treatments. Comparison with past surveys demonstrates that this hyperendemic area is stable from the disease transmission point of view. Therefore, the control design should prioritize sheep and cattle within the One Health action. Studies performed in the Bolivian Altiplano furnish a baseline for future initiatives to assess the transmission and epidemiological characteristics of fascioliasis in the way for its control in other high altitude Andean endemic areas.The Northern Bolivian Altiplano is the human fascioliasis hyperendemic area where the highest prevalences and intensities of infection by  Fasciola, Fasciola hepatica, and Fasciola gigantica, which infect herbivorous mammals and humans by ingestion of the infective stage of encysted metacercaria attached to freshwater plants but also floating metacercariae together with drinking water  launched a wide fascioliasis control initiative in this area in 2007–2008. Results of surveys in humans , 37 and smission .F. hepatica transmission and infection risks. This One Health initiative is at present ongoing and covers all multidisciplinary aspects of fascioliasis, according to modern standards  organized a meeting in La Paz with the participation of the Bolivian Ministry of Health, the Ministry of “Desarrollo Rural y Tierras de Bolivia,” and the Servicio Departamental de Salud de La Paz, together with experts on human and animal fascioliasis, on November 10–12, 2014. In this meeting, the decision was taken to start a One Health action including long-term experimental studies, field monitoring assessments, and control activities with the aim of decreasing tandards –41. The F. hepatica, fecal samples from three naturally infected sheep individuals from the locality of Batallas and from three naturally infected cattle from Kallutaca were used  and logistics , an appropriate experimental approach was designed. Experiments were performed in a laboratory at low altitude. To minimize the influence of oxygen pressure , experimental studies were started immediately after each field mission and arrival to the laboratory, i.e., altiplanic F. hepatica eggs were similarly obtained from sheep from Batallas and from cattle from Kallutaca and Batallas  eggs in the phase of morula, showing vitelline granules and/or spheroidal cells (EM), (ii) eggs in the phase of outlined miracidium, in which a miracidial form begins to be observed (EOM), and (iii) eggs in the phase of developed miracidium, in which a fully developed miracidium is observed inside (EDM). For each 4-day study, a total of 33 eggs from each host individual were analyzed. Counting included not only EM, EOM, and EDM but also (i) degenerated eggs, (ii) empty eggs, and (iii) broken eggs. Egg counts were noted in percentages independently for sheep or cattle per observational day.Developed miracidia were forced to hatch by putting fully embryonated eggs under light and used for the experimental infection of snails , 44. OnlF. hepatica isolate was used for several infection assays under different experimental conditions concerning (i) miracidial dose and (ii) day/night temperature according to a photoperiod of 12-h light/12-h darkness in high-accuracy climatic chambers   . A similG. truncatula as only lymnaeid species inhabiting the Northern Bolivian Altiplano hyperendemic area has recently been confirmed by the sequencing of complete nuclear ribosomal DNA and mitochondrial DNA markers  aged 4–5 weeks were used throughout. A balanced commercial rodent diet (Panlab Chow A04) and water were provided escribed .F. hepatica metacercariae per rat was used  or not treated at all.2) after filtration and second with 50 ml water, and stained with methyl green according to a standard method . For this purpose, the whole sediment was microscopically analyzed in the positive samples, and the number of fluke eggs found was divided by 4 to obtain the epg value.Only coprological methods for qualitative and quantitative analyses were carried out. Fecal samples were placed in numbered plastic bags, transported to the laboratory of La Paz within the following 5 h, and maintained in a fridge at 4°C until examination. From each fecal sample, a quantity of 4 g was sedimented twice, first with 50 ml of detergent solution  in sheep and cattle isolates were compared by ANOVA test. Statistical comparison of categorical variables was carried out with the Chi-square test and Yates continuity corrected Chi-square test. Means obtained in data from experimental infections of lymnaeid snails were compared by F. hepatica are shown in The results of the experimental follow-up of the egg embryonation of the sheep and cattle isolates of altiplanic P = 0.015) in sheep isolate (38.93%) than in cattle isolate (26.63%).An outlined miracidium form begins to be observed inside eggs at 20 and 12 days, respectively, and the first fully developed miracidium already appears at day 24 in both isolates. Eggs including a fully developed miracidium were henceforth observed in each observational day until 143 and 60 days, respectively. Days in which analyzed eggs showed the highest percentage of fully developed miracidia were 56 and 36 days, respectively. The average egg embryonation  in the 60 days showed higher values (P = 0.9941) were found in snail infectivity, assessed by the percentage of snails successfully infected by a monomiracidial dose at 20/20°C in the sheep isolate and cattle isolate, although snail infectivity was higher in the sheep isolate under temperature conditions of both 20/20°C and 22/5°C. However, the result was the opposite in the assays performed at 20/20°C when comparing the infectivity of a bimiracidial dose in the sheep isolate with that of a trimiracidial dose in the cattle isolate.Results of the different experimental lymnaeid infection assays performed with the sheep and cattle isolates are shown in P = 0.0699) were found in the values of the pre-patent period. This period was shorter when bimiracidially infecting with the sheep isolate (P = 0.1039) than in the trimiracidial infections with the cattle isolate (P = 0.4661), although no statistically significant differences appeared. The cercarial shedding period was similarly long in all assays when comparing sheep with cattle isolates, as confirmed by the absence of statistically significant differences (P = 0.0998).The pre-patent period in monomiracidial infections was similar in both isolates both at 20/20°C and at 22/5°C, and no statistically significant differences  were found. However, the cattle isolate monomiracidial infection assay cannot be considered because of the very low number of successfully infected lymnaeids.Regarding the production of cercariae per infected snail, results were similar between the two altiplanic There were surprisingly long snail survivals after shedding ended. This aspect did not show significant differences between both isolates, although bimiracidial and trimiracidial infections appear to reduce lymnaeid survival. When comparing the longevity of successfully infected snails with that of the non-infected ones, the absence of differences is worth mentioning.P = 0.9999). The intensity of infection, measured by the number of flukes recovered per metacercariae inoculated and also per rat, however shows differences, although the number of rats inoculated was unfortunately too low as to reach a significant conclusion .Results of the experimental infection of Wistar rats with experimentally obtained metacercariae of the altiplanic sheep and cattle isolates are shown in Results of the coprological surveys on sheep and cattle populations inhabiting eight selected zones of the fascioliasis hyperendemic area are shown in F. hepatica infection in the Corriedale breed  when compared to Merino, Criollo, and Mestizo  (56.2–63.9%). Sheep aged <4 months appear to be the most infected (87.1%), whereas the prevalence decreases relatively with age, up to the 2–6-year-old group (56.9%).In sheep, prevalences found were high and very homogeneous, with a total prevalence of 63.1% and a range of 38.9–68.5% according to zones. Among the different sheep breeds studied, there appears to be a higher susceptibility for P = 0.0001). The prevalences were irregularly distributed, varying considerably according to zones (8.2–43.3%). Cattle aged <1 year proved to be infected only sporadically (9.4%). The liver fluke prevalence increases pronouncedly in bovines aged 1–2 years (37.5%) and appears to be maintained at a similar level in animals aged more than 2 years (28.7%). No conclusion about potential susceptibility differences according to cattle breeds could be obtained, as the Brown Swiss in the Achacachi zone are regularly treated, similarly as the Holstein breed in Kallutaca and Viacha. The Creole breed, distributed throughout the remaining zones, is irregularly treated depending on owners or not treated at all.In cattle, the total prevalence found in the Northern Bolivian Altiplano was 20.6%, significantly lower than the total prevalence found in sheep  decreases in oxygen and air density, (ii) low temperature and humidity, and (iii) an increase in solar radiation. These environmental factors exert an influence on the animals and humans born and living at high altitude , 52. Thuhe blood .F. hepatica, mainly because of its tissue migration and hematophagous diet  was experimentally demonstrated  of 51.8 and 25% monomiracidially obtained at 20/20°C with the altiplanic sheep and cattle isolates, respectively, fit the ranges of 32–57% and 14–56.8% experimentally detected in different n France , 71. Then France . The hign France . The 22/F. hepatica/G. truncatula demonstrated that the pre-patent period (from infection up to the shedding of the first cercaria) is dependent on temperature, higher temperatures reducing its length: 56–86 days at 15°C, 48–51 days at 20°C, and 38 days at 25°C  the Aymara concept of village or community usually concerns markedly dispersed dwellings which allow the animals to walk in between and around, (ii) 5–15-year-old children living in small communities not provided with a school are in need for a daily walk along rural unpaved roads to the nearest school which in many cases may be relatively distant, (iii) freshwater collections inhabited by lymnaeids and grazing free sheep and cattle used to be present in the neighborhood or besides these rural ways, and (iv) such home–school–home walks have been verified to represent a high infection risk for the children .Consequently, sheep and cattle should be considered priority within the One Health action, including management measures to decrease their infection probabilities and appropriate treatments of the infected animals to decrease field contamination and lymnaeid infection rates. Drugs different from triclabendazole, such as closantel, should be used to treat these animals, to avoid the appearance of a potential resistance in F. hepatica coproantigen detection techniques may facilitate the task in ruminants  (A1263 915389140), strictly following the institution's guidelines based on Directive 2010/63/EU. Permission for animal research was additionally obtained from the Servicio de Sanidad y Bienestar Animal, Dirección General de Producción Agraria y Ganadería, Consellería de Presidencia y Agricultura, Pesca, Alimentación y Agua, Generalitat Valenciana, Valencia, Spain (No. 2015/VSC/PEA/00001 tipo 2). Animal ethics guidelines regarding animal care were strictly adhered. The study was approved by the Comisión de Etica de la Investigación of the Comité Nacional de Bioética, La Paz (Certificate dated 10 September 2007), Comité de Etica y Bioética de la Facultad de Medicina de la Universidad Mayor de San Andres, UMSA, La Paz—COMETICA , and Comité de Revisión Etica (PAHOERC) of the Pan American Health Organization, PAHO, Washington DC . All investigations were made after permission was obtained from local Aymara Community Chiefs , and with the consent of animal owners.SM-C performed One Health studies, designed the protocols, wrote the manuscript, and obtained project funding. PB performed the surveys and diagnosis of animals. IRF