This study presents the results of an experimental and numerical program carried out on unreinforced masonry panels strengthened by textile-reinforced mortar (TRM) plastering. For this purpose, five panels were constructed, instrumented and tested in diagonal shear mode. Two panels were tested as reference. The first reference panel was left unstrengthened, while the second one was strengthened by a traditional self-supporting cement mortar matrix reinforced with steel meshes. The remaining three panels were strengthened by TRM plastering applied on one or both faces and connected with transversal composite anchors. The numerical and the experimental results evidenced a good effectiveness of the TRM systems, especially when applied on both panel facings. Unreinforced masonry (URM) has a long history of being the predominant building technique in Romania and in Europe. A large share of this existing building stock consists of historical monuments, which have been designed to resist only gravitational loads or, in many cases, have not been designed at all, but simply erected according to a few generic rules of common practice ,4,5,6,7.During seismic excitation, URM structures can develop two possible types of dominant failure modes, the in-plane shear or the out-of-plane bending mechanisms ,14. The Originally, the reinforced concrete strengthening techniques were adopted for URM elements as well . These tAccording to previous studies ,25,26,27By studying the data presented in The experimental campaigns cited in Moreover, despite the available studies carried out to investigate the shear structural behavior of the URM strengthened with TRM, which highlighted the effectiveness of this type of strengthening system, some important aspects have still not been fully investigated. The latter may refer to anchorage methods for masonry walls which enable intervention works on both faces (the side-to-side connection) or on a singular face (the middle-to-side connection); the increase in lateral capacity based on the type and configuration of the transversal connectors, pattern and effective length for various types of anchorages; the characteristic failure modes for the systems with additional reinforcement due to the radial ends of the anchorages; and the synchronization between the experimental and numerical analyses based on adapted modelling strategies.This paper reports the results obtained through a numerical and experimental program that was carried out at the Laboratory of Heavy Structure, located at the Faculty of Civil Engineering and Building Services in Iași. The masonry materials and the strengthening systems, both traditional and modern, are rather common in Romania. In particular, five URM panels were constructed, instrumented and tested in diagonal shear mode. In addition, all the specimens were micro-modelled and subjected to a nonlinear finite element analysis using the Ansys Workbench software. Two panels were tested as reference. The first reference panel was left unstrengthened, while the second one was strengthened through a traditional self-supporting cement mortar matrix reinforced with steel meshes. The remaining three panels were strengthened by TRM plastering applied on one or both faces and connected with transversal composite anchors. After a detailed experimental set-up and testing procedures, the main experimental results and the key features of each configuration of the strengthening system are reported. In addition, the experimental results are compared to the ones obtained through a micro-detailed finite element analysis and a general good agreement is found.The experimental program was performed on five 1200 × 1200 × 115 mm URM panels. The specimens were manufactured using brick clay masonry units  and normThe URM panels were left to cure for 28 days. Prior to strengthening and testing, the mechanical characteristics of the masonry material were determined and compared to the ones provided by the manufacturer. Bricks were the first component of the URM panels to be investigated.Tests to determine the compressive strength were performed on five real-size specimens , which wThe aim of the micro, non-linear analysis that is presented in Flexural and compression tests were performed to evaluate mortar strength, according to EN 1015-11  Figure . The flem,t—was approximately 3.66 MPa, while the average value of the compressive strength—fm,c—was 18.61 MPa. The computed standard deviations  indicate that the values tend to be close to the mean. Thus, it can be concluded that the experimentally determined values showed no statistically significant differences.The results were determined under the assumption of general elastic–brittle behavior of the masonry wall loaded in tension. The average value of the tensile strength—fThe experimental values were introduced as input data for the mechanical characteristics of the micro non-linear 3D model defined in As mentioned before, two masonry panels were manufactured and tested as reference (noted with URM and TSM). The first one (URM) was left unstrengthened, as shown in The remaining three URM panels were strengthened by plastering on one face (for one specimen—TRM1) or on both faces (for two specimens—TRM2 and TRM3) with a two-component ready-mixed, high ductility pozzolan-reaction mortar reinforced with an alkali-resistant (AR), pre-primed glass fiber mesh  47,48].,48.47,48m,t—was approximately 4.35 MPa, while the value given in the technical data sheet was equal with 8 MPa. Similarly, the average experimental value of the compressive strength—fm,c—was 19.77 MPa, while the producer indicated a minimum 25 MPa strength in compression at 28 days. The computed standard deviations  indicate that the values tend to be close to the mean. Thus, it can be concluded that the experimentally determined values showed no statistically significant differences.According to the manufacturer , the morAccording to the technical data sheets, the producer determined the compressive and the flexural strength according to the provisions given in the Norms EN 12190 and EN 196/1 ,50, whilThe textile meshes used to reinforce the mortar are part of the same custom design system for masonry strengthening. The meshes consist in multiple dry carbon high-strength fiber roving disposed in a square pattern . Due to As it can be observed in In order to achieve a full interaction among the cords and the specimens, a chemical anchor was manufactured by means of an adhesive product. A bi-component styrene-free adhesive made from polyester resin was poured so as to fill the holes through which the cords were inserted. The physical and mechanical characteristics of the adhesive, as provided by the manufacturer, are listed in The URM panels were constructed of bricks with nominal dimensions of 1200 by 1200 by 115 mm. The latter were assembled by an experienced mason using a mortar made of Portland cement and sand, in the proportion and quality that are commonly used in the traditional Romanian masonry buildings (one part Portland cement to three parts sand). The thickness of both the horizontal and vertical mortar joints was 10 mm. Since the bricks were stored in indoor conditions, each unit was wetted before usage. After all the specimen’s manufacturing processes were complete, the surfaces were cleaned of dust, pollutants or loose materials by sandblasting and low-pressure water jetting.For the second reference panel, two steel meshes of ɸ6 were disposed on each side . The mesThe remaining URM panels were strengthened with TRM systems. First, all the surfaces were wetted and the first layers of the strengthening mortar were overlaid. The two constituents of the mortar, component A (powder) and component B (liquid), were mixed according to the specifications of the manufacturer . The finAfter the first layer of the mortar was applied and while it was still wet, the meshes were placed over the surfaces of the panels. The meshes were lightly pressed down with a towel for them to adhere to the mortar. The adjacent pieces of the meshes were overlapped both widthways and lengthways by approximately 15 cm. Next, the second layer of the mortar was applied. The width of both mortar layers was approximately 5–6 mm.In order to reduce the occurrence and development of the de-bonding phenomena and to improve the static efficiency of the system, the faces of the TRM system were tied with composite connectors . For thiIt is worth mentioning that, in the case of the URM panel strengthened only on one face, the anchoring was performed starting from the middle of the element to its face. This simulated a common situation for monumental buildings, when strengthening system needs to be applied without imposing any interventions to one side of the wall. The construction stages of the TRM1, TRM2 and TRM3 specimens are illustrated in 0 and positioned on the transportation carriage in such a way that no disturbance was caused either to the unstrengthened or the strengthened panels  assembled with or without mortar. The mechanical models that were developed to evaluate the structural behavior of the URM structures fall under the category of no-tension material models. From the theoretical point of view, URM walls are considered to behave as a linear elastic material when subjected to compressive stresses. However, based on the mechanical and elastic properties of the components, the mechanical model and, by default, the numerical one may be adapted from a typical linear–elastic to an elastic–plastic one.Generally, three distinct modelling approaches can be adopted to