An evolutionary algorithm is developed and used to search for shape persistent porous organic cages. The chemical and structural space of possible molecular materials is enormous, as they can, in principle, be built from any combination of organic building blocks. Here we have developed an evolutionary algorithm (EA) that can assist in the efficient exploration of chemical space for molecular materials, helping to guide synthesis to materials with promising applications. We demonstrate the utility of our EA to porous organic cages, predicting both promising targets and identifying the chemical features that emerge as important for a cage to be shape persistent or to adopt a particular cavity size. We identify that shape persistent cages require a low percentage of rotatable bonds in their precursors (<20%) and that the higher topicity building block in particular should use double bonds for rigidity. We can use the EA to explore what size ranges for precursors are required for achieving a given pore size in a cage and show that 16 Å pores, which are absent in the literature, should be synthetically achievable. Our EA implementation is adaptable and easily extendable, not only to target specific properties of porous organic cages, such as optimal encapsulants or molecular separation materials, but also to any easily calculable property of other molecular materials. Simple changes to the precursors can double the mass of the product, changing its molecular topology and consequently its properties, for example losing any potential porosity through loss of shape persistency.Porous molecular materials still only number in their hundreds, compared to, for example, the many hundreds of thousands of MOFs reported.et al. have developed the software HostDesigner, which designs molecular receptors for guests.At the solid-state level, it has been shown that the low energy polymorphs and therefore crystal packing of porous molecular materials can be predicted using crystal structure prediction techniques originally developed for pharmaceuticals.5 total aldehyde and amine precursors. If you combine these into two-component cages in all possible topologies, then you already have 107 potential host molecules to consider. For this reason, we have developed an evolutionary algorithm (EA) for the prediction of molecular materials, and demonstrate here its application to the discovery of porous molecular materials. EAs are inspired by biological evolution, where candidates are evaluated for their ‘fitness’, which determines their likelihood of proceeding to a subsequent generation. At each generation, several candidates will undergo random mutations and pairs of candidates will ‘reproduce’, with their chromosomes undergoing crossover. EAs, such as genetic algorithms, have been widely applied in chemistry, including for structure predictionThe chemical and structural space of hypothetical porous molecular materials is enormous, given they are formed from building blocks of organic molecules. Most porous organic cage systems to date have been synthesised using dynamic covalent chemistry (DCC), where the reversible nature of the reaction allows error correction towards high symmetry, discrete structures. If we consider the most commonly used chemistry for cage synthesis, imine condensation, then inspection of only the online Reaxys database of previously reported molecules,Evolutionary methods have been developed both for experimentalstk),stk is written in python and makes use of utilities within the RDKit cheminformatics libraries.stk, each EA individual is defined as a Python object, which allows for the labelling and tracking of its compositional  and structural  properties throughout the evolutionary run. Whilst here we develop the EA for the discovery of cages, it is easily extendable to other (supra)molecular materials in the future. We focus on the molecular prediction of cages, having previously demonstrated the utility of this approach for property prediction,In this work, we report on the extension of our previously reported software, the supramolecular toolkit (stk),stk to the exploration of chemical space using an EA. Each organic cage  chromosome can be uniquely defined by a set of three variables (or genes): two building blocks (BB) and a topology, as shown in The overall structure of our EA is shown in Once all the individuals in the initial population have been generated, each cage in the population has its fitness value evaluated (as described below). The individuals are ranked according to their fitness value and, depending on the specific selection algorithm, a set of parent structures is chosen. New offspring structures are generated starting from the selected parents by applying the genetic operations of crossover and mutation, shown in In our EA, the BBs selected for the generation of the initial seed population (and during the mutation step) come from a chemical database. Since this work focuses on the generation of porous organic imine cages, our initial database only contained aldehydes and amines and, in order to restrict the size of the chemical space to be explored, we only allowed for tri-topic aldehydes and di-topic amines. Our database was generated by mixing the free eMolecules databaseThe 3D coordinates of each molecule within the chemical database were obtained starting from SMILES codes by using the ETKDGBy completely substituting one of the three genes of a chromosome, a mutation step strongly modifies the chemical/structural properties of a cage, possibly losing all the evolutionary advantage that has been developed up to that specific generation in a EA run. For this reason, we developed the similarity mutation, as shown in 4Di6Tri topology that relates to a tetrahedron and the 8Di12Tri topology that relates to a cube. The superscripts from the nomenclature represent the number of precursor molecules with the specific topicity (Tri for tri-topic or Di for di-topic) included in the assembly. For example, the 4Di6Tri molecule consists of four tri-topic and six di-topic building blocks. The nomenclature was recently suggested by us7 possible molecular cages.In this work, we investigate the family of molecular cages built by a combination of a tri-topic and di-topic BBs, where the BBs are combined in a 2 : 3 ratio.Once each individual in the current population has been generated and geometry optimised, the evaluation of the fitness function allows for their ranking. The higher the fitness value of a candidate, the higher its rank within the current population. During an EA run, we should observe an increase in the maximum and average fitness values. We have developed a generalisable multi-objective fitness function, which can be used for a series of optimisation problems for porous organic cages, and is easily modifiable and extensible to include other terms and to other molecular materials. In our case, we do not necessarily seek pareto-optimal solutions, rather we use a scalarisation approach where the fitness function aims to allow compromise between the different objectives.a, b, c in eqn (1)), so that the same general fitness function can be tuned for a variety of different applications or targets. More details on the specific implementation of the fitness function can be found in the ESI.Our fitness function, shown in eqn 1), can be specified from a series of three parameters that we believe are important for the design of reasonable candidate porous organic cages. We chose each parameter after a detailed analysis of the structures of the porous organic cages recently synthesized in the literature., can be For porous molecular materials, the material's overall porosity can be linked to both intrinsic and extrinsic porosity. As already defined in the introduction, here we only investigate porous organic cages from a molecular perspective, not addressing the effects of packing multiple cages in the bulk. We have previously demonstrated that consideration of only the molecular structure can be effective even for screening for molecular separation applications of these materials.i.e. the diameter of largest sphere that can fit in the centre of the host molecule), whereas the diameter of a window is determined by the largest circle that can fit in the window.In a similar way, we can compare the window size of the candidate, calculated through the use of the software pywindow,The vast majority of organic cages experimentally synthesised in the literature exhibit a high level of symmetry.15stk software, as previously described.e.g. aldehydes and amines). The newly formed bonds and the atoms directly linked to them are initially relaxed while constraining the remaining atoms within the molecular cage. Following this, the geometry of the whole molecular cage is optimised. Finally, we employ high-temperature molecular dynamics (MD) to probe the flexibility of the optimized individual and its tendency to retain its original cavity – also known as its “shape persistency”. In this step, for each candidate, we perform the MD simulation and extract a series of conformers along the trajectory. We then geometry optimize all the extracted conformers and select the lowest energy conformer, which represents the best guess of the experimental geometry upon desolvation , assuring that different areas of the chemical space are explored. Similarly, the user can choose among five different selection functions for selecting molecules for the next generation, such as only selecting the fittest candidates at each iteration, or using roulette wheel (with or without elitism) or universal stochastic sampling.stk documentation.67In our current implementation of the EA, the user can choose between different types of functions for the initial population generation or for the genetic operations of selection, crossover and mutation. For example, when creating a new population of individuals, the user can decide to randomly sample BBs from the chemical database to generate the required number of cages. Alternatively, the user can employ a diverse initialization, where a random selection of BBs from the database is alternated with the selection of dissimilar BBs ,4Di6Tri imine cage, with a pore size of 5.72 Å and average window sizes of 3.91 Å when OPLS3 optimized.When facing this excess of functions and options, it can be difficult to know which settings, or combinations of settings, will lead the EA to have better or worse performance. To address this question, we ran the EA with 360 different input files, where each input file contained a distinct combination of functions available within our EA implementation. For each input file, 2500 individual runs of the EA were completed. We have run each setup multiple times as EAs are stochastic algorithms and the generation by which the global minimum is found is dependent on the initial population. Since the initial population and mutation operations are random – the generation by which the global minimum is found is given by a distribution of values. A run was considered complete whenever the global minimum structure was obtained or whenever 100 generations were reached. Each run employed a fitness function developed for the re-discovery