Anchialine ecosystems in the eastern Adriatic Sea are diverse both morphologically and biologically. In this study, for the first time, we explored the microeukaryotic and prokaryotic community of anchialine caves in the Mediterranean region using high-throughput sequencing. Four anchialine caves located on nearby islands with a well-pronounced salinity gradient were sampled at the surface freshwater area, halocline area, and seawater area. Sequencing revealed a surprisingly wide diversity of the microeukaryotic and prokaryotic community with the relative abundance of major phyla differing within the salinity gradient and between the caves. Interestingly, microeukaryotic and prokaryotic communities clustered into four groups based on location, pointing out that sampled anchialine caves have different microbial community patterns and high microbial endemism. Our results indicate that even with the halocline acting as a selecting barrier, the salinity is not the only community structuring factor. Despite the short geographical distance, the isolation of anchialine caves facilitated high microbial community adaptation and endemism. Our study suggests that anchialine caves represent reservoirs of new biodiversity, maintaining unique and complex microbial diversity influenced by biotic interactions and abiotic environmental conditions.The online version contains supplementary material available at 10.1007/s00248-021-01760-5. Aquatic microbial communities are affected by many environmental factors that determine their diversity and abundance , 2. ChanAnchialine ecosystems are defined as “tidally-influenced subterranean estuaries within crevicular and cavernous karst and volcanic terrains, that extend inland to the limit of seawater penetration” . Due to Thus far, we have begun to understand better the importance and function of microorganisms and how microbial diversity is distributed across environments, yet the microbial community of anchialine ecosystems is still poorly investigated compared to other aquatic environments. Most anchialine cave studies documented endemism among eukaryotes . RelativTo our knowledge, this study presents the first investigation of the microeukaryotic and prokaryotic plankton community across the halocline of anchialine caves in the Mediterranean region using amplicon sequencing. The primary objective was the identification of abiotic factors that are of importance for structuring microbial communities in four anchialine caves at three depths defined by sharp vertical salinity stratification. We hypothesized that microeukaryotic and prokaryotic communities in anchialine caves have a similar habitat pattern shifting through the halocline area and have high variations in diversity between the sampling depths.Sampling took place in National Park Kornati, situated in the eastern part of the Adriatic Sea, Croatia Fig. . Four an4+  + Norg), nitrogen-nitrite (N-NO2−), nitrogen-nitrate (N-NO3−), orthophosphate (PO43−), and total dissolved carbon (TOC) were determined in collected water samples (300 mL). The concentrations of nutrients were measured on a Perkin Elmer Lambda 25 UV/Vis spectrometer. Nitrate, nitrite, and ammonium were measured according to Zhang and Fischer [−1, 0.03 μmol L−1, and 0.4 μmol L−1, respectively, with overall precision ± 10%. Orthophosphate concentrations were analyzed according to ISO 6878:1998(E). The method is based on the formation of the phosphomolybdate complex, which is subsequently reduced with ascorbic acid to form a strongly colored blue molybdenum complex; the absorbance is measured spectrometrically at 880 nm. Detection limits in orthophosphate analyses were 0.04 μmol L−1 and 0.15 μmol L−1, respectively, while precision was typically better than ± 10%.Salinity stratification in the anchialine water column was determined by instant CTD probe , taken at each station before all other samples, to characterize the halocline. Physical parameters , and water temperature) were measured with depth in situ using diver-carried multiparameter data loggers Hach HQ40D Portable Multi-Parameter Meter . Concentrations of total nitrogen (TN), ammonium with organic nitrogen , and stored in the dark at 4 °C until analysis. Filters were dried to constant weight at 60 °C and then exposed to HCl fumes for 4 h to remove all inorganic carbon [2 [A sample aliquot was filtered on 25-mm glass filters  using an all-glass filtering system (Wheaton) under vacuum to determine the total organic carbon content. All glass equipment  was calcined at 450 °C for 4 h before use. The resulting dissolved fraction was stored in a 24-mL glass tube equipped with a Teflon/silicone septum (Wheaton), poisoned with 50 μL of 1 mol Lc carbon . The disarbon [2 , calibraWater samples (1 L) were filtered on 0.2-µm pore size polycarbonate filters. According to the manufacturer’s guidelines, total genomic DNA was extracted from filters with the DNeasy PowerWater Kit . The hypervariable V9 region of the eukaryotic SSU rRNA gene was amplified using the primer pair 1391F (5′-GTACACACCGCCCGTC-3′) and EukB (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) following the protocol of Stoeck et al. . For thehttps://sourceforge.net/projects/bbmap/) and quality-filtered using the split_libraries.py script implemented in QIIME v. 1.8.0 to remove low-quality reads [d = 1, clustering amplicons by using a local clustering threshold. For the microeukaryotic dataset, the taxonomic assignment was done using blastn in BLAST v. 2.9.0 [rrarefy function of the R package “vegan” [Paired-end reads were quality trimmed using the bbduk function and merged using bbmerge function of the BBMap package  based on the PCoA results was used to evaluate the correlation of microbial communities using the “ade4” package [https://github.com/kkajan/anchialine-miceco).All statistical analyses were performed in the R environment (v. 4.0.4)  and visu package . CIA res−1). The highest TN at the surface was measured in VG cave (7 mg L−1) and the lowest in BP (0.39 mg L−1) (Table −1).The water in all caves during the sampling was heavily stratified due to a strong salinity gradient Fig. . Salinit1) Table . The lown = 463) and the greatest richness above the halocline in BP cave  followed by the richness of the halocline in ZVP (n = 914)  or were assigned to the non-target OTUs . The total number of assigned reads ranged between 57,334  and 979,181 . Taxonomic richness varied notably along the salinity gradient with the lowest number of OTUs detected below the halocline in cave VG (14) Fig. . The higAltogether, 22 higher taxonomical levels were recorded wherein an average microeukaryotic community of anchialine caves consisted of Alveolata 44.37%), Fungi (29.02%), Stramenopiles (21.94%), Rhizaria (1.29%), and Viridiplantae (1.26%) Fig. . On aver4.37%, Fun = 36,528) followed by the sample below the halocline of VG cave . In contrast to BP cave, the sample above the halocline in GKV cave was presented by the highest number of reads , while below, the halocline with the highest number of OTUs . The prokaryotic community richness showed a similar pattern in both the VG and BP cave with the highest richness recorded in the halocline, while in the ZVP cave, the greatest richness was recorded below the halocline . The communities were only affected by temperature, while no correlation was observed with other measured environmental parameters on the OTU level. CIA resulted in a high significant correlation of microeukaryotic and prokaryotic communities of anchialine caves  dominated the area with the lowest salinity concentration in the BP cave. Despite being known as globally distributed euryhaline and eurythermal genus with prevalence in intertidal pools and estuaries, it was reported that reduced salinity, caused by freshwater inflow, may stimulate blooms of this genus [Only in the BP cave, the richness and the Shannon index of the microeukaryotic community had not followed a similar pattern. This cave is the deepest among other sampled caves and the most branched with speleothems in the submerged part. The highest microeukaryotic richness was detected in the area below the halocline in GKV and ZVP cave, while the lowest in the area above and below the halocline in the VG cave. Alveolata and Stramenopiles were the most diverse and abundant groups, as observed in other ecosystems , 41. A d VG cave . In and is genus Uronema (Scuticociliatia) produces proteases responsible for the digestion of the host’s tissues and proteins responsible for the high mortality rates of fish [Paraphysomonas contributed with a high relative abundance (83.5%). Usually known as important feeders on bacteria, Paraphysomonas can be found as a freely swimming cell and also occurring attached to bacterial mats or other surfaces [In the GKV cave, the geographically most distant cave that during the Roman time was used as a freshwater source, Alveolata were highly dominated by Ciliophora. The area above the halocline was dominated by the subclass Scuticociliatia. This subclass gathers free-living ciliates in fresh, brackish, and marine water together with opportunistic or facultative parasitic ciliates of aquatic animals . Parasit of fish . Howeversurfaces .Poterioochromonas (4.6%) [Malassezia (Basidiomycota) to Verrucoconiothyrium (Ascomycota). Ciliates (scuticocilate Protocyclidium and Holotrichous ciliate Prorodon) dominated in the low-nutrient conditions in and below the halocline. Fungi were numerous in all layers with the highest abundance (69.1%) in the hypoxic marine-like area, dominated by genera Malassezia, Cladosporium, and Pseudobensingtonia. Species of these genera often have pathogenic or saprophytic lifestyles, e.g., Malassezia is a lipid-dependent basidiomycetous yeast accounting for the majority of the eukaryotic diversity in deep-sea subsurface sediments [Compared to other sampled caves, the highest microeukaryotic diversity based on the higher taxon groups was established in the ZVP cave, the shallowest sampled cave, with the dominance of Stramenopiles, Alveolata, Fungi, Viridiplantae, Heterolobosea, and Rhizaria in the surface area. The relative abundance of Stramenopiles and Fungi has followed the decrease by the depth within the salinity gradient, whereas the relative abundance of Alveolata increased, ranging from 30.6 to 71.0%. Above the halocline, OTUs affiliated to Chrysophyceae and Synurophyceae reached a total relative abundance of 40.5%, with the highest contribution of uncultured Chrysophyceae (32%) and s (4.6%) . The funediments .