A method for the quality evaluation of Atractylodis Macrocephalae Rhizoma (AMR) based on high-performance liquid chromatography (HPLC) fingerprint, HPLC quantification, and chemical pattern recognition analysis was developed and validated. The fingerprint similarity of the 27 batches of AMR samples was 0.887–0.999, which indicates there was very limited variance between the batches. The 27 batches of samples were divided into two categories according to cluster analysis (CA) and principal component analysis (PCA). A total of six differential components of AMR were identified in the partial least-squares discriminant analysis (PLS-DA), among which atractylenolide I, II, III, and atractylone counted 0.003–0.045%, 0.006–0.023%, 0.001–0.058%, and 0.307–1.175%, respectively. The results indicate that the quality evaluation method could be used for quality control and authentication of AMR. Atractylodis Macrocephalae Rhizoma (AMR), named “Baizhu” or “Zhu”, is a member of Atractylodes and one of the “Eight Famous Herbs from Zhejiang Province”. AMR was first documented in “Shennong Ben Cao Jing” , where it is classified as “noble” or “upper herbs”. AMR is the dried rhizome of Atractylodis Macrocephalae, with a fresh fragrance and a sweet, slightly pungent taste. Its functions include invigorating the spleen and replenishing qi, drying dampness and diuresis, antiperspirant, and anti-fetus. It is used for treating spleen deficiency, no appetite, abdominal distension, diarrhea, phlegm, palpitations, edema, spontaneous perspiration, and fetal irritability . SeveralHigh-performance liquid chromatography (HPLC) fingerprinting has become the most widely used method because of its efficiency and convenience. Some scholars have also conducted exploratory work on the fingerprint of AMR of different origins ,18. StudThe full wavelength of AMR solution was scanned with an ultraviolet detector. In order to make the fingerprints informative and ensure that the multiple components do not interfere with each other at the maximum absorption wavelength, 235 nm was chosen as the detection wavelength in the final fingerprint. The detection wavelength of atractylenolide I was 280 nm, and the detection wavelength of atractylenolide II, atractylenolide III, and atractylone was 220 nm for multi-indicator content determination .−1 and 0.9 mL·min−1), different sampling volumes  were compared to evaluate the fitness of chromatographic conditions. It was found that the separation resolution of AMR was best under Kromasil 100-5C18 column with acetonitrile-water with gradient elution as the mobile phase, a flow rate of 1.0 mL·min−1, and sample volume of 20 µL, so these were set as the optimum chromatographic conditions for further use.The separation effects of different columns (Eclipse plus C18 and Kromasil 100-5C18), as well as different mobile phases (acetonitrile—0.1% formic acid and acetonitrile-water), different flow rates  were chosen as the reference, and the relative retention time (RRT) and the relative peak area (RPA) of the 17 common peaks of all the samples were calculated. The relative standard deviations (RSDs) of the RPA of each common peak were found to be less than 2.0%, and RRT of each common peak was found to be less than 1.0% , which sThe RPA and RRT of the 17 common peaks were calculated in the repeatability test. The RSDs of the RRT for each peak were less than 0.9%. The RSDs of the RPA were found to be less than 2.2%. The two RSDs indicated that the repeatability of the HPLC method was satisfactory.For the stability test, the sample solution was measured at 0, 2, 4, 8, 10, 16, and 24 h after preparation, and then the RRT and the RPA were calculated. The RSD of the RRT was found to be less than 0.7%. The RSD of the RPA was found to be less than 2.5%. The results showed that the AMR sample solution was stable within 24 h.The chromatographic profiles of 16 batches of raw AMR (S12–S27) samples were processed and a simulative mean chromatogram was generated. The similarities of different chromatographic fingerprints were compared with the simulative mean chromatogram. The similarity of 27 batch samples ranged from 0.887 to 0.999 , showingThe relative peak areas of 17 common peaks in the HPLC chromatogram fingerprint of 27 batches of AMR were used as variables, and systematic cluster analysis was performed with SIMCA-P; the results showed that the 27 batches of AMR could be divided into two categories, in which S1–11 were clustered into one category and S12–27 were clustered into the other category , indicatThe peak areas of the common peaks of the 27 batches of AMR were standardized by SIMCA-P software, and the eigenvalues of the correlation matrix were calculated as the variance contribution rate. Taking the feature value greater than 1 as the extraction standard, the cumulative variance contribution of the four principal components was 76.44%, which could basically represent most of the information on the HPLC fingerprint. As indicated by the results, the information on the first principal component was mainly derived from chromatographic peaks 2~5; the information on the second principal component was mainly derived from peaks 1, 2, 4, 6, and 11; the information on the third principal component was mainly derived from peak 8, and the information on the fourth principal component was mainly derived from peaks 9 and 10. The peak areas in 27 batches of AMR and AMR stir-fried with