Understanding drivers of ecosystem primary production is a foundational question in ecology that grows in importance with anthropogenic stresses . Traditionally, ecosystem production is considered to be abiotically controlled at large spatial scales , which underlies forecasting climate change impacts. Using a “common garden” experiment over 10 years at two sites with the same plant and grasshopper species, we show that primary production is strongly influenced by biotic factors (herbivory and plant adaptations to it) at finer spatial scales by creating positive feedbacks, which reverse relative productivity of ecosystems expected from abiotic conditions alone. Our results without herbivory indicate that one site has 26% less annual net primary production (ANPP) than the other site. With herbivory, the sites reverse in ANPP, so the site with lower ANPP without herbivory now is 15% greater than the site with higher ANPP without herbivory, as they respectively increase by 6% and decline by 33%. This reversal is due to changing nitrogen availability (N), as N becomes 16% greater at the higher ANPP site with herbivory, respectively a 3% increase and 41% decline in N. The ANPP and N changes are observed, even though the sites are a few kilometers apart and have the same grasshopper and plant species. However, this forecasting may be misleading if biotic effects are ignored. For example, there is mounting evidence supporting an early claim3 that herbivory strongly affects ecosystem primary production by modifying nutrient cycling9. This biotic influence can create two types of positive feedbacks: (1) herbivory may enhance nutrient cycling and thereby increase primary production, or (2) herbivory may diminish nutrient cycling and thereby decrease primary production. If these biotic positive feedbacks strongly impact nutrient cycling and primary production, they will reduce the impact of abiotic processes2 that are traditionally considered all important1. Therefore, how important this biotic effect is relative to abiotic effects is crucial for forecasting climate change impacts on ecosystem functioning and needs to be addressed. A productive way to distinguish between the roles played by abiotic and biotic influences is a “common garden” experiment. Starting with a plant/herbivore system that exhibits the alternative positive feedback effects between two sites, the system’s components  can be translocated between the sites and the ecosystem functioning (N and ANPP) can be compared with the site of origin.Abiotic factors  have traditionally been viewed as the main drivers of ecosystem primary productionPascopyrum smithii and Poa pratensis) and the same most common grasshopper , but each exhibits one of the alternative positive feedback effects on N-cycling and ANPP12. Previous experiments indicated that if the grasshopper prefers to feed on the slower decomposing plant (Pascopyrum smithii), then N-cycling and ANPP increase (Site A: UTM 713570 E 5248100N), while preferential feeding on the faster decomposing plant (Poa pratensis) leads to N-cycling and ANPP decreasing (Site B: UTM 706453 E 5249980N)12. Soils and grasses from the two sites can be translocated between the two sites creating different independent combinations  to which the grasshopper can be added to assess the effects of microclimate, soil, and plant differences on the previously observed positive feedback effects of herbivory on N-cycling and ANPP.An appropriate plant/herbivore system for this “common garden” experiment is provided by a grassland . Here two sites differing by <30 m in elevation and within <7.5 km of each other have the same plant species .Sites (B > A) and years (2006 > 2005) differed in ANPP (Table ANPP was measured in June 2005 and 2006 (before adding grasshoppers). Several patterns emerged for ANPP when pots containing that site’s soil and plants, pots containing the other site’s soil and plants, and controls were compared Fig. :Pots refAbiotic effects are expected given site, year and soil responses Table . Two comBiotic effects in the absence of grasshoppers can only emerge with plant source, but no main or interaction Tables  and S2.32, t = 3.94, df = 93, P < 0.0002).As without grasshoppers, ANPP with 0% feeding intensity (intercept) was greater for Site B than Site A plants  were examined in pots with the plant source from that site and controls Fig. :As withoTherefore, ANPP between sites was reversed with herbivory Fig.  vs. b, cAbiotic effects on ANPP, when grasshoppers were absent, appeared for soil moisture and growing season N Tables . Soil mo11, ANPP with grasshoppers rises slightly for Site A’s plants at Site A (3%), but falls dramatically for Site B’s plants at Site B (41%), a 44% net change  increases Fig. . With grP. pratensis at Site A = 42.5%: χ2 = 2.40, P < 0.14; at Site B = 48.9%: χ2 = 0.02, P < 0.88), and the sites did not differ . With grasshoppers, the faster decomposing P. pratensis increased in relative abundance at both sites, but much more for plants from Site A . While the faster decomposing plant relative abundance did not differ between sites for plants from Site B (P < 0.99), it was 27% greater for Site A plants at A than B. Therefore, species composition of the litter changes with grasshoppers, which accounts for decomposition rate and N differences, as previously suggested10.Without grasshoppers, the initial equal relative abundance of the two plants did not change , so herbivory may be a stronger selection pressure at Site A. Local adaptation to herbivory can be examined by plotting relative ANPP (with ÷ without grasshoppers) vs. grasshopper density. The plot increases and then decreases as grasshopper density increases for both Site A and B plants  and soil between sites, while removing or allowing herbivory, to examine ecosystem functioning (N cycling and ANPP). While experimental ecosystems have been studied, e.g.1, N cycling and ANPP were driven solely by abiotic conditions  and the only available biotic factor, plant source, had no effect. One site (B) was 26% more productive due to having greater nitrogen availability, while inter-annual variability in productivity and soil effects were driven by moisture. N may limit ANPP more than moisture, as the more productive site (B) had greater N, but less moisture, than the other site (A). This is consistent with experimental supplementation of N at both sites, that found N to limit ANPP11. In summary, without herbivory (no grasshoppers), the traditional view that ANPP primarily is driven abiotically by microclimate (site), annual weather (year), and soil is supported.Without herbivory, as traditionally expectedWith herbivory (grasshoppers), the relative productivity of the two sites reversed; so the less productive site in the absence of herbivory had a 6% increase in ANPP but was now 15% more productive than the more productive site in the absence of herbivory, which had a 33% decline in ANPP. This occurred even though the more productive site in the absence of herbivory experienced 55% less herbivory (lower grasshopper density).P. pratensis litter decomposes faster than P. smithii11 litter, so percent of litter remaining will decrease and N will increase as relative abundance of P. pratensis litter increases. Grasshopper selective feeding enhanced N at the originally less productive site (A) by increasing the relative abundance of faster decomposing plants (P. pratensis), and diminished N at the originally more productive site (B) by increasing the relative abundance of slower decomposing plants (P. smithii). Preferential feeding arises because grasshoppers prefer green vegetation that is lower in water content, so P. pratensis at Site B is preferred, while P. smithii is preferred at Site A11.The reversal was due to changes in litter decomposition due to herbivory. Litter decomposition rate is biotically affected by the plant species comprising the litter. 17, and at least one study has hypothesized that this might affect nutrient dynamics18.With herbivory, we also observed that the plant species from the different sites were locally adapted to herbivory, even though they are the same species, with the plants from the site (A) where the herbivore was consistently more abundant being able to cope better with herbivory. A number of field experiments have reported on the importance of local adaptation of plants to herbivory, e.g.27. While this did not negate abiotic influences, it surpassed their effect, as hypothesized earlier3.In summary, grasshoppers affect ANPP through plant source, plant species composition and level of herbivory. Counter to tradition that ANPP is driven by abiotic effects, biotic effects emerge stronger, increasing ANPP at one site and decreasing it at another beyond abiotic limits. This is driven by herbivory creating positive feedbacks that affect productivity, as reported elsewhere, e.g.29. This requires better and more detailed ecological insights30.Our study indicates that focusing solely on the abiotic effects, and ignoring biotic influences, can be very misleading in understanding ecosystem functioning. This includes misrepresenting system productivity by as much as 33%, and misidentifying the most productive system. Furthermore, the biotic influences operate on a local spatial scale (few kilometers). Consequently, we suggest that characterizing ecosystem functioning based solely on large spatial scale abiotic conditions , as commonly conducted, especially in making climate change projections, may be misleading. Furthermore, environmental changes often change species abundances, including lost and added species, so that novel biotic effects may arise2: diameter = 26 cm, depth = 30 cm) with the bottom cut out were buried (28 cm) in the ground. Holes closely fit a pot and the soil that was removed to make the hole was saved, maintaining its profile and the surface was labelled. Pots were installed 2 m apart in a 4 × 10 grid.The “common garden” experimental ecosystems were created in June 2004. Forty large plastic pots (0.053 mPoa pratensis and Pascopyrum smithii) were planted in a pot. Plants were carefully dug up to maintain root mass (~20 cm around plant) and soil was gently washed away. To help establish plants, 0.25 L of water/pot was provided weekly from June–Sept 2004 and any other plants were removed. To exclude grasshoppers, pots were covered by a nylon insect screen bag , which was supported by wire and attached to the pot with plastic clips.Pots were randomly assigned to 4 treatments  with 10 replicates for each. Each pot’s soil treatment was provided by soil saved from a hole cut for a pot at the appropriate site. Each pot’s plant treatment was provided by transplanting plants from the appropriate site. Based on abundance and plant size at the sites in June 2004, five 20 cm (±10%) tall individuals of each of two grass species (Melanoplus sanguinipes) at that year’s field density were allowed to feed in half of the pots in each treatment. This was accomplished by placing the appropriate number of grasshoppers in a pot for 1–2 days every 2 weeks, where the number of grasshoppers and length of time the grasshoppers were allowed to feed in a pot was based upon their mean field density over the preceding 2 weeks (See Table 2 ringsPots had grasshoppers excluded during 2004–2005 (late June–September). Starting in late June 2006, grasshoppers (2 (0.33 m × 0.33 m) control areas were randomly placed immediately outside the grid of 40 pots at each site, but within the experimental area. Grasshoppers were excluded from each control in years 2004–2005, using a 0.5 m high box of aluminum insect screen supported by wooden stakes and attached to garden edging by folding32. From 2006–2012, the insect screen box was removed, so that grasshoppers had access.Five 0.11 m34. The radiometer was positioned to measure a 0.05 m2 area. Live plant biomass was computed from radiometer readings using site and date specific regressions. Each regression was based on readings from 3–5 0.1 m2 plots adjacent to the grid of pots, which were selected to range from low to high plant abundances, and from the plant biomasses of these 3–5 0.1 m2 plots. Green vegetation in each of these plots was clipped, dried for 48 hours at 60 °C, and weighed. Regressions averaged an r of 0.93 . ANPP is the sum of all increases in green plant biomass between consecutive periods from June–Oct; however, we only report the highest values