−1) [−1) was measured below the halocline, where the Gammaproteobacteria, Firmicutes, and Actinobacteria had the highest relative abundance. The common kestrel nests and bats were detected in this cave that could have a possible contribution to the source of pathogenic bacterial strains below the halocline. The only genus contributing to the prokaryotic community below the halocline that is not corroborated as pathogenic was Lactobacillus.The prokaryotic community was partially dominated in all the investigated caves by Gammaproteobacteria, and it was not possible to identify a partially similar pattern in richness and Shannon index as was in the microeukaryotic community. In the shallowest cave (ZVP), where the sunlight enters into the surface layers of the cave, the highest richness was detected. Although this cave was not the richest in nutrients, this is the only cave where the influence of the light could have an impact on the community. In the VG cave, the abundance of Thaumarchaeota, Bacteroidetes, Gemmatimonadetes, and Nitrospirae decreased with the increasing salinity gradient, while the abundance of Gammaproteobacteria, Firmicutes, and Actinobacteria was greatest below the halocline. The ammonia-oxidizing archaea Nitrososphaeria, which relies solely on the energy generated from the oxidation of ammonia, was recorded in high abundance above (29%) and the area of halocline (12.8%), corresponding with the highest concentration of ammonium with organic nitrogen (4.3 mg L−1) . Bactero−1) , were al Nitrosopumilus and Nitrosoarchaeum, reached a relative abundance of 25.2% in the surface area despite the low concentration of ammonium with organic nitrogen (0.218 mg L−1). Bacteroidetes, including the strictly aerobic and chemoorganotrophic family Cryomorphaceae and Algoriphagus (Cyclobacteriaceae), were abundant in the area above the halocline. Although previous studies showed their prevalence in the productive ocean and coastal regions, no specific associations with organic matter of Cryomorphaceae are known [Aquilina and RS62 marine group dominated, while in the marine-like area, Rhodococcus and order Pseudomonadales. The highest OTU richness of the prokaryotic community in this cave was determined in the hypoxic halocline. This could be correlated to the higher bacterial activity in this layer [Rhodococcus, Sphingomonas, Arcobacter, Lawsonella, and Staphylococcus. The source of this pathogenic bacteria remains unclear.In the prokaryotic community of the BP cave, the most prominent archaeal lineage was Thaumarchaeota, with a similar contribution to the relative abundance and decreasing by the increasing salinity as in the VG cave. The ammonia-oxidizing archaea,re known . The trais layer . Below tAcinetobacter (11.5%) had the highest relative abundance among other Gammaproteobacteria together with ubiquitous gram-negative and aerobic or facultatively anaerobic Myroides (9.1%) from phyla Bacteroidetes. Acinetobacter species are widely distributed in nature and their growth may be enhanced by the contaminated environment such as hydrocarbon-contaminated areas, activated sludge, sewage [Myroides with proven high multi-drug resistance [The main prokaryotic groups detected in the surface area of the GKV cave were Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes. Ubiquitous gram-negative and non-fermenting coccobacilli , sewage , whereassistance . The lowsistance . Heterotsistance .Limnohabitans had the greatest relative abundance (18.8%) compared to other detected Betaproteobacteriales. This genus is characterized by a high growth rate and metabolic flexibility with a notably tight relationship to algae-derived organic substances [Sulfurimonas (Epsilonbacteraeota), Ectothiorhodospiraceae (Gammaproteobacteria), and Desulfatiglans (Deltaproteobacteria)). In depth with the highest salinity and lowest DO, archaeal phylum Epsilonbacteraeota, Omnitrophicaeota, and Gammaproteobacteria were detected [The highest concentration of ammonium with organic nitrogen and TOC was measured in the surface area of the ZVP cave, potentially contributing to the development of the prokaryotic community. The relative abundance of Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, and Actinobacteria decreased with the increasing salinity gradient, while Epsilonbacteraeota, Omnitrophicaeota, and Marinimicrobia (SAR406 clade) were most abundant in the marine-like layer. The genus bstances . Decreasdetected , 59.Our study identifies specific transitional boundaries for microeukaryotic and prokaryotic communities in the salinity gradient of anchialine caves. These transition boundaries are not restricted and it remains unclear if and to what extent the microeukaryotic and prokaryotic communities respond to the salinity gradient. Each anchialine cave had a unique microeukaryotic and prokaryotic community, indicating that cave niches play an important role in determining cave microbial diversity. This result also confirms the highly endemic character of anchialine environments and targeted studies should therefore be carried out to reveal the extent of the diversity and the ecological roles of the microeukaryotic and prokaryotic communities.Supplementary file1 (DOCX 407 KB)Below is the link to the electronic supplementary material.


Air pollution exposures have been suggested as risk factors for childhood respiratory diseases. We investigated proximity to major roads, an indicator of air pollution exposure, and its associations with childhood recurrent wheeze and asthma. We used data from a multicenter prospective cohort study of 921 infants hospitalized for bronchiolitis and recruited from 14 U.S. states. Primary exposure was residential proximity to the nearest major road at birth through age 3 years. Residential distance from nearest major road was divided into four categories: <100, 100–200, 201–300, and >300 m. Outcomes were parent-reported recurrent wheeze by age 3 years and asthma by age 5 years. Associations between residential proximity to major roads and respiratory outcomes were investigated using multivariable Cox proportional hazards modeling and logistic regression, adjusted for confounders. Out of 920 participants with home address data, pooled estimates identified 241 (26%) participants resided within 300 m of a major road, 296 (32%) developed recurrent wheeze by age 3, and 235 out of 858 participants (27%) developed asthma by 5 years. Participants who resided close to a major road had the highest risk of recurrent wheeze  and asthma , compared to those residing >300 m from a major road. Proximity to major roads is associated with increased risks of recurrent wheeze and asthma in young children. Respiratory problems in young children are highly prevalent and, in particular, recurrent wheeze and asthma are a significant worldwide public health issue ,2,3,4,5.The health effects of air pollution on the general population remains a worldwide public health concern and infants and young children are particularly vulnerable ,11. InfaResidential proximity to major roads (high traffic roads) is commonly used as a surrogate measure of exposure to TRAP ,18. WhilThis study is a secondary analysis of data from the Multicenter Airway Research Collaboration-35 study (MARC-35) cohort. The primary goal of MARC-35 study was to investigate infectious etiology of a child’s severe bronchiolitis, and subsequent development of recurrent wheeze by age 3 years and asthma by age 5 years. More details on the original cohort design, participants recruitment, general characteristics and primary results can be found elsewhere . BrieflyParticipant baseline data were predominantly collected through standardized parent and/or legal guardian interviews conducted at enrollment. Parent-reported variables included participant’s demographics, medical, family, and environmental history as well as details of acute illness . Medical2 land). The location of major and primary roads was obtained from the 2015 Topologically Integrated Geographic Encoding and Referencing (TIGER)/Line shapefiles published by the US Census Bureau. The 2015 road shapefile was chosen since it was the year that most participants stopped moving. Major roads, including both primary and secondary roads, were defined as roads with annual average total traffic count ≥10,000 vehicles per day. Primary roads were defined as interstate or state highways with average total traffic count ≥30,000 vehicles per day. Primary roads are distinguished by the presence of interchanges and speed limit of >50 miles per hour. These geocoded road shapefiles were used to obtain distance to the nearest primary road and major road from residential address of each participant in meters. Major road and primary density from each participant residential address within a 250-m radius of a circular buffer area were calculated using the road shapefiles to be in line with previous studies . In addiThe primary exposure measure of this study was the proximity to the nearest major road from participants’ residential addresses. Previous studies showed that TRAP concentration decreases with increasing distance from TRAP sources, and variables such as length of high traffic roads or traffic density within 100–300 m can explain spatial variation in TRAP ,29,30,31D” denotes duration spent in address “i” before age 3 years, and “E” is the pooled exposure measurement from that address, in this case, the proximity to major road, primary road, and also population density in the residential area. For cases of recurrent wheeze by age 3 years, we accounted for each participant’s address and duration of stay until the day they developed the recurrent wheeze outcome; thus, “iD” is either the day when the recurrent wheeze outcome had been achieved or, for non-cases, until the last date of the child’s observation period up to age 3 years. For the outcome of asthma by age 5 years, we accounted for all residential addresses until age 3 years. The number of residential moves for each participant varied from 1 to 6, including birth address. A weighted model according to length of stay in each address was used to pool exposures. The following formula was used to calculate the pooled exposure:The recurrent wheeze outcome was derived using parent-reported data from parent interviews in which they were asked to provide detailed information regarding all of the child’s breathing problems, from birth to age 3 years. Recurrent wheeze by age 3 years was defined by parental report of at least 2 corticosteroid-requiring breathing problems in 6 months or at least 4 breathing problem episodes in 1 year that last at least 1 day and affected sleep . Asthma Participants’ characteristics were described overall and by primary and secondary exposure to TRAP using counts and proportions, with bivariate associations tested using chi-square and Fisher’s exact test, as appropriate. Bivariate analysis of TRAP exposures and recurrent wheeze by age 3 years used log rank test of equality for continuous exposures and chi-square and Fisher’s exact test, as appropriate, for categorical exposures. Bivariate analysis of TRAP exposures and asthma by age 5 years used Wilcoxon rank sum for continuous exposures and used chi-square and Fisher’s exact test, as appropriate, for categorical exposures.We used multivariable Cox proportional hazard modeling to assess the association between proximity to major road and primary road with recurrent wheeze by age 3 years. Time-to-event was calculated in days from birth to the date when the recurrent wheeze outcome definition was met. Participants were censored at the date of their 3-year follow-up interview or at their last follow-up interview. The models were stratified by sex and the results were reported as hazard ratios (HRs) with 95% CI. We used multivariable logistic regression to assess the association between the primary exposures and asthma by age 5 years. The models accounted for potential clustering by sites using a clustered sandwich estimator for standard errors, and the results were reported as odds ratios (ORs) with 95% CI. All multivariable models were adjusted for sex, race/ethnicity, enrollment age (dichotomized by age 2 months), prematurity , medical insurance type, median household income estimated using ZIP code at enrollment, parental history of asthma, population at the birth residential area, and infant tobacco exposure at enrollment. Covariates were selected based on relevance to the TRAP exposures and respiratory outcomes of our study, with the goal of creating parsimonious models. Additionally, we conducted sensitivity analyses using multivariable logistic regression to model road exposures with two combination outcomes: recurrent wheeze by age 3 years without asthma by age 5 years and recurrent wheeze by age 3 years with asthma by age 5 years. For both outcomes, non-cases were defined as having neither recurrent wheeze nor asthma.p-values were two-tailed, with p < 0.05 considered statistically significant. Using cartographic tools of ArcGIS Pro 2.7 , we created maps depicting participants’ locations at enrollment by population density (2010 US Census) and ZIP code tabulation area, and by distribution of major roads and primary roads.Additionally, we plotted the distances between birth residence to major roads and pooled distances to major roads by incidence rates of recurrent wheeze by age 3 years. Incidence rates of recurrent wheeze were obtained from the summary statistics of the survival model for each of birth residence distance to major road and pooled distance to major road. Then we multiplied incidence rates by 100,000 and excluded distances >4000 m from each plot. All analyses were performed using Stata 14.1 . All the n = 920), the majority were enrolled at age ≥ 2 months (71%), male (60%), had non-private health insurance (60%), and no parental history of asthma (67%)  participated in the longitudinal data collection period. One participant was excluded from the analytical cohort because they