bran were carried on PCA analysis. As seen in Supervised PLS-DA was performed on 27 batches of AMR fingerprint data to establish a PLS-DA model that had good explanatory and predictive power . The PLS-DA score plot revealed  that AMR2) were calculated, and they are presented in 2 > 0.999). R values for the calibration curves higher than 0.99 verified that the linearity was adequate for the intended purpose.The range of linearity was established by injecting six different concentrations obtained by the dilution of a standard of atractylenolide III, atractylenolide II, atractylenolide I and atractylone. Analytical curves for each compound were obtained considering the correlation between the peak area and the respective concentration of the standard. The linearity data including slope, intercept, and correlation coefficient  for peak area of atractylenolide III, atractylenolide II, atractylenolide I, and atractylone, was 0.19%, 0.25%, 0.30%, and 0.13%, respectively.The repeatability of the method was tested by the determination of a sample of S9. The RSDs for the contents of atractylenolide III, atractylenolide II, atractylenolide I, and atractylone were 2.66%, 2.76%, 2.43%, and 2.41%, respectively.For the stability test, the RSDs of peak area of atractylenolide III, atractylenolide II, atractylenolide I, and atractylone were 0.34%, 0.45%, 0.33% and 0.11%, respectively, indicating the standard solution was stable for 24 h at ambient temperature.The recovery test was conducted to evaluate the accuracy of this method. The recovery test of solution was obtained by adding a known amount of atractylenolide III, atractylenolide II, atractylenolide I, and atractylone standard solution, respectively, to the six AMR solutions. As shown in The proposed HPLC method was successfully applied to the quantification of atractylenolide III, atractylenolide II, atractylenolide I, and atractylone in AMR. As shown in The content of atractylone in AMR stir-fried with bran was generally lower than that in raw AMR, while the contents of atractylenolide I~III showed the opposite results. Therefore, calculated as dry product, the total amount of Atractylodes I~III should not be less than 0.025%, the sum of the four should not be less than 0.6%, the total amount of Atractylodes I~III in AMR stir-fried with bran should not be less than 0.045%, and the sum of the four should not be less than 0.4%. The limit can well distinguish AMR from AMR stir-fried with bran.Atractylenolide I (batch number (BN):111975-201501, mass fraction (MF): 99.9%), Atractylenolide II  and Atractylenolide III  reference substances were all purchased from China Institute for Food and Drug Control; atractylone ; Acetonitrile, HPLC grade ; formic acid, HPLC grade ; methanol of analytical purity; ultrapure water, prepared by Milli-Q system.Atractylodis Macrocephalae Rhizoma (numbered S1 to S27) were purchased from pharmaceutical factories and Chinese medicine hospitals. The samples were collected from Zhejiang and Anhui of China for analysis, and the source information is listed in the The HPLC analysis was performed on a Dionex UltiMate 3000 LC series–diode array detector (DAD) system with a quaternary pump and an autosampler that could thermostat samples. Separation was achieved on Kromasil 100-5C18 . The detection wavelength was set at 235 nm for HPLC fingerprinting, and 220 nm and 280 nm for HPLC quantification. The injection volume was 20 µL.−1. The gradient program was set as follows: 0–35 min: 5% A–65% A; 35–40 min: isocratic 65% A; 40–45 min: 65% A–5% A; 45–50 min: 5% A–80% A.The chromatographic separation was performed using acetonitrile (solvent A) and water (solvent B) as mobile phase at a flow rate of 1 mL·minUnder the above conditions, resolution, and theoretical plates, the peak was calculated. Theoretical plates according to atractylone peak should not be less than 20,000, and the resolution between peak 16 and peak 17 should not be less than 1.0.Under the above conditions and theoretical plates, the peak of atractylone was calculated. It should not be less than 20,000.To establish the representative HPLC chromatogram, 16 batches of AMR as well as 11 batches of AMR stir-fried with bran were analyzed under the optimized HPLC conditions. Then, 17 common peaks were symbolized, among which four peaks were identified, namely atractylenolide III (P2), atractylenolide II (P6), atractylenolide I (P18), and atractylone (P14), as shown in According to the guidelines for analytical method validation in Pharmacopeia of People’s Republic of China (volume IV) (version 2020), the precision of the HPLC method was evaluated by sampling the replicated sample solution (S9) six times with successive injections. The stability test was determined by analyzing the same standard solution at 0, 2, 4, 8, 10, 16, and 24 h. The repeatability was determined by preparing six sample solutions (S9) independently and calculating the RSDs of relative peak area and relative peak retention time.According to the guidelines for analytical method validation in Pharmacopeia of People’s Republic of China (volume IV) (version 2020), the linearity regression curves for each component were obtained by plotting the peak areas (y) against the concentrations of each component standard solution. The precision of the HPLC method was evaluated by sampling each replicated component standard solution of the same sample six times with successive injections. The