for June as they always decreased afterwards.Annual aboveground net primary production (ANPP) was measured nondestructively using a ©CropScan radiometer every two weeks from June–OctP. pratensis and P. smithii) were determined using point frame sampling35. Twenty-five random points in each pot and control were identified in June and Sept as: bare ground, litter, moss, or plant species. Plant abundances were examined using the point frame “hits” on each species.Absolute and relative abundances of the two grasses  was measured in each pot and control twice a year  with an ion exchange resin bag (©Rexyn) buried 15 cm in each pot and control in June and Oct38, which was correlated with soil moisture (% H2O).Soil moisture was measured every 2 weeks as conductivity using a gypsum block buried 20 cm in each pot and controlLitter abundance in pots and controls was measured in Sept by removing it, drying it for 48 hours at 60 °C, weighing it and then returning it to its pot or control.Percentage of grass blades eaten was measured in Oct by examining up to 25 blades of each of the two grass species for feeding damage by grasshoppers. No attempt was made to quantify the amount of tissue removed from a blade, just damage or no damage was recorded.The following measurements were made from 2005–2013 in the pots and controls (see Table 2 analyses were conducted using ©SYSTAT 13. Data employed in GLMs and ANCOVAs were normally distributed. Data from each individual pot with and without grasshoppers were employed in GLMs and ANCOVAs for ANPP, N and soil moisture (Table 2 analysis employing the sum of all point samples with grasshoppers over the five years in the treatments being compared. Finally, the ANPP responses for Site A and B plants to grasshopper density were analyzed by regression for the mean of all pots (Fig. GLM, ANCOVA, regression and χre Table . Insuffire Table  and regrre Table . Effect re Table . For GLMre Table . Fast vsSupplementary Information


IBV-Flagellin-SAPN, a negative-control construct Flagellin-SAPN or a buffer control. The immunized chickens were challenged with 5x104.7 EID50 IBV M41 strain. High antibody responses were detected in chickens immunized with IBV-Flagellin-SAPN. In ex vivo proliferation tests, peripheral mononuclear cells (PBMCs) derived from IBV-Flagellin-SAPN immunized chickens had a significantly higher stimulation index than that of PBMCs from chickens receiving Flagellin-SAPN. Chickens immunized with IBV-Flagellin-SAPN had a significant reduction of tracheal virus shedding and lesser tracheal lesion scores than did negative control chickens. The data demonstrated that the IBV-Flagellin-SAPN holds promise as a vaccine for IBV.Infectious bronchitis virus (IBV) affects poultry respiratory, renal and reproductive systems. Currently the efficacy of available live attenuated or killed vaccines against IBV has been challenged. We designed a novel IBV vaccine alternative using a highly innovative platform called Self-Assembling Protein Nanoparticle (SAPN). In this vaccine, B cell epitopes derived from the second heptad repeat (HR2) region of IBV spike proteins were repetitively presented in its native trimeric conformation. In addition, flagellin was co-displayed in the SAPN to achieve a self-adjuvanted effect. Three groups of chickens were immunized at four weeks of age with the vaccine prototype,  E. coli is present [Infectious bronchitis (IB) is a highly contagious avian disease that causes significant economic losses to the poultry industry. Commercial losses are mainly due to decreased weight gain and egg production, and a poor egg quality . Infecti present .Vaccination is utilized as a major means of controlling IB. Both live attenuated and inactivated virus vaccines are used to protect chickens against IB. However, limitations of live attenuated IBV vaccines include; reversion to virulence, tissue damage which can lead to secondary bacterial infections. In addition, the potential interference of maternal antibody on vaccine efficacy should also be taken into consideration . MoreoveLately, there has been an increasing interest directed towards the use of nanoparticles as vaccines against infectious pathogens –21. ThesIn the present study, we developed a self-adjuvanted SAPN vaccine for the delivery of an antigenic polypeptide of IBV. IBV has a positive, non-segmented, single-stranded RNA genome 27 kb in length, coding for four structural proteins, the spike glycoprotein (S), the membrane protein (M), the envelope protein (E) and the nucleocapsid protein (N) . The spiin vitro infection inhibition assay [In a previous study, we reported that a recombinant DNA vaccine carrying IBV S protein with chicken interferon protected chickens against challenge with Massachusetts 41 (M41) field type IBV . Also, won assay  Based on2 in an inhalation chamber according to the approved protocol.Animal housing and all animal experiments were conducted strictly in accordance with Ag Guide (2010) and animal protocol approved by the Institutional Animal Care and Use Committee of University of Connecticut, Storrs, CT, USA (IACUC protocol #: A15-001). All animals were acclimated for one week prior to any experimental procedures. Chickens had ad libitum access to water and feed, and were monitored daily by attending veterinarians during the study. All animals were supervised daily by the attending veterinarians providing care to reduce pain and stress. All animals were humanely euthanized using COPlasmodium falciparum) epitope CelTOS (residues 24–83 and 84–182), amino acid residues were showed in Salmonella enterica serovar Typhimurium in place of the IBV antigen .The backbone of SAPN monomers were designed containing a pentameric and a trimeric coiled coil region held together by a linker and malaria  using a purifier, ÄKTApurifier 100 . Five column volumes of binding buffer of 8M urea, 100 mM NaH2PO4, 20 mM imidazole, 20mM Tris base, pH 8.0 were used to equilibrate HisTrap HP column. Clear lysate was loaded into the equilibrated column and then underwent washing with 5 volumes of binding buffer. Columns were then washed with 5 column volumes of pH gradient phosphate buffer pH 8.0 buffer , pH 6.5 buffer , pH 5.5 buffer , and pH 4.5 buffer , and finally a buffer 8M urea, 100 mM NaH2PO4, 20mM Tris base, pH 8.0. Subsequently, a 500 mM imidazole gradient was used to elute protein from the column. Fractions of the eluate were analyzed by SDS-PAGE. Fractions containing the target proteins were combined and dialyzed overnight to remove imidazole in a pre-refolding buffer .Expression and purification was carried out as previously described . BrieflyIBV-Flagellin-SAPN) a molar ratio of 58:2 IBV-SAPN monomer to Flagellin-SAPN monomer, was established based on protein concentration. After establishment of the molar ratios, SAPNs were refolded in a stepwise manner by slowly removing urea, in which the gradient urea concentration of refolding buffers were used 8M, 6M, 4M, 2M, 120 mM Urea in 20 mM Tris, 5% glycerol pH 8.0. SANPs were dialyzed for 4 hours in each concentration of urea.The protein concentration was determined by NanoDrop 2000 . To generate Self-Adjuvanted SAPNs . Chickens were bled once before vaccination (at days one of the experiment) and bled three times nine days after each immunization . The sera from the experimental chicken were stored at -80° C until tested. As negative controls, Flagellin-SAPN, or refolding buffer was used to immunize chicken (5 chickens for each group) with the same immunization protocol. Another non-vaccinated/non-challenged negative control group was included in the study for histopathology comparison. Chickens were challenged at intranasally with 5x104.7 EID50 IBV M41 strain per chicken at 58 days of age. The chickens were observed daily for clinical signs, tracheal swab samples were collected at two and four days post challenge and the chicken were euthanized four days post-challenge. Tracheal swabs were placed in brain heart infusion broth media, and stored at -80 °C until tested. Chicken groups were housed separately in isolators with controlled pressure, temperature and humidity in accordance with Ag Guide (2010). Any chicken showing lacking of interest in food and water, difficulty in breathing, or depression were brought to the attention of the veterinary staff at animal care office.Four-week-old SPF white Leghorn chickens (n = 5)  were given 200μL vaccine formulation containing 100 μg 5 cells/well) were plated in 96-well cell culture plates. The PBMCs were stimulated in triplicate wells with 10 μg/mL concanavalin A, 10 μg/mL protein nanoparticle constructs, or 10 μg/mL UV-inactivated IBV M41 strain, respectively. Plates were incubated at 41° C with 5% CO2 for 48 hours. The proliferation assay was conducted using a cell proliferation ELISA BrdU (colorimetric) kit . Briefly, BrdU was added into each well with a final concentration of 10 μM and incubated at 41° C with 5% CO2 for 21h. Plates were centrifuged at 300g for 10 min. Labeling medium was removed by flicking- off plates, which were air-dried in the dark overnight. 200 μL/well FixDenat was used to fix cells in the plates for 60 min at 15–25° C. The fixative was removed thoroughly by flicking and tapping. One-hundred μL/well anti-BrdU-POD working solution was added to the microplates and incubated for 90 min at 15–25 °C. Plates were washed three times using PBS. A substrate solution was added (100 μL/well TMB (tetramethyle-benzidine) solution) and incubated at 15–25° C for 5–30 min until color development was visible. The reaction was stopped by adding 25 μL of 1M sulfuric acid to each well. Absorbance was measured at 450nm with background reference wavelength 690nm. Stimulation index (SI) was calculated by the formula, SI = OD value (antigen-stimulated PBMCs)/OD value (medium treated PBMCs).Peripheral blood mononuclear cells (PBMC) were isolated using gradient centrifugation in Histopaque-1077 (Sigma). Briefly, 1 mL of blood was mixed with equal amount of 1X PBS. The blood-PBS mixture was overlaid on top of an equal volume of Histopaque-1077 and centrifuged for 30 min at 400 ×g at 20° C. The opaque interface was collected and washed twice with PBS with centrifugation at 300 xg for 5 min for each wash. The cell pellet was re-suspended, stained with trypan blue and counted with a hemocytometer. PBMCs  were coated with 50 μL containing 10 μg/mL synthetic HR2 sequence (FVRALSMQILDIDSEIDRIQGVIQGLNDSLIDLEKLSILKTYIKWPWY) in carbonate-bicarbonate buffer pH 9.6 overnight at 4°C. Plates were blocked with 100 μL of a 3% bovine serum albumin (BSA) in PBST solution (PBS plus 0.05% Tween 20). Chicken’s sera collected during the vaccine trial were serially diluted starting at 1/40 and were incubated in the plates at room temperature for 1 hour. Hyperimmune sera were used as positive control was included in each ELISA test. After washing the plates with PBST, secondary antibody  was added and incubated in the plates at a 1/1000 dilution at room temperature for 1 hour. The plates were washed and then 50 μL ELISA TMB (Thermo Scientific) substrate was applied to the wells at room temperature for 5 to 15 minutes until color developed. Reactions were stopped with 0.01M HCl. Plates were read for absorbance at 450 nm.Three nine day-old SPF embryonated chicken eggs were inoculated in the allantoic cavity with 200μl of each filter sterilized tracheal swab. Eggs were incubated at 37°C for five days. The eggs were observed daily for embryo mortality. All eggs were kept at 4°C on the fifth day post-inoculation. The allantoic fluids were harvested.ACA GGT TCT GGT GGT GTT TAG TG; Reverse: AGT TGT TCG GGA ATG TCT TTG G), 4 μL of cDNA template and nuclease-free water was added to bring the volume to 20 μL. Reaction was run using a Bio-Rad real time PCR system CFX 96 with a cycling profile, at 94° C for 2 min, and 40 cycles at 94° C for 20 s, 60° C for 20 s, and 72° C for 15 s. Melting temperature was determined from 65° C to 95° C with heating ramp of 0.5° C/s.RNAs were extracted from the tracheal swabs and from recovered allantoic fluids using QIAamp viral RNA mini kit . First strand cDNA was synthesized in a reverse transcription reaction (RT) using the high capacity cDNA reverse transcriptase kit of Applied Biosystems (USA). Total RNA 680 ng was used in a 20 μL reaction containing 0.8 μL dNTP, 2 μL reaction buffer, 2 μL random primer and 1 μL enzyme mix according to the manufacturer’s instructions. The RT reaction