only provided a PO Box number as the contact address during the entire study period. Among the remaining participants (ma (67%) . At the ma (67%) .The majority of participants had a pooled residential location away from major roads: 58 (6%) were located within <100 m, 82 (9%) were 100–200 m, 101 (11%) were 201–300 m, and 679 (74%) were >300 m from a major road. Participant characteristics were similar across pooled proximity to major road categories, but non-Hispanic black participants were more likely to live within ≤200 m of a major road compared to other participants. Pooled proximity to primary roads had a similar distribution compared to major roads, with 34 (4%) located within <400 m, 93 (10%) were 400–1000 m, and 793 (86%) were >1000 m from a primary road . A signi2). In addition, compared to non-cases, those who developed recurrent wheezing by age 3 years had borderline significantly higher proportions for birth residential distances within <100 m of a major road (8% vs. 13%) and pooled residential addresses with distance <100 m to major road (6% vs. 11%).Among participants in the analytical cohort, 296 (32%) had developed recurrent wheeze by age 3 years . By the Among participants in the analytical cohort, 858 (92%) had available data to identify the asthma by age 5 years outcome . Among pExamining associations between road exposures and recurrent wheeze by age 3 years using unadjusted survival models, distance to major roads from birth residence of <100 m  and pooled residential addresses with distance to major road <100 m  had a significantly higher risk of recurrent wheeze by age 3 years compared to residences >300 m from major roads . These aExamining associations between road exposures and asthma by age 5 years using unadjusted logistic regression models, distance to major roads from birth residences within 201–300 m  and pooled residential addresses with distance to major roads <100 m  and within 201–300 m  had a significantly higher odds of developing asthma by age 5 years compared to residences >300 m from major roads . In adjuAdditionally, the sensitivity analysis using the combined outcome of recurrent wheeze by age 3 years with asthma by age 5 years had attenuated findings for distance to major roads <100 m from birth residence , and pooled distance to major road <100 m  and within 201–300 m  .In this multicenter prospective cohort study, participants living within close birth and pooled proximity (<100 m) to major roads had higher risk of developing recurrent wheeze by age 3 years compared to those living >300 m away from their birth address. Risk of recurrent wheeze diminished as distance to major roads increased, suggesting a linear relationship with recurrent wheeze. Similarly, proximity to major roads of <100 m and 201–300 m to major roads from pooled address had higher odds of asthma by age 5 years. In addition, there was significant association between birth distance to major roads <100 m and asthma by age 5 years in an unadjusted model; however, the significance diminished after adjustment for covariates. There was no association between birth or pooled proximity to primary roads and risk of recurrent wheeze or asthma outcomes. As such, our results show that proximity to major roads, more than primary roads, have a significant association with the development of childhood airway disease. The current study highlights a knowledge gap in assessing TRAP exposure in high-risk populations, shows varying associations between TRAP and wheeze and asthma, and suggests that there are potential synergistic effects on health from TRAP proximity.A distinct feature of our study is that we assessed exposures within a pediatric population in which all participants had a bronchiolitis episode in infancy that resulted in hospitalization. From previous literature, it is well established that infants with bronchiolitis represent a high-risk group for development of wheeze and asthma, and bronchiolitis is a common viral respiratory illness in infants ,24,34. OCHEER study researchers also observed an exposure-response relationship among all children, with and without a history of bronchiolitis, aged 6–14 years living within 700 m of a main road . ResearcIn general-pediatric population studies that assessed risk of wheeze  or asthma and residential proximity exposure to TRAP, results have been inconsistent and often found weak or no association ,37,38,39In other cohorts that assessed long-term exposure, The German Infant Nutritional Intervention (GINI) study and the Influences of Life Style and Related Factors on the Immune System and the Development of Allergies in East and West Germany (LISA) cohorts found a significantly higher risk of wheeze and asthmatic/spastic/obstructive bronchitis from proximity to a main road (<50 m) exposed at birth, 2–3 years, and 6 years of age ,21. A maOur findings may also represent a possible association between synergistic TRAP effects and risk of respiratory disease. Our adjusted estimates for recurrent wheeze at pooled distance to major roads are higher compared to other studies that focused only on exposure to a primary pollutant and airway disease: where risk elevation is generally slightly significant in ranges of 3–20% ,34. AlthThe present study has potential limitations. Our study population is higher risk for both recurrent wheeze and asthma, which limits generality of the results to all children. Conversely, our results represent the challenges of multiple environmental risk factors and the advancement of airway disease in a susceptible population. Additionally, we measured TRAP exposure by using proximity to TRAP sources  as a surrogate. As such, our exposures did not account for specific air pollutants by measuring or modeling TRAP concentrations. However, evidence from multiple studies have shown that living within proximity to roads creates an exposure zone for a combination of TRAP and that pollutants from various vehicles are at elevated concentrations near major roads ,42. ThusOur study was strengthened by the population scope that including multiple major cities throughout the U.S, availability of detailed address histories due to biannual follow up interviews and confirming addresses through direct contact with parents/guardians, low attrition rate, and robust outcome data. Our exposure measurements highlight the importance of the first three years of life when assessing environmental exposures and their influence on respiratory function later in childhood. Results on any synergistic effects should be interpreted with caution and future studies are needed to assess a long-term cumulative TRAP effects on risk of recurrent wheeze and asthma through a combination of TRAP source proximity, concentration, and land use modeling. Furthermore, additional long-term and multifactorial exposure analyses on sensitive populations would be beneficial to add to the weight of evidence suggesting TRAP exposures are associated with respiratory illness.Our main results show that proximity to major roads increases risk of both recurrent wheeze and asthma in a high-risk pediatric cohort. Our findings are comparable to studies focusing on early-life TRAP exposure, adding to evidence that exposures surrounding birth affect respiratory health of a growing infant ,16,17,18


Klebsiella pneumoniae carbapenemase-producing multidrug-resistant Gram-negative pathogens. Physicochemical parameters such as pH, hydrogen peroxide, free acidity, lactonic acid, total phenols total flavonoids, free radical scavenging activities, tyrosinase enzyme inhibitory activity and kojic acid were examined. Moreover, the antimicrobial activity of 10 different honey types was evaluated in five consecutive dilutions  against the clinical isolates by the well diffusion method, as well as by the determination of the minimum inhibition concentration after the addition of catalase and protease. Almost all the physicochemical parameters varied significantly among the different honeys. Fir and manuka honey showed the highest values in pH and H2O2, while the free acidity and lactonic acid levels were higher in chestnut honey. Total phenols, total flavonoids and free radical scavenging activities were found higher in cotton, arbutus and manuka honey, and finally, manuka and oregano honeys showed higher tyrosinase inhibition activity and kojic acid levels. The antimicrobial susceptibility depended on the type of honey, on its dilution, on the treatment methodology and on the microorganism. Arbutus honey was the most potent against VIM-producing Enterobacter cloacae subsp. dissolvens in 75% concentration, while fir honey was more lethal for the same microorganism in the 25% concentration. Many honeys outperformed manuka honey in their antibacterial potency. It is of interest that, for any given concentration in the well diffusion method and for any given type of honey, significant differences were not detected among the four multidrug-resistant pathogens, which explains that the damaging effect to the bacterial cells was the same regardless of the bacterial species or strain. Although the antimicrobial potency of different honey varieties dependents on their geographical origin and on their compositional differences, the exact underlying mechanism remains yet unclear.The development of antibiotic resistance is a major public health issue, as infections are increasingly unresponsive to antibiotics. Emerging antimicrobial resistance has raised researchers’ interest in the development of alternative strategies using natural compounds with antibacterial activity, like honey, which has emerged as an agent to treat several infections and wound injuries. Nevertheless, the antibacterial effect of honey was mostly evaluated against Gram-positive bacteria. Hence, the objective of our study was to evaluate the antibacterial activity, as well as the physicochemical parameters, of genuine Greek honeys against multidrug-resistant Gram-negative pathogenic bacteria. In this vein, we aimed to study the in vitro antibacterial potential of rare Greek honeys against Verona integron-encoded metallo-β-lactamase (VIM)- or  Antibiotics are used as prophylactic or therapeutic agents to treat human infections. Definitively, their proper clinical use increased life expectancy and reduced mortality. However, the misuse of antibiotics has led to the emergence of multidrug-resistant bacterial strains . Under sKlebsiella pneumoniae carbapenemases (KPCs), and metallo-β-lactamases (MBLs), such as Verona integron-encoded metallo-β-lactamases (VIMs). KPC and VIM carbapenemases pose a serious threat in human health and particularly in health institution environments , diethyl ether , ethyl acetate , chloroform  and distilled water.The following solvents were used in our study to the order of increasing polarity; n-Hexane [CH(CH2)The Separation Funnel Method was to determine the antibacterial activity of the different types of extraction of raw honey samples based on polarity. Initially, 100 g of crude honey was diluted in 150 of sterile distilled water, placed in a Stomacher sterile bag and mixed. Transfer to a 500-mL separatory funnel, shake and allow to precipitate. 50 mL of the less polar solvent n-hexane was added and shaken for 15 min and reprecipitated to allow the solvent layers to separate. Through the bottom of the funnel, the aqueous layer was removed, and the top layer was collected to obtain a n-hexane fraction. Three successive extracts were performed by applying an equal volume of n-hexane followed by shaking and separation. In the same manner, diethyl ether, ethyl acetate and chloroform were extracted to obtain diethyl ether, ethyl acetate and chloroform fractions. All 3 successive layers were thoroughly mixed and the water contaminating extracts were ward off by filtration over anhydrous sodium sulfate. The last step involves collecting the organic solvent extract and concentrating it by evaporation under reduced pressure using a rotary evaporator  at 40 °C, 30 °C, 60 °C and 50 °C for n-hexane, diethyl ether, ethyl acetate and chloroform, respectively ,117.2O solution. Each experiment was performed in triplicate.The samples were diluted in sterile saline at different concentrations. 