stability test was performed by analyzing the same standard solution at 0, 2, 5, 8, 15, 20 and 24 h. The repeatability was determined by preparing six sample solutions (S9) independently and calculating the RSDs of the contents. The recovery test was conducted to evaluate the accuracy of this method. The recovery test of a solution was performed by adding a known amount of atractylenolide III, atractylenolide II, atractylenolide I, and atractylone standard solution, respectively, to the six AMR solutions (S9) and calculating the recovery rate and the RSDs.The HPLC chromatograms of 27 batches of AMR samples were analyzed by “the similarity evaluation system for chromatographic fingerprint of TCM” software (version 2012). Chemical pattern recognition analysis was performed with SIMCA-P software (version 14.0).The proposed HPLC fingerprinting and multi-component content determination method for AMR is easy to carry out with a sound level of repeatability and a high level of reliability. It provides a scientific basis for the comprehensive quality evaluation of AMR. As shown in the experimental results, the overall chemical compositions of the 27 batches of AMR processed by the two different processing methods were not identical. The similarity analysis was performed using the established HPLC fingerprint. The results showed that the 27 batches of sample had a high level of similarity. The similarity of the 17 selected common peaks was above 0.90. This study combined HPLC fingerprinting with quantitative analysis of multi-components, and PCA revealed that there were significant differences in AMR quality from different processing methods. Peaks 16, 14 (atractylone), 15, 13, 17, and 6 (atractylenolide I) were screened out by combining the CA, PCA, and LS-DA methods, which contributed to the differentiation between the raw and stir-baked AMR. In addition, the content levels of four components in AMR  were identified and compared with the spectra and retention times of the standards. The aim was to explore further the medicinal value of AMR and expand its clinical application. In conclusion, a quality evaluation method for AMR was established in this study to provide a reference for the quality control and clinical applications of AMR.


Several experts have expressed their concerns regarding the potential increase in antimicrobial resistance (AMR) during the COVID-19 pandemic as a consequence of the increase in antimicrobial and biocide use in humans globally. However, the impact of the pandemic on antimicrobial use (AMU) and AMR in animals has yet to be discussed and evaluated. Indeed, veterinary practices have been hugely impacted by the pandemic and its restrictive measures around the world. In this perspective, we call for more research to estimate the impact of COVID-19 on AMU and AMR in both humans and animals, as well as on the environment, in coherence with the One Health approach. In addition, we argue that the current pandemic is an opportunity to accelerate the implementation of a One Health approach to tackle the AMR crisis at the global scale. Indeed, the momentum created by the increased general awareness of both the public and decision-makers for the development and maintenance of effective drugs to treat human infections, as well as for the importance of a One Health approach to prevent the emergence of infectious diseases, should be used as a lever to implement global collaborative and sustainable solutions to the complex challenges of AMR. When first identified in December 2019, in Wuhan , coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was described as a mystery viral pneumonia outbreak . MoreoveMultiple countries experienced multiple waves of cases and hospitalizations, pushing authorities to implement strict public health measures. This unprecedented global health issue is exerting a colossal pressure on scientists and physicians to develop and use therapeutic and prophylactic strategies to counter the severe consequences of this infection, so that societies can return to some form of normality and the economy can be restarted. Increasing evidence suggests that this context has resulted in an increase in both AMU and AMR in humans, followed by an increase in AMR bacteria shed into the environment ,6,7. It In this perspective we: (i) give an overview of the trends in AMU during this pandemic and its consequences on humans and the environment; (ii) explore the potential consequences of the pandemic on AMU/AMR in veterinary medicine; and (iii) explain how the pandemic is an opportunity to strengthen the One Health approach for AMR prevention and control.Clostridioides difficile infection) [In May 2016, an independent review on AMR reported that AMR infections in humans were estimated to cause at least 700,000 deaths per year and this number is projected to increase up to 10 million deaths per year by 2050 globally, overtaking the number of people dying from cancer (8.2 million each year) . In the fection) ,18. Consfection) . While sE. coli, Salmonella and Pseudomonas strains [Pseudomonas aeruginosa isolates to increasing concentrations of BAC selected for mutations in the polymyxin resistance (pmrB) gene, as well as for some physiological adaptations (including an overexpression of mexCD-oprJ multidrug efflux pump genes), contributing to a higher tolerance to polymyxin B and to other antimicrobials  [In addition, considerable amounts of