simulate the structural behavior of a URM wall loaded in shear ,63,64,65In this study, a detailed micro non-linear 3D model was developed to accurately simulate the laboratory test conditions . The nonFor the numerical 3D micro-models, the bricks and the mortar were simulated using SOLID187 tetrahedron finite elements a 55]. T. T55]. TThe bricks were assumed to behave as linear elastic isotropic materials and the plasticity properties were entirely assessed to the mortar, which was modelled as an elastic–plastic material. In this approach, the structural characteristics of the mortar are capable to account for the global non-linear behavior of the URM module. In addition, by applying this modelling strategy, it is assured that failure occurs in a characteristic mode, which is defined by a continuous diagonal crack that passes through the head and the bed mortar joints. The plasticity of the mortar was defined using the Drucker–Prager formulation. According to the latter, the material is characterized by a specific domain of yield stresses. When the yield surface is reached, the mortar starts to deform plastically and, upon further increase in load, the plastic flow is initiated.As mentioned before, the complete test specimens were modelled, including the interface levels. For this purpose, the augmented Lagrange formulation was used to define the contacts. The penetration points were determined for both bricks and mortar based on the integration point detection.Since the stresses vary with a high gradient between distinct components of the 3D model, the mesh size was set differently for bricks, mortar, TRM and composite cords. For the bricks, the maximum mesh size was limited to 30 mm; for the mortar and the composite cords, the mesh size was limited to maximum 2 mm; whereas, for the TRM system, a maximum length of 80 mm was imposed in the model .The nonlinear 3D micro-detailed models were analyzed by using the Newton–Raphson iteration method. More specifically, the method was applied by gradually increasing the load, up to the limit where the convergence criterion was satisfied. Furthermore, whenever required, various sub-steps were defined so as to divide the load ratio. Thus, the final step/sub-step corresponded to the ultimate load. This value was reached at the end of the elastic branch, immediately before an abrupt change in displacement was recorded. This change also indicated that the convergence criterion was no longer satisfied.In the case of the URM reference panel, the failure occurred in the mortar layer by splitting the specimen along the horizontal and vertical joints . BesidesBy analyzing the load–displacement curve (on both faces of the panel) of the URM reference panel, depicted based on both experimentally and numerical results, it can be observed that, in the first stage, the specimen’s response was quasi-linear–elastic with almost constant stiffness value. In the second plateau of the graph, the load–displacement curve illustrates a plastic phase which represents the degraded stiffness state of the URM panel . The plaAs it can be observed, the numerical and the experimental results are in good agreement both for ultimate values and distributions. It should also be mentioned that a sign convention was adopted in order to represent, in the same graph, both the horizontal and vertical displacements. For the evaluation of the shear stress–shear strain state, all the data were taken as absolute values.In the case of the TSM panel, even though the overall thickness of the strengthening systems is around 12 cm, the typical pattern for crack development (across the masonry mortar joints) can be easily identified . When thUnlike the reference URM panels, the TRM-strengthened ones  evidenced a gradual failure mode, which is highly advantageous for the masonry structures located in earthquake-vulnerable areas. For the TRM1 panel, the crack pattern is similar to the ones developed for the reference panels a. Howevediatones). On the contrary, in the case of similar experimental works, that were performed on masonry panels tied with punctual transversal connectors, the sliding phenomena was more intense, as reported in [The TRM 2 and TRM3 panels developed a particular crack pattern which followed the border line of the composite anchors . Besidesorted in .As it can be observed in s—shear stress (MPa); P—load measured along the diagonal pattern; An—net area of the panel; The shear stress–shear strain distributions of all panels are presented in According to ASTM E519/E519M—15, the shear strain is computed using Equation (2) .(2)Ɣ=ΔVAs it can be observed in In the cases of the URM panel and of the traditional, strengthened panel (URM and TSM panels), in the plastic phase, a substantial degraded stiffness can be observed. On the other hand, in the cases of the TRM-strengthened panels , the deformation capacity is considerably improved and the characteristic (yield plateau) that was previously mentioned for the load–displacement distribution can be easily identified. Thus, TRM systems provide significant additional ductility that enables the shear stresses to remain constant while the shear strains continue to increase, meaning that the elongation and contraction continues as long as the load is not removed.The maximum shear stress recorded for the URM panels strengthened on both faces (TRM2 and TRM3) were 20% higher than the one measured for the URM panel strengthened only on one face. When it comes to the numerical values, similarly, an increase of 33% was recorded for the maximum shear stress for the panels strengthened on both faces when compared to the one of the panel strengthened on one face. The experimentally obtained value for the shear strain was 23% higher for the TRM2 and TRM3 specimens when compared to the one measured for the TRM1 specimen. However, the numerical values of the shear strains were almost equal in both cases.The ultimate values of both the shear stress and shear strain that were experimentally recorded for the TSM panel were almost equal to the ones recorded for the TRM2 and TRM3 panels. However, the structural improvement of the TSM panel was obtained by increasing the specimen overall thickness by approximately 12 cm, while the overall thickness of the TRM2 and TRM3 panels was increased by only 2 cm. Thus, the total weight of the traditional strengthened system was considerably higher than the one of the modern TRM system.s—and the shear strain—Ɣ—[According to ASTM E519/E519M—15 , the modstrain—Ɣ—. The stistrain—Ɣ—,60.(3)EThe ductility of the URM panels can be quantified by the drift ratio—δ—Equation (4).The experimental and the numerical results are listed in As presented in The ductility of the URM panel, expressed as the drift ratio, is considerably lower when compared to the values determined for the strengthened panels. Thus, increases in ductility of 64% for the TSM panel, 54% for the TRM1 panel, 66% for the TRM2 panel and 34% for the TRM3 panel were recorded with respect to the experimentally determined value of the URM panel. Similarly, increases in ductility of 73% for the TSM panel and 67% for the TRM1, TRM2 and TRM 3 panels were recorded with respect to the numerically determined value of the URM panel.This paper presents the main outcomes of a numerical and experimental study which relates to the structural efficiency of a modern strengthening technique designed for URM panels. The latter consists of a TRM jacketing that can be applied either on one side of the wall or on both sides. The structural TRM layers can be anchored both by the side-to-side method and the middle-to-side one.The observed damaged patterns both for the URM and TSM specimens matched the characteristic crack path, which consisted in a continuous-propagation step-like line that spread along the compressed diagonal, through the horizontal and vertical mortar joints.The TRM2 and TRM3 panels developed a particular crack pattern which followed the border line of the composite anchors.The TRM systems evidenced a general and significant increase in the original shear capability and ductility of the URM panel. They also enabled the URM panels to largely deform before failure.The selected type of anchorage system provided sufficient lateral capacity to avoid the possible out-of-plane failure.The ultimate loads that were experimentally determined increased by 53% for the TSM panel, 41% for the TRM1 panel, 56% and 57% for the TRM2 and TRM3 panels, when compared to the ultimate load recorded for the URM panel.Similarly, the numerical results evidenced an increase of 56% for the ultimate load of the traditional strengthened panel, 45% for the panel plastered with TRM on one face and 57% for the 3D micro-model with TRM jacketing on both faces, when compared to the ultimate load recorded for the URM reference panel.The ultimate values of the mechanical properties that were experimentally and numerically investigated were almost equal for the TSM panel and the TRM2 and TRM3 panels.The numerical and the experimental results validate the efficiency of the TRM systems that are applied only on one face of the wall, a case which often comes across in the strengthening applications of monumental masonry buildings.From the findings summarized above, the following can be concluded:Although further experimental studies are required for an adequate assessment of the investigated strengthening techniques  and for the validation of the micro-numerical model, the studies discussed in this paper represent a background for the general structural efficiency of the TRM strengthening systems.