75%, 50%, 25%, 12.5% and 6% following the agar well diffusion test method ,123,124.8 CFU/mL). Approximately 5 × 104 CFUs in 10 µL Mueller–Hinton broth were added to 190 µL of 2-fold diluted test honey (honey concentration ranged from 75 to 0.58% w/v) in Mueller–Hinton broth. Two-fold serial dilutions of the same range of manuka honey and artificial honey were included for comparison. The control wells contained only Mueller–Hinton broth-inoculated with bacteria. The optical density (OD) was determined at 630 nm using a microplate reader , just prior to incubation (t = 0) and after 24 h of incubation (t = 24) at 37 °C. MIC was determined as the lowest honey concentration that results in 100% growth inhibition.The minimum inhibitory concentration (MIC) of the honey types was determined in sterile 96-well polystyrene microtiter plates  by using a spectrophotometric bioassay, as previously described ,124. Bri(a)Honey samples were treated with catalase which degrades hydrogen peroxide, allowing evaluation of the contribution of hydrogen peroxide production to antibacterial activity .(b)The addition of proteinase K permits assessment of antimicrobial activity due to proteins and peptides .This determination was performed in two ways: v/v (750 µL honey and 750 µL Muller Hinton Broth), 28 µL of the stock solution were added to obtain a final concentration of 600 U/mL. Diluted honey was shacked in an incubator for 16 h at 37 °C in 210 rounds. The proteinase K stock solution was set by dilution of proteinase powder (10mg)  in 1-mL distilled water to obtain a final concentration of 10 mg/mL. Then, all treated honeys solutions by catalase and proteinase K were determined the MIC values, as reported previously in the section “Determination of Minimum Inhibitory Concentration (MIC)”. Controls were prepared; no honey control as a positive control and no honey control with catalase or proteinase K  in order to evaluate their effect upon the bacterial growth. MIC values of the treated honeys when compared to the untreated honey reveal the presence of hydrogen peroxide and/or proteinaceous aggregates, which redound to the antibacterial activity of honeys. All experiments were done in triplicate.The catalase solution was set by dilution of catalase powder (30 mg) from bovine liver  to 10 mL phosphate buffer  were performed equally, as well as the solvent control, which showed no activity. The diameter of the inhibition zones was measured (mm).The antibacterial efficacy of honey extracts from n-hexane, diethyl ether, chloroform and ethyl acetate were evaluated in vitro using the agar well diffusion assay. Honey samples proved to be most active at 75% v/v) were placed. Multi-resistant bacterial strains were tenfold diluted with sterile saline solution and 20 µL of a bacterial suspension (1 × 108 colony-forming units (CFUs)/mL, 0.5 McFarland standards) was placed in each well and then sealed and incubated at 37 °C for 72 h. In the same manner, they were prepared for comparisons two-folds Manuka and artificial honey. Mueller-Hinton was poured into control wells and inoculated with bacteria.All 4 crude honey extracts were determined their minimum inhibitory concentration (MIC) by microtiter method using a spectrophotometer  . The wel®), the optical density (OD) was measured at 630 nm before and after 24 h incubation at 37 °C. Finally, the MIC was recognized as the lowest concentration of honey resulting in 100% growth inhibition.By the aid of a microplate reader Crude honey: Honey sample (10 g) was extracted in a condenser with 50 mL of distilled water at 60 °C for more than 6 h. The obtained extract was filtered to remove particles and volume was adjusted with ultra-pure water.(ii)Dried honey: After extraction with diethyl ether, dried honey was applied in the MIC assay as following; weights extracts were diluted (400 mg/mL honey/ultrapure water solution) for 24 h, vortexed at 1500 rpm for 3 min and filtered through a 0.45 Whatman TM syringe filter .Then, serial dilutions were prepared at the following concentrations .Analysis of variance with one way ANOVA and Tukey’s HSD post hoc comparison was used to compare more than two groups of samples, while in the case of two groups of samples, the Mann–Whitney (Wilcoxon) nonparametric test was applied. Spearman Rho correlation coefficient was estimated when the degree of correlation was required. All analyses were performed with Microsoft Excel at an alpha of 95%.Although honey is known to have multiple successful uses in medicine, food and cosmetics, there is still missing information on the mechanisms of antimicrobial action, as it seems to be a multifactorial process. While some authors have reported the effects of honey directly upon the bacterial membrane by impacting the membrane integrity and metabolic activity, others have reported the potential role of the enzymatic production of hydrogen peroxide, its acidic pH, the phenolic content, its minerals, the levels of MGO, some biodrastic proteins and, eventually, certain beneficial microorganisms secreting antimicrobial substances in combat bacteria. Our results showed that there are significant variations in the physical and chemical characteristics of the various types of honey, depending on their floral origin. Furthermore, the different honeys showed important differentiations concerning their antibacterial effects, as demonstrated by the well diffusion assay, and many of them outperformed the antimicrobial potency of manuka honey. The addition of catalase caused significant differences among the types of honeys in their antibacterial activity, assessed by the MIC method, implying that hydrogen peroxide is involved in the pathways that lead to the formation of compounds with antibacterial properties. The extraction process of the honeys by four organic solvents demonstrated that each solvent extracted different mixtures of compounds, and hence, the antibacterial activities of the extracts were different. This finding implies that there is more than one responsible substance and that the final antibacterial effect is probably dose-responsive.


Gmelina arborea Roxb is a fast-growing tree species of commercial importance for tropical countries due to multiple industrial uses of its wood. Wood is primarily composed of thick secondary cell walls of xylem cells which imparts the strength to the wood. Identification of the genes involved in the secondary cell wall biosynthesis as well as their cognate regulators is crucial to understand how the production of wood occurs and serves as a starting point for developing breeding strategies to produce varieties with improved wood quality, better paper pulping or new potential uses such as biofuel production.In order to gain knowledge on the molecular mechanisms and gene regulation related with wood development in white teak, a de novo sequencing and transcriptome assembly approach was used employing secondary cell wall synthesizing cells from young white teak trees., RNA-seq reads were assembled into 110,992 transcripts and 49,364 genes were functionally annotated using plant databases; 5071 GO terms and 25,460 SSR markers were identified within xylem transcripts and 10,256 unigenes were assigned to KEGG database in 130 pathways. Among transcription factor families, C2H2, C3H, bLHLH and MYB were the most represented in xylem. Differential gene expression analysis using leaves as a reference was carried out and a total of 20,954 differentially expressed genes were identified including monolignol biosynthetic pathway genes. The differential expression of selected genes  was validated using qPCR.For generation of transcriptomeWe report the very first de novo transcriptome of xylem-related genes in this tropical timber species of commercial importance and constitutes a valuable extension of the publicly available transcriptomic resource aimed at fostering both basic and breeding studies.The online version contains supplementary material available at 10.1186/s12864-021-07777-x. Gmelina arborea Roxb.  is a fast-growing tree species belonging to the lamiaceae family, with tremendous economic importance in several tropical and subtropical areas of southeastern Asia, Africa and America. Its introduction and excellent adaptation to the American tropics  is due to the traits like fast growth, high biomass production (20–25 m3/ha/year), less susceptibility to the local pests and high yields in addition to the versatility of its wood use which allow a faster investment return [Tree wood is considered as a sustainable alternative source for biofuel production  in additt return . Therefot return , 7.3 of biomass/ha/year [White teak has also shown natural tolerance to water stress and resistance to fire, both characteristics of high interest in the context of climate change. This species has been considered as a tree with higher bioenergetics production, generating an average of 265 m/ha/year . White t/ha/year –10. Wood/ha/year .Formation of wood xylem cells involves two basic processes occurring simultaneously i.e. formation of the secondary cell-wall and programmed cell death . The secp-coumaryl alcohol, sinapyl alcohol and coniferyl alcohol [Lignin biosynthetic pathway involves eleven enzymes in order to produce three monolignols;  alcohol . Polymer alcohol . The gym alcohol , 21.Various transcription factors have been identified and characterized as key players of wood development, primarily members of NAC and MYB families involved in the regulation of monolignol pathway and lignin polymerization , 22, 23.The monolignol pathway has been mainly studied in model plant species such as Arabidopsis and poplar , 27. TheG. arborea xylem library resulted in approximately 165 million paired reads. Quality filtration for the low-quality reads (Q < 20) and contaminants such as reads of ribosomal and organellar origin resulted in the removal of total of 18,968 paired sequences. The cleaned reads were assembled using Trinity software to obtain the reference transcriptome with 110,992 transcripts. The assembled transcripts showed a considerably higher N50 value of 1466 bases with the average transcript length of 864 bases  database, 10,256 genes were assigned to 130 metabolic pathways , Homeobox (54 genes), NAC (45 genes), WRKY (43 genes) and bZIP (42 genes) TF families. Nine biosynthetic genes of the monolignol pathway and transcription factors of different levels of regulation were identified from the reference transcriptome.The main families of transcription factors identified in the reference transcriptome are presented in Fig. VND7, SND2 and NST1, reported as “master” regulators, were identified. In the case of MYB transcription factors, MYB46 and MYB83, which were classified as regulators of second level, and MYB20, MYB69 and MYB85 which are directly related with the activation of monolignol biosynthetic genes, were identified. Other important transcription factor encoding genes were found like MYB7, MYB4, MYB32 and KNAT7, all reported as negative regulators, or BES1 a specific activator of the synthesis of celluloses  and trinucleotides  , 147,130,884 sequences were obtained. For generation of combined transcriptome, the sequences obtained from leaves were fused with the sequences obtained from xylem. The mapping of reads against this transcriptome indicated an average alignment percentage of 95%, which is indicative of a good representability of expressed transcripts in the transcriptome. Metrics related with the assembly and annotation of this transcriptome are shown in supplementary Table With the goal to perform the differential expression analysis between xylem and leaves, we first generated a unique combined transcriptome using the reads from both tissues, since no reference genome sequence is available for p value < 0.05), out of which 20,964 showed log 2 fold change (Log2FC) absolute values higher than 2 as a threshold: 9011 transcripts showed an induction pattern whereas 11,953 were repressed in xylem compared to leaf tissue  was performed using leaf tissue as a control. Principal component analysis (PCA) of transcript expression levels revealed a clear differentiation of the samples according to the tissue type . Figure To identify overall changes in xylem metabolic pathways encoded by these DEGs, the Mapman tool was