these antimicrobials could reach the environment in their active forms and might exert selection for resistant bacteria ,22. More strains ,29. In f strains ,29. Moreifampin) . Common ifampin) ,31. In aIn farm animals, antimicrobials are used therapeutically  for prophylaxis , for metaphylaxis , and some are still used for growth promoting purposes ,32. In 2The impact of the COVID-19 pandemic on the global efforts to control AMU and AMR in animals is unknown. The following factors could have contributed to an increase in AMU and ultimately an increase in AMR. First, veterinary services were not considered essential in several countries implementing lockdowns, at least during the first wave of the pandemic, and consequently veterinarians were not able to offer preventative care, including vaccination, to their clients . This could have resulted in animals developing infections which would normally be prevented and hence an increase in AMU in animal production. Second, the unavailability of veterinary services, particularly during the first lockdown, could increase self-medication as well as off-label use of antimicrobials on farms. Furthermore, the pandemic has also resulted in instances where animals were kept on farms for longer than usual due to large outbreaks in slaughterhouses and the disruption of inter-regional and international transportation. This situation might lead to an increase in AMU in animal production as a consequence of the increase in animal density on farms, which could facilitate the spread of infectious diseases. In addition, all research and development activities of alternatives to antimicrobials and vaccines in animals could be disrupted worldwide during this pandemic, which could create more pressure on the AMU in animals in the long term. Salmonella, Campylobacter, E. coli) were greatly altered by the pandemic and particularly during the first lockdown. The extent to which the pandemic led to an increase or a decrease in AMU and ultimately its impact on AMR in animal populations should be urgently assessed in future studies.On the other hand, the pandemic may have also resulted in a decrease in AMU in farm animals. Indeed, access to antimicrobials (or their molecules or ingredients) could have been disrupted by commerce and transport limitations. Moreover, the pandemic may have caused a breach in surveillance for AMU and AMR in food systems globally. For example, in several countries , the majority of animal health laboratories were redirected towards COVID-19 diagnoses in support of the public health laboratories. Food safety regulatory agencies also faced a huge challenge related to staff shortages, resulting in a reorientation of the inspection and the sampling activities towards certain sectors , rather than sampling for AMR monitoring, to avoid further overloading of the human health systems with foodborne infections. Sampling along the food chain  and the analysis for the surveillance of AMR for some foodborne pathogenic bacteria  in Enterobacteriaceae [mcr) genes may have originated in aquatic environments as a result of aquaculture activities, and these genes could have earned terrestrial bacteria by horizontal gene transfer to yield colistin-resistant bacteria in humans and animals [Environmental changes and ecosystem degradation across the planet  increased the frequency of contacts between wild animals, domestic animals and humans, contributing to a zoonotic transfer of diseases . Therebyeriaceae . Cabello animals . It shou animals . It is t animals . Finally, by joining the few episodes of infectious diseases that have deeply shaped human history, the COVID-19 pandemic had succeeded, in an unprecedented way, in increasing awareness of both the public and decision-makers regarding the development and maintenance of effective drugs to treat human infections as well as for the importance of the One Health approach to prevent the emergence of infectious diseases. We believe that these prerequisites should be used as a lever to accelerate both the development of global collaborations and the implementation of sustainable solutions for the management of the current AMR crisis.Antimicrobial resistance is a cross-sectoral complex problem affecting the health of humans, animals and the environment. We strongly believe that COVID-19 pandemic has generated a powerful incentive and momentum to address the AMR crisis by accelerating collaborations and interdisciplinary communication between concerned stakeholders , while taking into account the specificity of each sector during and beyond this pandemic. More research and retrospective analysis of surveillance data are needed in both humans and animals as well as in the environment to estimate the impact of COVID-19 on AMU and AMR in coherence with the One Health approach. One of the lessons of the COVID-19 pandemic is that acting too late carries serious costs to both health and the economy. Thereby, the colossal budgets allocated to human medicine worldwide for the control of this pandemic should not compromise efforts conducted to manage the current AMR crisis at the human–animal–environment interface. We stress here the paramount importance of the One Health approach to face the increasing threat of AMR, and we believe that this pandemic could be an excellent opportunity to accelerate its implementation for an effective surveillance, prevention and control of AMR at the global scale.