of Covalent Cage 3 (CC3 cage (pore size and window size used in eqn (1)). This means that for each EA run, the entire search space was loaded into memory and no calculations needed to be performed. Using this setup, 100 EA generations could be completed in approximately 1 s. It also meant that the individual with the largest fitness value, the global minimum CC3 could be found ahead of time. The selected mock chemical space contained 142 trialdehyde BBs and 350 diamine BBs selected randomly from the initial chemical database, as well as the BBs for CC3. For this mock chemical space, we assembled, optimized and calculated the properties of all the possible 4Di6Tri cages, which amounted to a total of 49 700 individuals.To allow for the consecutive execution of 900 000 EA runs (360 × 2500), we defined a smaller chemical space, for which we could pre-generate and optimize all the possible molecular cages. The fitness value of each individual was then pre-calculated with the fitness function targeting the CC3 cage that ranges between 0 and 59% of the times (over the 2500 runs). The best performing setups show a huge improvement when the EA is compared to randomly picking BBs from the chemical database, which only gives a probability of 5% of finding the global minimum. Among the best performing setups, we chose the one including the diverse initial population as we believe that this type of initialization function will perform even better with the large chemical space used for the following case studies. All the functions used for the EA setup can be found in the ESI.In each EA run, we explored an initial population of 25 individuals, allowing 20 possible crossover operations and 5 mutations per generation for up to 100 generations. This means that in a EA run which successfully reaches 100 generations, 2500 possible individuals are explored, corresponding to only the 5% of the total chemical space. As shown in Table S2,a = 10, b = 10, c = 1 from eqn (1), where we defined ideal pore and window diameters of 5.72 and 3.91 Å, respectively. We wanted to put a strong emphasis on the pore and window sizes in order for the global minimum to precisely match the properties of the OPLS3 optimized CC3 cage. We also gave some importance to the level of symmetry of the cage.The fitness function for this investigation employed the coefficients CC3 (triformylbenzene and cyclohexane diamine). Molecular similarity is calculated by means of Dice similarity through the use of Morgan fingerprints with a standard radius of 1 within RDKit.CC3, located on the top-right point of the plot (CC3). However, a few other individuals have medium fitness values . For most of those cases, at least one of the BBs shows high Dice similarity with the CC3 BBs  triformylbenzene, whereas for cage C, triformylbenzene is mixed with a different diamine. Some interesting cages still exhibit a medium fitness value even if their precursors are very different to the ones employed for CC3 . Here we use a fitness function with the coefficients a = 5, b = 1, c = 10, where in this case the strongest evolutionary pressure comes from the asymmetry parameter, while the pore and window size  play a minor role.As already discussed in the introduction, finding porous organic cages that are ‘shape persistent’ is a challenging task. Shape persistency, which is the tendency of a porous cage to retain its original cavity upon desolvation, strongly correlates with the rigidity of the assembly. From the analysis of the cages generated in the previous section, we calculated that only approximately 2% of the total cages were shape persistent  are equivalent to the ones used for the versus those with low asymmetry values. The weak penalty for pores that are not at the target value is the likely reason why a larger variation is observed in the average pore diameters compared to average asymmetry between generations, as the pore size does not significantly affect the fitness value. The right plots (green) show that the asymmetry parameter converges steadily towards 0 . Both the asymmetry of the best individual and of the population average are very low at the end of the EA runs.In 4Di6Tri topology, suggesting that this topology offers better control of the asymmetry when dealing with smaller cages (pore diameter of 4–11 Å), and thus offering better shape persistency. A non-negligible percentage of 8Di12Tri is only observed in run B, where around 20% of the individuals in the final population possess the 8Di12Tri topology.It is of interest whether a certain topology dominates when targeting a specific cage property. 4Di6Tri individuals with pore diameters that fall within 0.5 Å of the target pore size of 5.0 Å. All cages share a very similar shape to that of CC3, typically with some external functionalisation. Some of the external functionalisation or heteroatom substitution on the rings may increase the complexity of the synthesis, however, these hypothetical cages could be simplified prior to any synthesis attempts. In this instance, the external functionalisation has limited or no influence on the fitness function, as this only depends on the pore size, window size and window symmetry. We only observed 8Di12Tri individuals for two of the runs, at rank #16 and #19 within the runs . This suggests that 4Di6Tri cages are preferred when looking for rigid cages with smaller (5 Å) pore diameters.i.e. amine nitrogen to amine nitrogen distance in a diamine and average aldehyde carbon to aldehyde carbon distance in a trialdehyde) is shown on the right-hand side of 4Di6Tri cages of this size in the literature. The mean distance for the tri-topic BBs is larger, at ∼5 Å.To conclude this case study, we analysed the emerging patterns for the molecular properties of the BBs for the individuals in the population. In 4Di6Tri porous organic cages with a cavity diameter of 16 Å. We have selected this cavity size as we found a clear absence of this pore size in the previously reported experimental pore sizes of 116 cages from the literature, as shown in et al., which we calculate to have an internal spherical cavity of 21.9 Å in diameter.CC3 rediscovery section , as shown in Fig. S1.a = 10, b = 0, c = 5, where the ideal pore diameters matched 16.0 Å. This specific set of parameters was chosen in order to give a clear advantage to the pore size diameter, making it the driving force for the evolutionary pressure. All the other setup details are equivalent to the ones from the previous case study.In this case study, we explore our full chemical space with the EA to find The plots in As would be expected, the size of the BBs required to target a large pore (16 Å) shape persistent porous organic cage has considerably increased compared to the previous case study of a small pore cage (5 Å). The mean distance between end groups here is 9–11 Å for tri-topic BBs and 6–9 Å for di-topic BBs (compared to ∼5 and 3.5 Å in case study 1). It is also interesting to see that each of the runs evolves towards slightly different size BBs , suggesting that there are multiple different solutions for shape persistent cages of 16 Å from the database of BBs we explored. It is likely this is due to the interplay between the size of the two BBs, where a larger di-topic BB could compensate for a small tri-topic BB than an alternative solution. We believe that this is an encouraging sign that porous organic cages with an internal cavity diameter of 16 Å are synthetically achievable, despite the absence of synthetic reports to date.CC3, we then carried out two case studies to demonstrate our EA's utility.We have developed an evolutionary algorithm for the prediction of molecular materials with desirable properties. Here, we have applied this to the field of porous molecular cages, where the enormous chemical and structural space of possible molecules makes an efficient sampling crucial. In our flexible implementation, a crossover operation is performed by switching the BBs of two candidate molecules or their topology, and a mutation operation is performed by switching in a new BB from the library. We found that our exploration of hypothetical cages was far more efficient when we included the possibility for mutation to a molecularly similar BB, rather than losing a large portion of the chemical information of a cage in a single mutation step. After demonstrating the effectiveness of our software for the rediscovery of a known cage, 4Di6Tri cages requiring a higher percentage (25–30%) than the di-topic BBs (15–20%). The best candidate from each of the two case studies is shown in 4Di6Tri topology cages were strongly selected for, and all bore a visual similarity to the CC3 series of cages, and were built using di-topic BBs with the typical nitrogen–nitrogen separation of a 1,2-diamine.Our two case studies allowed us to not only generate both specific targets, but further to explore the emerging trends of candidates with the desired properties, so we can identify key chemical features for a given property. We have identified that shape persistent porous organic cages require BBs with a very low percentage of rotatable bonds (<20%), and high percentages of double bonds, with tri-topic BBs in 4Di6Tri cages typically chose a large tritopic building block, for instance the boronate structure shown in In our second case study, we targeted a cage with a cavity of 16 Å diameter, as we found that this was a cavity size absent in previously synthesised cage molecules. The best candidates among stk, is designed to be flexible. The fitness function can be adapted for new design challenges by adding additional parameters. For porous cages, the next obvious step would be to use our EA for targeting specific properties, such as encapsulation of a particular guest, by, for example, seeking to maximise the host–guest binding energy, or improving the separation performance by optimising window size or the diffusion barrier. If a given precursor is known to have a desirable property, such as fluorescence, which could be used for sensing, then the EA could be used to find a BB partner that forms a cage molecule of desired structure. Any designed molecules could also be used as a first step for crystal structure prediction, in order to produce optimal packing and properties in the solid-state. The EA can easily be applied to alternative molecular materials – indeed stk can already automatically assemble linear polymers, covalent organic frameworks (COFs), and small molecules built from multiple BBs. These molecular materials could then be explored with the EA if a fitness function for them is defined.The evolutionary algorithm we have included into our supramolecular toolkit, In the future, we also wish to extend the EA to make the predictions more facile to synthetically realise. Already, by using a database of known BBs and combining them in what would equate experimentally to a one-pot synthesis, we increase the likelihood of the candidates being synthetically achievable. Of course, much of the Reaxys and eMolecules databases feature molecules of high chemical complexity, this could be countered by including a score of synthetic accessibilityThere are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.