performed the snail cultures and experimental snail infections. RA coordinated local research and logistics and participated in surveys. PA participated in surveys and diagnosis of animals. MAV performed the experimental mammal infections and statistical analyses and obtained project funding. MDB performed the egg development studies, designed and participated in the experimental snail infections, participated in surveys, and obtained project funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The National Institutes of Health Stroke Scale (NIHSS) score is the most frequently used score worldwide for assessing the clinical severity of a stroke. Prior research suggested an association between acute symptomatic seizures after stroke and poorer outcome. We determined the frequency of acute seizures after ischemic stroke in a large population-based registry in a central European region between 2004 and 2016 and identified risk factors for acute seizures in univariate and multivariate analyses. Additionally, we determined the influence of seizures on morbidity and mortality in a matched case–control design. Our analysis of 135,117 cases demonstrated a seizure frequency of 1.3%. Seizure risk was 0.6% with an NIHSS score at admission <3 points and increased up to 7.0% with >31 score points. Seizure risk was significantly higher in the presence of acute non-neurological infections . A lower premorbid functional level also significantly increased seizure risk . Mortality in patients with acute symptomatic seizures was almost doubled when compared to controls matched for age, gender, and stroke severity. Acute symptomatic seizures increase morbidity and mortality in ischemic stroke. Their odds increase with a higher NIHSS score at admission. In the elderly population, strokes may represent a cause of epilepsy in more than 30% of cases3.Ischemic stroke has long been recognized as one of the major risk factors for the development of structural epilepsy, particularly among the elderly4. Between 1.1% and 10% of stroke patients end up suffering from acute symptomatic seizures, while 3% to 25.2% develop poststroke epilepsy12. While the occurrence of acute symptomatic seizures during the stroke episode can predict the occurrence of poststroke epilepsy13, late seizures in patients with a structural lesion due to the stroke by definition result in the diagnosis of epilepsy14.Epileptic seizures can occur early (known as acute symptomatic seizures) or late after a stroke, with the threshold usually set at seven days16. Previously, stroke severity was assessed by using the Scandinavian Stroke Scale (SSS), Canadian Neurological Score (CNS), modified Rankin Scale (mRS) at admission and the NIHSS in two smaller studies18. Predictive scores have also been constructed for the occurrence of late seizures following stroke and poststroke epilepsy20. The majority of relevant studies have prospectively analyzed a comparatively small number of patients, so there exists a dearth of large population-based studies. Further, the influence of acute symptomatic seizures on mortality is controversial22.The risk factors for the occurrence of acute symptomatic seizures after ischemic stroke have been investigated in several studies to date and include anterior hemisphere location, cortical localization, stroke severity, and hemorrhagic transformation23. The aim of this study was to investigate the frequency of acute symptomatic seizures following ischemic stroke in a defined, large population in central Europe and to determine if and to what extent a higher National Institutes of Health Stroke Scale (NIHSS) score at admission predicts seizure risk. In a second step, we assessed risk factors for the occurrence of acute symptomatic seizures. Furthermore, we examined the influence of acute symptomatic seizures on mortality in a matched case–control analysis. Patients with acute symptomatic seizures in intracerebral and subarachnoid hemorrhage were evaluated in an independent studyp = 0.5]. In total, 66,157 patients had an NIHSS score at admission of zero to five and 40,005 had an NIHSS score of more than five. For detailed results, please refer to Table The GQH database  contains information on 297,120 patients with ischemic stroke, transient ischemic attack, and intracerebral and subarachnoid hemorrhage collected between January 2004 and December 2016. After application of the inclusion criteria for ischemic stroke, 135,117 patients remained. During the evaluation period, acute symptomatic seizures occurred in a total of 1,787 (1.3%) ischemic stroke patients. Seizure incidence did not change significantly between 2004 and 2016  over a 13-year period (2004–2016). The GQH stroke registry is used to investigate the treatment quality of patients with strokes in more than 100 clinics in Hessen, a federal state of the Federal Republic of Germany with more than six million inhabitants . The data is entered by the treating physicians or a coding specialist and the validity of the entries is checked by regular audits. Data entry is mandatory for all hospitals in Hessen, and cross-validation with billing data according to §21 Krankenhausentgeltgesetz  shows more than 99% completeness. Data analysis for research purposes was approved by the ethical committee of the medical faculty of the Goethe-University Frankfurt.We evaluated data from the stroke registry of the Institute of Quality Assurance Hessen [Geschäftsstelle Qualitätssicherung Hessen (GQH), Eschborn, Germany; th revision code I63.x), defined as an acute loss of neurological function due to a cerebrovascular cause persisting for more than 24 hours with exclusion of an intracerebral bleeding23 on an adequate imaging modality  or imaging findings of ischemic stroke, and admission to the hospital within 24 hours after symptom onset. We extracted age; sex; time from onset of symptoms to admission; overall incidence of epileptic seizures, presence of arterial hypertension, diabetes mellitus, hypercholesterolemia, and atrial fibrillation; the modified Rankin Scale (mRS) score; NIHSS score; the occurrence of urinary tract infection, pneumonia, or sepsis; and preexisting treatment with anticoagulants or antiplatelet agents.Inclusion criteria were age of 18 years or older, ischemic stroke .The mRS score was dichotomized into zero to two points (living independently) and three to five points (at least some dependency in everyday life), respectively, while the NIHSS score was dichotomized into less than or equal to five points (mild to no impairment) and more than five points (moderate to severe impairment); for a secondary analysis, the NIHSS was treated as a numerical variable. The infection variables were combined under the umbrella of “acute non-neurological infection.” We used the chi-squared test for categorical variables and the Mann–Whitney U test for continuous variables in a univariate analysis. Univariate logistic regression was employed to evaluate the relation between NIHSS score and acute symptomatic seizures. We used multivariate forced entry logistic regression to determine the occurrence of acute symptomatic seizures. Additionally, we performed a matched case–control analysis. Complete-case analyses were used exclusively. Patients were matched based on age, gender, and NIHSS score at admission as well as pre-morbid mRS score. Out of 1,787 patients, a total of 34 patients could not be matched. Statistical analyses were performed using the SPSS version 26.0 software program . Trend analysis was performed using JoinPoint version 4.7.0.0 . Statistical significance was assumed at a


Childhood asthma and allergic diseases are a significant global problem. There are inconsistent findings on the associations of delivery mode, the number of children in the household and breastfeeding with childhood asthma and allergic diseases. We assessed these associations and examined whether breastfeeding modified the effects of neonatal and familial risk factors on childhood asthma and allergic diseases.A population-based cross-sectional study was conducted in Shanghai, China. A total of 17 primary schools were randomly selected from 13 districts of Shanghai in this study. The International Study of Asthma and Allergies in Childhood questionnaire was adopted to assess the childhood asthma and allergic diseases. Multivariable logistic regression models were used to evaluate the associations between neonatal and familial factors and childhood asthma and allergic diseases, and to examine the modification effects of breastfeeding on the associations assessed.Of 10,464 primary school children aged 6–11 years, the overall prevalence of childhood asthma, allergic rhinitis, urticaria, food allergy and drug allergy was 13.9, 22.7, 15.3, 8.1 and 4.6%, respectively. Male sex, high socioeconomic status, cesarean section delivery, only one child in the household and having family history of allergy were associated with increased odds ratio (OR) of childhood asthma and allergic diseases while longer breastfeeding duration (> 6 months) was inversely associated with these diseases. Longer breastfeeding duration also attenuated the OR of neonatal and familial risk factors on childhood asthma and allergic diseases. For instance, the adjusted OR of childhood asthma in the group of vaginal delivery and breastfeeding duration > 6 months was lowest .Longer breastfeeding duration was inversely associated with childhood asthma and allergic diseases, and also reduced the OR of neonatal and familial risk factors on these diseases. Giving the prevalence of childhood asthma and allergic diseases is rapidly rising across the globe, these findings may have important clinical and public health implications. The prevalence of childhood asthma and allergic diseases has elevated worldwide in the past 3 decades. For instance, the International Study of Asthma and Allergies in Childhood (ISAAC) phase 3 showed a significant increase in the prevalence of childhood asthma and allergic rhinitis (AR) in most of Asia–Pacific region from 2001 to 2010 . These cMounting evidence suggests that prenatal and neonatal factors are associated with the development of childhood asthma and allergic diseases –8. In ChKnowledge gaps exist in the associations between prenatal and neonatal factors and development of childhood asthma and allergic diseases. To assess these associations, and to examine whether breastfeeding duration modifies these associations, we conduct a population-based cross-sectional study among primary school children aged 6–11 years in Shanghai, the largest metropolitan city in China.Shanghai is located in the Yangtze River Delta region, east of China, E 120°52′-122°12′, N 30°40′-31°53′, which has over 27 million residents in 2020 . A city-The ethical application and the consent procedure of this study were approved by the Ethics Committees of Shanghai Jiao Tong University School of Medicine and Shanghai Children’s Medical Center.The questionnaire included questions on childhood asthma and allergic diseases, general characteristics of children and parents, socioeconomic status (SES), allergy history of children and their family members, exposure during pregnancy, children’s lifestyle habits, and home environmental exposures in early life. The Cronbach's alpha coefficient of the ISAAC allergic questionnaire in our sampled children was 0.91. The intra-class correlation coefficient of retest reliability at intervals of 2 weeks was 0.94. Validity presented by Kaiser–Meyer–Olkin test was 0.94. These results show the high validity of the questionnaire for the primary school children.The core question of the ISAAC questionnaire was applied to assess asthma and allergic diseases status . Asthma:Mode of delivery included vaginal delivery (VD) and caesarean section (CS). Breastfeeding duration was divided into two groups according to WHO recommendations. One was exclusive breastfeeding duration ≤ 6 months, and another was exclusive breastfeeding for 6 months, and continued breast feeding (breastfeeding duration > 6 months). The number of children in household was categorized as: only one child or not. A question about family history of allergy was: Does anyone at home have the following allergic diseases ? Socioeconomic status (SES) was measured as: Do you own the apartment or not?