To distinguish between these two theories, we compared an ionic liquid with its uncharged, isoelectronic, isostructural molecular mimic. We measured the viscosity of the molecular mimic at high pressure to emulate the high densities in ionic liquids, which result from the Coulomb interactions in the latter. We were thus able to reveal that the relative contributions of coulombic compaction and the charge network interactions are of similar magnitude. We therefore suggest that the optimisation of the viscosity in room temperature ionic liquids must follow a dual approach.Room temperature ionic liquids are considered to have huge potential for practical applications such as batteries. However, their high viscosity presents a significant challenge to their use changing from niche to ubiquitous. The modelling and prediction of viscosity in ionic liquids is the subject of an ongoing debate involving two competing hypotheses: molecular and local mechanisms  We use an experimental approach to compare an ionic liquid with a molecular mimic, focusing on viscosities. Charge network and coulombic compaction contribute significantly to the high viscosity of ionic liquids; we discuss the implications on their design and optimisation. This is a key aspect for applications such as batteries, gas separation or biomass processing. In order to optimise the viscosity, it is necessary to develop a mechanistic understanding of the difference between how viscosity arises in ionic liquids and conventional molecular solvents.In recent years, ionic liquids have transformed from a scientific curiosity to extensively used functional fluids, both in academia and industry.6,7 and is a mixture of neutral analogues of the anionic and cationic molecular constituents.6–8 To ensure similarity, the molecular mimic and the corresponding ionic liquid should ideally be isoelectronic and isostructural to each other. 9 Crucially, the viscosity of the ionic liquid is almost 30 times that of the molecular mimic, despite the similar molecular structures. One might interpret this factor of 30 as the difference between conventional molecular solvent and ionic liquid, i.e. the isolated effect of the added charge. However, the ionic liquid also has a higher density than the molecular mimic, which still constitutes a bias. The higher density is the result of coulombic compaction, i.e. strong (attractive) coulombic interactions which reduce the volume of the liquid phase.9A fair, unbiased comparison between ionic liquids and conventional molecular solvents necessitates two systems which are as similar as possible; one charged, and one neutral. The neutral system has been called the ‘molecular mimic’i.e. pressure) generally leads to an increase in viscosity.10,11 Indeed, the viscosity of both molecular and ionic liquids can often be expressed as a function of ργ/T, where ρ is the density, T the temperature and γ a material parameter.12–15 Consequently, the comparison between a molecular mimic and an ionic liquid should be made under isodensity conditions. In other words, the molecular mimic must be subjected to pressure high enough so that its density becomes equal to that of the ionic liquid under ambient pressure at the same temperature. It is conceivable that the two systems would then also have similar viscosities.16 If this were the case, it would imply that the high density itself was the cause of the high viscosity.The differences in density and viscosity lead to an important question: what is the degree to which coulombic compaction causes the high viscosity of ionic liquids? It is well known that an increase in density  and 7.5 (from the isodense molecular mimic to the ionic liquid).44 Data at lower temperatures are not available due to the high melting point of [NBu4][BBu4] of about 110 °C.45 However, comparison with the viscosity ratios at higher temperatures shows that the relative difference in viscosity between the ionic liquid and the isodense molecular mimic increases significantly at lower temperatures. At 163 °C, the viscosity ratios were 2.4 (from the molecular mimic to the isodense molecular mimic) and 5.1 (from the isodense molecular mimic to the ionic liquid).The concept of experimentally measuring molecular mimics under isodensity conditions was explored in 1968 by Morrison and Lind,et al. performed molecular dynamics simulations on a coarse grained model of 1-butyl-3-methylimidazolium hexafluorophosphate.47 In order to realise the molecular mimic, the authors simply removed electrostatic interactions. At a simulation temperature of 450 K, the viscosity increases by a factor of 75 from the isodense molecular mimic (1.1 mPa s) to the ionic liquid (83 mPa s).47 Unfortunately a comparison between the molecular mimic under ambient pressure and the isodense molecular mimic is not feasible since at this temperature the molecular mimic is a gas (under ambient pressure).Molecular dynamics simulations can also be employed to directly compare ionic liquids and their molecular mimics. Roy et al. observed only a factor of 30 between their molecular mimic under ambient conditions and the ionic liquid. Critically, if the experimental