used, using the same LogPAL, C4H, COMT and CCoAOMT genes in xylem, while the HCT gene was repressed compared to leaves. In the case of 4CL, F5H, CCR and CAD different transcripts (associated in various cases with possible splicing isoforms) of the same gene presented a higher expression in one or other tissue.As expected, genes involved in photosynthetic light reactions were clearly repressed in xylem compared to leaves, whereas those related to respiration were induced. Accordingly, genes related to cell wall synthesis tend to show an induction pattern in stem compared to leaves. Analysis of nine genes of the monolignol pathway showed a clear differential expression between leaves and xylem Fig. . A generVND7 homolog showed a repression pattern in xylem. Finally, KNAT7, a member of the homeobox family, was also induced in xylem tissue. Other genes involved in the development of secondary cell wall also showed differences between leaves and xylem  and the last (CAD) ones to be involved in the monolignol pathway and are key players for the lignin biosynthesis. In the case of PAL, one variant induced in stem (putative PAL1) was selected, while for CAD, two variants were included: one upregulated in stem  and another one upregulated in leaf   Fig. A and B.Scutellaria baicalensi, and also with PAL1 of Coffea arabica (Rubiaceae). For CAD protein, the two evaluated white teak members appeared in different but closely located clusters where CADS was most related with CAD of Salvia miltiorrhiza and Sinopodophyllum hexandrum, while CADL was most related with CAD of Sesamum indicum and CAD4 of Tectona grandis (teak), two species belonging to lamiales order . CAD1 and CAD4 from T. grandis (Lamiaceae), a species closely related to G. arborea, were located in distant clusters, indicating a high degree of divergence amongst homologous members of this protein family.In the case of PAL, white teak protein formed a single cluster with another possible ortholog of a Lamiaceae family member, 2FC values obtained previously. Comparing the values between the fold change observed in RT-qPCR and the fold change of gene expression obtained by RNA-seq, a concordance was found between the values for the COMT, CCR and NST1 genes. A similar trend in the expression pattern was found for CCoAOMT, 4CL, HCT and CAD genes (induced in leaf) . Correlation analysis between the values of Log2FC of genes with concordant patterns indicated a moderate general correlation coefficient of 0.50.In order to validate the patterns of differential expression observed, a total of 12 genes (10 upregulated and 2 downregulated) were selected for qPCR validation: seven from metabolic genes of the monolignol pathway, two from regulatory genes (transcription factors) and three genes related with synthesis of celluloses and hemicelluloses. For each case, the genes were selected based on the LogEucalyptus sp [Populus sp [Pinus radiata [G. arborea Roxb.In Colombia, white teak plantations are located mainly in the dry tropical Caribbean zone area, characterized by the presence of a bimodal rainfall pattern, in which the plants are frequently subjected to drought periods that can affect the establishment of new plantations and yields . During yptus sp , Populuspulus sp  and Pinu radiata  with rep radiata  and teak radiata –39. HencThe xylem transcriptome contains 110,992 transcripts, up to 60% of these could be annotated using different annotation methods , and also generated a high percentage of transcripts with full length ORFs (16%) and quasi full length ORFs . GO annotation revealed binding and catalysis as main enriched molecular functions. In the binding category, genes related to transcription factors predominated, indicating that this function is critical for the development of white teak’s xylem, while in the catalysis GO category, the importance of different metabolic processes ocurring in this tissue is reflected. One of the most represented category in KEGG pathway was the phenylpropanoid pathway which gives rise to secondary metabolites that are important for different biological processes like pigmentation, UV protection, or responses to pathogens . AdditioIdentification of genetic polymorphisms from transcriptomic data, like SSRs markers, is also relevant for a non-model species as it can be used in future studies for associating genotype/phenotype oriented towards germplasm bank characterization and breeding processes. The analysis of SSRs markers in the white teak xylem-transcriptome indicated a predominance of the dinucleotides AT and AG, which is in accordance with studies in different dicot and gymnosperms species . The xylKAN family members that have been related to the abaxial identity [MYB-like related to foliar senescence [ELF3 related to development and flowering [C2H2 and C3H, which are involved in the hormonal signal transduction process and different processes of development and response to stress in plants were the most abundant [MYB and NAC families, which are involved in different biological processes like response to biotic and abiotic stress, cell cycle control, amongst others [NAC family of transcription factors such as SND2, VND7 and NST1 act as activators in the third and second level of the regulatory network [In order to identify genes more specifically related with the wood development in white teak, the transcriptional profiles of growing trunks (secondary xylem) and leaves from young trees were contrasted. Differential expression analysis evidenced that, in the case of leaves, various transcription factors, predominantly upregulated, were related to leaf development and photomorphogenesis processes such as identity , MYB-liknescence  and ELF3lowering . In the abundant , 48. Furt others , 50 were network . The MYB network , 53. IntNST1 and SND2 genes, whereas the transcript annotated as VND7 was downregulated. NST1 is involved mainly in the regulation of development of xylem fibers as has been reported for different species like Arabidopsis and Poplar [MYB20, MYB63, MYB69 and MYB85. Interestingly, the repressor genes KNAT7 and MYB4 were also found to be significantly induced in stem, which suggests the presence of negative feedback control loops induced along the development processes of G. arborea secondary cell wall.The secondary cell wall master regulator NAC transcription factors showed a general significant pattern of induction in stems was observed for d Poplar , 54. In d Poplar , its lowd Poplar , as wellPAL, C4H, COMT and CCoAOMT genes  [PAL2 is more related to processes of development of leaves and flowers as well as pollen viability [COMT and C4H were identified and both were significantly upregulated in stem, whereas F5H and 4CL were expressed in both tissues, which does not exclude the possible presence of other variants or multi-functionality of a same variant in other tissues or developmental process.Specifically, the phenylpropanoids pathway showed a clear pattern of upregulation in xylem compared to leaves, as exemplified by nes Fig. . Howevernd PAL4) , all of iability . In our , some of them with different patterns of expression among different types of tissues [CAD gene were identified, among which CAD3 showed a predominant expression in stem and CAD9 was equally expressed in both tissues, which could indicate a multifunctional role for this gene. CAD9 has been related mainly to the lignification processes [CAD members was not determined, however both of them were predominantly expressed in leaves of white teak. Besides, some Arabidopsis variants of CAD (i.e. CAD2 and CAD3) are poorly or not expressed during lignification processes, thereby indicating probable different roles in other biological processes [S. milthiorrhiza CAD, whereas CADL grouped together with CAD4 of T. grandis and CAD of S. indicum, indicating its possible relation with other members of the lamiales order. However, a more in-depth analysis is necessary to determine the specific identity, ortholog relationship, and biological function of all these members found in white teak.In the case of CAD enzyme, which catalyzes the last step of the biosynthesis of monolignols for the formation of the alcoholic forms, 9 different members have been reported in Arabidopsis and 12 in rice tissues , 56. In rocesses , with a rocesses , althougrocesses –60. The rocesses . PhylogeHCT gene was significantly upregulated in white teak leaves. According to Besseau et al. [HCT may have a key role in the synthesis of flavonoids which may be the case in the leaves of white teak. Xylem expression of HCT, although lower, could be enough to maintain the lignification process.Differential expression analysis showed that a unique u et al. , under cIRX (Irregular xylem) genes, whose mutations affect the phenotypic development at the level of xylem cells [PGSIP (plant glycogenin-like starch initiation proteins) genes, also known as Gux, that constitute a group of genes involved in xylan synthesis and whose function has been specifically related with secondary wall formation [IRXs genes are involved in synthesis of celluloses and hemicelluloses: IRX1, IRX3 and IRX5, for example, are cellulose synthases (CesA) specifically expressed in secondary cell wall [XCP1, XCP2 and VPE, known to be involved in programmed cell death during xylem development, have been identified amongst xylem upregulated genes [Development of the xylem cells requires coordinated synthesis of the different elements constituting the secondary cell wall and programmed cell death. Some of the genes involved in these processes showed highly specific expression patterns. This is the case of em cells  as well ormation . The IRXell wall . Interesed genes . Furthered genes .LAC4, LAC11, LAC17, LAC10 and LAC2, known to be involved in monolignol polymerization [FLA11 gene in xylem is in accordance with previous reports of its induction during the biosynthesis of the secondary cell wall in Arabidopsis and Eucalyptus, where a key role for these fasciclin-like arabinogalactan proteins in cell wall development biomechanics and development has been proposed [On the other hand, specific activation of laccase genes such as rization –68, may proposed .CesA), and cellulose synthase-like proteins (CSL), for which a significant downregulation in stem was observed. This could be related to fluctuations in the expression of these genes according to the type of cell wall, and the developmental stage. In Arabidopsis for example, expression of CesA1, CesA2, CesA3, CesA5, CesA6 and CesA9 genes was shown to be related to formation of primary cell wall, rather than secondary cell wall [Eucalyptus camaldulensis, differences in the patterns of expression of some CesA were found in different types of tissue,cell wall or development stages [CSL genes, in white teak most of them presented a predominant expression in leaves. About this, Lerouxel et al. [Other key genes showed a different pattern of expression like those coding for cellulose synthases  trees was carried out, which constitutes an important genomic resource for this tropical timber. Differential expression analysis allowed to identify, for the first time in this species, major genes related with lignin biosynthesis and other components of the secondary cell wall, as well as the main transcription factors implicated in its regulation. Also, a catalog of intragenic microsatellite markers was obtained that may be useful in the future establishment of strategies for marker assisted selection of traits related with lignin formation, wood and/or secondary cell wall development in this economically important tree species. The transcriptome obtained could contribute significantly to increase the knowledge on wood and lignin formation that is still scarce in white teak, and will be highly useful for other non-model tropical wood tree species.Transcriptomic profiling of leaves and wood of young white teak . Leaves and stem cuttings from six different individual plants were collected and stored in liquid nitrogen. For RNA isolation from stem (with secondary xylem), external tissues that constitute the bark (phloem and periderm), and pith were removed from stems. Wood was chopped into small pieces using a sterile scalpel and grounded in liquid nitrogen. Total RNA was obtained