Core-shell nanofibers with the ability to carry multiple drugs are attracting the attention to develop appropriate drug delivery systems for wounds dressing applications. In this study, biocompatible core-shell nanofibers have been designed as a promising dual-drug carrier with the capability of delivering both water-soluble and organic solvent-soluble drugs simultaneously. With the aim of fabricating the core-shell nanofibers, the dipping method has been employed. For this propose, core nanofibers made from polyvinyl alcohol (PVA) were immersed in various concentrations of polyacrylonitrile (PAN) and cross-linked by dipping into ethanol. Diclofenac sodium salt (DSs) and gentamicin sulfate (GENs) have been loaded into the core and shell nanofibers as models of the drug, respectively. The morphology study of core-shell nanofibers showed that the concentrations between 1% w/w up to 2% w/w PAN/GENs, with deep penetration into the internal layers of PAV/DSs nanofibers could lead to the core-shell structure. The cytotoxicity results showed the competency of designed core-shell nanofibers for wound dressing application. Also, the release profile exhibits the controllable behavior of drug release. Different kind of nanofibers, such as core-shell nanofibers for drug delivery applications, have been employed as an effective and innovative architecture6. In the last years, dual drug delivery systems based on water-soluble and organic solvent-soluble drugs remaining the challenges when specific target required multiple medications9. Among the advanced methods, appropriate drug delivery systems have been designed based on co-axial or core-shell electrospun nanofibers11. However, the envisaged issues to prepare core-shell nanofibers are adjusting the supplied electrical voltage and polymers viscosity to obtain the uniformed nanofibers13. Therefore, designing the facile methods as well as dipping methods could help to fabricate the core-shell nanofibers for dual drug delivery system17.Nanofibers with simulating the extracellular matrix (ECM)21. However, polyacrylonitrile (PAN) is soluble in the organic solvents, and nanofibers from PAN in addition to carrying the organic solvent soluble drugs are showingthe biocompatibility against the fibroblast cells22.On the other hand, for designing the dual drug delivery systems based on water-soluble and organic solvent-soluble drugs, the polymers should meet the requirements such as biocompatibility and solubility in water and organic solvent. PVA is a biocompatible and hydrophilic polymer, which recently has been used as an undertaking material for scaffold nanofibers preparations regarding tissue engineering and drug delivery applications after cross-linking via glutaraldehyde (GA)Therefore, we developed a dual drug delivery system for wound dressing application based on PVA-PAN core-shell nanofibers with the ability to caring DSs and GENs as anti-inflammation and antibiotic drugs. The DSs and GENs are well-known drugs as anti-inflammatory and antibiotic to reducing inflammatory for the regeneration of wound. Figure 23. From the morphology study of nanofibers, it was obtained that GA did not have a significant effect on the morphology of nanofibers after cross-linking as presented in Fig. 24.The scanning electron microscopy (SEM) was used for the morphology study of the prepared nanofibers in all steps, as shown in Fig. To prepare the final core-shell nanofibers, five different concentrations of PAN solution containing GENs were applied. The SEM images showed that the morphology of PVA/DSs nanofibers affected by 0.5% w/w of PAN/GENs solution due to the high concentration of dimethylformamide (DMF). On the other hand, increasing the concentration of PAN/GENs to more than 2% w/w showed that the solutions with blocking the pores on the surface caused to prevent the PAN/GENs solution penetration into the PVA/DSs nanofibers mat.The mean diameter for the final nanofibers has been calculated as below in Table However, the results showed that submersing the nanofibers into the PAN/GENs solution and coating the surface of PVA/DSs electrospun nanofibers, has changed the mean diameter of nanofibers as presented in Table The transmission electron microscopy (TEM) was used for observing the inner structural nanofibers Fig. . The TEMPreserving the structure of nanofibers are essential due to simulating the extracellular matrix (ECM), especially for biomedical applications. Therefore, in the current research emphasis was on preserving the original structures of nanofibers after achieving the final core-shell structure. The SEM and LSCM images results showed that the structure of nanofibers and porosity was preserved after coating the nanofibers by PAN/GENs solution with concentrations of 1% and 2% w/w and followed by TEM monitoring. Also, the TEM images showed that the best core-shell nanofibers were formed by using 1% and 2% w/w of PAN/GENs solution successfully.The Bunner-Emmett-Teller (BET) was used to evaluate the PAN/GENs penetration into the PVA/DSs nanofibers. As shown in Fig. Also, Fig. −1 due to the presence of N-H. The band related to phenyl group appeared at 1580 cm−1 and substituted phenyl group stretch at 750 cm−1 in Fig. 5(a)25. The spectrum of pure GENs showed the band at 1615 cm−1, and 1512 cm−1 corresponding to an amide group. However, the peak at 1125 cm−1 represents the HSO4 group, 1068 cm−1 related to C-O-C and sharp band at 621 cm−1 appeared due to SO2 in the structure of GENs26. The PVA spectrum in Fig. −1 response to the presence of OH functional group that the intensity of the same peak was decreased and shifted to higher wavelength due to a chemical reaction between PVA and GA after crosslinking24. Therefore, from the results, as summarized in Table −1. The functional groups related to PAN layer appeared at 2241 cm−1 due to C≡N group13.Fourier-transform infrared spectroscopy (FT-IR) was carried out to investigate the chemical functional groups, as shown in Fig. 27. On the other hand, the spectra from O 1s shown the two group of the peak at 530 eV and 531.5 eV as well as C 1s attribute to different spices of oxygen and carbon bonding28. From the N 1s spectra result, it was confirmed that increasing the concentration of PAN/GENs may lead to decreasing the intensity due to the aggregation of PAN/GENs polymer. While the distribution and penetration of PAN/GENs polymer related to the concentrations less than 2% w/w into the PVA/DSs nanofibers caused to increase the intensity in comparison to the high concentrations of PAN/GENs. The results showed that core-shell nanofibers formed with the maximum yield by using the concentrations from 0.5% w/w to 2% w/w.In addition to FT-IR, the X-ray photoelectron spectroscopy (XPS) has utilized to characterize the chemical composition of the core-shell nanofibers prepared by using the various concentration of PAN/GENs from 0.5% w/w to 5% w/w. As shown in Fig. The crosslinking reactions for PVA and PAN have been shown in Fig. As shown in the polymerization reaction in Fig. 18 and decomposition of nanofibers continued up to 350 °C. The final result has summarised in Table 29. Decreasing the thermal stability could be related to morphology change of core-shell nanofiber with 0.5% w/w PAN/GENs concentration as showed in SEM results  spectra were studied to investigate the thermal stability of core-shell nanofibers. TGA and DTGA thermos-gram for different core nanofibers are shown in Fig. lts Fig. . To obsein-vitro degradation, core-shell nanofibers were examined for 21 days in water at 37 °C. As reported in previous degradation studies related to PAN/GENs nanofibers, prepared core-shell nanofibers did not indicate significant degradation during the initial seven days30. From the results, as presented in Fig. For further investigation of 31. As results are shown in Fig. PVA and PAN polymers are well known biocompatible materials for fabricating nanofibers, but there was a concern that drug interactions with polymers may lead to the toxicity in contact with the wound. To determine the toxicity of the final product, samples were examined based on ISO 10993-5 and evaluated from grade zero (nontoxic) to grade four (severe toxic) in comparison to the positive (latex glove) and negative (absence of any sample) controlThe cell proliferation has been don for evaluation of each sample in terms of long-term contact with the wound. The results, as shown in Fig. The results showed that prepared core-shell nanofibers meet the requirements for the preparation of appropriate wound dressing. While the cell adhesion occurred on the surface of all samples, it seems that for the chronic wound such as diabetic wounds, loading the grows factors may help to improve the cell adhesion and cell migration for better regeneration of wound.in-vitro drug release. As shown in Fig. 32. Also, the DSs release profile from PVA nanofibers after coating with PAN/GENs was significantly decreased due to two-stage release theory which introduced by S. Khansari et al.The nanofibers, when used for wound healing, need to evaluate drug release. Therefore, the produced core-shell nanofibers loaded by DSs/GENs with a ratio of 1:1 was studied for 34. Therefore, the thickness of the shell had a significant effect on DSs release from the core and the nanofibers coated with PAN/GENs solution with higher concentrations in the core-shell structure caused to improve extended-release of DSs from PVA nanofibers.Based on two-stage release theory, the PAN polymer containing the GENs is playing the substantial active role to control the DSs release from core nanofibers with covering the core part35.On the other hand from the release profile related to GENs, as shown in Fig. 36. On the other hand, the release from core nanofibers showed that DSs release was extended up to 60 hrs, while in the previous research the release time for DSs has been reported about 24 hrs when DSs had been loaded into the PVA nanofibers37.The release of GENs has compared with previous studies. The results showed that the release of GENs had been completed after 12 hrs, that release duration was similar as reported for core-shell nanofibers in the current researchThe results showed that part of the drug was released during the washing process, but the core-shell nanofibers with concentrations of 1% to 2% PAN showed relevant results use as a carrier for drugs and current structure meet the demands to be used as local drug delivery systems for wound dressing.We are reported a facile method to obtain the advanced core-shell architecture for dual drug delivery system. The core-shell nanofibers were successfully designed and prepared for carrying both water-soluble and organic solvent soluble drugs at the same time. In comparison to traditional methods as well as co-axial electrospinning to obtain the core-shell nanofibers, this method was exhibited a high performance for a dual drug delivery system and wound dressing. Attributed to results the PAN concentration from 1% w/w up to 2% w/w were appropriate concentrations to achieve the unformed core-shell nanofibers. Prepared core-shell nanofibers showed the excellent biocompatibility in addition to optimized dual drug delivery, simultaneously. Also, it is believed that the method used in this study could extend to various drugs. Furthermore, this method could be the beginning of initial design for preparation of multi-layer nanofibers architecture for multiple drug delivery. Also, the prepared core-shell nanofibers showed the enormous potential of various fields such as protein delivery, sensors, photocatalysis, and tissue engineering in addition to drug delivery with changing the functionality instead of drugs.Sodium hydroxide with a purity of 97% (NaOH), acetone, ethanol (99.5), hydrochloric acid, purity of 35–37% and glutaraldehyde (GA) (50%) was purchased from Wako Chemical Industries, Ltd Japan. Gentamicin sulfate salt (GENs), diclofenac sodium salt, polyacrylonitrile (PAN) with , polyvinyl alcohol (PVA), , Dimethylformamide (DMF) were purchased from Aldrich Chemical.The PVA solution with a ratio of 11% w/w was prepared under stirring at 60 °C. The 5 mg/mL DSs was added to the obtained solution and mixed until a white suspension of PVA/DSs formed. The pH of viscose suspension was increased up to 9 by NaOH 0.5 M to obtain a transparent PVA/DSs solution. The clear PVA/DSs solution was subjected to a 20 mL syringe with a capillary tip gage of 20 with a 12 cm