χ2) test was used to assess the differences among the prevalence of asthma and allergic diseases between different groups. Univariate logistic regression model was used to calculate the unadjusted odds ratios (OR) and 95% confidence interval (CI) of child’s sex, age, SES, mode of delivery, breastfeeding duration, only one child in the household and family history of allergy. Then, multivariable logistic regression model was performed to calculate adjusted OR (AOR) and 95% CI after adjustment for putative confounders. Furthermore, the modification effects of breastfeeding duration on the associations between neonatal and familial factors and children’s allergic diseases were assessed using stratified analyses. All statistical analyses were conducted using R .Statistical descriptions were made utilizing the frequency and percentage for categorical variables. Pearson’s chi-squared (p < 0.001) in the group with higher SES (having the home ownership) or with family history of allergy or with only one child in the household. The prevalence of childhood asthma and allergic diseases was significantly higher in urban or suburban area group compared with the group of rural.In this study, the overall prevalence of childhood asthma, AR, urticaria, FA and DA was 13.9, 22.7, 15.3, 8.1 and 4.6%, respectively. Table Univariate and multivariate logistic regression analyses between childhood allergy and independent variables Table  suggest Longer breastfeeding duration (> 6 months) might reduce the associations of delivery mode, family history of allergy, and only one child in the household with childhood asthma and allergic diseases Figs. , 2, 3. FSimilarly, the AOR in the group of breastfeeding duration > 6 months and more than one child in the household was lowest for childhood asthma , AR  and DA  compared to the reference group of breastfeeding duration ≤ 6 months and only one child in the household , AR , urticarial , FA  and DA  compared with the reference group of breastfeeding duration ≤ 6 months and having family history of allergy  was inversely associated with childhood asthma and allergic diseases, and could attenuate the effects of CS delivery, only one child in the household and having family history of allergy on childhood asthma and allergic diseases.Our findings are consistent with most previous studies in which CS delivery, only one child in the household and formula feeding were associated with an increased risk of allergic diseases among children –8, 12–18Hygiene or old friends hypotheses involved gut microbiota exposure might partly explain the associations of CS, only one child in the household, and breastfeeding with childhood asthma and allergic diseases . A meta-Similar to previous studies , 27–29, There are three major strengths in this study. First, a representative sample was obtained through a stringent multi-stage and multi-strata random sampling approach. Second, this large scale population-based cross-sectional study achieved a high response rate (> 95%), so selection bias was probably minimal. Third, a wide range of childhood allergic diseases including asthma, AR, urticaria, FA and DA were considered in this study to explore their associations with a range of neonatal and familial risk factors.This study also has several limitations. First, all the information was obtained from questionnaires, and some bias  was inevitable to some extent. For example, the diagnoses of childhood allergy were recalled but were not made by objective allergic testing. The information on breastfeeding duration was not perfect because it was collected retrospectively rather than prospectively. Second, the nature and severity of childhood asthma and allergic diseases was not measured in this study, and therefore, the determinants of mild and severe cases cannot be distinguished. Finally, a causal relationship cannot be established due to the cross-sectional study design. However, the independent variables included in the multivariable model  were significantly earlier than the diagnosis of childhood asthma and allergic diseases, so a certain temporal relationship exists.Despite these limitations, the results from this study have demonstrated the contemporary prevalence of asthma and allergic diseases among children aged 6–11 years and their associations with CS, only one child in the household, family history of allergy and breastfeeding duration. Longer breastfeeding duration appeared to decrease the effects of the neonatal and familial factors on childhood asthma and allergic diseases. Our findings may be used to develop appropriate strategies to prevent and control childhood asthma and allergic diseases if they are confirmed by further research.Among primary school children aged 6–11 years in Shanghai, born by CS delivery, only one child in the household, and having family history of allergy were associated with increased OR of childhood asthma and allergic diseases. Longer breastfeeding duration was associated with reduced OR of childhood asthma and allergic diseases, and seemed to attenuate the OR of neonatal and familial factors on childhood allergic diseases. These findings may have important clinical and public health implications.Additional file 1: Table S1. Sensitivity analysis of multivariate logistic regression after adjustment for residential area and whether the address changed after birth.


A head injury is a very dangerous condition that threatens human life. This study examines the use of caffeic acid phenethyl ester (CAPE) in reducing cerebral edema in cases of head injury. The purpose of this study is to demonstrate whether CAPE can improve various parameters related to the expression of Aquaporin-4 (AQP4) mRNA and the serum AQP4 levels in rat subjects.Rattus norvegicus (Sprague Dawley strain) rats aged 10–12 weeks and weighing 200–300 g. This study used a head injury model according to Marmarou (1994) with minor modifications to the animal model fixation tool. The parameters of the AQP4 mRNA were examined with real-time PCR, while serum AQP4 levels were examined with sandwich ELISA.This is a randomized controlled study using a posttest-only control group design that uses experimental animals—specifically, male The AQP4 mRNA expression in rats that were given CAPE was lower than those not given CAPE, both on the fourth and seventh days; serum AQP4 levels in rats that were given CAPE were also lower than those not given CAPE, both on the fourth and seventh days.Administration of CAPE in a rat model with head injury can reduce cerebral edema, mediated by AQP4. •Head injury is a major cause of mechanical brain cell injury and also catalyzes secondary damage.•Caffeic Acid Phenethyl Ester (CAPE) can reduce inflammatory processes, brain lipid peroxidation, and free radical damage.•CAPE treatment can cause a decrease in cerebral edema, mediated by Aquaporin-4 (AQP4). This sec release from mitochondria ), and a p-value of <0.05 was considered significant.Data were processed and analyzed using SPSS version 23 . The effects of CAPE administration—in this study, on the expression of AQP4 mRNA—was tested by independent 33.1Sprague Dawley), this study aimed to explain the benefits of CAPE in relation to the process of cerebral edema. The characteristics of the animal subjects, such as body weight, are listed in This study examined the role of CAPE in inhibiting AQP4 mRNA. By using a head injury model on test rats . As seen in The results in these brain-injured rats on days 4 and 7 showed a significantly lower average mRNA expression in rats treated with CAPE compared to rats that were not treated with CAPE (t-test. The average AQP4 expression in rats treated with CAPE was significantly lower than in rats not treated with CAPE (p < 0.05)  . AQP4 ex < 0.05) .Table 3EBased on the trauma models, experimental analysis on brain tissue damage showed an effect of CAPE in a seventh-day result of decreased proliferation and dilation of blood vessels .Fig. 4Co4The definition of head injury is an injury to brain tissue not due to degenerative or congenital processes but instead due to external impacts that can result in a decrease or change in the status of consciousness. Head injury is the leading cause of accident-related death for people under the age of 40 years. Every year approximately 10 million people are hospitalized due to head injuries worldwide .Head injury is a major cause of mechanical brain cell injury and is also a catalyst for secondary damage, which develops immediately after the primary damage occurs . SecondaThis study examined the use of CAPE in reducing the formation of cerebral edema in a head injury. This study aimed to prove whether CAPE plays a role in reducing AQP4 levels and decreasing mRNA expression of AQP4 mRNA.Sprague Dawley strain of Rattus norvegicus, with an average body weight of 290.07 (±10.48) grams; no significant difference was found in the distribution of body weight of these rat subjects. Consequently, any differences in expression of the dependent variables are expected to be the result of CAPE treatment after the induced head injury.The experimental animal in this study was a This study used a head injury model that modified the methods used by Marmarou . To provThe process of cerebral edema is mediated by several mediators, including AQP4 and cytokines. AQP4 is crucial in the development of cerebral edema . SeveralIn this study, AQP4 levels increased until the 7th day. With CAPE treatment, AQP4 levels were found to be lower than without CAPE treatment. This process shows that CAPE can reduce the occurrence of cerebral edema. This process is in line with the reduction in edema symptoms and the improvement in functional status associated with a return of AQP4 to normal levels. The speed of improvement of AQP4 levels after CAPE treatment indicates post-translational modification, which plays a role in protein synthesis .Increased levels of AQP4 were also observed at the level of AQP4 mRNA expression. Real-time quantitative PCR found a significant increase in mRNA expression in the control group compared to the treatment group. This indicates an effectiveness of CAPE in the process of decreasing cerebral edema caused by a head injury—especially the cytotoxic process of edema characterized by the release of AQP4 by astrocytes, which undergoes a neuroinflammatory process .This study examined AQP4 expression, which was observed by assessing the serum levels and the mRNA amplification. The results of post-trauma AQP4 expression after CAPE administration showed a significant decrease in serum levels.Most brain diseases, including traumatic head injuries, present the characteristic feature of edema, which is the accumulation of fluid produced by the brain as a result of dysfunction of osmotic homeostasis. The main consequence of edema is brain swelling, which worsens other secondary injuries, such as decreased brain perfusion . Edema h5Administration of CAPE in a rat subject with a head injury can reduce cerebral edema, mediated by AQP4. These findings warrant further research that observes cerebral edema in rat subjects, measured directly by computed tomography (CT) scans and anti-AQP4 immunohistochemistry expression.6Not commissioned, externally peer reviewed.All procedure for Animal experiment has been approved by Ethics Commission Faculty of Medicine, Hasanuddin University Number: 771/UN4.6.4.5.31/PP36/2019.No funding or sponsorship.RAN, AAI, MH, PP, DHL, and ISM wrote the manuscript and participated in the study design. RAN, AAI, PP, DHL, ISM, HW, WS, MNM, and KIN drafted and revised the manuscript. RAN, AAI, PP, DHL, and ISM, performed head trauma treatment and surgery. RAN, AAI, MH and PP performed bioinformatics analyses and revised the manuscript. All authors read and approved the final manuscript.None.Rizha Anshori Nasution.The authors declare that they have no conflict of interests.