using the protocol developed for RNA extraction from the pine wood by Chang et al. . The leaRNA samples with best integrity and concentration values were further validated using a bioanalyzer  and samples with a RIN value > 7 were selected for sequencing. Nine RNA samples of each, xylem and leaves, were used to make 3 pools of 3 different individuals for each tissue type. From each pool of RNA, sequencing libraries were generated using the TruSeq library prep kit , obtaining six indexed libraries with three replicates for each tissue. All the libraries were sequenced using the NextSeq500 platform  to generate paired-end reads of 2 × 150 bases length.Raw RNA-seq reads were evaluated for quality, and sequences with a Q score < 20 were eliminated. Adapters were eliminated by trimming the 10 bp from the 5′ ends of the reads using Trimmomatic (version 0.36) . AdditioT. grandis). Thus, transcriptome assembly was performed using Trinity (version 2.1.1) [A de novo transcriptome assembly strategy was chosen discarding the alternative of reference genome-guided assembly, because the most closely related genome sequence available belongs to a relatively distant member of the Lamiaceae family, and a different genus (n 2.1.1) , settingTranscriptome annotation was performed using the BLASTX similarity search program  against The xylem reference transcriptome was further analyzed for the presence of microsatellite markers using the MISA tool , consideFor differential expression analysis, the transcriptome obtained from the assembly of pooled reads from xylem (stem) and leaf tissues was used as the reference transcriptome. Reads from each replicate and tissue were aligned against this reference transcriptome using bowtie2 and samtools  and the p-value (p < 0.05) and values of logarithmic to base 2 of expression fold change (log2 FC) ≥ 2 or ≤ − 2, indicating up- or down-regulation of the xylem genes in comparison with leaves. The functional annotation of differentially expressed transcripts was performed using the Mercator [Selection of differentially expressed genes between xylem and leaf tissue was done using a binomial test with an adjusted Mercator  and TRAPMercator  tools. VMercator .UBIQUITIN5 (UBQ5), β-TUBULIN (β-TUB) and HISTONE3 (HIS3) genes were evaluated as reference genes based on the transcriptome data and finally UBIQUITIN5 (UBQ5) was used for the normalization of RT-qPCR data. Primer3 tool was used for the primer designing taking into account the criteria for qPCR primers [To validate the differential expression of a selection of genes upregulated in xylem, RT-qPCR was performed. cDNA of xylem (stem) and leaves were prepared using the Transcriptor first strand cDNA synthesis kit : 1 μg of total RNA per 40 μl final reaction volume was used following manufacturer operating procedure. Primers were designed for the selected 13 candidate genes related to the monolignol biosynthetic pathway, cellulose and hemicellulose synthesis, and transcription factors involved in the regulation of secondary cell wall biosynthesis.  primers   using the Fast start™ SYBR green  and primers at a final concentration of 0.5 pmol/μl. Three negative template controls per primer pair were included in each plate. Running conditions were: a pre incubation phase at 95 °C for 10 min, 45 cycles of amplification with 3 steps: 95 °C for 10 s, 58 °C for 10 s and 72 °C for 10 s, a melting phase with 3 steps: 95 °C for 10 s, 65 °C for 60 s and 97 °C for 1 s, finally a cooling phase at 37 °C for 30 s. Melting curves were analyzed to verify the presence of only one product and the absence of primer dimers. The ΔΔCt comparative method [Additional file 1: Supplementary Fig. 1. Principal component analysis (PCA) of G. arborea expressed transcripts. Transcript read counts obtained in each sample were used. Difference between plant tissues (condition) is highlighted.Additional file 2: Supplementary Fig. 2. Main functional categories represented by DEG.Additional file 3: Supplementary Table 1. Summary of G. arborea de novo transcriptome assembly metrics combining RNA-seq data from leaves and xylem.Additional file 4: Supplementary Table 2. Frequency in the number of repetitions found for SSRs microsatellite markers.Additional file 5: Supplementary Table 3. Primers used for RT-qPCR validation of differentially expressed genes between xylem and leaf tissues.


CRYBB3) has the smallest number of reported variants. Clinical ophthalmological and genetic-dysmorphological evaluation were performed in three autosomal dominant family members with pediatric cataract and microphthalmia, as well as one unaffected family member. Peripheral blood was collected from all participating family members and next-generation sequencing was performed. Bioinformatics analysis revealed a novel missense variant c.467G>A/p.Gly156Glu in CRYBB3 in all family members with childhood cataract. This variant is classified as likely pathogenic by ACMG, and no previous descriptions of it were found in ClinVar, HGMD or Cat-Map. The only other mutation previously described in the fifth exon of CRYBB3 is a missense variant that causes a change in amino acid from the same 156th amino acid to arginine and has been associated with pediatric cataract and microphthalmia. To the best of our knowledge, this is the first time the c.467G>A/p.Gly156Glu variant is reported and the second time a mutation in CRYBB3 has been associated with microphthalmia.Up to 25% of pediatric cataract cases are inherited, with half of the known mutant genes belonging to the crystallin family. Within these, crystallin beta B3 ( Cataract is defined as lens opacity that results in light scattering, reduced image clarity and visual acuity, as well as impaired contrast sensitivity ,2. In ch(CRYBB3) [CRYBB3, as well as phenotypes, family origins and inheritance patterns.Pediatric cataract can be isolated or associated with anterior chamber developmental anomalies, such as microcornea, microphthalmia and aniridia. They can also be related to multisystem genetic conditions, including chromosome disorders, developmental disorders or metabolic disorders . Inherit(CRYBB3) . SupplemWe aimed to determine the genetic cause of pediatric cataract related to microphthalmia in three members of a family with an autosomal dominant pattern of inheritance of pediatric cataract and microphthalmia phenotype using next-generation sequencing.A case report with clinical and next-generation sequencing of three members of the same autosomal dominant family (father and two children) with a history of pediatric cataract and microphthalmia, as well as one family member who did not present any ocular pathologies.At evaluation, all three patients had already been submitted to cataract surgery, and detailed medical records were available for the two youngest. History of congenital TORCH infections , use of corticosteroids and ocular trauma were ruled out.Ophthalmic exam with slit-lamp biomicroscopy, intraocular pressure (IOP), indirect ophthalmoscopy, ophthalmic ultrasonography and biometry for axial length measurement were performed in all individuals.Genetic evaluation of family history and origins and pedigree, as well as existence of other systemic conditions, and clinic-morphological evaluation were performed by a clinical geneticist.® Genomic DNA Mini Kit Thermofisher (USA), according to the manufacturer’s protocol. DNA concentration in samples were determined by fluorometry using Invitrogen Qubit® 4 Fluorometer. To assess DNA purity, a Spectrophotometer NanoDrop® 2000 was used to evaluate ratio of the absorbance at 260/280 nm  and at 260/230 nm . DNA samples were stored at 4 °C prior to use.Peripheral blood samples were collected in EDTA tubes from the four family members. Genomic DNA was extracted from peripheral blood leucocytes using PureLink® according to the manufacturer’s instructions . This clinical exome solution covers 4493 genes related to the most common inherited diseases. Sequencing was performed using the NextSeq® 500 system  using a multiplex system with 16 samples per run with the NextSeq® kit. During library preparation, DNA fragments of 400 bp long on average were evaluated using the Bioanalyzer by Agilent.DNA libraries from both parents and children were prepared with 50 ng of DNA using Clinical Exome Solution V2® software (Switzerland), and the genetic variant calls were performed against the reference sequence of hg19 from the University of California Santa Cruz (UCSC) Genome Browser. The analysis strategy started with a virtual panel based on Human Phenotype Ontology (HPO) where 658 genes were found related to cataract in general , 1000 Genomes Project database, gnomAD and ABraOM, an online archive of Brazilian mutations . Twelve ® (Invitrogen™) and sequenced on an automated sequencer .In order to confirm the variant identified by clinical exome analysis, PCR amplification and bi-directional direct Sanger sequencing were performed using the oligonucleotide primers 5′CCTCCTTGACCTCTGTTCTGG3′ and 5′ GGCACTGATTCTGTTTGGAGC3′. Briefly, primers and PCR products were purified using PureLinkProband was examined at 3 months old and had an unremarkable gestational and peripartum history. An ophthalmological exam revealed nystagmus and bilateral anterior polar cataract, as well as small corneas (7 mm diameter OU) and a reduced axial length  for her age. Ultrasonography showed no alterations other than lens opacification and a reduced axial length. A diagnosis of bilateral microphthalmia and congenital cataracts was made and lensectomy with anterior vitrectomy was performed on both eyes. Aphakic glaucoma developed four years after surgery on both eyes and was managed with topical antiglaucoma drugs. Family history revealed that the proband’s father was diagnosed with bilateral cataracts at three years old, as well as microphthalmia. The eldest brother also presented with nystagmus and microphthalmia and was diagnosed with cataracts at 18 months old. Two years after lensectomy, the brother presented pain in the left eye. Ophthalmological examination revealed aphakia with a shallow anterior chamber and high intraocular pressure. A diagnosis of aqueous misdirection was made and a surgical approach indicated. Since both the father and eldest son were not initially operated on at our facilities, the records of their cataract phenotype prior to surgery were not available. At examination, the father was microphthalmic, with nystagmus and aphakic bilaterally. CRYBB3 in all family members with childhood cataract. No previous descriptions of it were found in ClinVar, HGMD or Cat-Map. Not only is p.Gly156 highly conserved among several species, the variant c.467G>A/p.Gly156Glu in CRYBB3 segregates accordingly in family members  and account for its optical transparency and high refractive index ,13. TheyCRYBB3) [CRYBB3, 23 in CRYBB1 and 35 in CRYBB2 [beta-crystallins can also be subdivided into four acidic  and three basic  isoforms, depending on the isoelectric point and the terminal extension . The leaCRYBB3) . There an CRYBB2 .CRYBB3 mutations reported in the Cat-Map are also isolated [CRYBB3 missense variant c.466G>A (p.Gly156Arg) has included microphthalmia as an additional phenotype of congenital cataract [Seventy percent of pediatric cataract are isolated , and, asisolated . Howeverisolated . Anothercataract .The phenotype of all these c.466G>A (p.Gly156Arg) mutations was reported as bilateral nuclear cataract ,9,18. CoInterestingly, glaucoma also developed after surgery in the patient with microphthalmia and congenital cataract described by Sekeroglu et al. , as was Genetic diagnosis of developmental eye disorders remains a challenge worldwide due to diverse phenotypes and genetic heterogeneity . HoweverTimely pediatric cataract surgical treatment is efficient and does not depend on molecular diagnosis. Nonetheless, the latter is important since it may provide knowledge toward the development of new therapeutic possibilities. Once the causative mechanism of the cataract is identified, the degenerative progressive damage could be delayed and surgical treatment avoided.Therefore, we believe that our finding can contribute to better understand the role of the CRYBB3 protein in inherited childhood cataract associated with microphthalmia.