distance from the collector and supplying 17 kV, and continuously electrospinning continued for 24 hrs to obtain the PVA sheet nanofibers with the thickness of 0.16 mm. The viscosity of PVA polymer has been measured as 7.51 gr/cm/s with the surface tension of 39.85 mN/m and conductivity of 1.12 ms/cm.The obtained PVA/DSs nanofibers mat was immersed for 10 minutes into the cross-linking solution prepared by a mixture of (GA): ethanol: hydrochloric acid (2:1:0.2 gm) in a 50 mL glass round bottom flask and the volume was adjusted to 50 mL by adding acetone. The unreacted HCl and GA washed out from cross-linked PVA/DSs nanofibers mat with deionized water for 5 minutes and dried at 60 °C for 30 minutes.The different concentrations of PAN solution  were prepared in the DMF solution under stirring for 12 hrs, and 5 mg/mL GENs was added to the obtained solutions. The PVA/DSs nanofibers from the previous step were immersed in each PAN/GENs solution for 1 minute separately. The PVA/DSs nanofibers containing the PAN/GENs were immersed into the ethanol immediately for 30 seconds for crosslinking the PAN solution.The final product was immersed in deionized water to wash out the ethanol and DMF for 10 minutes, and the obtained mat was dried at 37 °C for one hr. Figure −1. The thermal stability of each sample studied by the thermal- plus TG-8120  from 20 to 350 °C under air temperature at the heating rate of 10 °C. The toxicity of core-shell nanofibers was evaluated by using (ISO 10993-5). Briefly, the samples were sterilized by ethanol 70% and washed with PBS 1X for 5 minutes. The fibroblast cell from rat was obtained from Riken Bioresource Center (ID: RCB1862), Tsukuba, Japan and cultured in accordance with the guidelines set forth by the Riken Bioresource Center for the use for a cell culture test. The cells were cultured in 24 multi-well containing DMEM: FBS (85:15) until cells covered the 80% of the bottom at 37 °C with 5% CO2. The sterilized samples were placed on the cells layer and incubated for 24 hrs at the same conditions. In addition to toxicity evaluation, cell proliferation and cell adhesion assay were studied. The cell proliferation was evaluated via the WST-1 assay for viable cell percentage. Briefly, 1000 cell per well have been cultured in the presence of samples in the 96 multiwall plates and placed in an incubator at 37 °C for 1, 3, and 7 days respectively. After incubation, the WST-1 solution was added into each well with a ratio of 20% and incubate for 2 hrs. The plates were read at 560 nm with a microplate reader. For cell adhesion study, the cells were cultured for seven days in the presence of each sample, respectively. The cells fixed on the surface of samples, after washing with PBS and immersion into the glutaraldehyde 25% for 30 minutes. Dehydrate with ethanol series from 50% to ethanol absolute has been done for 2 hours and fixed cells were rated from nontoxic (grade 0) to severe toxically (grade 4), and samples have been used for morphology study via SEM. The Bunner-Emmett-Teller (BET) surface area of prepared samples was measured by using Micromeritics TriStar II 3020 version 3.02 with a degasser accessory Teledyne Hastings Instruments. The internal diameter of the sample tube was 3/8 inch, and 0.1 g of each sample was transferred in sample tubes, respectively. BET surface area was calculated by the amount of nitrogen adsorbed by the samples. The UV-visible Spectrophotometer  was employed to study the release behavior for 72 hrs. For this propose, the final core-shell nanofibers containing the models of drugs were immersed into the 20 mL deionized water under the shaking with a speed of 60 rpm. During the specified times  the 3 mL of solution from each sample was removed and exchanged with 3 mL fresh deionized water and kept in the refrigerator. The absorbance of removed solutions from each sample has been measured by UV-visible Spectrophotometer based on λ (max) for each drug. The λ (max) was measured 203 and 277 nm for gentamicin sulfate and diclofenac sodium salt, respectively. The amount of drug release compared with the standard curve and the cumulative release has been measured by the following formula:The Scanning Electron Microscope (SEM)  with an accelerating voltage of 15 kV has been employed for morphology observation of nanofibers after coating with platinum for 90 seconds. The Transmission Electron Microscope  was conducted to observe the interface between core and shell nanofibers. Attenuated Total Reflectance (ATR) spectroscopy  was used from 400 to 4000 cm


The molecular framework is planar within 0.19 Å. The molecules form a head-to-tail type of π-stacking dimer with an interplanar distance is 3.50(1) Å, where methylthio groups face away from each other. The molecules are also linked by weak intermolecular S···S heteroatom interactions [3.497(1) and 3.572(1) Å] to construct a unique one-dimensional molecular tape structure.The title compound, 5--1,3-diaza-2,4,6-trithiapentalene (4,5-dimethylthiothiadiazolotetrathiafulvalene, molecular formula C The geoadiazole . The moladiazole  and bis [3.382(5) Å] hydrogen bond and an interdimer distance is 3.71(1) Å. Between the dimers, the S7–C8 methylthio groups are placed across from each other. The overlap modes of the dimer of 1 are shown in π-conjugated system is observed in the interdimer overlapping. This is due to steric hindrance of the S7–C8 methylthio group. The intradimer arrangement of the molecules corresponds to the overlap of HOMO and LUMO for 1. As illustrated in In the dimer, an interplanar distance is 3.50(1) Å and the S7–C8 methylthio groups face away from each other. On the other hand, the dimers are linked by an intermolecular C8–H8A···N1 for S2···S7 and 3.572(1) Å for S5···S6] in the crystalline state. The S···S interactions are 0.8-2.9 % shorter than the sum of the corresponding van der Waals radii and build up a unique one-dimensional molecular tape structure  Å, where methylthio groups face away from each other. A unique one-dimensional molecular tape network can be built by the usage of intermolecular S···S heteroatom interactions [3.497(1) and 3.572(1) Å] as a connection tool.We have determined the crystal structure of the unsymmetrical tetrathiafulvalene derivative with a fused 1,2,5-thiadiazole ring and methylthio groups by X-ray diffraction. The molecular framework is planar within 0.19 Å. The molecules form a head-to-tail type of 1 was synthesized by the reaction of thiadiazolo-1,3-dithiol-2-one with 4,5-dimethylthio-1,3-dithiole-2-thione according to the literature method  and 156 variable parameters, converged with R1 = 0.055 and wR2 = 0.125. Crystal data and refinement details are summarized in viahttp://www.ccdc.cam.ac.uk/data_request/cif, by e-mailing data_request@ccdc.cam.ac.uk or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336033.The data of a single crystal with a size of 0.35 × 0.05 × 0.05 mm was collected at 295 K on a Rigaku Mercury CCD diffractometer. No absorption correction was applied. The crystal structure was solved by direct methods and refined by full-matrix least-squares on SHELXL97 . All non1 were evaluated in single point 3-21G calculations using the crystallographic geometry of 1 with Gaussian 98 [The molecular orbital shapes of HOMO and LUMO of ssian 98  and weressian 98 .


Molecular shuttles are the basis of some of the most advanced synthetic molecular machines. In these devices a macrocycle threaded onto a linear component shuttles between different portions of the thread in response to external stimuli. Here, we use optical tweezers to measure the mechanics and dynamics of individual molecular shuttles in aqueous conditions. Using DNA as a handle and as a single molecule reporter, we measure thousands of individual shuttling events and determine the force-dependent kinetic rates of the macrocycle motion and the main parameters governing the energy landscape of the system. Our findings could open avenues for the real-time characterization of synthetic devices at the single molecule level, and provide crucial information for designing molecular machinery able to operate under physiological conditions. Molecular shuttles are bi-stable and stimuli-responsive systems that are considered potential elements for molecular machinery. Here, the authors use optical tweezers to measure the force dependent real-time kinetics of individual molecular shuttles under aqueous conditions. Many of these molecular devices are currently the subject of intense research interest due to their many potential applications in diverse fields from biomedicine to nanorobotics and their kinetics and thermodynamics in bulk are well understood3. However, quantitative information on the dynamics and mechanistic principles behind the operation of individual synthetic molecular devices is still lacking4. Single-molecule force spectroscopy techniques, such as atomic force microscopy (AFM), optical and magnetic tweezers, have revolutionized our understanding of the dynamics of biological motors6, but they have not yet had impact on the field of synthetic molecular machines. To date, AFM has been the most widely used technique to pull on synthetic mechanically interlocked molecules in order to investigate their mechanochemical properties12. In recent years, optical tweezers have revealed themselves as a versatile, powerful single-molecule manipulation technique to study biology at the single-molecule level. By attaching the system under study to an external probe (a microscopic bead acted upon by a photonic field) it is possible to measure and control in real-time the nanometer displacements  and picoNewton forces (<10 pN) that define the operational dynamics of biological systems under aqueous conditions14.Recent advances in synthetic molecular machinery have led to systems with the ability to work as molecular motors17. These architectures are the basis for some of the most advanced examples of synthetic molecular machines22. We show that coupling of a molecular shuttle with double-stranded DNA (dsDNA) enables its single-molecule manipulation under aqueous conditions. This strategy enables us to determine key parameters of the operation of this synthetic device under near-physiological conditions, including information that cannot be obtained from bulk approaches. First, we measure the mechanical strength of the macrocycle at each station and the free energy of the shuttling reaction. Second, we quantify the force-dependent real-time shuttling kinetics of the macrocycle between the two stations and calculate the energy landscape of the system under our experimental conditions.Here, we exploit the properties of optical tweezers to quantify the real-time operation of individual synthetic molecular shuttles. In molecular shuttles, a macrocycle trapped onto a linear component (axle) can be moved reversibly between two or more portions of the axle (stations), in response to external stimulifum) and succinic amide-ester (succ) stations, each of which can establish up to four hydrogen bonds with the macrocycle with sufficiently different affinities to yield a fum:succ occupancy ratio biased towards the fum station, even in the strongly competing solvent d6-DMSO (Supplementary Methods). To interface the synthetic device with the optical tweezers, a single shuttle was connected between two functionalized beads using two dsDNA molecules, Fig. fum station to a bead on top of a micropipette, providing physical separation (~282 nm) between them. Experiments were performed under aqueous conditions  at 22 ± 1 °C.We synthetized a hydrogen-bonded Leigh-type molecular shuttle, featuring a tetraamide macrocycle locked onto a oligoethyleneglycol axle by two diphenylethyl groups (stoppers) situated at either end of the axle, Fig. −1  and ΔLc, giving ΔG = 31 ± 2 kBT 28. ΔG equals the free energy of shuttling from the fum to the succ station at zero force plus the free energy for stretching the rotaxane-DNA hybrid from a force of 0 pN to f1/2, which we measured as 11.8 kBT .In order to investigate the mechanical strength of the interaction of the macrocycle at each station, individual rotaxane-DNA hybrids were subjected to pulling-relaxing cycles by moving the optical trap relative to the micropipette at a fixed pulling rate of 200 nm s−1 Fig. . At low  pN Fig. , which af1/2 with a feed-back stabilized protocol capable of maintaining a preset force within ~0.05 pN. Under these conditions, hundreds of shuttling