Drosophila model, we investigated the effects of dopamine administration on intestinal physiology and gut motility to gain new insights into what could be a potential future promise candidate for GI pharmacology. We investigated whether giving a dopamine-supplemented food medium to adult flies modified the gut contents, defecation rate, and excreta nature. The effects of dopamine on adult gut spontaneous contraction and motility were also studied. We discovered significant gender differences in the effect of dopamine. Drosophila dopamine D1-like receptors (Dop1R1 and Dop1R2) were also displayed by immunohistochemistry to be expressed in all smooth muscles in both larval and adult flies. Furthermore, we showed for the first time that dopamine drives phospholipase C Beta (PLC-β) translocation from the cytoplasm to the plasma membrane in enterocytes. Overall, the data provided new insights into the epidemiology and clinical aspects of neurodegenerative diseases associated with dopamine deficiency, as well as what may be a potential future prospect for GI pharmacotherapy.Dopamine is involved in a variety of physiological functions in the gastrointestinal tract (GI). Using a Drosophila dopamine D1-like receptors (DARs) and/or adrenergic receptors and has been confirmed as one of the enteric neurotransmitters. To gain new insights into what could be a potential future promise for GI pharmacology, we used Drosophila as a model organism to investigate the effects of dopamine on intestinal physiology and gut motility. GAL4/UAS system was utilized to knock down specific dopamine receptors using specialized GAL4 driver lines targeting neurons or enterocytes cells to identify which dopamine receptor controls stomach contractions. DARs (Dop1R1 and Dop1R2) were shown by immunohistochemistry to be strongly expressed in all smooth muscles in both larval and adult flies, which could explain the inhibitory effect of dopamine on GI motility. Adult males’ gut peristalsis was significantly inhibited by knocking down dopamine receptors Dop1R1, Dop1R2, and Dop2R, but female flies’ gut peristalsis was significantly repressed by knocking down only Dop1R1 and Dop1R2. Our findings also showed that dopamine drives PLC-β translocation from the cytoplasm to the plasma membrane in enterocytes for the first time. Overall, these data revealed the role of dopamine in modulating Drosophila gut physiology, offering us new insights for the future gastrointestinal pharmacotherapy of neurodegenerative diseases associated with dopamine deficiency.Dopamine has a variety of physiological roles in the gastrointestinal tract (GI) through binding to  The gut plays an important role in nutrient digestion and absorption, and the digestive tract’s inner lining also serves as the first line of defense against pathogens. It is also a primary source of neuronal and endocrine signals generated by the functionally important peptidergic brain–gut axis . The latCatecholamines are physiologically active chemicals that include dopamine, norepinephrine, and epinephrine . DopaminIn vertebrates, the food intake, digestion, and nutrient absorption through the GI tract wall are all important physiological events. Food enters the body through the GI system, and it is widely recognized that GI motility regulates feeding habits and aids in energy homeostasis regulation. Dopamine has been shown to suppress stomach motility in vitro and in vivo via binding to certain dopamine receptors (DARs) ,7,8; it Dopamine has been identified as an enteric neurotransmitter and plays a variety of physiological roles in the GI tract after binding to DARs and/or adrenergic receptors ,11. It h2+ mobilizing  and other (DAG) messengers responsible for the initial and sustained phases of gut contraction, respectively [Signal transmission in gastric and intestinal smooth muscle is facilitated by receptors that are linked to various effector enzymes via different G proteins, such as phosphoinositide-specific phospholipase C (PLC-1 and PLC-3) and phosphatidylcholine (PC)-specific PLC, PLD, and PLA2. These enzymes are activated differently in circular and longitudinal muscle cells, resulting in Caectively . Accordiectively ). HoweveDrosophila, ~60 percent of the genes linked to human disorders have a Drosophila counterpart. The intestines of humans and flies have similar tissue, morphology, and physiological functions [Drosophila melanogaster a good model. One of these tools is the GAL4/UAS system, which consists of two parts: the GAL4 gene, which encodes the yeast transcription activator protein Gal4, and the UAS (Upstream Activation Sequence), an enhancer to which GAL4 specifically binds to activate gene transcription, allowing researchers to study gene functions both temporally and spatially [Drosophila model, we investigated the effects of dopamine on the intestinal physiology and gut motility to gain new insights into what could be a potential future promise for GI pharmacology. Our main question is whether feeding Drosophila flies food medium supplemented with dopamine will modulate the gut content, defecation rate, the nature of excreta, and gut motility. We also employed the GAL4/UAS system to knock down particular dopamine receptors in various neurons or enterocytes cells using specific GAL4 driver lines that help us to determine which dopamine receptor plays a primary role in controlling gut contractions.In unctions ,18. Furtpatially . In thisDrosophila melanogaster lines were purchased from the Bloomington Drosophila Stock Center  and utilized in all the experiments: w1118, Dop1R1-RNAi (#31765), Dop1R2-RNAi (#26018), and Dop2R-RNAi (#26001) and GAL4 driver lines: Dop1R1-GAL4 (#39609), Dop1R2-GAL4 (#46640), Dop2R-GAL4(#49416), NP1-GAL4 (#84307), nSyb-GAL4 (#51941), and UAS-GFP (#5428). The flies were maintained on standard Drosophila food media comprising cornmeal agar  with an 18/6-h light/dark cycle) [The following k cycle) ,21,22.Dopamine  was dissolved in ddH2O to obtain a stock solution with a concentration of 10 M, and the desired working solutions were then prepared. The primary antibody was anti-GFP (green fluorescent protein) rat monoclonal IgG from Santa Cruz Biotechnology , and the secondary antibody was donkey anti-Rat IgG from Jackson Immuno Research Europe .n = 5) were fed for 24 h on blue media spiked with 300 μL of dopamine concentration ranging from 0.001 to 1 M on top of the normal fly food. The food vials were kept at 25 °C at least 6–8 h until the dopamine was completely absorbed through the media. For the control food (without dopamine), ddH2O alone was added. The guts of three individuals were then dissected in distilled water and centrifuged; the first supernatant was discarded, then centrifuged again, and the blue dye in the second supernatant was measured at 590 nm using a nanodrop ND-1000 spectrophotometer. The measurements were repeated six times. Adults fed on blue media without dopamine were used as the control.To test whether feeding adult flies  food media supplemented with dopamine would increase the gut contents or not, we quantified the gut content according to Reference . Brieflyn = 5) were starved for 2 h prior to the onset of the experiment. Each group was fed for 24 h on blue media supplied with a dopamine concentration ranging from 10−5 to 10−3 M in a normal food vial or in a Petri dish. All 24-h fecal deposits at a distance of 4 cm from the surface of the food in the vial or in the Petri dishes lids were recorded. The experiments were done in 6 replicas. Using ImageJ, the fecal pellets area, perimeter, and integrated dye intensity were calculated. The integrated dye intensity was calculated as the product of the Area and Mean Gray Value , which were calculated by converting each pixel to grayscale using the formula: gray = 0.299 red + 0.587 green + 0.114 blue. The results were expressed as the mean ± S.E.M.The fecal deposits were counted on the clear walls of the dopamine-supplemented blue media . To do tDrosophila excreta, we use phenol red as the pH indicator to get colored fecal deposits [−5 to 10−3 M and phenol red (0.5 M in 10% glucose) as a