was conducted in a thermocycler (Applied Biosystems) with a programmed profile of 26° C for 10 min, 42° C for 45 min, and 75° C for 10 min. Real time RT-PCR was performed as previously described . The reaTracheal tissues were harvested after euthanasia and necropsy four days post challenge, and fixed in 10% neutral formalin. Hematoxylin and eosin staining was performed on cross sections of tracheal tissues. A scoring scale was established with modification of previously described scoring method .Data was analyzed using one or two-way ANOVA with Bonferroni post-test in GraphPad prism 6.0. Statistical significance was achieved at levels of P values <0.05 (*), <0.01 (**), and <0.001 (***).IBV-Flagellin-SAPN and Flagellin-SAPN showed high purity and expected molecular weights of 37.8 kDa and 64.5 kDa for the two types of protein chains . This demonstrated the induction of cellular immune responses and generation of a memory pool by the vaccine.The stimulation of PBMCs from chickens immunized with th SAPNs  or inactth SAPNs  resultedIBV-Flagllin-SAPN as compared to Flagellin-SAPN and Buffer groups  was lower than that of the buffer control group, indicating a level of restriction of virus replication in chickens by SAPN treatment itself . Also the viral RNA loads in tracheas were significantly lower as compared to control groups four days after challenge. This was consistent with significantly lesser tracheal lesion scores four days after challenge in chickens vaccinated with IBV-Flagellin-SAPN than Flagellin- SAPN and buffer groups. Microscopic evaluation revealed that, there were no major pathologic changes in the tracheas of IBV-Flagellin SAPN vaccinated chicken and the histology was comparable to that non-vaccinated/non-challenged control group. This is a clear indication of vaccine induced protection. Our results are consistent to those reported previously in chickens vaccinated with a recombinant S-ectodomain protein vaccine which elicited protection against IBV challenge and suggested that, S2 domain has a crucial role in persuading protective immunity In the current study, protection was higher in the group of chickens vaccinated with IBV-Flagellin-SAPN vaccine has shown here a clear potential to protect chickens against challenge with IBV M41 without co-administration of commercial adjuvants. However, it has to be kept in mind that this is an experimental vaccine and no previous data exist with the use of the same platform with this particular IBV antigen as to make comparisons. Therefore, this work conducted here provides proof of principle where vaccine immunogenicity was demonstrated as shown by measurable antibody and cellular responses and a level of protection were clearly demonstrated in vaccinated chickens.On the basis of the viral RNA load in the trachea and tracheal histopathological assessments, the Flagellin-SAPN is able to significantly reduce viral shedding in trachea of inoculated chickens after challenge with IBV ; Color scheme: Red, malaria protein; Green: a pentameric coiled coil; yellow: trimeric coiled coil; Blue: HR2 B cell epitope; Black: linker sequences; Pink: CD4 T cell epitopes; Purple: Sequence of the flagellin D0 and D1 domains.(A) IBV-SAPN monomer (Monomer A). (B) (TIF)Click here for additional data file.S2 FigIBV-Flagellin-SAPN assembled from Monomer A and Monomer B at a 58:2 molar ratio; and 3- Flagellin-SAPN monomer ; 2- (TIF)Click here for additional data file.


The successful inhibition of sexual thoughts, desires, and behaviors represents an essential ability for adequate functioning in our daily life. Evidence derived from lesion studies indicates a link between sexual inhibition and the general ability for behavioral and cognitive control. This is further supported by the high comorbidity of sexual compulsivity with other inhibition-related disorders. Here, we aimed at investigating whether sexual and general inhibition recruit overlapping or distinct neural correlates in the brain. Furthermore, we investigated the specificity of two different kinds of sexual inhibition: inhibition of sexually driven motor responses and inhibition of sexual incoming information. To this end, 22 healthy participants underwent functional Magnetic Resonance Imaging (fMRI) while performing a task requiring general response inhibition (Go/No-go), as well as cognitive and motivational sexual inhibition (Negative Affective Priming and Approach-Avoidance task). Our within-subject within-session design enabled the direct statistical comparison between general and sexual inhibitory mechanisms. The general inhibition task recruited mainly prefrontal and insular regions, replicating previous findings. In contrast, the two types of sexual inhibition activated both common and distinct neural networks. Whereas cognitive sexual inhibition engaged the inferior frontal gyrus, the orbitofrontal cortex and the fusiform gyrus, motivational sexual inhibition was characterized by a hypoactivation in the anterolateral prefrontal cortex. Both types of sexual inhibition recruited the inferior frontal gyrus and the inferotemporal cortex. However, the activity of the inferior frontal gyrus did not correlate with behavioral inhibitory scores. These results support the hypothesis of inhibitory processing being an emergent property of a functional network. Sexuality is one of the main driving forces underlying human behavior. Being essential for the individual’s ability to reproduce, sexuality is a deeply rooted and highly rewarding behavior. Nonetheless, the ability to inhibit sexual stimuli and control sexual behavior is of utter importance to convey to societal norms or to prevent harmful consequences. The failure to control sexual impulses and behavior may lead to a wide range of undesired consequences such as compulsive masturbation and pornography watching, undesired pregnancy, sexual transmitted diseases contagion, relationships problems, and sexual offending. It is, thus, plausible that specific sexual inhibitory mechanisms underlie the ability to control sexual responding.The lack of control over sexual behavior has been studied in the context of compulsivity and addiction models. Hypersexual patients show typical addiction or compulsive symptoms, such as the interference with important occupational and social goals, and the repetitive and unsuccessful efforts in controlling their urges .The high prevalence of substance abuse, impulse-control, and obsessive-compulsive spectrum disorders among hypersexual patients , 3 raiseOther studies have shown that the failure to respond to the Go stimulus (i.e. misses in the Go/No-go task) was associated with sexual arousability and sexual compulsivity , 12. MorThe link between general and sexual inhibition is further supported by observations from lesion studies, in which frontal damage often leads to general disinhibition as well as sexual inhibition deficits . HoweverPrevious neuroimaging studies have proposed the left lateral orbitofrontal cortex, the gyrus rectus, and the inferotemporal cortex to be tonic inhibitors of sexual arousal, showing distinct deactivation during the presentation of sexual stimuli . In addiOn the other hand, neuroimaging studies investigating the neural correlates of general response inhibition using the Go/No-go paradigm, consistently describe mostly right-lateralized activation of the superior, middle, and inferior frontal gyri, the pre-supplementary motor area, the ACC, the inferior parietal lobe, the angular gyrus, basal ganglia, and anterior insula during the deliberate inhibition of a preponderant response ,19–21. Smotivational sexual inhibition assessed by an Approach-Avoidance paradigm) behaviorally differs from the attentional control over sexual inputs  [Here, we aimed to characterize the neural substrates of sexual inhibition using functional Magnetic Resonance Imaging (fMRI) with two experimentally controlled paradigms assessing the motivational and cognitive aspects of sexual inhibition: Negative Affective Priming and Approach-Avoidance. We previously showed that the control of motivationally driven motor action tendencies (aradigm) . In addiTwenty-four healthy male participants (18–34 years old) without neurological or psychiatric disorders took part in this study. One participant was excluded due to extensive head movements, and a second participant due to technical difficulties leading to an incomplete data set . Participants gave written informed consent and at the end of the session they received twenty euro in vouchers for their participation. The study was approved by the local Ethical Committee of the Faculty of Psychology and Neuroscience at Maastricht University.Participants performed two sexual (Approach-Avoidance and Negative Affective Priming task) and one non-sexual (Go/No-go task) paradigm in an MR scanner. The order in which the two sexual tasks were presented was counterbalanced. The Go/No-go task was always presented between the sexual tasks to prevent habituation to sexual stimuli.motivational sexual inhibition as it targets approach-avoidance tendencies towards stimuli with affective or neutral content  and to avoid the non-sexual stimuli. For the other two blocks, participants were instructed to do the opposite. To approach, participants had to pull a joystick towards them. This action doubled the image size. To avoid, they had to push the joystick away from them, which in turn halved the image size. In every trial, the presentation of the stimulus lasted 1700 milliseconds. To avoid variability in timing across trials and participants, the resizing always occurred 1300 ms after stimulus onset. Between trials, a fixation cross was presented for 3400, 4250, or 5100 ms . The fouSexual stimuli were color photographs displaying sexual intercourse or oral sex between one woman and one man; 95% of these pictures were previously evaluated and validated in terms of valence and arousability . The remcognitive sexual inhibition (This paradigm (adapted from ) addresshibition . ParticiThe task consisted of four types of trial-sequences: a) Sex Priming, b) Sex No Priming, c) Non-Sex Priming, and d) Non-Sex No Priming. A trial-sequence contained a prime and a probe trial; during each, two pictures were presented simultaneously. The pictures were displayed one above the other; one surrounded by a black and the other by a gray frame. The instruction was to attend only to the picture with the black frame (target), therefore ignoring the one with the gray frame (distractor) and to indicate whether the target displayed sexual or non-sexual content. Participants responded by pressing a left or right button on a button box located below their right hand. During the priming trial-sequences, the content type of the distractor in the prime trial was the same as in the target picture of the probe trial. In the control trial-sequences (No Priming) the content type of the distractor in the primer trial and the target in the probe trial was different. The target of the probe trial could be sexual or non-sexual. The four types of trial-sequences were presented randomly in equal proportions throughout each of three blocks. Each block contained 32 trial-sequences and there were 24 trial-sequences in total for each condition. The prime and probe trials were presented for 1700 ms each. A fixation cross was displayed for 1700 ms between the prime trial and probe trial of the same sequence, and for 3400, 4250, or 5100 ms between different trial-sequences . The sexgeneral inhibition. Participants were instructed to respond to a frequent Go stimulus and to not respond to an infrequent No-go stimulus. They responded with the right index finger on a button-box , eight for the Negative Affective Priming task (NAP), and ten for the Go/No-go task. The practice trials involved different stimuli than the actual tasks .3). Online-scanner reconstruction was performed using the slice-GRAPPA algorithm [https://www.nmr.mgh.harvard.edu/software/c2p/sms). This GRAPPA sequence was selected to optimize the BOLD signal in frontoventral regions.Participants performed all paradigms inside the MR Scanner. Data were acquired at the 3 T Siemens Prisma Scanner at the Maastricht Brain Imaging Center, Maastricht University. Functional EPI images were collected using an in-house developed multi-echo multi-band sequence . The acquisition of anatomical images was done after the Go/No-go task (see above) to avoid cognitive