Blastocystis sp., the most common intestinal protozoa, remains a public health problem among people in many countries, particularly in rural areas of developing countries. The infection usually reflects poor sanitation in communities by waterborne, zoonotic, and person-to-person transmission. Interestingly, at least 17 subtypes (STs) have been reported and are associated with a broad range of animal hosts, including humans. In this study, we reported potential evidence of zoonotic transmission of Blastocystis ST1 in rural communities of eastern Thailand where the overall prevalence of Blastocystis infection was 15.7%. Two major and three minor subtypes were found to be distributed unequally in this region. Of 5 STs, only ST1 was found to be associated with pig feces in an open farm system that produced organic fertilizer for agriculture uses in the community. This finding suggests that properly protective contact and standard production of organic fertilizer from pig feces by-products could be key factors for reducing the prevalence of Blastocystis infection and prevent Blastocystis reinfection among people in the community.IMPORTANCEBlastocystis sp. remains a public health problem among people, particularly in rural areas of many developing countries. The infection usually reflects poor sanitation in communities by waterborne, zoonotic, and person-to-person transmission. In this study, we reported potential evidence of zoonotic transmission of Blastocystis subtype 1 (ST1) in rural communities of eastern Thailand. Two major and three minor subtypes were found to be unequally distributed in this region. Interestingly, only ST1 was found to be associated with pig feces in an open farm system that produced organic fertilizer for agriculture uses in the community. The finding makes significant contributions to genetic and molecular investigations of microbial topics of practical value and suggest that properly protective contact and standard production of organic fertilizer from pig feces by-products could be key factors for reducing the prevalence of Blastocystis infection and prevent Blastocystis reinfection among people in the community. Blastocystis sp. is an anaerobic enteric parasite, a member of the Stramenopiles or Heterokonta branch of the Eukarya . The prevalence of Blastocystis infection significantly differed regarding age group (P < 0.01), occupation (P < 0.01), education (P = 0.03) and among participants residing in different villages (P = 0.01). No significant difference was found regarding sex. The populations or households were mostly farmers and those graduated at primary school level or below.In January 2018, 902 participants were enrolled in this study and 745 stool samples were collected with a response rate of 82.6%. The characteristics of the enrolled subjects and the prevalence of Blastocystis infection in this community showed that those at middle age from 40 to 49 years old , living in Non Sa Ard or Tun Geang Village , working in animal husbandry , and raising and breeding pigs and pig farming,  had a higher risk of acquiring Blastocystis infection, whereas those not working in agriculture  and washing hands after defecation  had a lower risk of the infection  and pigs (n = 9) showed a 98% to 99% genetic identity match with the SSU rRNA gene of Blastocystis species based on BLASTN. A phylogenetic tree of Blastocystis sp. based on 1,032 bp of the SSU rRNA gene was constructed using 33 DNA sequences in this study and 34 Blastocystis references retrieved from GenBank (Proteromonas lacertae was used as an outgroup. The rooted randomized axelerated maximum likelihood (RAxML) tree identified 12 clades called ST1, ST2, ST3, ST4, ST5, ST6, ST7, ST8, ST9, ST11, ST12, and ST13 that were strongly supported by 80% to 100% bootstrap values. The tree topology revealed that Blastocystis subtypes observed among human samples in these two communities could be classified in four subtypes, i.e., ST1, ST3, ST6, and ST7, and formed a polyphyletic evolution. Subtypes 1 and 3 were predominant among people , whereas other subtypes were comparatively less common, namely, subtypes 6  and 7 . Interestingly, Blastocystis isolates of pig feces were found to be associated mostly with human subtypes , otherwise, 1 isolate of Blastocystis from pig feces was clustered in the animal subtype . Three owners of the open pig farm were infected with Blastocystis belonging to ST1, ST3, and ST6.Of 117 cultured positive human stool samples, PCR amplification was successful for 95 samples (81.2%). Small subunit (SSU) rRNA amplicons of 24 samples associated with residing in a village  and pig livestock were chosen randomly for sequencing. In addition, nine positive pig feces samples from an open pig farm of infected owners were amplified successfully and sequenced. In total, 33 sequences from humans  and fouBlastocystis isolates based on the SSU rRNA gene was 0.915 ± 0.028 and 0.056 ± 0.004, respectively  implied that small differences between haplotypes were found within a population. In all, 26 haplotypes of Blastocystis infection were found among people in these communities where the highest number of haplotypes was found within the population of Moo 11 . In addition, the haplotype network clearly demonstrated two distinct groups of haplotypes related with ST1 and ST3  indicating an excess of high- and low-frequency polymorphisms causing population contraction or balancing selection  and nucleotide diversity  among 48 ectively . The hig and ST3 . The comelection . NonetheBlastocystis infection was apparently high compared with that of related community studies (15.7% versus 6.1% to 7.8%). The Phra Ploeng community was located next to these communities , similar to related findings  and open pig farm owners (n = 1) as well as people living in same (Moo 11) and different communities (Moo 18 and 19). Thus, the zoonotic subtype 1 could cause an infection across these three communities of the Thakradan subdistrict where pig feces by-products from open farm systems are commonly used for producing organic fertilizer. The pig feces fertilizer is distributed normally for plant breeding and cultivation within the communities owing to ecotourism and sufficiency economy theory. Nevertheless, isolation and characterization of Blastocystis in the fertilizer are further needed to confirm the existence of fertile cysts that could be the causative sources of reinfection and distribution of ST1 in these communities.Based on the SSU rRNA gene, subtypes –40. The ubtypes – in that findings ; howeverommunity . Many stommunity , 39 denoBlastocystis contaminant related to the modes of transmission of Blastocystis sp. According to the large number of potential zoonotic hosts, host origins and transmission routes remain difficult to identify and control, unlike the control of waterborne transmission by untreated water and poor sanitary conditions  at Thakradan Subdistrict, Chachoengsao Province, eastern Thailand in January 2018, 153 km from Bangkok and 50 km from the Cambodian border. The research protocol was reviewed and approved by the Ethics Committee of the Royal Thai Army Medical Department (reference number S025q/61_Exp). Informed consent was obtained from the enrolled participants (n = 902). Demographic information was collected, including associated risk factors (1421–Blastocystis infection. A face-to-face interview was conducted for every household visit using electronic standardized questionnaires. Each participant received a prelabeled container and was advised regarding the method of a single stool collection. Stool specimens (n = 745) were examined for intestinal parasitic infections under a light microscope by wet preparation, the Kato-Katz technique, and formalin ethyl acetate concentration at the microscopic lab station in the field.A cross-sectional study of actors 1416, 21–24n = 12) were collected from two local pig farms at Moo 11 associated with a Blastocystis risk factor. Each fecal sample was collected carefully from the upper portion of fresh defecated material in order to avoid ground contamination, which is the same as the stool collection technique in humans; then, samples were kept individually in a sterile container. Before collecting fecal samples, the pigs’ owners were informed verbally about the project and the collecting protocol. All owners provided their verbal informed consent personally and permission to collect feces from their pigs. The pigs were raised freely and bred under an open system, and their feces by-products were further used to produce organic fertilizer for organic sufficiency agriculture and agricultural heritage/agrotourism in the community. Fecal samples were examined in a manner similar to the stool examination of the human participants as described previously.In addition to human feces, pig feces  were centrifuged at 2,300 × g for 10 min to precipitate Blastocystis pellets before extracting DNA. The pellet samples were extracted using the DNeasy blood and tissue kit  as described by the manufacturer instructions. The extracted DNA was eluted to a 100-μl final volumes and stored at −20°C until used. PCR amplicons of the small subunit rRNA (SSU rRNA) gene of Blastocystis that were approximately 1,790 bp were amplified using the MJ Mini thermal cycler  according to Yoshikawa et al.  for direct sequencing. The sequencing chromatograms were validated and edited manually using BioEdit version 7.2.5 (Blastocystis infection using NCBI BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi)  . To geneoutgroup , which woutgroup  using Raoutgroup  where clBlastocystis isolates   , 52.Blastocystis infection was analyzed using STATA/SE version 14 . The prevalence was reported with percentage, and risks were assessed with odds ratio (OR) with 95% confidence interval (CI) and P value. A univariate analysis was performed initially to determine relationships between the study covariate and infection status (yes/no). Logistic regression was performed for the multivariate analysis to assess the independent association of risk factors and Blastocystis infection.The association between potential risk factors and MZ164983 to MZ165006 and MZ165024 to MZ165032.All sequences in this study were submitted to GenBank under accession no.