nonabsorbable pH marker in 50-mm Petri dishes. After 24 h, digital images of Petri dish lids were obtained using an Olympus BX61 light microscope. The mean red–green–blue values obtained from ImageJ were converted to hue–saturation–brightness and the corresponding color patches using a free RGB-to-HSB tool. The H value (hue) is computed as the difference between the two lowest values divided by the maximum value and expressed in degrees (0–360). Hue values refer to the pH of the fecal pellets. Patch colors are based on hexadecimal HTML codes. The hue histograms were obtained using ImageJ software with color mode HSB.To test whether dopamine affects the nature of deposits . Six groD. melanogaster was pinned dorsal-side down onto a dissecting Petri dish, and the animal was covered with physiological saline solution . The cuticle at the thorax–abdomen junction was removed, revealing the underlying gut tissue. Then, the contractions were recorded for 30 s at the base of the crop duct, followed by a 30-s pause. This paradigm was repeated five times in a 5-min period to measure the basal contraction rate. The saline was then removed and replaced with either saline (control) or saline containing 10−3-M dopamine, and the number of contractions was counted for 30 s followed by a 30-s interval, repeating this paradigm ten times over a 10-min period to determine the experimental contraction rate. This procedure was carried out five times . The in vivo effect of dopamine on spontaneous contractions of the larval midgut was studied using a modified version of Reference [−3-M dopamine-containing saline was administered to the top immediately, and the contractions were monitored for 30 s followed by a 30-s pause, repeating this paradigm ten times.The gut contraction assay was used to assess the ex vivo effect of dopamine on adult gut spontaneous contraction and motility . Brieflyeference . To avoi1118 were used as the control for the RNAi-mediated gene silencing experiments.We then investigated whether any possible dopamine-induced responses in gut motility are mediated by various neurons, including those innervating the gut or through other pathways, such as gut enterocytes cells, using the loss-of-function (RNAi knockdown) of the corresponding dopamine receptors. Specific GAL4 driver lines either directed in various neurons (nsyb driver line) or in enterocytes cells (NP1 driver line) were used. The guts of F1 individuals were dissected in saline; then, the gut contractions were calculated as mentioned above. F1 of the GAL4 driver lines crossed with Wn = 10) had their stomachs dissected in a hemolymph-like (HL3) buffer. Dopamine (10–3 M in HL3) was given to the top of the tissue immediately after dissection, and the translocation of PLC-β particles was observed using a SP5 Leica confocal microscope. In this experiment, the guts of F1 given a HL3 buffer without dopamine were employed as the control.To determine whether dopamine, an endogenous ligand of dopaminergic receptors, stimulates PLC activity, we crossed UAS-PLC-mRFP with NP1-GAL4 and performed an ex vivo assay in the gut of F1 adult flies. The adult flies . The primary antibody  was incubated overnight at 4 °C, followed by three 5-min washes in PBST (0.1 percent Triton-100). The secondary antibody  was incubated for 1–3 h at room temperature with TRITC-labeled Phalloidin (0.5 g/mL). The tissues were then washed three times in PBS-T for five minutes each time. Glycerol was used as a mounting medium for the preparations, which were then analyzed using a Leica SP5 confocal microscope.t-test was performed to assess the gastrointestinal motility in larvae and adult flies following dopamine treatment to the control. A liner regression analysis was used to see if there was a link between the gut content and the number of fecal pellets in relation to varied dopamine concentrations. The Pearson correlations coefficient was also used to assess the relationship between the effect of dopamine on the gut contents and fecal pellet counts in male and female flies to determine if the change in the gut contents is due to a change in the defecation or food consumption rates. The differences were considered significant at p < 0.05.To analyze the data, we utilized GraphPad Prism v. 8.0 for Windows. The data were changed to log10 when necessary to better estimate the variance’s normality and homogeneity. To investigate the differences in the gut content, number of fecal pellets, perimeter, and integrated dye intensity, a one-way analysis of variance (ANOVA), followed by Tukey’s Multiple Comparison test, were performed. The Student’s p = 0.002; 2 = 0.26, p = 0.01; 2 = 0.14, p = 0.03; Feeding adult flies  media supplemented with dopamine increased the gut contents of only males at the least conc. of dopamine (0.001 M) as compared to control flies fed on normal food media without dopamine  had substantially more fecal pellets than the control male flies fed standard food media without dopamine   of both larvae  and adulosophila  using thosophila  and Dop1osophila  are stroosophila .−3 M) rapidly activates the translocation of PLC-β from the cytoplasm to the plasma membrane and, as a result, is expected to enhance the intracellular Ca+2 mobilization in enterocytes as opposed to the control, in which PLC-β remains in the cytoplasm  via the hypothalamus are known to modulate the activity of dopamine cells and their projections into regions involved in the rewarding processes behind food intake . Only maChronic constipation is the most common symptom, affecting up to 89% of PD patients . LevodopIn vitro and in vivo studies have demonstrated that dopamine inhibits stomach motility via binding to specific DARs ,7,8. SimAlthough biological sex variations in the nigrostriatal dopaminergic (NSDA) system, which are regulated by hormonal, genetic, and environmental variables, are mostly responsible for sex differences in PD , knowingSimilarly, cocaine-induced dopamine increases lowered DAR signaling considerably, changing the balance between D1R and D2R signaling , affecti2+ mobilization in Drosophila enterocytes that may activate the subsequent signal pathways, including those involved in the inhibition of gut contractions. In the same context, intracellular calcium increases have been shown to play a role in the initial and persistent phases of gut contraction  and on eference . Furthereference . As a reDrosophila flies, and in dopamine receptor-defective adults, which provide new insights into the epidemiology and clinical aspects of PD. Immunohistochemical investigations revealed the existence of dopaminergic receptors in gut muscles, while functional studies demonstrated dopamine effects on the gut motility, elucidating dopamine’s role in the gut physiology. Our study also indicated that dopamine induces PLC to translocate from the cytoplasm to the plasma membrane for the first time in enterocytes, potentially activating downstream signal pathways, such as those involved in gut contraction inhibition. Overall, these data provided new information on what could be a potential future prospect for GI pharmacotherapy patients and PD patients.Taken together, we discovered significant gender differences in effects of dopamine on the gut content, defecation rate, excreta pH in