fatigue.High-resolution anatomical images were acquired with a MPRAGE sequence .Prior to the pre-processing, the echo images were combined using an optimized echo weighting method . The imaTo analyze the activation pattern of every task an event-related approach was implemented using a GLM model and a random-effects group analysis. For the sexual tasks, we executed 2 x 2 ANOVA analyses with Stimuli (Sex vs Non-Sex) and Inhibitory/Non-Inhibitory  conditions as factors. Further basic contrast analyses were executed to specifically compare sexual inhibitory conditions against the non-sexual inhibitory conditions  and the sexual non-inhibitory conditions against the non-sexual non-inhibitory conditions . In the case of the Approach-Avoidance task, the resizing of the stimuli was the same in the conditions within each contrast , therefore keeping the intensity of the stimuli constant. Motion correction parameters were included as confound variables in the GLM. In the case of the Go/No-go task, we calculated the No-go > Go contrast to identify regions active in motor response inhibition. For the three tasks, only correct trials were analyzed, excluding the trials where the participants did not respond or committed errors. The resulting maps were corrected for multiple comparisons by means of cluster threshold level estimation . Only cConjunction analyses were conducted to investigate the common neural substrates underlying the different inhibition processes. We looked at the conjunction of the three processes as compared to their respective control condition (Sex Avoid > Dance Avoid ^ Sex Priming > Non-Sex Priming ^ No-go > Hits) and of the paired combinations . In addition we performed the conjunction analyses for the non-inhibitory conditions from the sexual tasks (Sex Approach > Dance Approach ^ Sex No Priming > Non-Sex No Priming). = 12.11, p = .002, η2 = .35) and an interaction between Stimulus Type X Response  = 10.84, p = .01, η2 = .37) but no main effect of Response  = .07, p = .79, η2 = .002). Consistent with previous studies [A two-way repeated measures ANOVA  revealed a significant effect of stimulus type  = 4.9, p = .04, η2 = .16; Priming: M = 867, SD = 150; No Priming: M = 842, SD = 171 ms), and to non-sexual trials compared to sexual trials  = 7.51, p = .01, η2 = .24; Sexual: M = 839, SD = 165 ms; Non-Sexual: M = 870, SD = 157 ms). No significant interaction was found between Stimulus Type and Priming condition  = .02; p = .88, η2 = .001; As expected, participants were slower to respond to Priming trials compared to No Priming trials (FParticipants committed on average 11 (SD = 14.46) misses and 7 (SD = 6.3) false alarms.A two-way ANOVA  showed a significant Stimulus Type X Response interaction effect in the the parahippocampal gyrus, the cuneus , and the lentiform nucleus extending to the amygdala  an increased activation was observed in the middle occipital gyrus, the fusiform gyrus, the posterior cingulate, the cerebellum, the hippocampus, and the parahippocampal gyrus extending to the amygdala and to the lentiform nucleus  showed a significant Stimulus Type X Priming interaction effect in the post-central gyrus  did not reveal any overlapping region.For the two sexual inhibitory processes (Sex Avoid > Dance Avoid ^ Sex Priming > Non-Sex Priming), the analysis revealed an overlap in the inferior frontal gyrus and in the inferior and middle temporal gyri only at a liberal threshold of significance (CLTC p < .05). We also performed the conjunction of the two non-inhibitory sexual conditions from the two sexual tasks (Sex Approach > Dance Approach ^ Sex No Priming > Non-Sex No Priming). This analysis showed a common activation in the anterior and posterior cingulate, in the thalamus, the precuneus, the inferior occipital gyrus and the lentiform nucleus  and motivational sexual inhibition (Approach-Avoidance), in addition to a classic Go/No-go paradigm to target general response inhibition. In a within-subject design, participants were required to execute all of these inhibition tasks while assessing their task-related whole-brain BOLD signal changes using fMRI.To our knowledge, this is the first neuroimaging study that directly compares the neural networks underlying general inhibition and two types of sexual inhibition with a within-subject within-session design. The paradigms that we selected to target general inhibition, cognitive sexual inhibition and motivational sexual inhibition differed considerably in their design. This is important as it has been argued that the neural network associated with classic inhibitory paradigms reflects non-inhibitory processes inherent to the design . Therefore, by using different designs we reduced the risk of finding common neural mechanisms of non-inhibitory psychological processes associated with one particular paradigm.Our findings demonstrate that whereas the motivational sexual inhibition is distinguished by a prefrontal hypoactivation pattern, cognitive sexual inhibition is characterized by activation in the ventromedial and inferolateral prefrontal regions. Nonetheless, both sexual inhibitory processes show a common activation in the inferior frontal gyrus and in the inferotemporal cortex. The general inhibition paradigm engaged the insula and the right inferior frontal gyrus, which is in accordance with previous literature , 20. We The inhibitory control to avoid sexual stimuli was characterized by a hypoactivation in the anterolateral prefrontal cortex, the inferior parietal lobe, the middle temporal gyrus, and the cuneus. Of particular relevance is the observed hypoactivation of the anterolateral prefrontal cortex. This region was previously engaged in a similar approach-avoidance paradigm, being active in incongruent conditions (approaching angry faces and avoiding happy faces) as compared to congruent conditions . It is iIt is possible that in our paradigm, a tonic inhibitory process was induced by the mere exposure of sexual pictures during the tasks, which was ‘released’ when participants deliberately aimed at avoiding the sexual images. Remarkably, the activation in this region correlated with the Approach-Avoidance main index. Individuals with a stronger inhibition of sexual stimuli (by avoiding them faster and/or taking longer to approach them) showed a stronger hypoactivation in the anterolateral prefrontal cortex during the sex avoiding trials. If it was the case that the anterolateral prefrontal cortex is sustaining a tonic inhibitory process, this would indeed explain why this mechanism was more active in individuals with a stronger motivational inhibition of sexual stimuli.In contrast to the inhibitory control condition, approaching sexual stimuli largely engaged subcortical areas  that have been associated with sexual arousal and penile tumescence . LesionsThe Negative Affective Priming paradigm allowed us to target the neural correlates of inhibiting sexual information at a cognitive level. The left orbitofrontal cortex, the fusiform gyrus, the middle temporal gyrus, the posterior cingulate, and the right inferior frontal gyrus were engaged during cognitive sexual inhibition. Although an inhibitory role has often been ascribed to the right inferior frontal gyrus and generally to the ventrolateral prefrontal cortex , previouThe left orbitofrontal cortex has been proposed to exert an inhibitory tonic control over sexual stimuli, as it has shown to be hypoactive during different sexual cognition paradigms. In addition, this proposed inhibitory mechanism is dependent on testosterone levels, as this pattern  is not observed in hypogonadal men and it is restored after testosterone administration , 40. In Finally, we found that the fusiform gyrus was also active during cognitive sexual inhibition. The inferotemporal cortex, where the fusiform gyrus is located, has been suggested to act as a tonic inhibitor in the control of sexual behavior, as its lesion or resection has led to hypersexuality , 41. HowWe posed the question whether inhibiting sexually driven motor actions and inhibiting sexually incoming information would share common neural substrates. The motivational sexual and cognitive sexual inhibitory conditions showed an overlap in the inferior frontal gyrus and in the inferior and middle temporal gyri. As was discussed before, although frequently associated with response inhibition , the excThe inferior and medial temporal gyri were also conjointly active during the two types of sexual inhibition. The posterior inferotemporal cortex has found to be active during the perception of sexual stimuli and to vary according to the levels of sexual arousal and penile tumescence –51. It iThe pattern of activation during the two sexual inhibitory conditions largely contrasts with the pattern of the sexual non-inhibitory conditions from the two tasks. The latter revealed a common activation in the anteromedial prefrontal cortex which is associated with the subjective experience of sexual arousal . In addiper se, general inhibition may influence non-sexual processes that ultimately relate to sexual behavior; for instance, a lack of inhibition in social contexts could lead to more sexual encounters.Given the coexistence of sexual and general inhibition deficits in different clinical conditions, we also investigated whether sexual inhibition shares common neural mechanisms with general inhibition. We did not find commonalities among cognitive sexual inhibition, motivational sexual inhibition, and general inhibition. The absence of common neural networks in sexual and general inhibition seems to contrast with clinical observations showing that frontal lobe damaged patients are unable to control both their sexual and non-sexual behavior. Different explanations may account for this finding: 1) The three inhibitory processes recruited different but adjacent portions of the ventrolateral prefrontal cortex, therefore an extensive lesion in the area would lead to a generalized impairment in different inhibitory modalities. 2) The Go/No-go paradigm lacks a socio-affective component; the general inhibition impairment reported in frontal lobe lesion patients often refers to a disregard for social or even moral norms –54. ThusAnother potential contradiction to our findings is that sexual compulsion is often comorbid with other disinhibition related behaviors such as substance abuse. Even if sexual and general inhibition recruited different neural substrates, some conditions could influence different networks indistinctively. For example, a deficit in dopamine would have an effect on cognitive, motor, and motivational circuits.While our study allowed the characterization of the neural circuits sustaining sexual inhibitory mechanisms, one should consider that a possible limitation of the study is that only male participants were tested. Since women are less vulnerable to sexual inhibition impairments such as in hypersexuality , and womRegarding the technical part of the method, future studies can benefit from two particular points in the acquisition and in the analysis of the data. First, future studies may benefit, as we did, from the use of fMRI sequences that optimizes the signal in frontoventral regions. These regions are generally susceptible to noise artifacts, but are nevertheless crucial in socio-affective processing. Second, although our design provided the spatial accuracy and functional specificity that was not given by other methods or paradigms, a better comprehension of the neural circuits sustaining sexual inhibition can be achieved by studying the network interactions through advanced methods such as dynamic causal modelling.Finally, it is noteworthy that the regions found to be common in the inhibitory and non-inhibitory conditions of the sexual tasks were only observed at a very liberal threshold of significance  which may lead to false positives. Therefore, these results and its interpretation should be considered with caution.In sum, this study did not support the existence of common general and sexual inhibitory networks. However, the inhibition of a sexually motivated driven action and the attentional inhibition of sexual information commonly engaged the inferior frontal gyrus and the posterior inferotemporal cortex. The specific functional properties of these regions, as well as those of the individual networks  remain to be studied in order to understand the distinct symptomatology and comorbidity of sexual disorders.