Ictalurus punctatus). Overall, we found that the concentration of total PCBs and PCB homologs measured in whole body, fillet, and liver tissues declined more slowly in food-deprived fish, with slowest depuration observed in the liver. Additionally, fish that were exposed to PCBs had lower plasma cortisol concentrations, reduced phagocytic oxidative burst activity, and lower cytotoxic activity, suggesting that PCBs can influence stress and immune responses. However, for most measures of immune function, the effects of food deprivation had a larger effect on immune response than did PCB exposure. Taken together, these results suggest that short-term dietary exposure to PCBs can increase toxicity of consumable fish tissues for several weeks, and that PCB mixtures modulate immune and stress responses via multiple pathways. These results may inform development of human consumption advisories and can help predict and understand the influence of PCBs on fish health.Although most countries banned manufacturing of polychlorinated biphenyls (PCBs) over 40 years ago, PCBs remain a global concern for wildlife and human health due to high bioaccumulation and biopersistance. PCB uptake mechanisms have been well studied in many taxa; however, less is known about depuration rates and how post-exposure diet can influence PCB concentrations and immune response in fish and wildlife populations. In a controlled laboratory environment, we investigated the influence of subchronic dietary exposure to two PCB Aroclors and food deprivation on tissue-specific concentrations of total PCBs and PCB homologs and innate immune function in channel catfish ( Polychlorinated biphenyls (PCBs) are a group of synthetic chemical compounds that were globally produced and distributed for use in electrical and industrial applications . Due to PCBs also continue to be released into the environment through improper waste disposal, leaky infrastructure, and burning of industrial materials . DelayedCYP1A1 gene [Long-term persistence of PCBs in aquatic environments poses significant threats to fish and human health, worldwide. Although PCBs have not been causally linked to declines in fish populations, PCB exposure is associated with acute and chronic immunosuppression ,16,17 an1A1 gene . However1A1 gene ,22. ConsFor humans, PCBs are a probable carcinogen, and consumption of contaminated tissues is associated with birth defects and disruption to endocrine and lymphatic function ,24. GiveGiven concerns about the effects of PCBs on wildlife and human health, there is interest in determining how PCBs assimilate into consumable fish tissues and how exposure can influence long-term fish population demography. However, it is difficult to determine the effects of PCBs when studying wild populations due to the presence of other contaminants and the inability to experimentally control PCB exposure and diet. For example, periods of food deprivation in wild fish stocks have been shown to promote the mobilization of lipids and lipophilic toxicants (including PCBs) from adipose tissue, which can increase tissue toxicity even in the absence of active PCB exposure ,28,29,30While laboratory studies provide the opportunity for more controlled exposure and diet, to date most studies have exposed fish to PCBs using intraperitoneal injections and/or use PCB concentrations that are several orders of magnitude higher than those found in natural systems ,34,35. AIctalurus punctatus) and to document immunomodulation immediately following PCB exposure. Channel catfish are an ideal model organism for this study due to their widespread distribution across several continents, and because the U.S. Environmental Protection Agency (EPA) designated channel catfish as a target species for PCB monitoring due to the species’ high rate of recreational harvest and consumption, history of PCB bioaccumulation, and widespread distribution [The objectives of this study were twofold. The first was to determine how subchronic dietary exposure to two common PCB mixtures influenced tissue-specific concentrations of total PCBs and PCB homologs in channel catfish  Northern Appalachian Research Branch in Wellsboro, Pennsylvania. We used 16,814 L flow-through tank systems that were supplied with water by a nearby spring at a rate of 11 L/min. Water was maintained at 20 °C and dissolved oxygen was maintained above 5 ppm with atmospheric gas delivered via air stones.The concentration of background PCBs in the spring water influent was measured using semipermeable membrane devices . SPMDs were deployed in a tank with no fish, but that received the same influent as the study tanks. Each SPMD was replaced monthly and tested for total PCBs and homolog groups  or unspiked control (C) feed. In the second phase, herein referred to as the food restriction phase, fish were fed either PCB-free (“clean”) food (F) or not fed (NF). Thus, we had four treatments: (1) PCB exposed and clean fed (PF), (2) PCB exposed and not fed (PNF), (3) control fed and clean fed (CF), and (4) control fed and not fed (CNF). This combination of treatments allowed us to fully control for the effects of PCB exposure and food deprivation when comparing responses among treatment groups.n = 576 fish) from Zett’s Fish Farm in Drifting, Pennsylvania. There were four tanks for large fish (average weight = 0.76 kg), eight tanks for intermediate-sized fish (average weight = 0.45 kg), and four tanks of small fish (average weight = 0.22 kg). Thus, each of the four treatments was randomly applied to one tank of large fish, two tanks of intermediate-sized fish, and one tank of small fish, giving four replicates for each treatment.To account for potential effects of fish size, treatments were randomly assigned to tanks of fish using a randomized complete block design with fish size as the blocking variable. Each tank contained 36 randomly selected adult hatchery-sourced channel catfish  and immune response. The first sampling event  occurred before the PCB exposure phase to document baseline concentrations of PCBs in fish tissues. We did not complete fish health assessments at T0, as fish in the CNF and CF treatments served as adequate controls for determining the effect of PCB exposure on immune system responses. The second sampling event (T1) occurred immediately after the 11-week PCB exposure phase to determine tissue-specific PCB concentrations and immunological response of fish immediately after PCB exposure. The last two samples occurred four weeks (T2) and seven weeks (T3) after the beginning of the food-restriction phase to determine how a prior history of PCB exposure and active food deprivation influenced tissue-specific PCB concentrations and immune response. All testing for total PCBs and PCB homologs was completed using a GC–MS/MS system consisting of an Agilent 6890N GC equipped with a 30 m × 25 mm × 25 µm column coupled to a Waters Autospec Premier MS. Testing was completed by PACE Analytical Laboratories  in accordance with EPA 1668A guidelines.All procedures and fish handling were conducted in agreement with the Pennsylvania State University Institutional Animal Care and Use Committee .The goal of the PCB exposure phase was to feed fish in the PF and PNF treatments PCB-spiked food until they reached a body burden of 5 mg/kg while feeding fish in the CF and CNF treatments unspiked food. A target concentration of 5 mg/kg was chosen because it is representative of the total body burden found in tissues of wild fish in PCB-contaminated environments . As descFish were fed commercial-grade pellet feed recommended for large channel catfish. All feed was obtained from the same production number to minimize potential lot-to-lot variation in background PCB contamination. Prior to PCB spiking, three composite feed samples were tested for total PCBs to ensure that background total PCB concentrations were negligible.PCB-spiked feed was prepared by dissolving a composition of 90% Aroclor 1254 and 10% Aroclor 1260 in fish oil at a concentration of 80 μg/mL total PCB by Accustandard . Aroclors 1254 and 1260 were two of the most prevalent Aroclors used in industrial applications and in the environment they often occur at the relative concentrations used in this study. Importantly, these Aroclors are mostly comprised of more highly chlorinated homologs, including both planar and non-coplanar congeners. Subsamples of feed were saturated with PCB-spiked fish oil and had a total PCB concentration of 5.03 ± 0.21 mg/kg, while control feed was saturated in regular, unspiked fish oil and had a total PCB concentration of 0.02 ± 0.0003 mg/kg. Feed was stored in separate, treatment-specific sealed stainless steel containers at 10 °C.We periodically sampled one fish from each of the PF and PNF tanks to monitor increases in PCB body burden. To maintain constant stocking densities across tanks, we also removed one randomly selected fish from each of the CF and CNF tanks. Five weeks into the PCB exposure phase we determined that the concentration of PCBs in the spiked feed was not high enough to reach the desired 5 mg/kg body burden in a reasonable timeframe. Therefore, the concentration of PCBs in the fish oil was increased to 720 μg/mL (while maintaining 90% Aroclor 1254 and 10% Aroclor 1260) to reach mean total PCB concentrations in spiked feed of 11.7 ± 0.25 mg/kg. After 11 weeks, body burden of PN and PNF fish reached approximately 5 mg/kg, and T1 samples were collected from all tanks.At the conclusion of the PCB exposure phase, fish in the PNF and CNF treatments were food deprived, and fish in the PF and CF treatments were fed non-spiked feed that had not been saturated in fish oil. The food deprivation phase lasted for seven weeks, which was deemed an appropriate amount of time for lipid mobilization while avoiding serious adverse health effects for fish in the no-food treatments. Tissue samples were taken four weeks (T2) and seven weeks (T3) after the start of the food deprivation phase.Four fish were randomly sampled from each tank during each sampling event  and euthanized with a lethal dose of MS-222. Prior to tissue sampling we withheld food for 48 h to ensure there was no residual undigested feed in the digestive system that could bias our measurements of PCB concentrations. For each sampled fish, we measured fork length and body weight and examined fish for superficial external abnormalities. Using a heparinized 5-cc syringe, we drew blood from the caudal vasculature and transferred samples to a heparinized vacutainer. All samples were collected within 3 min of capture and placed on wet ice before separating the cellular fraction of the blood from plasma by centrifugation. Plasma was removed, transferred to cryovials, and frozen on dry ice.