events of the macrocycle between the two stations were monitored in real-time during minutes . These experiments yield a direct calculation of ΔG based on the population distributions, and agree well with the value for ΔG obtained with the pulling-relaxing experiments described above. In addition, the results of the fits rendered the relative distances from the fum and succ stations to the transition state at f1/2, xfum = 10 ± 2 nm and xsucc = 6 ± 2 nm, respectively. Therefore, for these particular experimental conditions, the distance to the transition state at the coexistence force is asymmetric: the macrocycle needs to travel a longer distance to reach the point where the probability of completing the shuttling process is 50% after abandoning the stronger fum station, compared to the weaker succ station.To investigate the force dependence of the shuttling rates (inverse of residence times) from 3. This experimental system may enable the study of the combined effect of force and other variables  on the operation of individual molecular shuttles under aqueous conditions. The results from these studies facilitate more reliable comparisons with biological devices  and, therefore, help to design more efficient synthetic and hybrid molecular devices. From a more general point of view, once the synthetic and technological challenges to study the thermodynamics and real-time kinetics of individual synthetic molecular devices have been surmounted, the atomic precision of synthetic chemistry will allow addressing fundamental questions, such as the structural basis for microscopic reversibility or the controversial presence (or absence) of quantum effects in nanoscopic engines36.In summary, our experimental design allowed the measurement of hundreds of pulling-relaxing cycles and real-time shuttling events on tens of individual molecular shuttles. From these data, we obtain crucial information that is not accessible from bulk experiments, such as the mechanical stability of the macrocycle at each station, the dynamics of shuttling, and a clear picture of the energy landscape at different forces, which reveals broad energy minima separated by a well-defined transition state, positioned asymmetrically with respect to each station. This experimental picture is significantly different from idealized theoretical energy profiles1H: 400 MHz; 13C: 100 MHz; COSY: 400 MHz; HSQC: 400 MHz), spectrometer at 298 K, unless otherwise stated, using partially deuterated solvents as internal standards. Coupling constants (J) are denoted in Hz and chemical shifts (δ) in ppm. Multiplicities are denoted as follows: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad. Fast Atom Bombardment (FAB) and Matrix-assisted Laser desorption ionization  experiments (MALDI-TOF) were recorded on a VS AutoSpec spectrometer and a Bruker ULTRAFLEX III spectrometer, respectively. A detailed description of the synthesis of the molecular shuttle is provided in Supplementary Methods.All solvents were dried according to standard procedures. Reagents were used as purchased. All air-sensitive reactions were carried out under argon atmosphere. Flash chromatography was performed using silica gel . Analytical thin layer chromatographies (TLC) were performed using aluminum-coated Merck Kieselgel 60 F254 plates. NMR spectra were recorded on a BrukerAvance 400 (2 in THF/Py/water 7/2/1). For the capping of residual 5′-OH-groups, a mixture of solution A  and solution B (10% 1-methylimidazole in THF) was used. After completion of the synthesis, the oligonucleotides were cleaved from the solid support with concomitant removal of the Fmoc and β-cyanoethyl protecting groups by reacting the oligonucleotide-charged solid support with 28% aq. NH3 at 55 °C for 20 h. The solution was filtered and the filtrate was concentrated in vacuum. The residue was dissolved in 750 μL water. For purification of this crude oligonucleotide solution, a volume containing ~40 nmol crude oligonucleotide was applied to gel electrophoresis . The oligonucleotide-containing segments of the gel were visualized by UV-light (260 nm) and separated from the rest of the gel. Oligonucleotides  were extracted from the gel using an elutrap system . The solutions were desalted using a NAP-10 column and concentrated in an evaporating centrifuge.The syntheses of oligonucleotides were performed on a MerMade 4 synthesizer (BioAutomation Corporation). For each oligonucleotide synthesis, columns were filled with the corresponding Controlled Pore Glass (CPG) solid support. Anhydrous MeCN was used as solvent. For the cleavage of DMTr protecting groups, the resin was purged with 3% trichloroacetic acid in anhydrous DCM. The removal of the acid was carried out by purging with anhydrous MeCN. The activation of the phosphoramidite functionality was effected by a 0.25 M benzylthiotetrazole solution in anhydrous MeCN. The coupling time for standard phosphoramidites was 2 min and for cyclooctine derivative 5 min. Oxidation of P(III)-species was attained by alkaline iodine solution  was added rotaxane 15 (60 µM) in H2O/DMF . The mixture was allowed to stir overnight at room temperature. Followed and purified by gel electrophoresis . The oligonucleotide-containing segments of the gel were visualized by UV-light (260 nm) and separated from the rest of the gel. Oligonucleotides were extracted from the gel using an elutrap system . The solutions were desalted using a NAP-10 column and concentrated in an evaporating centrifuge, to give the oligonucleotide 3  in Hfum and succ stations of the relaxed axle in the gas phase was calculated as 11.2 nm by geometry optimization with the molecular mechanics , labeled with streptavidin in one end and Dig in the other, connects the biotin labeled stopper to an anti-Dig covered bead held by suction on a micropipette.For optical tweezers experiments a single shuttle was connected between two functionalized beads using two dsDNA spacers, Fig. BamHI and SacI restriction endonucleases (NEB). BamHI end was labeled with multiple Dig as described before. The 830 bp dsDNA spacer connecting the biotin labeled stopper to the bead on the micropipette was synthetized by PCR amplification of the poly-linker section of the puc19 vector with two DNA oligonucleotides: one labeled with biotin and the other with digoxigenin at the 5′-end. The spacer was then incubated with saturated amounts of streptavidin and attached to anti-digoxigenin covered polystyrene micron-beads .The 2686 dsDNA handle was derived from the puc19 vector (Novagen). The plasmid was cut with 39, with a limiting spatial resolution of ≤ 1 nm. In the optical tweezers the molecular shuttle is attached between two beads thought two dsDNA molecules as described above and in the main text , which is different from the end-to-end distance of the molecular system we are interested in (Xend–end). The end-to-end distance of the system is calculated as follows:F is the force applied to the system, k the stiffness of the trap, and F/k corresponds to the distance (nm) moved by the bead out of the trap center (bead position). We assume a linear spring restoring force. The stiffness of the trap depends on the power of the lasers, the size and the index of refraction of the bead on the trap and the viscosity of the surrounding medium. The value of the stiffness obtained in our experimental conditions is k = 0.135 ± 0.0043 pN nm−1 (for 3 μm beads with a laser power of 60 mW).Optical tweezers experiments were performed with a highly stable miniaturized dual-beam optical tweezersConstant force measurements were performed with a feed-back stabilized protocol capable of maintaining a preset force within ~0.05 pN by moving the beads closer or further apart. All experiments were performed under near-physiological conditions  at 22 ± 1 °C and data was taken at 1 kHz.In the hopping experiments the residence times at each station were calculated by partitioning each trace into two states using a threshold set at the midpoint between the histogram peaks.fum to succ station, kfum, and its reverse ksucc, were found to depend exponentially on force  is the shuttling rate at zero force; xfum and xsucc are the relative distances between the fum and succ stations to the transition state at f1/2, respectively, and ΔG is the shuttling free-energy of the molecular switch at zero force, defined as ΔG = f1/2·Δx (where Δx is the distance between the fum and succ stations).The shuttling rates from rce Fig. . Accordiρ(x) corresponds to the probability distribution of an extension (x) and was obtained from the extension distributions shown in Fig. The shuttling potential energy profile at a given force can be computed according to Boltzmann distribution as follows: Supplementary InformationPeer Review File


Mikania micrantha is one of the top 100 worst invasive species that can cause serious damage to natural ecosystems and substantial economic losses. Here, we present its 1.79 Gb chromosome-scale reference genome. Half of the genome is composed of long terminal repeat retrotransposons, 80% of which have been derived from a significant expansion in the past one million years. We identify a whole genome duplication event and recent segmental duplications, which may be responsible for its rapid environmental adaptation. Additionally, we show that M. micrantha achieves higher photosynthetic capacity by CO2 absorption at night to supplement the carbon fixation during the day, as well as enhanced stem photosynthesis efficiency. Furthermore, the metabolites of M. micrantha can increase the availability of nitrogen by enriching the microbes that participate in nitrogen cycling pathways. These findings collectively provide insights into the rapid growth and invasive adaptation. Mikania micrantha is an extremely fast-growing invasive plant species that can cause serious damage to natural ecosystems. Here, the authors assemble its chromosome-scale reference genome and explore possible mechanisms that contribute to its rapid growth. The vine is listed as one of the top 100 worst invasive species by the International Union for Conservation of Nature (IUCN)2. It is problematic in tropical Asia, in parts of Papua New Guinea, on Indian Ocean islands and Pacific Ocean islands and in Florida in the United States4, causing serious damage to the natural ecosystems5. The invasion of M. micrantha has caused economic losses related to forests and crop production of up to US$4000 ha−17 by climbing plants to the canopy, and blocking the sunlight and has led to a loss of genetic and species diversity, a decline in soil and food web stability, and altered nutrient cycling11.MM. micrantha possesses a variety of biological characteristics associated with successful invasive plant species. It germinates early in the growing season; grows extremely fast (with a maximum mean growth rate of 20 cm day−1)12; roots from each vine node; has a smothering habit; produces very large numbers of widely dispersed seeds 13, which are very small (8.92 × 10−5 g 1000 grains−1)15; and has an ability to survive harsh conditions15. M. micrantha also exhibits a high degree of morphological and physiological plasticity in response to different light environments16. In addition, M. micrantha has strong allelopathic effects on other plants and soil microbes18. Allelopathy is the influence of a plant over a target species, through the release into the environment of compounds that influence the growth and development of biological systems19. Various allelochemicals, that are produced by a plant must escape into the environment and subsequently influence the growth and development of other plants, such as Mikania sesquiterpene lactones (STLs)22 and phenolic compounds23 have been isolated from different M. micrantha plant tissues. However, the molecular mechanism underlying the fast growth of M. micrantha and the biosynthetic pathways of its characteristic allelochemicals are not yet clear.24, sunflower25, artichoke26, and horseweed27, have been sequenced, deepening our understanding of Asteraceae evolution and promoting biological studies of plants in Asteraceae. Although some studies have examined the biological characteristics of M. micrantha, genomic and molecular data are scarce, which limits in-depth studies of this invasive species. Here, we generate a reference genome of M. micrantha and perform analyses of transcriptomic, metabolomics, and metagenomic data to explore possible reasons causing its rapid