In addition to the antitumor effect alone, maprotiline could also enhance the sensitivity of HCC cells to sorafenib. The depth studies revealed that maprotiline substantially decreased the phosphorylation of sterol regulatory element-binding protein 2 (SREBP2) through the ERK signaling pathway, which resulted in decreased cholesterol biosynthesis and eventually impeded HCC cell growth. Furthermore, we identified cellular retinoic acid binding protein 1 (CRABP1) as a direct target of maprotiline. In conclusion, our study provided the first evidence showing that maprotiline could attenuate cholesterol biosynthesis to inhibit the proliferation and metastasis of HCC cells through the ERK-SREBP2 signaling pathway by directly binding to CRABP1, which supports the strategy of repurposing maprotiline in the treatment of HCC.Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related death and has a poor prognosis worldwide, thus, more effective drugs are urgently needed. In this article, a small molecule drug library composed of 1,056 approved medicines from the FDA was used to screen for anticancer drugs. The tetracyclic compound maprotiline, a highly selective noradrenergic reuptake blocker, has strong antidepressant efficacy. However, the anticancer effect of maprotiline remains unclear. Here, we investigated the anticancer potential of maprotiline in the HCC cell lines Huh7 and HepG2. We found that maprotiline not only significantly restrained cell proliferation, colony formation and metastasis  According to Global Cancer Statistics in 2018, liver cancer ranks sixth and fourth in terms of incidence and mortality worldwide, with approximately 841,000 new cases and 782,000 deaths . HepatocCompared with creating a new drug from scratch, repurposing approved drugs can reduce the cost and substantially shorten the research cycle. Since drug toxicology, pharmacokinetic studies and clinical trials have already been finished, these drugs with confirmed efficacy can benefit patients faster. Thus, the new usage of existing drugs has become a trend in the field of drug development, and the discovered new efficacy of approved drugs constantly expands the indications of drugs. In this paper, we hope to identify anti-HCC drugs from 1056 small molecule drugs. Maprotiline, an FDA-approved tetracyclic antidepressant, blocks the reuptake of norepinephrine by the central presynaptic membrane, which can ameliorate symptoms to achieve antidepressant function and eliminate depressed mood . HoweverCellular cholesterol metabolism plays an important role in numerous biological activities through multifarious intermediates . TherefoThe aim of our research was to investigate the anticancer effect of the FDA-approved drug maprotiline in HCC cells and further elucidate the molecular mechanism. In this article, we demonstrated that maprotiline could restrain the growth of HCC cells through the ERK-SREBP2 signaling pathway, which suppresses cholesterol biosynthesis by interacting with CRABP1.2.The human HCC cell lines Huh7 and HepG2 were purchased from the Cell Bank of the Chinese Academy of Sciences  and cultured in DMEM  with 10% fetal bovine serum  in a 37°C humidified incubator with 5% COMaprotiline, 5-FU (Fluorouracil), oxaliplatin and sorafenib were obtained from Selleck Chemicals . All plasmids were designed and constructed by TranSheepBio Co., Ltd. , and HCC cells were transfected with CRABP1 or vector control using Lipofectamine 3000 reagent  according to the manufacturer’s instructions.3 cells per 50 μL. After the corresponding time of cultivation, CCK-8 assays  were used to measure the absorbance of the well to reflect cell growth by using the microplate spectrophotometer .Cells were digested and plated into each well of a 96-well plate after adjusting the cell suspension to the appropriate concentration of 1×10Cells were seeded in the wells of 6-well culture plates with 2 mL of complete DMEM, replacing the previous medium with new complete DMEM containing different concentrations of maprotiline on the second day. After two weeks of subculture, the colonies were fixed with methanol and stained with crystal violet. Finally, the cell clones were dried and photographed at ×100 magnification.The migration assay was performed as described previously . Cells wCells were gently collected with EDTA-free trypsin, and re-suspended with binding buffer. Under light-proof conditions, Annexin Ⅴ-FITC and Propidium Iodide (PI)  were added to the cell suspension and mixed, followed by incubation for 5–15 min at room temperature to ensure dye binding. The number of apoptotic cells was determined by flow cytometry with a FACSCalibur system (BD Biosciences) within 1 h.The total cell lysates were extracted by RIPA lysis buffer , and BCA protein assay kit (Thermo Scientific) was used to determine protein concentration. The proteins were separated by sodium dodecyl sulfate salt-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking in 5% nonfat dried milk for 2 h, washing the membrane to remove the milk. The primary antibody was incubated overnight at 4°C. The membrane was washed five times for 10 min each time in TBST, after which HRP-labeled secondary antibody was added to the membrane for 1 h at room temperature. The washing operation was repeated, and the reaction was assessed using chemiluminescence (ECL) western blotting kit. The primary antibodies for EMT, cleaved PARP, cleaved caspase-6, cleaved caspase-9, p-ERK, ERK, SREBP2, p-SREBP2, CRABP1, and β-actin and secondary antibodies were purchased from Cell Signaling Technology .The extraction of total RNA was carried out by using the HiPure Total RNA Mini Kit . RNA sequencing (RNA-seq) was performed by the Beijing Genomics Institute Tech . The gene expression in the treatment group was more than 1.5 times that of the control group, and these genes were defined as differentially expressed. IPA software  was used to search for potential targeted pathways. The Raw data of the project were deposited at SRA database with the access number SRR14191074 for non-treatment, and SRR14191072 for maprotiline-treatment.3, the mice were randomly divided into three groups (n = 5) and treated with intraperitoneal injection of either PBS or maprotiline  twice a week for three weeks. The mice were sacrificed after the treatment course, the tumors and blood for further tests were collected and properly stored. Cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), red blood cells (RBCs), white blood cells (WBCs), neutrophils, lymphocytes, hemoglobin (HGB) and platelets (PLTs) were measured by Servicebio Biotechnology Company .The use and handling of animals were reviewed and approved by the Ethics Committee for Animal Experiments of Jinan University. All experiments were approved by the Animal Research and Use Committee of Jinan University. The details were the same as those of the tumor xenograft experiments presented in our previous studies . Nude mihttps://cloud.yinfotek.com/). A virtual screening library of protein targets was built based on the sc-PDB (http://www.rdkit.org) were calculated to compare the docked poses with the crystal ligand. Finally, a set of top ranked potential targets was selected.Potential targets of the compound maprotiline were determined using the Inverse Docking protocol in the Yinfo Cloud Platform (e sc-PDB  and UniPe sc-PDB  program Human CRABP1 cDNA was inserted into pET-28a (+) by TranSheepBio Co., Ltd., with a His-tag. The plasmid was transformed into BL21 competent cells, and then, a single colony containing the pET-28a (+)/CRABP1 plasmid was picked and cultured at a temperature of 25°C and a rotation speed of 180 rpm/min for 6 h. IPTG was added to the bacterial fluid at a concentration of 0.5 mM and cultured overnight. The cells were collected by centrifugation, washed twice with deionized water and suspended in PBS, after which the target protein was harvested by ultrapressure lysis and centrifugation. The protein was purified by a Ni2-Sepharose plate column, eluted in 250 M gradient imidazole buffer and eluted completely in PBS (pH 7.4) buffer. The concentration of purified protein was determined by a Nanodrop 2000/2000c system.−1 for 600 s for binding. Oxaliplatin and 5-FU were used as the negative controls. The combination was analyzed by admin@Biacore X100 Evaluation Software.To confirm the binding of the CRABP1 protein and maprotiline, we carried out surface plasmon resonance analysis on a Biacore T100 biosensor system . CRABP1 was diluted to 10 mM with acetate (pH 5.0) and immobilized onto an active CM7 chip  as described previously . The mapt test. For all analyses in this article, statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.All experiments were independently performed three times, and the data are presented as the mean ± SD. All statistical analyses and figures were performed by using GraphPad Prism software  with Student’s Based on a drug library consisting of 1056 FDA-approved small molecules, cell viability assays were performed to identify drugs with anti-proliferative effects on HCC cells. After several rounds of large-scale experimental screening, we finally confirmed that maprotiline, which has never been previously reported to have anticancer function, has a good killing effect on HCC cells . In thisNext, to explore the effect of maprotiline on the invasion of HCC cells, we conducted Transwell assays in Huh7 and HepG2 cells. Cell migration assays indicated that maprotiline significantly restrained HCC cells migration ; moreoveSorafenib is the standard treatment in advanced hepatocellular carcinoma, however, chemotherapy resistance remains a crucial challenge for patients . To explThen, to investigate the molecular mechanism by which maprotiline inhibits tumor growth and metastasis in HCC, we conducted RNA sequencing to compare gene expression profiles between 20 μM maprotiline-treated HepG2 cells and nontreated HepG2 cells. The results indicated that there was a significant difference in the cholesterol biosynthesis pathway between the maprotiline-treated HepG2 cells and the negative control cells, suggesting that maprotiline possibly affect the survival of HCC cells via cholesterol metabolism . Based oin vitro, which suggested that maprotiline probably produces anticancer effects by directly targeting CRABP1  through intraperitoneal injection twice a week. As shown in Based on our in vivo through subcutaneous xenografts in nude mice  in Burkitt lymphoma, which provides a basis for us to study the anticancer function of this drug. In terms of mechanism, it has been reported that maprotiline can activate the JNK-related caspase-3 pathway to induce apoptosis, which provides a reference for the apoptosis induced by maprotiline in our study. We further confirmed that maprotiline can reduce the level of phosphorylated ERK in treated HCC cells and weaken signal transduction. The roles of the ERK pathway have been widely reported in the process of cancer development. In our previous studies, the ERK pathway was confirmed to promote tumor growth, yet the effects of maprotiline on ERK pathways have not been reported. In this paper, we revealed that the ERK pathway probably affect cholesterol synthesis through SREBP2. SREBP2 has been proven to be one of the important regulatory elements of cholesterol metabolism in many studies, and cholesterol metabolism mediated by SREBP2 has been proven to have a tumor-promoting effect. Many studies have shown that CRABP1 is closely related to the ERK pathway, suggesting we should focus on the role of CRABP1 when exploring the molecular mechanism of maprotiline's anticancer effect . In prevThe major obstacle of chemotherapy is drug resistance, and this condition continues to worsen. The molecular mechanisms of tumor resistance are extraordinarily complex, and the drug resistance mechanisms of different drugs are different, even the same drug has various drug resistance mechanisms. At present, it is difficult to reverse chemotherapy resistance, thus, finding new drugs for effective treatment of tumors may be a good choice. The existing FDA drugs have been proven to have pharmacokinetic characteristics and safety. Reinterpretation of drug efficacy will avoid the high clinical failure rate caused by ADMET, which reduces the R&D cost and shortens the time of manufacture. Celecoxib was initially approved by the Food and Drug Administration (FDA) as an analgesic, anti-inflammatory and antipyretic drug, followed by a large amount of preclinical evidence showing that celecoxib has a strong killing effect on cancer . Therefoin vitro and in vivo through cell experiments and nude mouse experiments. Mechanistically, we proposed that maprotiline may restrain cholesterol biosynthesis to inhibit the growth and metastasis of HCC cells through the ERK-SREBP2 signaling pathway by interacting with CRABP1. These results will support maprotiline as a candidate anticancer drug.In general, we first verified the antitumor function of maprotiline