−1 penicillin, 100 µg mL−1 streptomycin, and 10 U mL−1 sodium heparin) and stored on wet ice prior to cell processing. We weighed the gonads and calculated the hepatosomatic index (HSI) for all sexually mature individuals. HSI was calculated as the liver weight/ × 100 [After collecting blood samples, we preserved one fish for total body burden analysis. For the remaining three fish, we aseptically dissected the anterior kidney, gonads, liver, and right fillet. The anterior kidney tissue was processed as described by . Brieflyt) × 100 ,40. We p6 leukocytes suspended in AM were added in quadruplicate to the wells of a 96-well plate for bacterial challenge and in triplicate on the same plate for subsequent adherent cell enumeration. Leukocytes from fish from all treatments were included on all plates to minimize the effect of inter-plate variability.Assessment of adherent anterior kidney leukocytes (AKLs) ability to kill the salmonid pathogen Yersinia ruckeri was determined using the method described by . In shorY. ruckeri  washed and suspended in Hanks’ balanced salt solution  was added to the treatment wells and to a row of cell-free control wells in a volume of 25 μL. The plates were incubated in a humidified chamber at 20 °C for 4 h. Media was subsequently removed from the treatment and control wells, cells were lysed with lysis buffer (0.2% Tween 20 in dH2O), and the lysate was immediately serially diluted in serocluster plates containing tryptic soy broth. Diluted lysates were plated onto tryptic soy agar plates, and we counted the number of colony forming units (CFUs). Bactericidal activity was expressed as % CFU reduction (1 − (CFU treated/CFU control) × 100) where CFU treated = mean CFU value for replicate wells with adherent leukocytes and CFU control = mean CFU value for replicate wells with media only. A corrected % CFU reduction was defined as % CFU reduction x cell density correction, where cell density correction = mean number of adherent cells from the same cell source used to determine bactericidal activity/1 × 106 cells.After incubating plates at 20 °C for 2 h, we replaced the media in all wells with culture media, and then cultured plates at 20 °C in a humidified chamber for 36 h to allow activated leukocytes to reach a resting state. We then removed the culture media from all wells, washed wells with antibiotic-free unsupplemented L-15 medium, and added 100 μL of antibiotic-free supplemented L-15 with 5% FBS. A 48 h culture of 6 leukocytes suspended in AM were added to the wells of a clear 96-well plate, incubated for adherence, washed, and subsequently incubated in CM at 20 °C for 48 h to allow cells to reach a resting state. Culture medium was removed, and cells were gently washed with room-temperature HBSS. Cells were then activated with 50 ng/mL of PMA in CM containing 1 mg/mL NBT, and control cells were incubated in the presence of 1 mg/mL NBT without PMA. Cells were incubated at 20 °C for 30 min and then the supernatant was removed and cells were washed with HBSS to remove unreduced NBT. Reduced NBT was solubilized with the sequential addition of 60 µL of 2M KOH and 70 µL of DMSO followed by rigorous pipetting. A volume of 100 µL of solubilized NBT was transferred to a new multiwell plate and absorbance was measured at 600 nm. We calculated the stimulation index (SI) as the replicate mean OD600 for a given set of PMA-stimulated leukocytes divided by the replicate mean OD600 of the associated PMA-free control leukocytes.The production of reactive oxygen species (ROS) from anterior kidney samples was determined using the nitroblue tetrazolium (NBT) method . In shor5 cells well−1 in 96-well plates were incubated with 5 µM calcein AM in CM for 5 h. Cells were then washed four times with room-temperature DPBS and cultured in CM for an additional 30 min. Media was removed and effector AKLs suspended in CM were added in quadruplicate at effector to target ratios of 10:1, 2:1 and 1:1. Lysis buffer  or CM alone was added in quadruplicate to another set of wells to determine total and spontaneous release, respectively. Plates were centrifuged at 50 g for 5 min and incubated at 20 °C for 8 h in the dark. A volume of 50 µL supernatant was removed from all wells and added to a 96-well black plate pre-loaded with 2× lysis buffer. Supernatant from the total release wells were added to 1× lysis buffer. Fluorescence intensity (FI) was measured by reading the plates from the bottom using a SpectraFluorPlus . We quantified percent cytotoxic cell activity as / × 100.The ability of channel catfish anterior kidney leukocytes to lyse virally transformed epithelioma papulosum cyprini (EPC) cells was determined using the calcein AM release-based cytotoxic cell assay as described by . Brieflymax Kinetic Microplate Reader , and plasma cortisol concentrations were determined via interpolation to a 4-parameter standard curve. The range of the assay defined by the standard curve was 1–400 ng/mL.Plasma cortisol was determined using a competitive enzyme-linked immunoassay modified from . Adaptatiy is the response for observation i and l. The model included the fixed effects of time , treatment , and a time x treatment interaction. A random effect for tank j, e transformed prior to analysis to accommodate the assumption of normality. Additionally, to accommodate the assumption of homogeneity of variance, a separate residual variance parameter was estimated for each treatment, k  was as follows:e scale were back-transformed when reporting results.Uninformative normal priors were used for  package  using R  package ). For eaPCB exposure did not influence fish weight as, across all sample events, the posterior mean weights of fish in the PF and PNF treatments were similar to the posterior mean weights of fish in the CF and CNF treatments, respectively. At the end of the food restriction phase (T3) the posterior mean weights of fish in the PNF and CNF treatments were lower than the posterior mean weights of fish in the PF and CF treatments; however, the effect was not statistically significant A.Subchronic PCB exposure had no immediate effect on HSI, as fish in all treatments had a similar HSI at T1 (posterior mean HSI (and 90% CI) at T1 for CF = 1.51 , CNF = 1.59 , PF = 1.37 , PNF = 1.63 . However, food deprivation significantly decreased HSI, with the strongest effect observed at T3. Comparing fish with the same history of PCB exposure, the posterior mean differences in HSI between fish in the CF and CNF treatments = 0.79  and PF and PNF treatments = 0.81 . Conversely, posterior mean HSI was similar in fish in the CNF and PNF treatments  and CF and PF treatments , suggesting that food deprivation, and not PCB exposure, had the larger effect on HSI.Microscopic examination of liver tissue at sample times T2 and T3 showed reduction of both glycogen and lipids in hepatocytes of CNF and PNF. Hepatocytes of fed catfish were highly vacuolated and there were few to no macrophage aggregates within the hepatic tissue. Hepatocytes in livers from the CNF and PNF groups were reduced in size and showed a lack of vacuolization. In the PNF group, many of the livers also had areas of darkly staining apoptotic and regenerative cells and small accumulations of ceroid/lipofuscin-containing cells. One liver from this group contained a vacuolated preneoplastic focus .Total PCB concentrations in whole fish, fillet, and liver tissues for fish in the CN and CNF treatments did not exceed baseline measurements at any sample point . By the end of the PCB exposure phase (T1), posterior mean whole body total PCB concentrations for fish in the PF and PNF treatments increased to 4.11 mg/kg  and 5.24 mg/kg , respectively. There was no significant change in whole body PCB concentrations by T2. However, by T3, whole body total PCB concentrations declined by 47% to 2.16 mg/kg  in fish from the PF treatment, whereas whole fish total PCB concentrations in fish from the PNF treatment declined by only 27% to 3.79 mg/kg . Notably, at this time, PCB depuration was faster for fish in the PF treatment  and 2.22 mg/kg , respectively, at T1. However, unlike whole body samples, total PCB concentrations in the fillet were similar between fish in the PF and PNF treatments at all sample points. Further, we did not observe a significant decline in total PCBs in the fillet through time, as PCB concentrations decreased by only 25% and 21% in the PF and PNF treatments, respectively B.Food deprivation significantly influenced the rate of total PCB depuration in the liver. For fish in the PF treatment, the posterior mean total PCB concentration in the liver was 0.32 mg/kg  at T1, and declined by 46% to 0.17 mg/kg  by T3 . In contrast, the posterior mean total PCB concentration in liver tissue for fish in the PNF treatment reached 0.34 mg/kg  after PCB exposure, and then increased by 27% to 0.43 mg/kg  by T3. While this increase was not statistically significant, it is in contrast to the significant declines in total PCBs found in the liver of fish in the PF treatment C.Across tissue types, posterior mean total PCB concentrations were significantly lower in the liver compared to the fillet and whole body for fish in both the PF and PNF treatments and across all sample periods. The fillet also had significantly lower posterior mean total PCB concentrations until T3, at which point faster depuration in the whole body resulted in no statistically significant difference in PCB concentrations between the whole body and fillet.Posterior mean concentrations of each of the ten homolog groups (mono-through decachlorobiphenyl) generally followed similar tissue-specific patterns of depuration as those described for total PCB concentrations. Specifically, fish from the PF treatment had faster rates of depuration than fish in the PNF treatment and, for most homolog groups, there was a decline in posterior mean PCB concentrations in whole body and fillet tissues by T3. However, declines in PCB concentrations were significant only for less-chlorinated homolog groups. As with total PCBs, by T3 posterior mean PCB concentrations in the liver increased in PNF fish, but decreased in PF fish, particularly for lower-chlorinated homolog groups .At T1, plasma cortisol concentrations were marginally lower in fish from the PF and PNF treatments; however, the effect was not statistically significant , CNF = 9.57 ng/L , PF = 7.22 ng/L , PNF = 7.71 ng/L . Food deprivation significantly influenced plasma cortisol concentrations, but the direction of the effect was dependent on prior PCB exposure. Specifically, while posterior mean plasma cortisol concentrations in fish from the CNF treatment significantly increased by T3 to 19.94 ng/L  plasma cortisol concentrations for fish in the PNF treatment significantly decreased to 4.96 ng/L . Plasma cortisol concentrations remained stable for fish in the CF and PF treatments A.PCB exposure