growth.Asteraceae is not only one of the largest, most valuable plant families, but also contains the largest number of invasive alien species worldwide. Genomes of some important species of Asteraceae, including lettuceM. micrantha is 1.86 Gb  of contigs was anchored and oriented into the 19 linkage groups  of single molecule real-time (SMRT) sequences on the PacBio RS II and Sequel platform (Supplementary Table http://www.orthodb.org) were identified as complete in the reference gene set by BUSCO . In total, more than three quarters of the M. micrantha genome consists of TEs  , lettuce (Lactuca sativa), artichoke thistle (Cynara cardunculus), chrysanthemum (Dendranthema morifolium), Artemisia annua and coffee (Coffea canephora) with grape (Vitis vinifera) as an outgroup, showing that the M. micrantha diverged from grape ~109 million years ago (Mya), from artichoke ~51 Mya, and from lettuce ~43 Mya. Notably, M. micrantha and sunflower were diverged ~31 Mya  in dicotyledons, the whole-genome triplication WGT-1 (approximately 53–62 Mya) shared with asterids II, and the whole-genome duplication WGD-2 (approximately 32–36 Mya) shared with sunflower. In contrast to the genomes of other species in Asteraceae, the M. micrantha genome exhibits a large number of recent segmental duplications. With Ks values lower than 0.2, we identified 127 syntenic blocks ranging from 46 to 8955 kb in length and containing 23% of all syntenic genes, indicating that the M. micrantha genome experienced a recent explosion of lineage-specific segmental duplications  changes in M. micrantha leaves were not significantly different between day and night based on analysis of the carbon isotope ratio  photosynthesis, but the genes regulating the CAM pathway are often silent or inactivated in non-CAM plants28. However, the expression of genes related to CAM photosynthesis is activated in response to changes in the external environment or in certain growth stages30. To adapt to environmental changes, a few plants have evolved to use both the C3 and CAM photosynthesis pathways, with interconversion between growth stages31.In this study, the diurnal carbon isotope δC changesM. micrantha genome contains 51 candidate genes involved in the carbon fixation module of CAM, including carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxykinase (PEPCK), malic enzyme (ME), malate dehydrogenase (MDH), and pyruvate orthophosphate dikinase (PPDK)  and sunflower (a C3 plant), respectively. In addition, the activity of PEPC in the leaves of M. micrantha (2.8 μmol mg−1 protein) was higher than that in pineapple leaves (2.0 μmol mg−1 protein).Here, we identified genes related to the CAM pathway based on homology to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The DK) Fig. . The genM. micrantha  and 9:00 P.M. (night) from leaf tissues of potted ion Fig. . The eigion Fig. . In addiday Fig. . Carbon day Fig. . The sto3 and C4 plants open to absorb carbon dioxide during the day, while they close at night, except for in CAM plants, whose stomata exhibit a unique pattern of diurnal variation. Surprisingly, the stomata of M. micrantha leaves were not only always open during the day , respiration and gross photosynthetic rate and compared these values with those of five other species. The Pn of the M. micrantha stem was approximately 1.5 μmol CO2 m−2 s−1, and was significantly higher than that in Paederia scandens, Pueraria lobata, Stephania longa, Merremia hederacea, and Ipomoea cairica , which was more than ten times lower than that in the stems of the undefoliated samples , carbon fixation , and plant hormone signal transduction pathways  position and stereoselective hydroxylations of GAA, which can result in STLs with four unique configurations 37. Ih8H homolog is important enzyme convert to 7, 8-trans lactone from GAA. As all the detected STLs in this study had a 7, 8-trans lactone, we were investigating for a homolog of Ih8H, an enzyme capable of synthesizing 7, 8-trans inunolide, the putative precursor of these M. micrantha STLs.STLs, which are mainly derived from the precursor germacrene A acid (GAA), are known for their allelopathic effects and are characteristic of the family AsteraceaeM. micrantha and focused on the metabolic pathways involved in inunolide synthesis, yielding a total of 38 genes in this pathway  showed a significant higher STLs content in the leaf and flower , but almost no STLs were detected in stem and root  and leaching  , assimilatory nitrogen reduction (nirB and nasA), dissimilatory nitrogen reduction (napA and nirB/D), and ammonification (ureC) in M. micrantha soil was higher than that in the soil of its two neighboring native species (P. chinense and P. scandens) soil and the control  in M. micrantha soil was significantly lower than that in P. chinense and P. scandens soil  separately to the bare soil near an M. micrantha monoculture to investigate the metabolite-microbe interactions. The STLs significantly increased not only the concentration of carbon dioxide  using the RNA extraction kit . The mRNA from all samples was purified using the Truseq RNA Sample Prep Kit (illumina), followed to be sequenced on an Illumina HiSeq 2500 sequencer.Genomic DNA was extracted from a https://github.com/fanagislab/assembly_2ndGeneration/tree/master/clean_illumina), resulting in a clean and high-quality reads data with average error rate <0.001. The raw PacBio reads were assembled by the software canu-1.652, and contaminating heterozygous fragments were filtered by using the result of falcon/falcon-unzip53. We used the alternative heterozygous haplotype sequence (hapotig) assembled by falcon/falcon-unzip to align to the contigs assembled by canu and d filtered out the heterozygous contigs from canu assembled result  with E-value cutoff of 10−5. The pathway analysis and functional classification were conducted based on KEGG database. InterProScan60 was used to assign preliminary GO terms, Pfam domains and IPR domains to the gene models.The gene models were predicted by EVidence Modeler v1.1.1M. micrantha was constructed by RepeatModeler . TEs were identified by RepeatMasker  using both Repbase library and the de novo library. Tandem repeats were predicted using Tandem Repeats Finder v4.07b61. The LTR-RTs were annotated with an in-house pipeline that uses LTR-finder62 and LTR-retriever63. The LTR insert time is estimated by LTR-retriever.A de novo repeat library for 64, requiring at least ten paralogous gene pairs per collinear block, and the duplicate_gene_classifier in MCscanX was implemented to classify the origins of the duplicate genes into different types. Orthologous and paralogous gene families were assigned by OrthoFinder65 with default parameters.Duplicated genomic fragments were identified by MCscanX66 and then concatenated into one super-sequence. The phylogenetic tree was constructed by maximum likelihood (ML) using PhyML v3.067 with best-fit model (MtMAN) that was estimated by ProtTest368. The Bayesian Relaxed Molecular Clock (BRMC) approach was adopted to estimate the neutral evolutionary rate and species divergence time using the program MCMCTree, implemented in PAML v4.9 package69. The calibration time interval (110–124 Mya) of grape was adopted from TimeTree (http://www.timetree.org). We used the the inter- and intra-syntentic gene pairs to calculate the synonymous mutation rate (Ks) values by KaKs_Calculator 2.0 with default parameter70.Gene families that contain only one gene for each species were selected to construct the phylogenetic tree. The protein sequences of each gene family were independently aligned by muscle v3.8.31https://github.com/fanagislab/assembly_2ndGeneration/tree/master/clean_illumina) and then mapped to the reference genome of M. micrantha using TopHat v. 2.1.071 with default settings. Reads count were calculated with HTSeq v. 0.9.172 using BAM results from TopHat v. 2.1.0, and TPM values were then calculated for every gene in the samples. We continue to identify the differentially expressed genes by DEseq273. Co-expression network analysis was performed by WGCNA package74 using all transcriptomes. WGCNA network construction and module detection was identified using an unsigned type of topological overlap matrix with a soft-thresholding power β of 28 (correlation coefficient R2 > 0.9).Transcriptome reads were filtered by in-house software clean_adapter and clean_lowqual . Soil chemical characteristics, nitrogen, phosphorus, potassium content of plant tissue and the population density of soil cultured bacteria were, respectively, compared using one-way ANOVA, followed by least significant difference tests at Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The Thailand National Strategic Plan on Antimicrobial Resistance (AMR) 2017–2021, endorsed by the Thai Cabinet in 2016, aims to increase public knowledge about antibiotics and AMR awareness by 20% by 2021. This study assesses the prevalence of antibiotics use, clinical indications and sources; knowledge and access to information related to antibiotics and AMR; and factors related to level of knowledge and access to information among Thai adult population.An AMR module was developed and embedded into the 2017 Health and Welfare Survey; a cross-sectional, two-stage stratified sampling, nationally representative household survey carried out biannually by National Statistical Office. The survey applied a structured interview questionnaire. The survey was conducted in March 2017 where 27,762 Thai adults were interviewed of the AMR module. Data were analyzed using descriptive and inferential statistics.The one-month prevalence of antibiotic use was 7.9% for three common conditions; flu (27.0%), fever (19.2%) and sore throat (16.8%). The majority of antibiotics (70.3%) were provided by public or private healthcare facilities, and 26.7% by pharmacies. Thai adults have low levels of knowledge about antibiotics; only 2.6 gave correct answers to all six statements related to antibiotics, while 13.5% gave wrong answers to all six statements. A few factors associated with knowledge and having received information on antibiotics were assessed. People who have higher education levels, and belong to richer wealth quintiles, and receive antibiotics and AMR information have significantly higher levels of knowledge about antibiotics. In the last 12 months, only 17.8% of respondents had heard information about the proper use of antibiotics and AMR; mostly from doctors (36.1%), health workers (24.8%) and pharmacists (17.7%).There is a large gap of public knowledge about the use of antibiotics. The main communication channel is through healthcare professionals, which indicates they are key persons in communicating information about the proper use of antibiotics to the public. In order to improve awareness and understandings about antimicrobial resistance (AMR) as recommended by the World Health Organization (WHO) , countriThe Thailand National Strategic Plan on AMR 2017–2021 (NSP-AMR) was endorsed by the cabinet in August 2016 . One of Due to lack of knowledge among lay people, lack of access to qualified and affordable health services, self-medication of antibiotics is a widespread phenomenon worldwide ; which cRecognizing the complexity of antibiotics self-medication, government should develop effective multifaceted interventions that target healthcare professionals and patients simultaneously; while in parallel, ensure provision of adequate and affordable access to health care services can prevent self-medication with antibiotics.In response to the gap of understanding about the use of antibiotics in the population; this study aimed to generate baseline evidence on the one-month prevalence of antibiotic use, clinical indications and sources of antibiotics; the levels of knowledge about antibiotics and AMR among the Thai adult population, and assessed factors associated with knowledge and having received public information on proper use of antibiotics. This evidence is essential to assess progress in implementing the NSP-AMR.The AMR module was modified from the “Antimicrobial Resistance Eurobarometer Survey” with additional questions on knowledge of antibiotics specifically designed to suit the national context.The AMR module had three sections. The first section asked about the use of antibiotics in the last month, the sources of antibiotics and the reasons for using them. The second section asked about knowledge of antibiotics, which was assessed