results from Morrison and Lind were universally transferable, then the relative viscosity increases observed by Roy et al. should be smaller than those observed by Shirota et al. since the latter are (a) at a lower temperature and (b) additionally include the change from the molecular mimic under ambient pressure to the molecular mimic under isodensity conditions. Importantly, Roy et al. revised their model to more closely match experimental values.48 In the revised model, the viscosity of the ionic liquid is only 3.4 mPa s at 450 K,48 compared to 83 mPa s in the original model.47 A direct comparison at 450 K across the two models would not be meaningful due to various changes to simulation conditions and the force field itself. However, the authors provide the required data at 350 K: here, the viscosity changes by a factor of 22 from the isodense molecular mimic (1.2 mPa s) to the ionic liquid (26 mPa s).48 In addition, the authors provide viscosity data under ambient pressure and temperature. At 298 K, the molecular mimic has a viscosity of 0.42 mPa s, compared to the ionic liquid with a viscosity of 330 mPa s (with a reported uncertainty of ±100 mPa s).48 This corresponds to a change in viscosity by a factor of 785, however there is no information regarding the relative contributions of charge network and coulombic compaction.The large difference between isodense molecular mimics and ionic liquids is surprising, given that Shirota et al. who performed MD simulations on a generalised coarse grained model based on ionic liquids such as 1-butyl-3-methylimidazolium hexafluorophosphate.49 The authors provide diffusion coefficients, from which the viscosity ratios can be estimated (see ESI Section 1A similar approach has been followed by Park Due to recent advances in the field of (room temperature) ionic liquids, a study at ambient temperatures is now possible. Here, we demonstrate a proof of concept for the entire approach, from the selection of an experimental system to the characterisation under high pressure. Our results have far-reaching implications for the development of viscosity models, which must account for the diverse nature of ionic liquids.The selection of an appropriate experimental system is non-trivial, since a multitude of requirements have to be simultaneously fulfilled:(1) The molecular mimic should be structurally similar to the ionic liquid.(2) Both molecular mimic and ionic liquid must be experimentally accessible.(3) The two components of the molecular mimic must not react with each other, even under pressure.(4) The components must be miscible, and the molecular mimic must remain homogeneous and liquid under isodensity conditions.(5) The equipment must be resistant to the involved compounds.9 Hydrogen bonds between ether groups and the C–H acidic malononitrile have been reported in the literature using both experimental and theoretical methods.50–53 An alternative would be cyanogen oxide O(CN)2, but this violates at least the second and third requirements. Similarly, one trivial neutral analogue of the acetate anion is the rather hazardous and volatile acetyl fluoride. For our preliminary studies, therefore, we considered acetyl chloride, acetic acid and nitromethane as neutral analogues.The similarity of the ionic liquid and the molecular mimic is an obvious requirement, but some cases require compromises. For example, the malononitrile chosen by Shirota and Castner as a neutral analogue to the dicyanamide anion may introduce hydrogen bonding to the ether group present in the cation's neutral analogue.4 and SF6, which are the trivial neutral analogues for Cl−, [BF4]− and [PF6]−. These neutral analogues are, while stable, all gaseous under ambient conditions, presenting a significant obstacle for experimental investigations. In contrast, neutral analogues of this type are predestined for computer simulation. Even if the experimental setup can be prepared with an equimolar mixture of, say, propane and tetrafluoromethane, it would be preferable to have an estimate for required pressures and liquid–liquid critical points beforehand.The second point applies for compounds such as Ar, CF4C1im][NTf2]. To this end, the imidazolium cation could be replaced with a furan or pyrrole based neutral analogue, and the anion could be replaced with triflic anhydride OTf2, the free acid HNTf2 or methylene ditriflone CH2Tf2. However, as these compounds are rather reactive, they are not compatible with each other and indeed many other neutral analogues. Phosphonium and ammonium ionic liquids are much more benign in this respect, as they may be mimicked by thermodynamically stable silanes and hydrocarbons.We initially considered the commonly used ionic liquid −)/D([P222(3O1)]+) = 1.09 in the ionic liquid. Hence, the diffusion coefficients of the two constituents are much more similar in the ionic liquid than in the molecular mimic, which can be rationalised by the electrostatic interactions coupling the motion of cations and anions. The degree to which this coupling of motion already occurs under isodensity conditions, if at all, would be an interesting topic for future experimental and theoretical