Human carboxylesterases (hCES) are key serine hydrolases responsible for the hydrolysis of a wide range of endogenous and xenobiotic esters. Although it has been reported that some ginsenosides can modulate the activities of various enzymes, the inhibitory effects of ginsenosides on hCES have not been well-investigated.In this study, more than 20 ginsenosides were collected and their inhibitory effects on hCES1A and hCES2A were assayed using the highly specific fluorescent probe substrates for each isoenzyme. Molecular docking simulations were also performed to investigate the interactions between ginsenosides and hCES.O-β-(d-glucosyl)-dammarenediol II (DMG) displayed potent inhibition against both hCES1A and hCES2A, while protopanaxadiol (PPD) and protopanaxatriol (PPT) exhibited strong inhibition on hCES2A and high selectivity over hCES1A. Introduction of O-glycosyl groups at the core skeleton decreased hCES inhibition activity, while the hydroxyl groups at different sites might also effect hCES inhibition. Inhibition kinetic analyses demonstrated that DM and DMG functioned as competitive inhibitors against hCES1A-mediated d-luciferin methyl ester (DME) hydrolysis. In contrast, DM, DMG, PPD and PPT inhibit hCES2A-mediated fluorescein diacetate (FD) hydrolysis via a mixed manner.Among all tested ginsenosides, Dammarenediol II (DM) and 20S-The structure–inhibition relationships of ginsenosides as hCES inhibitors was investigated for the first time. Our results revealed that DM and DMG were potent inhibitors against both hCES1A and hCES2A, while PPD and PPT were selective and strong inhibitors against hCES2A. Panax ginseng Meyer, one of the most popular edible herbs used in both eastern and western countries, has been found with many beneficial effects for human health. Modern pharmacological and pharmacodynamic researches have demonstrated that both ginseng extract and its major constituents (ginsenosides) can enhance memory, improve immunity, improve cardiovascular functions, delay aging and anti-tumor, etc. [Panax ginseng, and most of them are 20(S)-protopanaxadiol (PPD)-type and 20(S)-protopanaxatriol (PPT)-type ginsenosides [Ginseng, the root or rhizome of or, etc. –6. Over or, etc. –10. Ginsor, etc. , anti-tuor, etc. –14 and aor, etc. , 16. Untenosides  In many Asia countries, ginseng has been widely used as a key material for preparing dietary supplements, herbal medicines and cosmetics , 19. CurMammalian carboxylesterases (CES) are key hydrolases in most organs, which catalyze the hydrolysis of various esters (including endogenous and xenobiotic ones) into the corresponding alcohols and carboxylic acids –24. In tOver the past decade, ginseng-related products have been widely used in combination with a variety of therapeutic drugs to treat various types of cancer in clinical settings. In addition to enhancing efficacy and reducing drug toxicity, the accumulating evidence has indicated that ginseng products may bring benefits to the cancer patients, including increasing immunity and tolerance of cancer patient , 39. NotN--2-chloroacetamide (NCEN) and d-luciferin methyl ester (DME) was synthesized by us and the synthetic schemes have been reported previously [Twenty-five ginsenosides were purchased from Chengdu Pufei Biotech Co., Ltd.  and used in this study. Luciferin Detection Reagent (LDR) was obtained from Promega Biotech . Fluorescein diacetate (FD) was from TCI , eviously , 42. FD d-luciferin (the hydrolytic metabolite of DME) was measured (excitation wavelength 580 nm) by a multi-mode microplate reader . The calculation of residual activities of different inhibitors against hCES1A was performed as described previously [DME hydrolysis was used as the probe reaction to assay the inhibitory effects of 25 ginsenosides against hCES1A, oleanic acid was used as a positive control of inhibition assays . The inceviously .® iD3 multi-mode microplate reader. The kinetic parameters were set at 30 reads, with an interval of 30 s (30 min) and shake during 5 s before the first read at 37 °C. The calculation method for residual activities of different inhibitors against hCES2A have been reported previously [To determine the inhibitory effects of ginsenosides against hCES2A, FD was used as the probe substrate, while loperamide (LPA) was used as a positive control of hCES2A inhibition assays. The incubation system contains 2 μL HLM , 194 μL PBS (0.1 M pH 7.4) and 2 μL corresponding inhibitor. After pre-incubating the incubation mixture for 3 min at 37 °C, the reaction was started by adding FD  with a total volume of 200 μL, in which the final content of DMSO was limited to 2% (v/v). The hydrolytic metabolite of FD was quantified  by a SpectraMaxKi) and inhibition kinetic types of ginsenosides were determined. The experimental details used to determine the inhibition constants have been reported previously [According to the inhibition kinetics of both hCES1A and hCES2A, which were investigated by using varying concentrations of probe substrates in the presence of different concentrations of ginseng compounds (inhibitors), the inhibition constant  on hCES2A were investigated in living cells. The detailed processes for hCES2A inhibition assays in HepG2 cells have been depicted previously .Docking simulations were executed to simulate the interactions between ginseng constituents and hCES by using Discovery Studio . The detailed processes for docking simulation were depicted previously , 43.50 and Ki values with GraphPad Prism 7.0 software .All inhibition assays were performed in triplicate, nonlinear regression was used to determine the ICO-glycosyl groups on the core skeleton , were hardly inhibit the hCES1A-mediated DME hydrolysis and hCES2A-mediated FD hydrolysis in HLM even at high dosage (100 μM). By contrast, the ginsenosides without O-glycosyl groups (such as Dammarenediol II (DM), panaxadiol, panaxatriol, PPD and PPT), exhibited strong or moderate inhibition on both hCES1A and hCES2A. Of note, 20S-O-β-(d-glucosyl)-dammarenediol II (DMG), a ginsenoside with a O-glycosyl group at the Rʹ site, also displayed strong inhibition on both hCES1A and hCES2A.First of all, the inhibitory potentials of 25 ginsenosides against hCES1A and hCES2A were preliminarily screened. As shown in Fig. 50 values of each ginsenoside against hCES1A and hCES2A were depicted using varying inhibitor concentrations  against hCES1A in HLM was also determined as 0.10 μM  against hCES2A in HLM was determined as 1.46 μM  on the skeleton  will lead to the loss of hCES1A inhibition, with the exception of DMG (IC50 = 1.76 μM).For the ginsenosides with skeleton A or skeleton C, introduction of any O-glycosyl group or introduction of a O-glycosyl group at the R2 site will abolish the hCES2A inhibition, such as ginsenoside C-K (IC50 = 15.46 μM) VS ginsenoside Re (IC50 > 100 μM), ginsenoside Rg1 (IC50 > 100 μM) and ginsenoside R1 (IC50 > 100 μM), PPD (IC50 = 1.34 μM) VS ginsenoside Rg2 (IC50 > 100 μM) and ginsenoside Rh1 (IC50 > 100 μM).For the ginsenosides with skeleton A, replacing the hydroxyl groupby a 3 site of the ginsenosides with skeleton A significantly reduces the inhibitory effects on hCES1A, such as DM (IC50 = 1.99 μM) VS PPD (IC50 > 100 μM), and DMG (IC50 = 1.76 μM) VS ginsenoside C-K (IC50 > 100 μM).Addition of a hydroxyl group at the R2 site reduces inhibitory effects on both hCES1A and hCES2A (about 3.8–5.0-folds), such as panaxadiol (IC50 = 6.95 μM) VS panaxatriol (IC50 = 34.93 μM) for hCES1A, and panaxadiol (IC50 = 3.78 μM) VS panaxatriol (IC50 = 14.59 μM) for hCES2A.For the ginsenosides with skeleton B, introduction of a hydroxyl group at the RO-glycosyl group does not affect hCES1A inhibition but reduces hCES2A inhibition, such as DM (IC50 = 0.69 μM) VS DMG (IC50 = 1.06 μM), PPT (IC50 = 0.86 μM) VS ginsenoside F1 (IC50 = 37.08 μM), Rh2 (IC50 = 5.35 μM) VS F2 (IC50 = 14.37 μM), as well as PPD (IC50 = 1.34 μM) VS C-K (IC50 = 15.46 μM).For the ginsenosides with skeleton A, replacing the hydroxyl group at the Rʹ site by a 1 site is essential for hCES2A inhibition, the presence of a monosaccharide or polysaccharide group at this site will decrease hCES2A inhibition activity, such as PPD (IC50 = 1.34 μM) VS Rg3 (IC50 = 10.37 μM), PPD (IC50 = 1.34 μM) VS Rh2 (IC50 = 5.35 μM).For the ginsenosides with skeleton A and skeleton B, the hydroxyl group at the R3 site will decrease hCES2A inhibition, such as DM (IC50 = 0.69 μM) VS PPD (IC50 = 1.34 μM), and DMG (IC50 = 1.06 μM) VS C-K (IC50 = 15.46 μM).For the ginsenosides with skeleton A, introduction of a hydroxyl group at the RAfter comprehensively analyzing the inhibitory effects of all tested ginsenosides on both hCES1A and hCES2A, the structure–inhibition relationships could be summarized as follows,O-glycosyl group(s) on the ginsenoside basic skeleton  is unbeneficial for hCES1A inhibition, while introduction of the O-glycosyl group(s) at the R2 site on skeleton A will lead to the loss of hCES2A inhibition. Replacing the O-glycosyl group at the Rʹ site by a hydroxyl group will enhance the inhibition potency of hCES2A and the specificity over hCES1A, but introduction of a hydroxyl group at the R3 site will decrease hCES2A inhibition. These findings will be of advantage to the design and development of more potent and specific ginsenoside-type inhibitors against hCES2A.Collectively, introduction of the Ki values of 2.10 μM and 2.40 μM, respectively  and a modeling hCES2A structure (accession number O00748) as basic models , 47. As 309 via hydrogen bonding in the active site, while the hydroxyl groups at C-2 site and C-7 site of PPT could create strong interactions with several key residuals located in the active site or the Z site of hCES2A, such as MET309, PRO260 or GLN408, via hydrogen bonding. But the similar effects were not shown between hCES1A and hydroxyl groups at C-2 site and C-7 site. As a result, the LibDockScores of these four ginsenosides on hCES2A binding on two distinct ligand-binding sites are similar to each other  on the skeleton A or skeleton C (including the C-2 and C-7 sites) may lead to the loss of hCES1A inhibition, suggesting that the O-glycosyl group at the C-2 and C-7 sites should be replaced by other groups that may create strong interactions with hCES1A. Furthermore, in view of the naturally occurring ginsenosides could be easily available via isolation from herbs or using synthetic biology techniques [O-β-carboxypropionyl or ethyl ester on these ginsenosides, should be used for the development of next generation hCES2A inhibitors bearing the ginsenoside skeleton [From the viewpoint of chemical structure, the ginsenosides have several hydroxyl groups that could be easily modified to generate a series of derivatives. On the basis of SAR of ginsenosides as hCES2A inhibitors, the hydroxyl group at the C-13 site is beneficial for the substrate specificity on hCES2A over hCES1A, which can be used for the development of novel ginsenoside-type