using true, false statements and one question. To ensure accuracy of Yes and No answer, a “do not know” answer also provided. Section three explored whether respondents had received information during the last twelve months about antibiotics and AMR, the sources of such information and whether the information changed their view and future practice of using antibiotics. See The content validity was assessed by five experts from different fields  on the logic and clarity of the content. The pilot testing was conducted with a sample of 40 individuals for improving the quality of questions. These 40 individuals were randomly selected from general staffs in the Ministry of Public Health (representing lay people); with an aim to improve the content validity. We found the contents are easily understood and accurate with no controversy. There is minor amendment of the questionnaire after piloting.The HWS, established by the NSO since 1974, is a national representative cross-sectional household interview survey carried out biannually by the NSO. The survey was conducted in March 2017.The HWS applied a stratified two-stage sampling. Greater Bangkok and the remaining provinces constituted strata, with 77 strata altogether. Each stratum was divided into municipal (urban) and non-municipal  areas. In the first stage, sampling enumeration areas (EAs) from urban and rural area were selected using probability proportional to size based on total household numbers. In the second stage, private households were the sampling units. In each sampled EA, a systematic random sample of private households was selected.The HWS contains comprehensive sets of questions categorized into modules, including 1) socio-economic and demographic parameters; 2) types of insurance coverage; 3) illness and use of services: 3.1—illness in the last month, use and choices of services and out-of-pocket payments by households; 3.2—use of prevention and health promotion services in the last 12 months; 3.3—admission in the last 12 months, uses, choices and payment; 3.4—use of dental services in the last 12 months; 4) unmet healthcare needs, 5) food consumption; 6) AMR module; and 7) housing characteristics and ownership of durables for computing wealth index and quintiles.The objective of the sample design for HWS 2017 was to produce statistically reliable estimates for each indicator, represented at national level, urban and rural areas, and five geographical regions. The sample size is not design to represent provincial level.Stratified two-stage sampling approach was used. The first stratum is all 77 provinces (including Bangkok); the second stratum in each province has two sub-strata, namely urban and rural areas. Enumeration areas (EA) for urban and rural were calculated based on proportional probability to size of population. Total 1,990 sample EAs were selected from the total national 127,460 EA. From the sampling frame in each of the selected EA, 16 and 8 households were systematically randomly selected from urban and rural EAs. This resulted in total 27,960 sample households for HWS.Of these 27,960 samples households, only 23,411 households had responded, with a response rate of 83.7%. The remaining are emptied houses or cannot be identified due to error of addresses. In these 23,411 households, there were a total of 65,781 members of all ages who participated in the HWS. Of the total 65,781 household members, only 27,762 who are adult members, age 15 years or above who presented on the survey time and date, had completed the face-to-face interview of AMR module. The interview was conducted in Thai and took about 60 minutes. Data were collected using a software program.Prior to the survey, all NSO interviewers across 77 provinces were trained through video conference on the AMR manual produced by the researchers. The contents include background, how antibiotics are different from other medicines, global and national concern of AMR, issues around self-medicated antibiotics, clinical conditions, potential sources of antibiotics distribution and the contents of true and false statements. The goal of training is to ensure high quality, reliable and valid information from the survey.On definition of urban and rural areas, NSO adheres to Ministry of Interior’s definition; that urban area means municipality according to the Municipality Act, B.E. 2496 (1953). Urban area also includes special local government established by its own legislation such as Bangkok Metropolitan and Pattaya City. Areas outside municipality are rural areas.Prior to interview, NSO interviewers provided survey background and objective and assured confidentiality to respondents. According to Section 15 of The Statistics Act, B.E. 2550 (2007), personal information obtained under this Act shall be strictly kept confidential. A person who performs duty or a person who has the duty of maintaining such information cannot disclose it to anyone who does not have a duty according to this Act. No consent is required by Section 9 of the Act, for surveys conducted by national statistical authority. Further, participants can withdraw from the interview at any time they so wish.Data were analyzed using STATA/IC (version 14.2). Descriptive measures were presented in percentages. Differences in distribution between groups were compared using logistic regression with an estimate of 95% confidence intervals (CIs). For all tests, p-values of 0.05 or less were used to determine the level of significant difference. Multivariate analysis was employed to assess the relationship between levels of knowledge about antibiotics and receiving information and demographic data such as gender, age, area of residence, education level and wealth status as shown in The prevalence of antibiotic use in the preceding month among the adult population was 7.9% of the total 27,762 adults individual who were successfully interviewed of the AMR module, while 12.3% of respondents could not confirm whether the drugs they used in the previous month were antibiotics or not. Among the 2,024 individuals who reported antibiotics use, 50.3% received it from public health facilities, 20.0% from private health facilities, 26.7% from retail pharmacies and 3.0% from grocery stores. Both retail pharmacies and grocery stores were categorized as self-medication of antibiotics; hence the prevalence of self-medicated antibiotics was 29.7% in Thai adult population.In The majority of respondents (63.6%) correctly recognized that unnecessary or inappropriate use of antibiotics can result in ineffective treatment or resistance; 61.7% agreed that they should stop antibiotics after completing a full course of treatment; 42.9% gave the correct answer that antibiotics are not anti-inflammatory drugs. However, a half of respondents (52.3%) gave the wrong answer to the statement that antibiotics can cure common cold and flu symptoms; and 49.8% of respondents wrongly thought that antibiotics can kill viruses. Almost half of respondents (47.4%) did not know that excessive use of antibiotics can result in side effects such as diarrhea. See Only 2.6% of all adult respondents gave correct answers to all six statements; 8.2% of respondents gave five or more correct answers; less than a quarter of respondents (23.7%) gave four or more correct answers; less than a half of respondents (46.6%) gave three or more correct answers. Alarmingly, 13.5% of Thai adults gave wrong answers to all six statements see .The bi-variate analysis showed that age, area of residence, educational level and wealth quintile were significantly associated with level of knowledge. However, previous study showed that age and receiving information also influenced the knowledge about antibiotics , 15. TheOn the right panel of Only 17.8% of respondents had received information about the appropriate use of antibiotics and AMR in last 12 months. Three common sources of information were doctors (36.1%), health workers (24.8%) and pharmacists (17.7%). Other sources such as conventional media  played a minor role contributing 8.3% and 3.5% respectively, while 7.2% of respondents received information from family and friends see .This is the first national level survey on public knowledge about antibiotics in Thailand. Most other studies have been conducted in specific groups of the population such as patients, migrants, consumers or at sub-national levels , 17. A fThe results of the survey demonstrated that the one-month prevalence of antibiotics use among the Thai population was lower than other studies; compared with the general population in Malaysia (16.5% in the last four weeks) . In the Sources of antibiotics vary according to the health systems context, financing medicines and regulatory conditions around access to antibiotics. In high-income countries where antibiotics are prescription-only medicines, the source of antibiotics is almost always through physicians’ prescriptions; while in most developing countries, access to antibiotics over the counter and self-medication is common. Thailand achieved universal health coverage in 2002  and all The results on indications of antibiotic use were different from Eurobarometer 445 on the top three conditions which were bronchitis (18%), flu (16%) and sore throat (14%) in European Union countries . The thrThe design of true and false statements in our study, similar to that of Euro-barometer and WHO, facilitated international comparison. The special Euro-barometer 478 conducted in September 2018 showed that the level of knowledge about antibiotics among Europeans was higher than among Thais. Our findings on the levels of knowledge about antibiotics and AMR were much lower than in European countries; we measured by percentages of wrong answers including “Do not know the answer” to the six statements. To a statement on “Antibiotics can kill virus”, 80.6% of Thais and 57.0% of European respondents provided the wrong answer including “Do not know”. 79.8% of Thais and 34.0% of European respondents gave wrong answers and did not know the answer to a statement on “Antibiotics can cure common colds and flu symptoms”. 70.8% of Thais and 32% of European respondents gave wrong answers and did not know the answer to a statement on “Excessive use of antibiotics can result in side effects such as diarrhea”. 38.2% of Thais and 16% of European respondents gave wrong answers and did not know the answer to a statement on “Only stop antibiotics after completing a full course”. Finally, 36.4% of Thais and 15.0% of European respondents did so to a statement on “Unnecessary or inappropriate use of antibiotics can result in ineffective treatment or resistance” [In Thailand, there is a common misunderstanding that antibiotics can treat inflammatory conditions in humans, so we added the following statement: “Antibiotics are not anti-inflammatory drugs”. From the results, we found that 42.9% gave the correct answer to this statement, which was higher than respondents in Hong Kong; one study showed that 14% of the general population in Hong Kong provided the correct answer to this statement . The genOur findings about the factors associated with levels of knowledge about antibiotics were similar to other studies; the higher the education levels, the higher level of knowledge about antibiotics , 27. A sOnly 17.8% of Thai respondents had received information about antibiotic use and AMR in the past twelve months, almost half that of those in the European study, where 33% had received information . PromotiThe common sources of information about antibiotics and AMR in this study were doctors 36.1%), other health professionals (24.8%) and pharmacists (17.7%) while other sources such as the media were uncommon. Sources of information vary across countries due to different contexts and policy interventions. For example, a study in Senegal showed that family or friends are the most common source (58.5%), followed by pharmacy staff (54.5%) and doctors or nurses (25%) whereas the Eurobarometer 445 showed that the most common sources were doctors (32%), television advertisements (27%) and television news (26%) 6.1%, oth. As healTo improve the rational use of antibiotics, there should be policies which limit easy access to antibiotics. In Thailand, most antibiotics are classified by the Thai Food and Drug Administration as “dangerous medicines” for which prescriptions are not required at retail pharmacies but can only be dispensed by licensed pharmacists. This