led to reduced oxidative burst activity, as fish from the PF and PNF treatments had significantly lower oxidative burst activity than fish in the CF and CNF treatments at T1 . By T2, fish from all treatments had similar oxidative burst activity, and by T3, food-deprived fish had a significantly lower stimulation index than fish consuming PCB-free feed, regardless of PCB exposure history . Compared to fish from the CF treatment at T1, this represents a 34% and 30% decline in the stimulation index in fish from the CNF and PNF treatments, respectively B.At T1, posterior mean bactericidal activity was significantly lower in fish from the PF treatment compared to fish from the CF treatment . However, while fish from the PNF treatment had a lower bactericidal activity than fish from the CNF treatment, the effect was not statistically significant suggesting an overall weak effect of PCB exposure on bactericidal activity. Similar to oxidative burst activity, irrespective to PCB exposure history, food deprivation resulted in a significant decline in bactericidal activity by T3 . Compared to the posterior mean bactericidal activity at T1 from fish from the CN treatment, by T3 bactericidal activity declined by 93% and 91% in fish from the CNF and PNF treatments, respectively. Conversely, bactericidal activity significantly increased in PF fish by T3 . Cytotoxic cell activity started declining at T2 for fish from the CNF, PF, and PNF treatments, and by T3 fish from these treatments had significantly lower cytotoxic cell activity than fish from the CF treatment . Compared to fish from the CF treatment at T1, cytotoxic cell activity declined by 56% and 70% for fish from the CNF and PNF treatments, respectively, whereas cytotoxic cell activity declined by only 28% for fish from the PF treatment. Together, these results suggest that PCB exposure and food deprivation both decrease cytotoxic cell activity, with food deprivation having the stronger effect.This is one of the first studies to investigate the combined effects of dietary exposure to PCB mixtures and food deprivation on tissue-specific concentrations of total PCBs and homolog groups and functional immune response in a controlled setting. Similar to results of other studies, we observed no effect of PCB exposure alone on fish weight or hepatosomatic index ,48. HoweWhile PCB uptake mechanisms are well understood for many aquatic species, less is known about the timing and pathways of PCB elimination . Thus, wUnderstanding how PCB exposure influences specific tissue bioaccumulation and immune responses is difficult in natural environments due, in part, to the inability to control forage quality and quantity. Similar to , we showIt is important to consider that, due to animal welfare concerns, the duration of the food deprivation phase here was limited to seven weeks. While this design limits our ability to make inferences about the long-term effects of PCB exposure and food deprivation, our results still provide insights into how PCBs may influence fish and human health through consumption of contaminated fish tissues. For example, we found that PCB concentrations in the fillet, which is the tissue most likely to be consumed by humans, may depurate more slowly than concentrations in the whole fish. This information is important for establishing human consumption advisories and monitoring protocols and for understanding how PCBs bioaccumulate across trophic levels.Similarly, our results can help elucidate the immunological response of channel catfish following PCB exposure and food deprivation. While the effect of food deprivation was generally greater than that for PCB exposure, PCB exposure did decrease bactericidal and oxidative burst activity. This result is consistent with those of previous studies in ictalurids ,38,54, aWhile functional immune responses changed most significantly to food deprivation, the effects of PCB exposure and food deprivation appear to be additive for plasma cortisol. Fish in the PF treatment had marginally lower plasma cortisol concentrations than fish in the CF treatment at all sampling times, suggesting that PCB exposure can decrease cortisol concentrations even after several weeks of consuming a PCB-free diet. Moreover, by T3, fish in the PNF treatment had significantly lower plasma cortisol concentrations than fish in the other treatments. The most notable difference in cortisol at T3 was between fish in the PNF and CNF treatments, highlighting the effect of PCB exposure independent of food deprivation. This result is consistent with those of other studies, which found that fish sampled from contaminated environments lacked the ability to increase serum cortisol concentration in response to acute stress ,56, perhOverall, decreased immune function associated with food deprivation is not surprising, and may be associated with inadequate energy reserves for metabolically depending immunocyte responses , as wellIt is challenging to determine how PCB-induced tissue toxicity and immunosuppression translate into population- and ecosystem-level effects. Aquatic organisms are rarely exposed to a single toxicant, nor are the exposure mechanisms and concentrations well understood. Additionally, environmental and biological stressors, such as competition, predation, and environmental stochasticity, can work synergistically to further suppress immune function, with the strength of the synergism dependent on species-specific ecology . For exaGiven the ubiquity of PCBs in aquatic environments and the occurrences of fish kills worldwide, the development of species-specific depuration models and robust biomarkers for PCB toxicity would be advantageous for assessing consumption risk and for developing remediation timelines . Our conThese results suggest that short-term dietary exposure to PCBs can increase toxicity of consumable fish tissues for several weeks, and that PCB mixtures modulate immune and stress responses in fishes via multiple interacting pathways. While PCBs can quickly depurate below the concentration that warrants human consumption advisories, lack of adequate nutrition can delay depuration and prolong human exposure risks. Thus, it may be important to consider species’ ecology and food availability when assessing human health concerns associated with the consumption of fishes from PCB-contaminated ecosystems.Overall, these results can be informative in the development of global fish consumption advisories and can also be used to predict the risk of PCBs to human health. Additionally, our findings of immunomodulation following PCB exposure and food deprivation can help elucidate how PCBs influence fish and wildlife population demography.


Malus domestica Borkh.) cultivar Rubin was grafted on dwarfing rootstocks P60, planted in single rows spaced 1.25 m apart with 3.5 m between rows. In contrast to plant senescing reflectance index and nitrogen balance index, the photochemical reflectance index was significantly higher in 2018 compared with 2017. Such differences might be caused by drought stress on the summer and fast recovery before harvest time when measurements were made. The movement of nutrients and water disrupted by trunk incision had significantly negative effect on reflectance indices regardless on the year. Mechanical pruning with trunk incision and calcium-prohexadione lead to decreased dry to fresh weight ratio by 10–12% in first year of treatment. Mechanical pruning had significantly negative impact on photosynthetic rate, compared to pruning by super spindle it decreased 47%. Strong positive correlation between PRI and NBI 0,89–0,94, and strong negative correlations between PRI, NBI and PSRI −0.88 – (−0.91) were determined.The aim was to find out the impact of stress, caused by agrotechnological tools on photosynthetic behaviour of apple trees. The apple tree ( From 100% sun light only 40% is typical light interception by orchard systems and 15% (out of 40%) is limited by orchard design and leaf area2. During photosynthesis, solar energy binds to dry matter, so the photosynthetic behavior of the plant can be evaluated. Increasing photosynthesis goes to increase in biomass. Optimal plant photosynthesis system work is the main part of the biomass quantity3.Not only environmental factors but also plant canopy ant light penetration into canopy strongly influence photosynthetic productivity and plants’ growth. Photosynthesis productivity depends on light, water, CO5. Nitrogen (N) is one of the most important components of photosynthetic productivity is the also, it is a part of chlorophyll and it is one of the most important elements for biomass production6. The chlorophyll index (Chl) and the nitrogen balance index (NBI) measure the relative amount of chlorophyll and nitrogen at the same point on the leaf in the same moment and it may convey N dynamics in apple trees systems8. Plant synthesizes proteins and favors primary metabolism(nitrogen containing molecules) containing chlorophyll and just a few flavonols is shown by higher nitrogen balance index. NBI status of leaves has a relationship to light availability and light limitation as shown in literature9.Non-destructive measurement methods easily measure variable optical properties without affecting the leaf at various stages of its development. In one measurement several functionally relevant leaf parameters is done without interruption10. PRI is commonly used as an appropriate indicator of abiotic factors that limit photosynthesis11. PRI via the xanthophyll cycle is related with photosystem II (PSII) and it is using as an alternative for light use efficiency. Photochemical reflectance index was applied as an active probe of pigment conversion, it can reflect almost all changes in the pigment content of the xanthophyll cycle12. Other authors have investigated that changes in pigment content and ratio due to leaf growth, aging, or chronic stress play an important role along with xanthophyll pigment epoxidation in the PRI signal. The photochemical reflectance index responds to the chlorophyll / carotenoid ratio in leaves due to species, age, and environmental conditions13. While PRI characterizes the photosynthetic efficiency, the plant senescing reflectance index (PSRI) is sensitive to the changes of carotenoids and chlorophyll ratio and it was used as a quantitative measure of leaf senescence. They are both commonly used to describe the changing physiological state of vegetation15. Reflectance spectra in the visible and near infra‐red range of the spectrum, acquired for maple (Acer platanoides L.), chestnut (Aesculus hippocastanum L.), potato (Solanum tuberosum L.), coleus (Coleus blumei Benth.), leaves and lemon (Citrus limon L.) and apple  were studied and it was found that leaf senescence effect is depending on pigment