Mycobacterium bovis that mainly affects cattle. This pathology has a negative impact on animals and animal products trade. Unfortunately, in Burkina Faso where agriculture and livestock sectors represent around 80% of the socio-economic activities, the real situation of the disease is not well known especially in small ruminants and swine. Thus, our study focused on both the epidemiology and the microbiological diagnosis of tuberculosis (TB) in small ruminants and pigs slaughtered at Bobo-Dioulasso abattoir. A prospective study was conducted between August 2017 and December 2017. Epidemiological data collection was performed during routine meat inspection; moreover, samples were taken and transported to the Bacteriology laboratory of Centre Muraz for microbiological analyses. This diagnosis consisted in search of Acid Fast Bacilli (AFB) using the hot Ziehl–Neelsen staining. Out of a total of 14 648 small ruminants and 2430 pigs slaughtered during the study period, 156 and 17 had lesions suggestive of bTB with prevalence of 1.07% and 0.7%, respectively. Females and those between 2 and 4 years old were mainly infected. The most affected organs were: lungs, liver, spleen and lymph nodes. Finally, microscopy revealed 43.35% (75/173) of positive cases for AFB. These results confirm the presence of bTB in small ruminants and pigs in Burkina Faso. Efforts must still be made in the fight against this zoonosis in order to limit its economic and public health impacts.Bovine tuberculosis (bTB) is a zoonotic, infectious, chronic and contagious disease, caused by  Livestock plays a central role in the economy of the Sahel part of Africa, which is illustrated as an excellent region for animal breeding. It is indeed one of the main economic activities on which the regional populations are dependent as a source of food and monetary income (CSAO/OCDE Mycobacterium bovis (M. bovis), whose main victims are not only domestic cattle but it also affects other animals (domestic and wild) and human  out of 55 had a bTB control programme in cattle herds, using tuberculin tests and post-mortem inspection for the surveillance of this disease , foot-and-mouth disease, which decimate among cattle, and the lack of means and the insidious nature of bTB have led to continuous neglect of this disease in the country. The government meat inspection regulations however, provide for regular checks of animal carcasses before consumption. Thus, the only information available most often comes from suspicions during routine meat inspection at slaughterhouses and slaughter areas. Nevertheless, the existence of bTB has been known for a very long time in this country, where the highest rates from all French West African countries were observed in slaughterhouses Regnoult . NonetheThus, the study aims to provide epidemiological and microbiological data on bTB in small ruminants and pigs slaughtered at the Bobo-Dioulasso abattoir to contribute to public health and bTB control improvement in the country.This was a prospective cross-sectional study that took place at Bobo-Dioulasso abattoir  between August 2017 and December 2017. The study settings also included Centre MURAZ, where the laboratory analyses were carried out. In addition, the animals  studied potentially originate from different parts of the country.We did an exhaustive sampling. Our study focused on cases of organ seizures for suspected TB during the study period. The samples consisted of tissues from suspected organs such as lymph nodes , lungs, spleen, liver, udders …, following routine meat inspection at the slaughterhouse.Data collection was carried out using a sheet developed for this study. The data collected were epidemiological and bio-clinical information, in particular the registration number of the suspected animal, the type of seizure, age, sex and anatomo-pathological aspects of the carcass . This information was collected through observation by veterinary technicians during meat inspection.At Bobo-Dioulasso Abattoir, animals are mostly slaughtered daily from midnight to 6 am and during the day to ensure permanence. The carcasses were inspected by veterinary doctors and officers. Pieces of organs from suspect animals were transported at 4 °C to Centre MURAZ for microbiological analyses. Three bio-clinical samples per suspected animal were taken to increase the probability of bTB detection.Pre-treatment consistent of samples decontamination organ pieces and grinding. The samples taken were previously cleaned with sterile distilled water before treatment using the Saeng and Costil method  for 20 min. Step 3 was repeated 3 times.Step 4: After removal of the supernatant, a volume of 1.5 mL of sterile distilled water was added to the pellet and the tube was vigorously vortexed to until obtained an homogenous mixture, ready for microbiological analysis.One part of the pellet was used for microscopical analysis using Ziehl–Neelsen staining so as to detect the presence of Acid Fast Bacilli (AFB). In order to have optimal conditions, all the slides to be used must be sterilised with a fire source  applying the heat on both faces for a few seconds. A certain volume of the sample is extracted and spread on the slide making a thin uniform barely transparent smear with a sterile bacteriological sampling loop. Once this is done, the spread is fixed with a little heat from the spirit lamp, applied (only) under the slide before undergoing staining using the hot Ziehl–Neelsen method.p < 0.05 value was considered statistically significant.A double entry was used to record data using Sphinx Plus2 V5 software where appropriate, descriptive parameters such as sums, percentages and fractions were then computed. Statistical analysis was performed using Epi info version 7.2.2.6. Logistic regression was used to assess the relationship between lab results and animal characteristics. The Out of 14 648 total small ruminants and 2430 pigs slaughtered during the study period, 156 and 17 had TB suspected lesions giving prevalence of 1.07% and 0.70%, respectively, and an overall prevalence of 1.01% see .Of the 173 suspected bTB carcasses, 141 (81.5%) were females and 32 (18.5%) were males, that is, a sex ratio of 0.23 (32/141) see . The ageOf the 173 suspected animals, 95.38% presented localised lesions to some organs  against 4.62% with generalised lesions throughout the carcass. The most affected organ was the lung with a proportion of 36.07%. However, there were sub-populations specificities because the liver was the most affected organ for pigs. For affected organs and the relative numbers refer to Amongst the 173 carcasses with suspicious lesions, microscopy revealed the presence of bTB in 75 (43.35%) samples. The results of multivariate analysis of laboratory results according to animal characteristics are shown in The objective of this study was to explore the full breadth of bTB in small ruminants and swine populations in Burkina Faso, suspecting its presence based on its frequency in cattle slaughtered and routinely inspected in Burkina Faso slaughterhouses. The present study indicates the existence of bTB in Burkina Faso with a proportion of 1.01% of lesions suggestive of this pathology in small ruminants and swine inspected. The presence of bTB in small ruminants and pigs is not surprising; indeed, evidence of bTB existence dates back to the colonial period based on single cervical intradermal test in cattle Sere . In addiIn the present study, the prevalence of the disease was higher in small ruminants compared with pigs, with respective prevalence of 1.07% and 0.70%. Our results were higher than the 0.03% found in goats in Nigeria in 2016 (Danbirni et al. Regarding parameters such as age and sex of slaughtered animals, they are not routinely collected by veterinary officers. The data collected during the present study were only related to suspected bTB cases. They show that suspected animals were mainly females and between 2 years and 4 years of age even if sex, age are not significantly associated with the prevalence. Previous studies have shown a link between sex and bTB occurrence Lamireou . These sRegarding the lesions extent that caused carcass seizures during routine inspection, most were located on a given organ. Lung damage is the leading cause of bTB seizure in sub-Saharan Africa (Kulo & Seme The proportion of suspected bTB positive lesions in microscopy was 43.35%. Despite the microscopic examination low sensitivity, our study found a prevalence above the 13.33% found in Algeria in goats (Naima et al. This study showed the bTB occurrence in small ruminants and pigs slaughtered at Bobo-Dioulasso abattoir with a prevalence of 1.01% of suspicious lesions. The study found that lungs damage was the leading cause of bTB seizure at Bobo-Dioulasso slaughterhouse. Also, the female and old animals were the most infected. Additional control efforts are required for more effective control of this zoonosis in Burkina Faso.