studies.Diffusion coefficients, unlike the viscosity, can be measured separately for each constituent and thus give access to additional information. The diffusion coefficients can be compared by analogy to the viscosity values, Overall, this study clearly shows how valuable insight can be gained from a comparison of ionic liquids and their molecular mimics. Without doubt, the practical importance of understanding the transport properties in ionic liquids justifies the considerable effort of performing experiments at such high pressures. It would be intriguing to study more types of ionic liquids in the future to probe the generality of the conclusions drawn from the phosphonium butyrate in this work.Furthermore, during the selection process, we observed incompatibilities which in themselves are interesting. For example, in several cases, the cation and anion neutral analogues were not miscible. Naturally, in the ionic liquid, the two constituents must mix due to electroneutrality conditions. We hypothesise that this effect – known as nanosegregation – to some degree counteracts the effects of the charge network. The use of molecular mimics can help to identify such cases, using the mixing behaviour of the cation and anion neutral analogues as design element.19,63,64 Here, future work could explore the evolution of the dynamic structure factor of molecular mimics at wavenumbers corresponding to the charge network in the corresponding ionic liquid with the help of molecular dynamics simulations. The use of molecular dynamics has the added advantage that high pressures and even intermediates between molecular mimic and ionic liquid – with fractional charges on the molecules – are possible. Vice versa, approaches such as hole theory should factor in the presence of a charge network. In other words, a good model for any transport property in ionic liquids must be able to predict the difference between the isodense molecular mimic and the ionic liquid.The future development of physically sound models should consider both coulombic compaction and the charge network as the two main contributors to ionic liquid viscosity. For example, mode coupling is a good approach to study and describe the connection between viscous flow and the charge network.65,66 Machine learning algorithms use a number of input properties to produce an estimate of an output property. Easily accessible input properties, such as molecular weight or connectivity, are advantageous. Based on our observations, both coulombic compaction and charge network formation should be present in the input properties. Furthermore, it might be beneficial to introduce feature extraction with coulombic compaction and charge network formation as intermediate features to improve the performance of machine learning algorithms.From an engineering point of view, the results from this work can be used to select appropriate models. Good examples are machine learning approaches, which are becoming more and more popular recently for quantitative predictions of ionic liquid properties.67,68 This might enable the experimental comparison of those pairs of ionic liquid and molecular mimic where isodensity conditions are not attainable, but where common points of ργ/T can be found  and molecular mimic (at lower temperature).For future studies, it could also be worth expanding the measurements to several isotherms, which in combination with density scaling would allow the use of a ‘density–temperature superposition principle’, in analogy to the time–temperature superposition principle.4/[BF4]− may still be studied in silico by means of molecular dynamics simulation. Here, one of the key issues remains consistency of the parameterisation across charged and uncharged systems, for example with regards to polarisability. Our experimental results at room temperature could be used to benchmark force fields and simulation setups. Once a reliable  model has been identified it can be used to generate further valuable data points at different temperatures or pressures, with considerably lower expense and hazards compared to the experimental high pressure studies.Finally, experimentally challenging systems such as CF3 solution was used to confirm the absence of halides in the carboxylate ionic liquids. Syntheses and  physicochemical characterisation are described in detail in the ESI.†Samples were prepared under Schlenk conditions or in a glovebox, ionic liquids were dried in high vacuum before use. AgNO69 The pycnometer was dried in vacuum before use, weighed, and filled with sample to above the mark in a glovebox. The pycnometer was then sealed, placed in a thermostat bath (thermostatted to ±0.01 K), equilibrated for 20 min, the liquid level adjusted to the mark with a Pasteur pipette, cooled to room temperature, and weighed again.Densities as a function of temperature under ambient pressure were measured with a 5 mL  Reischauer pycnometer  calibrated with octane and confirmed with water.The density of the molecular mimic under high pressure was measured to find the isodensity conditions using a U111 high pressure pump and an MV1-30 pressure chamber, both provided by Unipress in Poland. The sample is loaded into a cylindrical container with a movable piston at one end and the container is then submerged into enclosed pressure fluid which is connected to a high-pressure pump. The increasing pressure moves the piston inwards, compressing the liquid sample, during which the displacement of the piston is recorded. The density change is then calculated using the absolute mass and the change in volume of the sample due to the increase in pressure. Measurements were performed from 0–350 MPa at 25 °C.