composition14.The Photochemical Reflectance Index (PRI), which is based on leaf reflectance at 531 nm, is suitable for tracking variations in photosynthetic activity from leaf16. Alarcon and Sassenrath17 showed cotton leaf photosynthesis efficient dependence from shapes and canopy architecture. This shows that the effectiveness of photosynthesis can be controlled through the shape of the canopy and the best results were based on the high number of small-to-medium sized leaves. Like all plants, the ability of apple trees to sustain higher photosynthetic rates (PN) depends on the whole tree canopy and its regulation18. Also, Zhang et al.19 showed that photosynthesis consider from temperature, CO2 and especially light intensity and chlorophyll content. Moreover, photosynthetic rates are sensitive to various stresses like salt-alkaline, salt, water deficiency, shading and other20. One of the reasons for the PN reduction is the stress of water drought and Ohashi et al. measured the decrease by up to 34%21. The inhibition of photosynthetic rate under water stress may be attributed not only to stomatal closure but also on several biochemical and photochemical processes23.Photosynthesis activity is associated not only with the yield of individual leaves, but also with the optimal use of light, considering the penetration of light through the canopyet al.24 showed that heavy crop load decreased fruit size and weight. Denser canopy also has negative impact on fruit mass and yield25. Although calcium-prohexadione is using for smaller terminal growth it also has the impact for fruit weight. Usually fruit weight decreases by use of calcium-prohexadion. As Privé et al.26 showen Calcium-prohexadion can increase light penetration into the inner canopy of fully-grown apple trees and thus improve flower bud formation and expected yield.‘Rubin’ is large fruit variety. De Salvador 29. Trunk girdling not only increase fruit yield, but also can increase the influence of other agrotechnical tools using foliar spray or fertilization32.Water and nutrients movement are disturbed by trunk girdling as well as by trunk incision. Trunk girdling and ringing significantly increase yield but most of the studies shown that photosynthetic behavior decreases in the same time as yields increaseManual pruning of orchards requires a lot of resources and manpower to reduce the cost of gardening through mechanical pruning, trunk cutting, chemicals and more. Despite the amount of research on the relationship between the optical properties of apple leaves and the rate of photosynthesis in different apple agrotechnology tools, there is little. Thus, the aim was to elucidate the influence of stress caused by light penetration on canopy and agro-technological measures on photosynthetic behavior of apple trees.Malus domestica Borkh.) cultivar Rubin was grafted on dwarfing rootstocks P60. Apple trees were planted in 2010 year in single rows spaced 1.25 m apart with 3.5 m between rows. Pest and disease management was carried out according to integrated plant protection practices and orchard was not irrigated. Soil conditions of the experimental orchard were following: clay loam, pH 7.3, humus 2.8%, P2O5 255 mg kg−1, K2O 230 mg kg−1. Three single trees were fully randomized. The non-destructive and biometric analysis were performed in harvest time in the end of September. The samples were taken from five leaves in two levels: at 1.0–1.2 m and 1.8–2.0 m above the ground. Five agrotechnological tools were used: (1) each year hand pruning forming slender spindle (control); (2) mechanical pruning (each year) with hand pruning every second year; (3) mechanical pruning (each year); (4) trunk incision using chain saw +mechanical pruning (each year); (5) mechanical pruning (each year) +spraying with calcium-prohexadione. Mechanical pruning goes with hand pruning ones in three years removing the oldest branches from the stem base. Reducing the movement of water and nutrients by cutting the trunk also suppresses tree growth, reduces the number of shoots and at the same time increases the lighting of the canopy at the top of the it. Mechanical pruning, on the other hand, only shortens the branches, without the use of manual pruning, increasing the number of large branches, which increases the darkening of the crown. Also, trees grow actively in the upper part of the canopy, densify and multiply branches, which cast heavy shadows on the entire tree. Using only mechanical pruning, the trees resemble a hedge. While in the case of manual pruning, the large branches are removed from the trunk, there are no large branches at the top of the canopy, and the canopy is easily lightened and does not darken the rest of the crown.A field experiment was carried out in an intensive orchard. The apple tree  from 9 to 12 am. Reflection spectra obtained from the leaves were used to calculate photochemical reflectance index (PRI) and plant senescing reflectance index (PSRI). PRI shows changes in the xanthophyll cycle, using the following formula:678, R500 and R570 represent the leaf reflectance integrated over a 10 nm wavelength band centred on 678, 500 and 570 nm, respectivelyPSRI was proposed as being sensitive to the senescence phase of plant development and it’s calculated by formula:NBI was evaluated using no-destructive measurement of leaf chlorophyll and flavonoid content  in the epidermis .2] (400 μmol mol−1), light intensity (1000 μmol m−2 s−1) and the flow rate of gas pump (500 mmol s −1) were set.Photosynthetic rate was determined at 9:00–12:00 am by using a LI-6400XT portable open flow gas exchange system . Reference air [CO2), twenty leaves were randomly sampled from all tree canopy and measured with a leaf area meter . The dry mass of twenty leaves was determined by drying apple tree leaves in 70 °C for 48 h.  to constant weight33.To determine the leaf area , The data were processed using two-way ant three-way analysis of variance (ANOVA) Analysis of variance was performed with Duncan’s least significant difference test (P < 0.05).Mechanical pruning results denser crown and light penetration into canopy. The lower light penetration into the crown resulted the decrease of PRI (25–100%) Fig. . HoweverNitrogen balance index showed significant differences only in the lower part of canopy. Mechanical pruning with manual pruning increased NBI by 38% compared to control. Meanwhile, using trunk cut, the NBI variation is insignificant compared to the control Fig. Figure 3NN). PN decreased by 47% using only mechanical pruning compared to pruning by super spindle. Mechanical pruning with hand pruning, trunk incision and calcium-prohexadione also resulted in significant decrease of PN 10–20% compared to control.Trunk incision and calcium-prohexadione significantly decreased DW/FW ratio to 10–12% Fig. . Compare−1 yield in 2018 . The first two components accounted for the majority of variation in the data set (88.59%). The variation of 82.60% could be explained by the first principal component (PC1) and 5.99% by the droughty principal component (PC2). Strong positive correlation between PRI and NBI 0,89–0.94, and strong negative correlations between PRI, NBI and PSRI −0.88 – (−0.91) were found , light intensity, but also the effect of temperature on PRI (PRI and PSII efficiency). In their research is shown that at lower temperatures and dark green leaves, PRI values increase. Similarly, Weng et al.35 found that the relationship between the efficiency of PRS II and PRI varies with temperature and leaf color, they argue that lower PRI values are associated with increased illumination of mango tree leaves. While getting into the details of other authors’ data, and considering that this research comes from autumn measurements, when daytime temperatures drop, we find that during mechanical pruning, PRI responds more to temperature than to light intensity  organs by response to drought. We see that NBI increased significantly only in the sunny and droughty years 43. However, Kozlowski and Pallardy44 observations of woody plants adaptation to drought stress showed that trees have developed mechanisms to deal with dehydration conditions that are different from herbaceous plants. The amount of unstructured carbohydrates may also reflect tree drought, which may lead to a decrease in the concentration of unstructured carbohydrates, but as Gaul et al. In45, this usually only occurs in the later stages of drought.Water deficit is one of the greatest limiting factor for photosynthesis and biomass accumulation47. Trees that have been touched after a short drought have no major changes, even some tree species are able to recover quickly after a drought49. During treatment years was one drought episode form May till July , then another drought episode from the end of August till harvest time appeared. Apple trees were affected by short-term drought in 2018 but form the PRI and PSRI values it can be stated that the trees quickly recovered and functioned even better than in normal years.Water intake increases with increased root fine growth, deep root formation, and solvent accumulation to reduce the water potential in the root tissue. Only then does the drought level become too severe, and the plants react by activating the mechanisms that protect tissues from cell damage, mainly by the action of protective solutes and proteinsN is up to 50% lower in shade leaves compared to sun leaves50. Negative shade impact for trees photosynthesis also was described in several research like for Acer rubrum and Betula papyrifera51, Fagus syltica52, Ginkgo biloba53, Malus domestica54. However, shading can have positive impact for photosynthesis when it goes with drought but in our research there was no significant impact of shading55.Denser canopy formed by mechanical pruning decreases another limiting factor – light penetration into canopy. P56 that calcium-prohexadione had no significant impact for fruit weight for apple cultivar ‘Rubin’ . Mechanical pruning increased apple yield upto 45% but decreased fruit size by 4% in two years average. In general, the recommendation for industrial orchards is hand pruning every second year, mechanical pruning combined with reduced manual pruning lowered orchard maintenance but retained apple yield and photosynthetic activity.Strong positive correlation between PRI and NBI 0,89–0,94, and strong negative correlations between PRI, NBI and PSRI −0.88 – (−0.91) were found. Trunk incision disturbed nutrients and water movement from roots to canopy that resulted the significant decrease of photochemical reflectance index, photosynthetic rate, fruit mass and accelerated leaf aging. The lower light penetration in denser canopy formed using mechanical pruning also resulted in significant decrease of photosynthetic indices (PRI, PSRI, PDataset 1.