In this paper, we present a novel approach called “Molecular Anatomy” as a flexible and unbiased molecular scaffold-based metrics to cluster large set of compounds. We introduce a set of nine molecular representations at different abstraction levels, combined with fragmentation rules, to define a multi-dimensional network of hierarchically interconnected molecular frameworks. We demonstrate that the introduction of a flexible scaffold definition and multiple pruning rules is an effective method to identify relevant chemical moieties. This approach allows to cluster together active molecules belonging to different molecular classes, capturing most of the structure activity information, in particular when libraries containing a huge number of singletons are analyzed. We also propose a procedure to derive a network visualization that allows a full graphical representation of compounds dataset, permitting an efficient navigation in the scaffold’s space and significantly contributing to perform high quality SAR analysis. The protocol is freely available as a web interface at The online version contains supplementary material available at 10.1186/s13321-021-00526-y. The generation of clusters organized as “series” in medicinal chemistry is an important asset of the scaffold-based techniques.High-throughput screening (HTS) of small-molecule libraries is routinely used in drug discovery process to identify novel leads against clinically relevant targets. Successful HTS require high quality, validated screening assays, but also an effective strategy for chemical structures selection is fundamental for the following hit-to-lead phase. HTS libraries, indeed, comprise some hits of interest, but also many compounds resulting in false positives or promiscuous hits, as well as number of compounds with no relevant biological activities at micromolar concentrations in biochemical assays . The fir. [A chemical scaffold, also referred to as ‘chemotype’ or ‘Markush structure’, can be defined as the common structure characterizing a group of molecules. Compounds sharing the same scaffold are likely to have a similar synthetic pathway as resulting from the concept of molecular template in combinatorial chemistry . Once a .  introduc. . The int. , of hier.  demonstr. , recentl. . In 2008.  proposed. , in 2010.  proposed.  introduc. , 20.All the above described methods share two major limitations; first a single ring system is represented, decorated with chains of various length, therefore, when pruned, all the molecules collapse into a degenerated cluster. Additionally, there is no relationship between scaffolds when the difference is represented by one or more ring systems. These issues are particularly limiting for the analysis and selection of vendor libraries to build diverse compound collections and, afterward, for HTS campaigns analysis in order to obtain preliminary SAR.Very recently, Bandyopadhyay et al. , in ordeHowever, the main limitation of the network connecting molecules and scaffolds generated with these implementations is that they are based on a unique scaffold representation, not sufficient to effectively map the chemical space of a heterogeneous ensemble of molecules, for example multi-scaffold libraries, and to capture relationships with the relative biological activity. The critical point is that it is not possible to define a priori the best representation of a molecule, because it mainly depends on the biological context and on the nearest-neighbors of the screened library.Here, we present a novel approach, called “Molecular Anatomy”, for the generation and analysis of correlated molecular frameworks aimed at overcoming the limitations of scaffold analysis based on a single predefined set of rules. In our experience, the here identified molecular frameworks and related fragments are able to capture most of the structure activity information from HTS campaigns, and are also useful for other applications, such as library design and analysis. In particular, the combination of fragments, correlated in frameworks and wireframes, identifies relevant chemical moieties in an efficient manner, clustering together many scaffolds with similar shapes despite, for example, different dispositions of heteroatoms or small differences in bond order. To the best of our knowledge, this is a distinctive feature of our approach, compared to other known methodologies, such as the widely accepted Maximum Common Substructure (MCS) .In the “COX-2 A dataset containing COX-2 inhibitors was prepared and used to illustrate the “Molecular Anatomy” approach. To this aim, the Integrity™ database was interrogated to search for COX-2 inhibitors, providing 2599 molecules in total. Of these, 816 were in preclinical phase or in a higher phase of clinical development. This subset was used in the following analysis to compare different scaffold representations. A Pipeline Pilot protocol [protocol  was usedHDAC7 dataset of 26,092 commercial compounds, tested as potential HDAC7 inhibitors during an HTS campaign performed internally within Dompé, was used as a more complex case study. The compounds were stratified in different activity classes according to the value of percent inhibition of the HDAC7 activity obtained at 10 μM concentration , containing activity data for ~ 8000 compounds from the primary screening against SARS-CoV-2 Main protease (Mpro) [Additional compound datasets were retrieved from release 28 of ChEMBL  for furte (Mpro) . In thisIn theory, it is possible to define an arbitrary number of scaffold's representations based on different levels of abstraction and pruning rules. Figure Panel 1a shows the most abstracted representation (hereafter 1a), obtained removing both bond and atom type. This representation is also known as “cyclic skeleton”. Representations 1b and 1c retain only bond and atom type, respectively, whereas the 1d representation corresponds to the Bemis-Murcko scaffold, containing all the rings and chains connecting them of the original molecule.By using the most abstracted representation 1a of the Polmacoxib scaffold, corresponding to the most common moiety of COX-2 inhibitors, a subset of 224 COX-2 inhibitors was identified. Figure These 224 molecules correspond to 84 different scaffolds if the less abstracted 1d representation is used (Table Basic Scaffold), which is generated by removing all side chains and terminal atoms. Then, we defined a set of nine molecular frameworks (MF) at different abstraction levels to match different side chain definitions, as showed in Fig. In “Molecular Anatomy” we used, as starting point, the widely accepted scaffold abstraction representation ; in a second step, the longest atom chain, considering also substitutions, is retained but all terminal non-carbon atoms, belonging to side chains, are iteratively pruned (Augmented Scaffold). In case that no terminal atoms remain removing all terminal non-carbon atom with a bond order equal to 1, decorated and augmented scaffold coincide. Some examples reported in Fig. The first set of rules is based on an increased level of structural information with respect to the basic scaffold. In a first step, terminal atoms with bond order greater than one are maintained , thus finally producing nine molecular representations with a hierarchical correlation.The second set of rules, conversely, increases chemical abstraction by removing the atom type label and then the bond order, generating, respectively, a To further overcome one general limitation of the scaffold based techniques , 14, 32 The here reported fragmentation and deconstruction introduce other hierarchies, meaning that each fragment of the original scaffold is related with all the other representations in a multi-dimensional space. As a result of this multi-dimensional hierarchical scaffold analysis, the entire set of generated molecular frameworks are highly interconnected, and it is possible to move from one to another following SAR. To clarify this concept, Fig. The software Cytoscape was used for creating and visualizing an MF-based network, which was also integrated with activity data for the SAR analysis. This network provides a full graphical representation of the dataset composition, allowing to easily navigate through the molecular frameworks and their hierarchical correlation. A Pipeline Pilot protocol was implemented to prepare the data matrix needed for the visualization, in the format required for the import process.Each molecule from the dataset of 816 COX-2 inhibitors was described according to the nine molecular representations implemented in the “Molecular Anatomy” approach  calculated, according to Eq. , for eacIn order to focus the network visualization on the most relevant dataset information, the nodes associated with EF = 0 can be filtered out, thus stepping through the nodes with increasingly higher EF values, as described in the HDAC7 case study see “”.wireframe > framework > scaffold, or augmented > decorated > basic or wireframe > ring disassembly > fragments and in all the other possible directions maximizing the SAR information of the dataset. The network visualization can be extended also to the fragments to obtain a fully connected network, considering that the smallest fragments (e.g. benzene ring) are shared by a huge number of the original molecules. In this implementation, the network nodes can be molecular frameworks, fragments or entire small molecules, and the direction of the edges, defined by the fragmentation rule, starts from the originator fragment and end up into the corresponding fragments.