hCES2A inhibitors with excellent specificity. Meanwhile, our findings show that the introduction of any chniques , 68, thechniques , 69, whiskeleton . In addiskeleton , 71, 72.O-glycosyl group(s) on the skeleton A or skeleton C (including the C-2 and the C-7 sites) would lead to the loss of hCES1A inhibition. Among all tested ginsenosides, DM and DMG displayed relatively strong inhibitions on both hCES1A and hCES2A. Moreover, the gut metabolites of naturally occurring ginsenosides (such as PPD and PPT) exhibited potent and highly specific inhibition on hCES2A, suggesting that these two compounds could serve as ideal lead compounds to develop novel hCES2A inhibitors. Molecular docking simulations demonstrated that PPD and PPT could be well-docked into both the catalytic cavity and the Z site of hCES2A. In short, our results demonstrated that some ginsenosides exhibit strong to moderate inhibition on hCES2A, which hold great promise in modulating the pharmacokinetic profiles and treatment outcomes of hCES2A-substrate drugs, such as irinotecan. Our findings provided novel insights into the interactions between ginsenosides and CES, which were very helpful for the rational use of traditional herb ginseng in clinic, in order to avoid the potential risks of HDI and HEI via inhibition of hCES.In summary, the inhibitory effects of 25 ginsenosides on hCES as well as the structure–inhibition relationships of ginsenosides as hCES inhibitors, were revealed for the first time. The results clearly demonstrated that the hydroxyl group at the C-13 site is beneficial for the substrate specificity on hCES2A, while the introduction of any Additional file 1: Table S1. The Docking simulations of DM, DMG, PPD and PPT on hCES2A. Fig S1. The dose-inhibition curves of DM, DMG, panaxadiol and panaxotriol against hCES1A-mediated DME hydrolysis in HLM. Fig S2. The dose-inhibition curves of oleanic acid and LPA. Fig S3. The dose-inhibition curves of ginsenosides against hCES2A-mediated FD hydrolysis in HLM. Fig S4. The inhibitory effect of PPT on hCES2A in living HepG2 cells. Fig S5. Docking simulations of DME into hCES1A in the active site. Fig S6. 2D representation of the interactions between DM, DMG and the residuals in the active site of hCES1A. Fig S7. The docking simulations of FD into hCES2A in the active site. Fig S8. 2D representation of the interactions between DM and the residuals in the active site or the Z site of hCES2A. Fig S9. 2D representation of the interactions between DMG and the residuals in the active site or the Z site of hCES2A. Fig S10. 2D representation of the interactions between PPD and the residuals in the active site or the Z site of hCES2A. Fig S11. 2D representation of the interactions between PPT and the residuals in the active site or the Z site of hCES2A. Fig S12. 2D representation of the interactions between PPD and the residuals in the active site or the Z site of hCES1A. Fig S13. 2D representation of the interactions between PPT and the residuals in the active site or the Z site of hCES1A.


Chondrosia reniformis Nardo, 1847  in the proximity of floating fish cages. In a trial series, survival and growth of cultured explants were monitored near a polluted fish farm and a pristine control site. Attachment methods, plate materials, and plate orientation were compared. In a first trial, chicken wire-covered polyvinyl chloride (PVC) was found to be the most suitable substrate for C. reniformis . During a second trial, survival on chicken wire-covered PVC, after six months, was 79% and 63% for polluted and pristine environments, respectively. Net growth was obtained only on culture plates that were oriented away from direct sunlight (39% increase in six months), whereas sponges decreased in size when sun-exposed. Chicken wire caused pressure on explants and it resulted in unwanted epibiont growth and was therefore considered to be unsuitable for long-term culture. In a final trial, sponges were glued to PVC plates and cultured for 13 months oriented away from direct sunlight. Both survival and growth were higher at the polluted site  than at the pristine site . These results represent a first successful step towards production of sponge collagen in integrated aquacultures.In this study, novel methods were tested to culture the collagen-rich sponge  Spongia officinalis (Linnaeus) and Hippospongia communis (Lamarck) [The first attempts to farm sponges date back to the 19th century, presumably as a consequence of periodical depletion of “bath-sponge” stocks , or—in mLamarck) ,4,5,6. SLamarck) ,8,9; seeLamarck) ,11,12), Lamarck) ,14). Spo3 cm−3 sponge s−1 . ,26. 3 cmDysidea avara  and Chondrosia reniformis Nardo, 1847 , were cultured at a pristine site  and an organically polluted fish farm site, the latter sponges being directly cultured underneath an open cage fish farm. D. avara was chosen since it produces the bioactive compound avarol, a potential anti-psoriasis agent [Chondrosia reniformis synthesizes large amounts of collagen, which is suitable for cosmetic and medical applications [C. reniformis [Chondrosia reniformis showed better growth and survival rates at the pristine site, whereas D. avara grew and survived better at the polluted site [C. reniformis at the polluted site were largely due to the farming protocol used. Chondrosia reniformis is a highly plastic sponge, being able to de-attach and move around [C. reniformis were put inside cages on the seafloor [In 2006–2007, an integrated mariculture approach using sponges was tested in the coastal waters around the Bodrum Peninsula, Turkey . Two Medis agent ,28. Chonications ,30,31. Tniformis ,33 and tted site . The lowe around , a phenoe around ,8,35,36.seafloor . HoweverC. reniformis in an integrated multi-trophic aquaculture approach, i.e., by culturing the sponges in the vicinity of offshore floating fish cages. Using thin plastic plates as substratum, a series of consecutive trials were executed, aimed at developing an optimal, species-specific culture method. We monitored survival and growth rates of cultured explants of C. reniformis, thereby comparing a polluted fish farm site to a pristine site. Variables studied included methods for attaching explants to plates, plate materials and plate orientation. The culture methods  were applied previously on other sponge species by several authors; for detailed information, see review by Duckworth et al. [This study describes progress towards the development of a raw collagen production pipeline using the sponge h et al. .C. reniformis was selected as a pre-culture and initial testing site . Based on water visibility  and org−1) to remove dissolved inorganic carbon species. The acidified samples were supplemented with sodium tetraborate and potassium persulphate and processed using segmented flow analysis (SFA) on a Continuous Flow Analyser . In SFA, TOC is first oxidized using UV light and then measured as CO2 while using infrared detection. The TOC detection limit of the method, as intercalibrated with other labs, is 25 μmol L−1, the average TOC variation among replicate measurements is 10 μmol L−1. The internal standards used were 3.3 μmol L−1 EDTA, 3.3 μmol L−1 of a humic acid, and 3.6 μmol L−1 phenylalanine.Water temperature  and visibility were measured 17 times during periodic visits at the polluted site throughout the experimental period from April 2011 to December 2013. To determine organic loading, three replicate water samples (50 mL) were taken within 10 m from the culture platforms by SCUBA diving from each location for TOC analysis using the wet oxidation method ). TOC sa2 with an average thickness of 2 cm, and all of the explants had at least one osculum . Explants were stored in perforated plastic containers that allowed water flow and they were left underwater until the seeding operations started. To enable sponges to attach and acclimatize after seeding, the seeding plates with the explants were left horizontally next to the culture platforms for three days before the plates were secured to their spots on the culture frame [Sponge specimens were collected by SCUBA at 5–10 m water depth in the Bay of Meteoroloji  Pr.1. Exre frame .C. reniformis.Within the period between April 2011 and December 2013, three subsequent mariculture trials were executed in order to develop and improve a culture method for n = 5 specimens per plate) were attached to four types of supports , black PVC, and cemented PVC) using six different combinations of attachment methods and substrates   were positioned on both sides of five 50 × 50 cm plates, with 25 explants on each side of every plate. In order to find the optimal positioning of the sponges, explants were cultured at nine different angles, under light (exposed side of plate) or dark (underside of plate) conditions, resulting in 10 different conditions (C. reniformis (5 per plate), attached using gel-based polyacrylate superglue. A gel-based polyacrylate superglue method was preferred over chicken wire to improve the handling time and reduce the weight and cost of the culture materials. In addition, horizontal blue polypropylene PP plates were placed underneath the 90° PVC plates to provide extra surface for the explants to attach and grow, should they fall  and a Sigma 10–20 mm wide-angle lens set , coupled with dedicated Ikelite housing and an DS160 substrobe . Survival was calculated from the initial and final number (N) of explants residing on the PVC and/or PP plates, as follows:For Trial 3, as described above, blue PP plates were used to collect detached sponges. To calculate survival data, sponges that had fallen onto the PP plates were pooled with the sponges that remained on the PVC plates. However, fallen sponges were excluded from the growth analysis, as PP may affect sponge growth differently from PVC.For Trial 2, explant growth rates were calculated by measuring wet weights by using a scale (Sinbo SKS 4514) at the start and end of the experiment. To reduce measurement error, the sponges were briefly wiped with clean paper to remove seawater for a duration of approximately 10 s. Growth was calculated from initial and final explant wet weights (WW) as follows:For Trial 3, growth was monitored by underwater photography using the same setup, as described above, for the monitoring of survival. Following each dive, recorded images were transferred to Photoshop CS5 software  and lens distortion was corrected using a Camera Raw 6.7.1 plug-in . The images were calibrated using known plate dimensions, peripheries of explants were marked, and surface area was calculated from pixel counts of the marked areas . Growth To assess the correlation between surface area growth to both biomass and volumetric growth, an additional 20 sponges of random size were collected from a neighboring site. For all 20 sponges, the wet weights were determined, as described above. In addition, volume was determined by measuring