The demand of lattice structures for medical applications is increasing due to their ability to accelerate the osseointegration process, to reduce the implant weight and the stiffness. Selective laser melting (SLM) process offers the possibility to manufacture directly complex lattice applications, but there are a few studies that have focused on biocompatible Ti6Al7Nb alloy. The purpose of this work was to investigate the physical–mechanical properties and the microstructure of three dissimilar lattice structures that were SLM-manufactured by using Ti6Al7Nb powder. In particular, the strut morphology, the fracture characterization, the metallographic structure, and the X-ray phase identification were analyzed. Additionally, the Gibson-Ashby prediction model was adapted for each lattice topology, indicating the theoretical compressive strength and Young modulus. The resulted porosity of these lattice structures was approximately 56%, and the pore size ranged from 0.40 to 0.91 mm. Under quasi-static compression test, three failure modes were recorded. Compared to fully solid specimens, the actual lattice structures reduce the elastic modulus from 104 to 6–28 GPa. The struts surfaces were covered by a large amount of partial melted grains. Some solidification defects were recorded in struts structure. The fractographs revealed a brittle rupture of struts, and their microstructure was mainly α’ martensite with columnar grains. The results demonstrate the suitability of manufacturing lattice structures made of Ti6Al7Nb powder having unique physical–mechanical properties which could meet the medical requirements. The evolution of additive manufacturing (AM) technology has led to the possibility of processing various macro-porous networks, so-called lattice structures. By integrating cellular gradients into medical applications, they can provide promising perspectives on the physical–mechanical behavior of implants . CompareFrom a material point of view, previous SLM research has predominantly focused on lattice specimens made of Ti6Al4V ,27,28,29Moreover, using the Ti6Al7Nb powder could lead to multiple issues, and proper process parameters are needed for a stable fabrication of lattice structures. The information regarding the process parameters for this powder is also limited.The purpose of this study was to investigate the physical–mechanical properties and the microstructure of three dissimilar lattice structures made of Ti6Al7Nb powder SLM processed. While maintaining the same porosity level, the considered designs of lattice structures were a commonly body-centered cubic structure and two new concepts. The lattice specimens were mechanical tested under static compression, and they were morphologically, metallographically, and structural analyzed. Additionally, the Gibson–Ashby mathematical model, which can estimate the Young modulus, and compressive strength was customized for each lattice topology. In order not to influence the mechanical results, the specimens were not subjected to any stress-relief treatment. Finally, a future approach is presented, describing a hybrid lattice implant which could satisfy the mechanical requirements of human bones.Due to the presence of vanadium in Ti6Al4V alloy, which can cause side effects around it after implantation, this study focused on Ti6Al7Nb, with a better expected biocompatibility ,36,37. TIn general, the lattice structures are characterized by periodic arrangements of open cells into a 3D model, providing distinct physical–mechanical properties. From a lattice-design point of view, the present topologies were generated by using the manual method. Manual generation means designing a lattice material by implementing beams and trusses with joints modified to create seamless transitions between unit cell elements . TypicalThe new lattice concepts detailed above were elaborated in a such manner that during the SLM process, no support structures should be needed to sustain their struts b,c. WhenBesides this preliminary manufacturing evaluation focused on porosity level, a finite element analysis (FEA) was elaborated. The FEA simulation can analytically predict the mechanical behavior of these lattice topologies. Because the numerical simulations are well documented for BCC structures ,27, the To obtain the cylinder specimens for mechanical trials, each lattice unit was multiplied several times, intersecting between them with 0.10 mm. The cylinder specimens had the following overall dimensions: the diameter of 12 mm and the height of 24 mm. The lattice cylinders were manufactured by a SLM 250 system . This equipment is capable of manufacturing medical grafts or implants directly from biometal powders, under the “slice by slice” principle. In this research, this technology can fuse the grains, using a solid laser up to 200 W power (type Nd:YAG). All the specimens were built randomly, being orientated vertical on the machine platform. The scanning strategy adopted was “X/Y”, which allows a scanning in X direction of “n” layer and in Y direction of “n + 1” layer. To anchor the bottom of lattice specimens, some support structures were prepared in RDesigner software . The laser parameters programmed to print the support structures were 120 W laser power and 500 mm/s scanning speed. A soft recoater made of dense carbon fibers with Ø 7.5 µm was used to spread the powder. Because the SLM process involves heat transfer, remelting, and other thermo-physical behaviors, typical defects could occur in the Ti6Al7Nb lattice structures. Some of the typical manufacturing defects refer to the balling effect, micro-cracks, heat-affected zone, and residual stress phenomena ,42,43. TP is the porosity, w a weight, r and h are dimensions of the cylinder (radius and height), and ρ0 is the density of bulk Ti6Al7Nb. The present density of bulk Ti6Al7Nb was 4.50 g/cm3, being calculated on fully dense specimens manufactured with laser parameters detailed in After the SLM fabrication, the unmelted powder from lattice pores was removed by ultrasounds, in an alcohol bath, at 40 °C for 1 h. The porosity of lattice cylinders was calculated by using Equation (1) :(1)P=1−wE, σ, and P are the Young modulus, compressive strength, and porosity of lattice structures. E0 and oσ are corresponding to fully dense samples and represent the elasticity and the compressive strength. For a wide range of alloys, n exponent has been shown to vary from 1.8 to 2.3 and often is assumed to be 2 [m coefficient, based on actual mechanical results. Thus, the m coefficient characterizes the dependence of elastic modulus and compressive strength on the porosity.The mechanical tests were performed on fully dense and lattice specimens, using an Instron 1255 system . This system has 500 kN maximum capacity, and the mechanical trials were completed at room temperature . The spe to be 2 ,21,26. AThe morphological aspects of lattice specimens were investigated by scanning electron microscopy (SEM). The microscope used was Quanta 200 3D , and the accelerating voltage set up was 30 kV. During the SEM investigations, the Everhart–Thornley detector (ETD) was used in a high vacuum mode. Metallographic analyses were conducted, using an Olympus GX51  microscope, on ground and polished specimens, subsequently etched by Kroll’s reagent, for 6–10 s, at room temperature. Metallographic analysis of specimens was performed onto two directions, perpendicular and parallel to the layers . The resulted lattice structures were studied by using an X-ray diffraction (XRD) system type Equinox 3000 . The XRD analysis was carried out via Cu Kα radiation (λ = 1.54 Å).Geometric accuracy is often a limitation in fabrication of cellular structures, as is the entrapment of powder in the small pores’ spaces of these structures. In general, the geometric accuracy of SLM parts ranged from ±0.05 to ±0.10 mm. Thus, it is necessary to measure the strut size because the geometrical deviations are influenced by the process parameters. The cross-sectional metallographic investigations allowed us to measure the struts dimensions. Their mean value and standard deviation are summarized in The porosity of lattice models was calculated theoretically by Creo Parametric program, and it was 60% for all of them. After SLM manufacturing, the lattice specimens have a real porosity approximately 56%. The porosity of specimens is reduced because of the actual strut diameter, which was higher than the designed one. Compared to fully dense specimens, the lattice structures can reduce the processing time and the amount of powder required, making the SLM manufacturing more efficient. Furthermore, the osseointegration of macro-porous implants is highly influenced by the porosity level . In vivoBefore testing the lattice specimens, the compression properties of bulk Ti6Al7Nb were determined . The fulcr.) is marked. Three failure modes were recorded: brittle fracturing of the struts, successive cell collapse, and fracture propagation. Similar compressive failure modes were reported on lattice structures made of Ti6Al4V [cr. was reached  and (3), we see that both compressive strength and Young modulus can be estimated ), respectively, between compressive strength–porosity (Equation (3)) is similar. For this reason, the m exponent has the same value in these equations, and it can be applied to estimate the theoretical compressive strength and Young modulus of each lattice topology. This fact proves that the elasticity and compressive strength of the lattice structures are dependent by the porosity level, in the same proportion. To predict the Young modulus and compressive strength of SLM implants made of Ti6Al7Nb with lattice structures integrated, the Gibson–Ashby mathematical model was adapted to each lattice type. This knowledge is useful for design analyses or optimizations.As explained in recent studies, the Gibson–Ashby mathematical model for open cell foams indicates a linear correlation between the elastic modulus and the square of porosity ,64,65. D and 4.0 . Here, tSEM analyses can provide details about issues or defects occurring on SLM specimens. The main manufacturing problems identified on strut surfaces were the stair-stepping effect, unmolten or half-attached particles on struts, and the balling phenomenon. Because the stair-stepping effect is dependent upon the build angle, it was clearly visible in BCC specimens. An example is presented in It can be seen from the SEM images that all the Ti6Al7Nb struts were covered by a large amount of unmolten or partially melted grains. These particles are bonded on the strut’s surfaces, being nearly spherical, with a diameter of 20 to 80 µm . Two maiRepresentative fractographs obtained on Ti6Al7Nb specimens are detailed in Due to staircase effect and particles stuck on struts, their micro-rough surfaces can also be observed in Large spherical pores can be seen, mainly in the center of the strut structure or lattice nodes. These gaseous bubbles are caused by the solidification shrinkage effect of the Ti alloy. Some gaseous bubbles are marked in The binding defects are irregular cavities associated with spherical particles, marked with green arrows in A representative micrograph of crack propagation is illustrated in Based on these findings, the following future studies are approachable. From a cell-design point of view, future research should use a mathematical algorithm to generate and optimize the PCI and PHS topologies, and to define the relationship between the relative density and the cell aspect ratio. The established process parameters led to a good and stable manufacturing, but to limit the solidification defects from Ti6Al7Nb struts, extensive investigations should be developed to optimize the laser parameters. Nevertheless, additional work is required to evaluate the fatigue resistance of these lattice structures made of Ti6Al7Nb.On the other hand, the obtained porosity, the reduced Young modulus of these lattice structures, and the micro-rough surface of struts could improve the osseointegration process. These three aspects could allow a fast and effective bone ingrowth, aiming toward the long-term stability of implants. Future in vitro and in vivo study is needed to evaluate the cells’ behavior and bone ingrowth, using the present lattice structures SLM-manufactured. This comparative study focused on three topologies of lattice structures made of Ti6Al7Nb, offering preliminary knowledge which could enhance the development of medical treatments in different sectors, such as orthopedic, craniomaxillofacial reconstructions, and regenerative medicine. The results could contribute to expand the concept of implant customization from a structural and mechanical point of view, according to patient’s age and health status.The knowledge exposed could be used to develop hybrid lattice implants. This concept means that the medical component will have distinct physical–mechanical properties for the cortical bone and trabecular tissue, avoiding the effect of stress shielding or overburdening. A practical example of this future perspective is presented in The established SLM parameters allowed for stable manufacturing, and the Ti6Al7Nb specimens were fabricated with 56% porosity. When we compare them with their fully solid counterparts, we see that the present lattice specimens reduced the processing time and powder consumption. Under compression tests, three failure modes were recorded. Depending on lattice topology, the compressive strength ranged from 38 to 279 MPa, and the elastic modulus was between 6 and 28 GPa, being 73–94% lower than for solid specimens. Even if the porosity level of lattice structures is similar, their compressive strength and Young modulus are influenced by the cell design and strut diameter. The results of this work proved that it is possible to SLM manufacture lattice structures made of Ti6Al7Nb with comparable physical–mechanical properties to Ti6Al4V specimens. To predict the Young modulus and compressive strength of Ti6Al7Nb specimens, the Gibson–Ashby mathematical model was adapted to each lattice topology.The dimensional deviations of struts are ±0.05 mm, and their surfaces are covered by a large amount of partial melted grains. Some solidification defects were recorded in struts’ microstructures. Because the specimens were tested in as-build condition, the fractographs revealed a brittle rupture of struts near to nodes and a crack orientation at 45°. The microstructure has shown mainly α’ martensite, with columnar grains. The diffraction peaks correspond to hcp titanium phase, and they are similar with raw powder pattern. To exploit the potential of these lattice structures made of Ti6Al7Nb, future work should investigate the osteoblast cell affinity, in vivo osseointegration feasibility, and design optimization.