“Molecular Anatomy” allows, as already described, to derive trees in multiple dimensions such as https://ma.exscalate.eu. The web interface was implemented using LAMP (Linux Apache MariaDB PHP), an open source Web development platform that allows a fluent and responsive user experience in displaying and handling the output data, which in this case are calculated on the fly in a completely automated Pipeline Pilot workflow.The above-described protocol is freely accessible at The Pipeline Pilot protocol for the preparation of the data matrix was also re-implemented in Knime 4.3.2 by means of in-house Python scripts and taking advantage of the available RDKit  and IndiAs shown in “Representation 1a clusters together most of the well-known marketed drugs, such as valdecoxib and celecoxib, as well as many others leads and experimental drugs, and collapses all the 142 active molecules with exactly 3 rings to a single cluster. This cluster likely includes also several inactive molecules. Interestingly, we can note that, even though this representation is used, still almost the 40% of the scaffold information, corresponding to the molecules with additional rings, would be lost in unrelated clusters, impairing the identification of the most relevant additional structural information.Using the less abstracted representation 1d, we can retrieve and distinguish the most diverse COX-2 inhibitor scaffolds, even if this information is distributed in 84 clusters considering both those with 3 or more rings. Furthermore, an intermediate representation as 1b, where only the atom type information is removed, could allow a more effective clustering of the relevant structural information, identifying only 11 different frameworks containing molecules with exactly 3 rings, instead of 31; but, almost the same number of clusters containing molecules with more than 3 rings is generated with the two representations (48 instead of 53).This example on COX-2 inhibitors clearly shows how this kind of analysis strongly depends on the nature of the dataset; each scaffold abstraction of Fig. Attempts to identify more relevant chemical moieties have been presented in the past, for example the rule-based decompositions proposed by Schuffenhauer et al. , schematThe method that we propose, involving the combination of correlated molecular frameworks and fragments, is able to efficiently identify relevant chemical moieties, and to cluster together active molecules  belonging to different molecular classes within HTS campaigns, capturing most of the SAR information.To fully exploit the hierarchical correlation among the molecular frameworks and to generate a full graphical representation of the analyzed dataset, we also propose a network visualization. Actually, the combination of the MF approach with a network representation provides a more convenient tool for SAR evaluation and visualization –38, usefFigure Furthermore, it is possible to retrieve the relationships among the diverse representations within this cluster and, focusing on the most interconnected frameworks, to identify the structural characteristic representative of the active molecules, as shown in Fig. Focusing on the fragments related to the basic wireframe representation, all the clusters identified in Fig. Furthermore, Additional file Some qualitative considerations about the obtained networks can be done. As a first point, it is reasonable that highly connected singletons tend to be small fragments shared by a large number of molecules included in the library  to the first decorated wireframe (EF = 7.8), allows to highlight interesting “hotspots”, identifying a specific feature of the active molecules structure (e.g. a protruding bond in position 1 of the spacer between the two rings).Furthermore, it is also possible to move to the less abstracted representation within the network, the decorated frameworks also reported in Fig. A first interesting consideration about these results concerns the introduction of decorations in our scaffold representations: defining a description level in which protruding bonds are added to the basic scaffold allows to better identify and distinguish the requirement essential for the activity. This point is clearly showed in Figs. Finally, we want to show how the most useful SAR information can be obtained extending the analysis and the network to the fragments. When the fragmentation rules are applied to the dataset, the network visualization of the fragmented library allows to interconnect all the molecular frameworks containing the same fragment and the EF can be recalculated for each fragment according to the activity data of all the molecules connected via the corresponding molecular frameworks.In particular, focusing the attention on the interesting structures above identified, Fig. Comparing Fig. To better explain the contribution of the integration of fragments and molecular frameworks in the SAR analysis, we report on top of Fig. This example shows how this approach allows to extract structural information from the different levels of scaffold's representations. The decorated wireframe on top of Fig. We can conclude that, even if in this particular case study the decorated wireframe seems the most informative representation, in general the integration of all molecular frameworks and fragments in the network visualization is crucial for capturing the most relevant information in compound libraries SAR analysis.In order to investigate which level of scaffold abstraction leads to the highest enrichment factors, we employed three further datasets from ChEMBL see “” sectionhttps://ma.exscalate.eu. The user can upload a text file containing one or more compounds, encoded as canonical SMILES, to generate the molecular frameworks related to the nine molecular representations at different abstraction levels.The “Molecular Anatomy” approach is available as a web application, freely accessible at The output consists of four tables, resembling Additional files The dataset of 816 COX-2 inhibitors used for this study has been provided within the web interface as template file to test the application (by clicking on the “Submit Example” button).We propose “Molecular Anatomy” as a fast and flexible method for the analysis of the chemical space, library design and SAR studies.This set of tools could be useful in the management of large compounds collections, for example in the analysis of HTS campaign results, as well as in focused libraries design. On the other side, this kind of data organization allows to efficiently analyze scaffold-activity relationship, identify relevant clusters and easily connect different chemotypes with biological activity. The limitation in the underestimation of the side chain effect can be easily circumvented combining the “Molecular Anatomy” approach with other techniques, such as matched molecular pairs (MMP) –44. In tAnother interesting application, still in an early phase of evaluation, is the possibility to annotate a library according to therapeutic areas information of classified drugs, in order to accelerate the identification of target-based or disease-based libraries, using for example annotated database such as MDDR, WOMBAT , or publFurthermore, molecular frameworks can be profitably used for compounds clustering and database indexing. One of the most critical tasks in the design of large diverse libraries is the comprehensive mapping of chemical space. The generation of groups that can be considered as “series” in a medicinal chemist’s perception represents an important asset of the scaffold-based techniques. However, the clusters generated by currently available approaches generally tend to contain an elevated number of scaffolds, hampering the selection of chemical series for follow-up activities. Some improvements have been introduced to overcome this problem, for example by means of Maximum Overlapping Set (MOS) . The maiFinally, NC makes relational databases ideal for chemical graph-based compound clustering applications. The composition of a specific cluster is independent from the chemical structure of the other clusters, and, once the scaffold abstraction is defined, isn’t needed to re-cluster the whole library.Organizing molecules within a database by means of clustered SQL indices (based on the MA), can dramatically reduce the time required for substructure searches, as reported by Wilkens et al.  and MascAll these prospective applications make the MA approach a valuable cheminformatics tool that can considerably improve structural data analysis.Additional file 1: Figure S1. Cytoscape network visualization of the 816 COX-2 inhibitors subset where nodes includes fragments related to the basic wireframe representation, contributing to create a fully connected unique network. Figure S2. The fragments extracted from the basic wireframe representation, with the highest number of connections (indegree) in the Cytoscape network visualization reported in Figure S1. Figure S3. Selection of the most interesting basic wireframe, corresponding to the most abstracted representation in common within each cluster of the network, filtered by the highest values of EF and number of connected active molecules of the corresponding cluster.Additional file 2: Table S1. Dataset of 816 COX-2 inhibitors in preclinical development or in a higher phase, described according to the nine molecular representations of “Molecular Anatomy”. Each compound and the related nine molecular representations are encoded as canonical SMILES and InChIKey, respectively, and are listed in the same row.Additional file 3: Table S2. Dataset of 816 COX-2 inhibitors reported in Table S1; each compound and the related nine molecular representations are encoded as canonical SMILES and InChIKey and are listed as single rows. From this list, Tables S3 and S4 are generated.Additional file 4: Table S3. List of unique frameworks obtained from the molecules in Table S2.Additional file 5: Table S4. List of parent–child relationships between the nine molecular frameworks of molecules listed in Table S2.