facilitates self-medication by the population. Antibiotics in the list of Critically Important Antibiotics  should bThe WHO Strategic Communication Framework for Effective Communications suggests tailored communication in terms of messages, contents and channels to specific target population . This stIn the context of rapid expansion and wide spread of internet and social media, the government can maximize use and capitalize their potential for the provision of authentic message and knowledge about proper use of antibiotics and create awareness of the emerging global health threats from AMR, and improve antibiotics stewardship . In the In consultation with partners, we developed three sub-indicators to monitor progresses. These are a) percentage of Thai adults who provide correct answers to more than 60% of the true false statements and one question; this demonstrates knowledge of antibiotics; b) mean score of adult population who are aware of the importance of antibiotic uses and AMR using a five Likert scale measurement; and c) percentage of adult population who have received information about AMR and antibiotics. Based on the 2017 HWS results, 23.7% of Thai adults who provided correct answers of more than 60% of knowledge of antibiotic part (sub-indicator a) and 17.8% of Thai adults have received information about AMR and antibiotics (sub-indicator c). Though we did not design to measure sub-indicator b in 2017 survey; it was decided to measure in the next round survey in 2019.Sub-indicator a is the 2017 baseline knowledge about antibiotics in Thai people for monitoring progress biannually through the AMR module in the HWS. We reviewed the WHO multi-Country AMR public awareness survey as a part of the monitoring and evaluation framework of the AMR global action plan; and added relevant questions for monitoring sub-indicator b in the upcoming 2019 HWS.A few limitations were experienced in this study. Respondents may have limited understandings about drugs to be able to differentiate antibiotics from other medicines leading to incorrect responses for the use of antibiotics; this affects the findings for the one-month prevalence of antibiotic uses. It is important to note that this is a self-reported survey, and this can lead to a degree of bias with respondents providing the answer they believe is expected by NSO interviewers. This bias is prevented by training of interviewers and a field manual. Also the designs of true and false statements as a factual statement are neutral which reduce such biases; for example “Antibiotics kill viruses” or “Antibiotics are effective against colds and flu”.Unlike a conventional research, this study had embedded AMR module to the national representative population-based survey. AMR is one of the many modules such as illnesses and injuries, health service uses such as outpatient, inpatient and dental care, health promotion and prevention; and out of pocket payment by households. It is not possible to add more independent parameters such as health literacy. Multiple regression is limited by the existing independent parameters in the HWS.Despite these challenges, the strengths are the integration of the AMR module in the biannual HWS, which is less costly and that knowledge of antibiotics and AMR awareness can be assessed against various independent parameters.This study generated evidence on one-month prevalence of antibiotic use in the Thai population and their knowledge about antibiotics and AMR. Findings from multivariate clearly showed that a few population groups are target for public campaign in order to improve knowledge about proper use of antibiotics; these are individuals with low education, members from poor households, men and younger persons.Further, results indicated that provisions of public information, knowledge about appropriate use of antibiotics and AMR awareness can be provided through health professionals as they are a major source of antibiotics dispensing and information.The results from the 2017 HWS serve as a baseline for monitoring the progress of NSP-AMR 2017–2021, which aims to increase public knowledge about antibiotics and AMR awareness by 20% by 2021. Moreover, evidence from this survey contributes to partners’ work in the design of an effective communications strategy to increase levels of knowledge and awareness of antibiotics and AMR among the Thai population.S1 File(PDF)Click here for additional data file.


Aldama discolor (syn.Viguiera discolor) is an endemic Asteraceae from the Brazilian “Cerrado”, which has not previously been investigated for its chemical constituents and biological activity. Diterpenes are common secondary metabolites found in Aldama species, some of which have been reported to present potential antiprotozoal and antimicrobial activities. In this study, the known ent-3-α-hydroxy-kaur-16-en-18-ol (1), as well as three new diterpenes, namely, ent-7-oxo-pimara-8,15-diene-18-ol (2), ent-2S,4S-2-19-epoxy-pimara-8(3),15-diene-7β-ol (3) and ent-7-oxo-pimara-8,15-diene-3β-ol (4), were isolated from the dichloromethane extract of A. discolor leaves and identified by means of MS and NMR. The compounds were assayed in vitro against Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, Plasmodium falciparum and also tested for cytotoxicity against mammalian cells (L6 cell line). The ent-kaurane 1 showed significant in vitro activity against both P. falciparum , which has been reported to possess a strong anti-T. b. rhodesiense activity, was identified as major compound in the A. discolor extract and explains its high activity against this parasite (100% growth inhibition at 2  Aldama La Llave ent-2S,4S-2-19-epoxy-pimara-8(3),15-diene-7β-ol (4): white amorphous solid; m/z): 227.1842, 267.2159 budlein A , T. cruzi , Leishmania donovani , Plasmodium falciparum , as well as for cytotoxicity against mammalian cells  were performed according to established protocols as reported previously, e.g, in [In vitro tests for the activity against  e.g, in .A. discolor leaves led to the isolation of four structurally and biosynthetically related diterpenes, namely three new ent-pimar-15-ene derivatives  as well as a known ent-kaurane (1). Furthermore, the known sesquiterpene lactone budlein A (5) was identified as a major compound. The strong growth inhibition of A. discolor extracts towards T. b. rhodesiense can be explained by the presence of budlein A as a major compound, since this STL reportedly possesses strong anti-T. b. rhodesiense activity [ent-pimarane 2 showed considerable in vitro activity against P. falciparum, while ent-kaurane 1 presented the highest in vitro activity against both P. falciparum and L. donovani. Quite importantly, they returned significant SI >10, which makes them interesting candidates for in vivo tests. In order to better understand their mechanism of action, further studies will be necessary.The investigation of the dichloromethane extract of activity . Among t


In the era of personalized medicine, the management of oncological patients requires a translational and multidisciplinary approach. During early phases of cancer development, biochemical alterations of cell metabolism occur much before the formation of detectable tumour masses. Current molecular imaging techniques, targeted to the study of molecular kinetics, employ molecular tracers capable of detecting cancer lesions with both high sensitivity and specificity while also providing essential information for both prognosis and therapy. On the contrary, complementary and crucial information is provided by histopathological examination and ancillary techniques such as immunohistochemistry. Thus, the successful collaboration between diagnostic imaging and anatomic pathology can represent a fundamental step in the “tortuous” but decisive path towards personalized medicine. Molecular imaging is quickly gaining importance in both biomedical research and clinical diagnostics. During the early phases of cancer development, biochemical alterations of cell metabolism occur before the formation of detectable tumour masses. Current molecular imaging techniques, targeted to the study of molecular kinetics, employ molecular tracers capable of detecting cancer lesions with both high sensitivity and specificity while also providing essential information for both prognosis and therapy. On the contrary, crucial information is provided by histopathological examination and ancillary techniques such as immunohistochemistry. Indeed, in situ analysis of cancer biomarkers by immunohistochemistry provides prognostic and predictive information related to the metabolic characteristics of the tumour mass. Therefore, a multidisciplinary approach based on the collaboration between diagnostic imaging and anatomic pathology could improve the management of oncological patients, further supporting the current goal of a completely personalized medicine approach in oncology. In detail, this new approach will allow clinical doctors to investigate the living body to identify disease, monitor progression, or treat medical conditions at a molecular level . In thisThe use of diagnostic imaging has increased steadily over the past decade, dramatically contributing to improved management of oncological patients. Indeed, high-resolution images provided by computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US) allow early detection of a vast number of tumour masses. In this context, combined imaging based on positron-emission tomography- (PET-) CT plays a central role in the diagnosis, staging, and posttreatment follow-up of patients with neoplastic diseases , and sevin vivo by immuno-PET analysis and ex vivo by immunohistochemistry . Cu. Cu11C-cpression . The comMolecular imaging is a rapidly developing translational discipline which involves molecular biology, chemistry, computer science, engineering, and medicine . It can in vivo HER2 expression in breast cancer patients. It is known that HER2 overexpression is related with a worse survival in both node-positive and node-negative breast cancer patients [Recently, diagnostic sciences have been rapidly changing under the influence of the concept of personalized medicine; thus, molecular diagnostic techniques are increasingly used for the identification of individualized therapy and prediction of treatment response . As a dipatients . In addipatients , 73 overpatients . This mepatients . Specifipatients –79 and Tpatients . Indeed,patients , 82. Sevpatients . This apOne of the more frequent joint imaging histopathological applications concerns the in situ expression of Ki67, the main immunohistochemical marker of cancer proliferation , and FDGIn conclusion, the mutual support between diagnostic imaging and anatomic pathology can assist the oncologists in the choice of more appropriate therapeutic protocols both for primary lesions and for recurrence. Also, image-processing strategies can be used to develop artificial intelligence algorithms which have the potential to aid diagnosis, prognosis, and therapeutic decision-making by integrating large-scale, multimodal information and presenting it to the pathologist and oncologist in an interpretable manner.While artificial intelligence (AI) in general and machine learning in particular have been increasingly used in studies presenting work applied to the biomedical sciences, during the last three or four years, one particular class of methods known as deep neural networks (DNNs) has provided disruptive innovation in a number of fields, including medical applications in general and decision support in complex problems based on multimodal data in particular. Of note, DNNs have had extremely high success rates in the field of oncology. Examples include using nuclear features to classify breast lesions , predictThis type of workflow would not only render pathology and imaging work quickly and more accurately but also redefine the role of pathologists to experts able to agglomerate and interpret genetic/molecular, morphological, and imaging information to produce a more integrated and accurate diagnosis . In thisThe scientific dissertation reported here highlights the possible positive interaction among medical disciplines that base their activity on acquisition and interpretation of cancer imaging. Furthermore, the successful collaboration between diagnostic imaging and anatomic pathology departments can represent a fundamental step in the “tortuous” path to personalized medicine.