displaced seawater in a graduated cylinder. Finally, surface area was determined by using photography and a ruler as a reference, and photographs were processed, as described above.p > 0.05). Student’s independent t-test was used to determine growth differences between the light and dark group in the second trial, with n = 5 plates for both groups. A two-way mixed factorial ANOVA was used to determine the main and interactive effects of culture site and time on C. reniformis growth in the third trial, with culture site as a between-subjects factor, and time as a within-subjects factor. In all analyses, the culture plate was considered as an experimental unit, i.e., data of explants growing on a single plate was pooled. To correlate surface area to mass and volume, Pearson’s product-moment correlation was used. A p-value of less than 0.05 was considered to be statistically significant. Statistical analysis was performed with SPSS Statistics 22.0 . Graphs were plotted with SigmaPlot 12 .The normality of data was tested by plotting the residuals of each dataset versus the predicted values, and by performing a Shapiro-Wilk test. Homogeneity of variances was determined using Levene’s test. All data were found to be normally distributed and showed homogeneity of variance after a log10 transformation (−1) were 2.4 times higher than the TOC levels at the pristine site (110 ± 0.01 µmol L−1).Visibility was on average 3.8 times lower at the polluted site  when compared with pristine site . The 90° plate was selected as the preferred culture orientation in Trial 3, based on the survival rate and the ease of operation  PVC plates were used in Trial 3 and superglue was selected as the attachment method.The explants at the polluted site  and prisC. reniformis explants is presented in 1,27 = 55.550; p < 0.001), and for the pristine site even stalled after six months. Surface increase was significantly higher at the polluted site as compared to the pristine site , irrespective of time.During the first week of the experiment, 69 (polluted) and 70 (pristine) out of 200 explants dropped off the PVC plates. Fortunately, the PP plates that were placed under the PVC plates were able to catch 55 (at polluted site) and nine (at pristine site) of these explants, which attached onto the PP plates and continued to increase their surface area. Because they could not be related anymore to their original size, explants that were attached on the PP plates were left out of the surface area increase analysis. However, they were included in calculation of survival rates, which were highly different between the pristine and polluted sites  and culture angle , the third trial showed low survival for the pristine site. This was probably due to occasional strong currents that prevail at this site, which may make the explants more prone to dropping of the plates and physical removal from the site. During the whole month of September 2013, flow velocities above 20 cm/s were recorded at this site by analyzing the velocity of neutrally buoyant particles . Chondrosia reniformis inhabits both nearly stagnant to occasional high flow waters , however the attachment of explants to PVC plates is probably less firm than attachment to natural substrates, especially during the acclimatization time after wounding them to explant the parent sponges. At the polluted site, the use of glue instead of chicken wire did slightly improve long-term survival rate, which shows that gluing is a suitable method to attach explants of C. reniformis. It is also the fastest and easiest method. A future recommendation is to perform the initial acclimatization  gave ambiguous results. Sandwiched mesh structures were designed to promote the explants to grow out of the pocket and to ease harvesting . Mesh cuays; see ) at a moDuring culture Trial 2, initial sponge survival was compromised by disease-like phenomena. Bacterial infections, which were possibly due to late seeding of explants in Mid-June with relatively higher water temperatures, might have been responsible for the initial losses at both sites. High water temperatures in summer have been reported to be a risk for sponge mariculture in temperate and subtropical climates ,39,41,43C. reniformis prefers shaded habitats.Culture angle directly affected explant survival, mainly in association with prevailing light levels. Lower light levels at the more turbid polluted site may, therefore, also explain the higher explant survival at the light-exposed angles at the polluted site. These results corroborate the findings of , which pC. reniformis showed a size-independent relationship with wet weight and volume, surface area can be used as a proxy for growth. This enables a direct comparison of growth data obtained for this species using different methods.Since surface area of C. reniformis has been considered to be difficult, to even unsuitable with the methods applied [C. reniformis was cultured under shaded conditions. Ref. [C. reniformis on the bottom of metal wire cages under pristine conditions, but this study failed to achieve such results at a fish farm site as the explants cultured were smothered by effluents from the fish farm. Conversely, the current study demonstrates that if cultured using an appropriate method, C. reniformis will survive and grow (up to 170% in 13 months), even in a fish farm environment with a considerable particle load. These growth rates are considerably higher than those reported for naturally growing specimen. Garrabou and Zabala [C. reniformis, which was an order of magnitude lower than the growth rate of three other Mediterranean sponge species in their study Hemimycale Ccolumella (Bowerbank), Oscarella lobularis, and Crambe Crambe (Schmidt). They ascribed the slow growth rate of C. reniformis to a greater energy investment in tissue production per unit area as a result of its thick collagenous cortex. However, the data found by Osinga et al. [C. reniformis exhibits growth rates that are nearly two orders of magnitude higher than the in situ rates reported by Garrabou and Zabala [C. reniformis. The highly variable growth of C. reniformis under different conditions and the high variability within treatments highlight the need for further optimization studies.Culture of  applied ,8. Wilki applied  reportedns. Ref.  obtainedd Zabala  reporteda et al.  and thosd Zabala . Under oC. reniformis surface area increase rates were significantly different between culture sites, with an approximate two-fold higher growth at the polluted site. This may relate to the higher food availability—i.e., higher TOC concentration as a result of fish farm activities—and, as mentioned earlier, correspondingly lower light levels at the polluted culture site. Hence, the combination with fish farming is potentially beneficial for culture success of this sponge species. The surface area increase of C. reniformis was clearly higher in the first six months after initiation of the cultures, regardless of culture site. Although this may partially be explained by seasonal effects , it d winter . This cod winter , which cd winter . Fast inC. reniformis during the first two trials. Chondrosia reniformis explants attached to PVC plates tend to move on the plate, thus obfuscating multiple genotypic comparisons on one plate . In addition, fusion and fission of explants makes the proper assessment of survival difficult. Even though attachment to the PVC plates has succeeded, some C. reniformis still found ways to divide their body into several parts, moved around the PVC plate (possibly in pursuit of shaded areas), or dropped themselves to the ground possibly in search for better living conditions. Survival and growth is best at culture angles of 90° and above, where explants are not being exposed to direct sunlight, as C. reniformis performs better at low illumination levels.As stated in Schippers et al. , initialIn experiments 2 & 3, the initial losses and/or droppings of explants were slightly high and unpredictable, despite the variety of methods applied. Once attached for a longer time, the explants would remain attached. Therefore, initial losses and/or droppings are the main problem to be solved to secure better culture performance. Restraining bacterial attack on freshly cut explants by initiation of cultures early in the season (spring) and preventing exposure to high currents during the first months should be practiced together with the best performing methods regarding attachment.C. reniformis in sea-based aquaculture under turbid conditions:Culture method: Sponge explants cut from parent sponges are glued to PVC plates using gel-based polyacrylate superglue. PVC plates are best positioned vertically onto frames and they should be extended with a basket on the bottom site to recover explants falling off the plates. Chicken wire may be applied during the first few weeks after explanting to prevent early losses but should be removed once the attachment is stable. Prolonged use of chicken wire cover tends to hold sediments and promotes epibiont growth and hence undesired space competition with the cultured sponge.C. reniformis on vertical plates, making this sponge an interesting candidate for integrated multitrophic mariculture. Daily fish feeding activities and occasional net replacing hinders the use of culture platforms inside the fish farm area. Thus, sponge culture platforms have to be placed outside boat traffic area. To eliminate this problem, one method that we consider for future applications is using layered scallop lanterns placed in between an anchor and a submerged buoy system (just outside the fish farm culture area), a method that was successfully applied by both Duckworth et al. [Latrunculia wellingtonensis, Polymastia croceus, and (Heterofibria) manipulatus, respectively.Site selection: Sites should not be prone to strong fluctuations in weather. The area should be secured and should be clear of boat traffic and anchoring ,11. Siteh et al.  and Kellh et al. , for LatC. reniformis in the Mediterranean is best done in either spring (April-May) or autumn (October-November) to prevent bacterial infections following cutting of explants from parent sponges.Seasonality: Initiating a culture of Based upon the three mariculture trials described above, the following best practices have been deduced for culturing Mycale (Carmia) hentscheli (Bergquist & Fromont) [−1 to 1355% year−1 from the first to the third culture period. Moreover, the growth rates of cloned sponges harvested from cultured explants should also be followed, as Page et al. [Culture success can be further improved by optimizing the period of culture. Optimal culture time can be determined by observing sponge growth rates over a period of two or more subsequent years. Page et al. , found rFromont)  over time et al. , found rOther important aspects to include in future studies are seasonality  and genotypic variability.