This situation is an emerging threat to public health and therefore the development of approaches to infer the presence of resistant strains is a topic of high importance. The resistome construction of an isolate microbial species could be considered a solved task with many state-of-the-art tools available. However, when it comes to the analysis of the resistome of a microbial community (metagenome), then there exist many challenges that influence the accuracy and precision of the predictions. For example, the prediction sensitivity of the existing tools suffer from the fragmented metagenomic assemblies due to interspecies repeats: usually it is impossible to recover conservative parts of antibiotic resistance genes that belong to different species that occur due to e.g., horizontal gene transfer or residing on a plasmid. The recent advances in development of new graph-based methods open a way to recover gene sequences of interest directly from the assembly graph without relying on cumbersome and incomplete metagenomic assembly. We present GraphAMR—a novel computational pipeline for recovery and identification of antibiotic resistance genes from fragmented metagenomic assemblies. The pipeline involves the alignment of profile hidden Markov models of target genes directly to the assembly graph of a metagenome with further dereplication and annotation of the results using state-of-the art tools. We show significant improvement of the quality of the results obtained (both in terms of accuracy and completeness) as compared to the analysis of an output of ordinary metagenomic assembly as well as different read mapping approaches. The pipeline is freely available from  Antimicrobial resistance (AMR) is a global health crisis resulting from widespread and uncontrolled use of antibiotics , RGI . Another approach involves the use of sequences obtained from raw reads after the genome assembly process. Genome assembly may overcome the difficulties connected with the lengths of short reads and allows for reconstruction of fuller gene sequences, however it still has some limitations on its own. Possible issues include possible assembly artifacts, increased computational processing time, etc. Nonetheless, all these issues could certainly be detected, most of them solved in automatic fashion and therefore AMR prediction on top of microbial isolate assembly could be considered a mostly solved problem.However, the overall situation is much worse when one would need to analyse a resistome from an environmental sample, such as water metagenome, or human-associated sample, e.g., gut metagenome. Such assemblies are often very fragmented due to vastly different species abundance, presence of multiple strains, interspecies repeats that arise from conservative genes or genes that underwent horizontal transfer, etc.   format be provided as an input. Such assembly graphs are readily produced by genome and metagenome assemblers including SPAdes  metagenomic equences . The firhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The resulting HTML report shows summary graphs with main characteristics for quality assessment. Metagenome assembly is done via metaSPAdes  or if there are multiple paths due to synonymous substitutions. To dereplicate the results, the complete ORFs are extracted and further clustered at 90% AA IDY using MMseqs2 , sraX .There is no need to design a completely new AMR prediction approach given that the major challenges of obtaining putative AMR sequences from fragmented metagenome assemblies are solved via the proper utilization of the assembly graph. Therefore, this step delegates the task of final AMR prediction, annotation, and result generation to state of the art tools that are well-known and respected by the bioinformatics community. The pipeline passes the output of the dereplication stage to abricate Install nextflow (https://conda.io/miniconda.html)Install any Nextflow-supported container engines, such as conda :*_R{1,2}.fastq.gz’nextflow run ablab/graphamr -profile conda --reads ‘Typical command for analysis starting from assembly graph (NCBI AMR database is used by default):nextflow run ablab/graphamr -profile conda --graph ‘assembly_graph_with_scaffolds.gfa’Typical command for analysis starting from assembly graph with one of pre-defined AMR databases:nextflow run ablab/graphamr -profile conda --graph ‘assembly_graph_with_scaffolds.gfa’ --db More examples, description of other command line options and produced results are available from the “Usage/Results” section of documentation in GraphAMR github repository.To demonstrate the performance of graph-based approach for AMR discovery we benchmarked GraphAMR pipeline on two different environmental datasets using two different databases: NCBI AMR HMMs and amino acid sequences from CARD.SRR5997540–SRR5997552 and analyzed using the pipeline. For the sake of simplicity only AMR predictions by Abricate are shown. URBAN is a collection of urban wastewater datasets from Ng et al. . Raw seqThe resulting AMR presence heatmap as produced by RGI is available as We note that HMM paths represent unique path sequences over the assembly graph and might be redundant: two different paths in the graph may yield the same amino acid gene sequence, for example, due to synonymous mutations or if the alignment ends in the node of the graph since edges have overlapping k-mers. This explains the higher number of predicted AMR gene sequences obtained from bare HMM paths as compared to dereplicated or clustered ORFs.The sample SRR5997545 looks like an outlier in To further compare the assembly graph-based approach with the read-based one we run SRST2 on the same collection of datasets. SOIL is groundwater metagenome sample SRR8931193 from Smith et al. . AbricatAs Another important task that could be solved using the assembly-graph based approach is AMR host association: sometimes it is not enough simply to detect the gene sequences, but also associate them with the particular species. This task is quite complex in case of metagenomic assemblies as a dedicated procedure called “binning” is required. However, typically binners ignore short contigs (shorter than 2–5 kbp) and therefore further detection of AMR gene sequences from MAGs could be quite limited (Maguire et al., GraphAMR could be used to improve the present results of AMR prediction of a metagenomic assembly if the assembly graph output was preserved, otherwise the pipeline allows for seamless reassembly and AMR prediction starting from the input sequencing reads.Publicly available datasets were analyzed in this study. This data can be found here: NCBI SRA: SRR5997540–SRR5997552, SRR8931193.AK contributed to conception and design of the study. AK, AC, and DS implemented the pipeline. AK and DS wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This reported study was funded by RFBR, project number 19-34-51017.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.