Macrolide antibiotics are commonly prescribed treatments for drug-resistant bacterial infections; however, many macrolides have been shown to cause liver enzyme elevations and one macrolide, telithromycin, has been pulled from the market by its provider due to liver toxicity. This work seeks to assess the mechanisms responsible for the toxicity of macrolide antibiotics.in vitro systems designed to test for bile acid transporter inhibition, mitochondrial dysfunction, and oxidative stress. The macrolides were then represented in DILIsym, a quantitative systems pharmacology (QST) model of drug-induced liver injury, placing the in vitro results in context with each compound’s predicted liver exposure and known biochemistry.Five macrolides were assessed in DILIsym results suggest that solithromycin and clarithromycin toxicity is primarily due to inhibition of the mitochondrial electron transport chain (ETC) while erythromycin toxicity is primarily due to bile acid transporter inhibition. Telithromycin and azithromycin toxicity was not predicted by DILIsym and may be caused by mechanisms not currently incorporated into DILIsym or by unknown metabolite effects.The mechanisms responsible for toxicity can be significantly different within a class of drugs, despite the structural similarity among the drugs. QST modeling can provide valuable insight into the nature of these mechanistic differences.The online version of this article (10.1007/s11095-019-2582-y) contains supplementary material, which is available to authorized users. Saccharopolyspora erythraea in the 1950s. Clarithromycin and azithromycin are the second generation macrolides; they are semi-synthetic derivatives of erythromycin. Erythromycin, clarithromycin, and azithromycin have been reported to cause mild, asymptomatic elevations in serum alanine aminotransferase (ALT) in 1–2% of the population  and otherwise drug-resistant bacterial infections. In multi-center phase III clinical trials with CABP patients, solithromycin showed promise by proving non-inferiority to moxifloxacin ,9. Howevin vitro mechanistic toxicity assays with estimates of in vivo exposure and known biochemistry to understand hepatotoxicity and the biochemical processes behind it  is a discipline of pharmacology that seeks to understand and ultimately predict the toxic effects of drugs/chemicals by integrating computational and experimental methods . DILIsym it Fig.  13–15).–15.in vi it Fig. ,16–18. T it Fig. ,19. In tDILIsym v5A was used to conduct the simulations in this paper. DILIsym is a software package that is available to members of the DILI-sim Initiative; academic and regulatory licensing is also available.PBPK models for the five macrolide antibiotics were constructed within DILIsym to describe disposition of macrolides in humans. For solithromycin, the PBPK model was based on plasma concentration-time data from clinical trials; literature reports of plasma time courses were used for azithromycin, telithromycin, erythromycin, and clarithromycin. The basic structure of the DILIsym PBPK sub-model has been discussed elsewhere ,17,19,20in vitro assays for three main hepatotoxicity mechanisms represented within DILIsym: mitochondrial dysfunction, oxidative stress, and bile acid transporter inhibition. To detect potential mitochondrial dysfunction signals, cellular respiration assays were conducted using a Seahorse XFe96 Flux Analyzer in HepG2 cells incubated with various concentrations of macrolides for 1 or 24 h. HepG2 cells were chosen in part because of their metabolic incompetence, since any effect observed in the HepG2 system can be attributed solely to the activity of the parent compound. Induction of oxidative stress was determined by high content screening using a fluorescent probe, dihydroethidium (DHE), in HepG2 cells incubated with various concentrations of macrolides for 1 or 24 h. In these whole cell-based assays, intracellular concentrations of macrolides were determined by LC/MS/MS analysis in parallel HepG2 cultures. Parameter values for macrolide mediated induction of mitochondrial dysfunction and oxidative stress were determined by reproducing the cellular respiration data and the oxidative stress data directly within DILIsym using measured intracellular concentrations. Inhibitory effects of macrolides for bile acid transporters were assessed experimentally using membrane vesicles overexpressing a bile acid efflux transporter  and CHO cells overexpressing NTCP or obtained from published literature. Detailed experimental methods are described in Supplement B. Mitochondrial dysfunction and oxidative stress assays were performed by Cyprotex, Inc. . Transporter inhibition assays were performed by Solvo Biotechnology .Five macrolides were assessed in 50 was used directly as the inhibition constant. Mode of inhibition was assumed to be mixed inhibition with α = 5. While competitive and non-competitive inhibition types may result in low and high extremes of potential bile acid accumulation, respectively, mixed inhibition with α = 5 leads to a median impact on bile acid accumulation. In addition, mixed inhibitors are more common compared to pure competitive or noncompetitive inhibitors. For mitochondrial dysfunction, the assay results comparing intracellular concentrations and OCR were recapitulated in MITOsym if the OCR decline was non-saturable and in DILIsym if the OCR decline was saturable; the resulting parameters were translated into DILIsym parameters using translation factors involving exemplar compounds, a process which has been reported elsewhere  included in DILIsym v5A. This SimPops represents variability in parameters related to bile acid homeostasis, mitochondrial function, oxidative stress, apoptosis, and regeneration. A list of parameters varied in the v4A_1 SimPops, as well as the sources used in the construction of the SimPops, are shown in Table Mechanistic Investigation simulations: These simulations were conducted on a subset of simulated individuals found to be most susceptible to the toxicity from each antibiotic. Then one of the three mechanisms is inactivated for each of the Mechanistic Investigation simulations while the other mechanisms remain active; if inactivating the mechanism leads to a decrease in the number of simulated individuals in which ALT elevations occur, the mechanism is determined to be contributing to the toxicity; the relative magnitude of the decrease in ALT elevation frequency represents the relative contribution of each mechanism to the overall simulated toxicity  in a concentration-dependent manner after 1 and 24 h incubation, whereas erythromycin did not inhibit cellular respiration at both time points. Telithromycin, clarithromycin, and azithromycin decreased basal OCR following 24 h incubation, but not after 1 h incubation . This is an interesting result because these five molecules are all in the same class of drug and all somewhat structurally similar; however, they are clearly mechanistically distinct from one another with regard to their hepatic effects. The simulation results therefore demonstrate that one should not draw conclusions about the mechanisms of toxicity – or about the frequency thereof – for a molecule based on the fact that it is part of the same class of drug as another molecule that causes toxicity via a known mechanism.Macrolide antibiotics have been associated with varying levels of liver injury, but the underlying mechanisms have not been elucidated. In the current study, QST modeling was employed to integrate For telithromycin, in particular, it was not surprising that DILIsym failed to predict ALT elevations. ALT elevations were rather rare in the clinic with telithromycin, and indeed ALT elevations were less frequent during treatment with telithromycin than with the other macrolides . The sevin vitro assays did not map directly to the mechanisms that were responsible for toxicity. For example, each of the five macrolides demonstrated some response in the oxidative stress assay, but ROS was found to contribute only to erythromycin toxicity by the simulations. The combination of in vitro assay results with measures of exposure has been shown to produce an increased ability to predict liver toxicity beyond that of the assay alone  process for the toxicity parameters is also a plausible reason for the lack of predictivity for telithromycin and azithromycin; if the estimate of intracellular:extracellular concentration ratio derived from the mass spectrometry assay is significantly different from that which occurs in vivo, for example, the toxicity parameter derivation may be affected as a result. However, the magnitude of the assay uncertainty necessary for this effect to be able to explain the missed predictions of telithromycin and azithromycin is quite large, as suggested by dose escalation simulations conducted on both compounds (data not shown). As a result, we view this as a less likely contributor than alternative mechanisms, patient effects, or metabolite effects.One limitation of this work is that the metabolites of the macrolides were not investigated for their potential toxic effects. The results suggest that it is unlikely that metabolites of solithromycin, clarithromycin, and erythromycin are contributing to the observed ALT elevations; the liver responses for these molecules were adequately explained by the effects of the parent compound. However, metabolite effects may be contributing to azithromycin and telithromycin toxicity, since these compounds’ toxic responses were not adequately explained by parent effects due to the mechanisms represented in DILIsym. Furthermore, the simulations were conducted in a SimPops intended to represent normal healthy volunteers; the ALT elevations were almost all observed in individuals with some sort of bacterial infection. It is unclear whether infected individuals demonstrate a different level of drug exposure for many of the macrolides, though elderly individuals and individuals with community-acquired bacterial pneumonia have been shown to have a higher plasma concentration of telithromycin than normal healthy volunteers . Differein vitro experiments and assess the likelihood that five macrolide antibiotics cause serum ALT elevations by oxidative stress, mitochondrial toxicity, and bile acid accumulation. DILIsym found that solithromycin and clarithromycin ALT elevations can be primarily accounted for by mitochondrial ETC inhibition whereas erythromycin ALT elevations can be primarily accounted for by inhibition of bile acid transporters. Bile acid transporter inhibition may also in part account for ALT elevations caused by telithromycin, but the model predictions were poor for both telithromycin and azithromycin. This may reflect effects of metabolites of these drugs or involvement of mechanisms not included in DILIsym. This research demonstrates that despite the fact that these five drugs are all in the same class, they are mechanistically distinct from one another concerning their hepatic adverse effects.In conclusion, DILIsym was used to contextualize novel ESM 1(MAT 82 kb)ESM 2(MAT 82 kb)ESM 3(MAT 82 kb)ESM 4(MAT 3384 kb)ESM 5(MAT 83 kb)ESM 6(DOCX 684 kb)