−1 in 30 linear steps. Newtonian behaviour was observed, and the viscosity obtained as an average over the shear rates. The temperature was allowed to equilibrate for 15 min before the measurements. The coaxial setup was used only for the molecular mimic with outer radius 14.4600 mm, inner radius 13.3292 mm, gap 1.1308 mm. The temperature was set to 25 °C with a Peltier heater.The viscosity under ambient pressure was measured with a cone-plate setup as well as with a coaxial setup. The cone-plate setup was used on a stress-controlled MCR 301 rheometer  with a CP50-1 cone  and a gap size of 0.101 mm. The measurements were performed under nitrogen atmosphere with shear rates varying from 5 to 150 s70 To this end, the sample is filled in a cartridge containing falling body (=sinker). One end of the cartridge is sealed with a plug, the other with a moveable piston to allow for pressure transfer. The cartridge is placed in a high pressure vessel surrounding by a pressurising medium. The high pressure vessel containing the cartridge is then rotated, causing the sinker to fall, the position of the latter is detected via a linear variable differential transformer. The viscosity is obtained from the falling time.Viscosity measurements under high pressure up to 500 MPa were performed at LaMCoS – INSA de Lyon using a falling body viscometer as described by Scott Bair.−1) using the pulsed field-gradient stimulated echo sequence with bipolar gradients and longitudinal eddy current delay (‘ledbpgp2s’ in the Bruker library) and smoothed rectangular gradient pulses similar to previous work.71 The individual self-diffusion coefficients of the constituents could be determined using resolved signals of the two species. The uncertainty of the measurement with this setup is approximately 2% of the absolute value (from our own repeat measurements on this setup and comparing different pulse sequences).Diffusion coefficients were measured on an Avance Neo 500 MHz NMR spectrometer  with Prodigy TCI CryoProbe , writing , visualization, project administration. Daniel Rauber: investigation . Kira Lieberkind Eliasen: validation, formal analysis (density fitting), investigation (pressure-dependent densitometry). Nathalie Bouscharain: validation, formal analysis (viscosity fitting), investigation (pressure-dependent rheology). Kristine Niss: resources (high pressure laboratory). Christopher W. M. Kay: resources . Tom Welton: supervision, funding acquisition, resources (facilities for syntheses).There are no conflicts to declare.SC-013-D1SC06857A-s001


Kaposi’s sarcoma (KS) is an angioproliferative tumor showing an increased frequency and aggressiveness in HIV-infected subjects (AIDS-KS), due to the combined effects of inflammatory cytokines (IC), angiogenic factors, and the HIV-1 Tat protein. While the introduction of effective combined antiretroviral regimens greatly improved AIDS-KS incidence and course, it continues to be an incurable disease and the development of new rational targeted therapies is warranted. We used the BKV/Tat transgenic mouse model to evaluate the effects of IC and anti-Tat antibodies (Abs) treatment on KS-like lesions arising in BKV/Tat mice. We demonstrated here that IC-treatment increases the severity and delays the regression of KS-like lesions. Further, anti-Tat Abs reduced KS-like lesion severity developing in IC-treated mice when anti-Tat Abs were administered at an early-stage of lesion development as compared to more advanced lesions. Early anti-Tat Abs treatment also accelerated KS-like lesion regression and reduced the rate of severe-grade lesions. This effect was more evident in the first weeks after Ab treatment, suggesting that a longer treatment with anti-Tat Abs might be even more effective, particularly if administered just after lesion development. Although preliminary, these results are encouraging, and the approach deserves further studies for the development of anti-Tat Ab-based therapies for AIDS-KS. Clinical studies specifically addressing the effect of anti-Tat antibodies in treating AIDS-KS are not yet available. Nevertheless, the effectiveness of anti-Tat antibodies in controlling HIV/AIDS progression, likely due to the neutralization of extracellular Tat activities, is suggested by several cross-sectional and longitudinal clinical studies, indicating that anti-Tat Ab treatment or Tat-based vaccines may be effective to treat AIDS-KS patients or prevent the tumor in individuals at risk. Kaposi’s sarcoma (KS) is an angioproliferative tumor first described in elderly men of the Mediterranean Eastern-European areas  and also observed in patients treated with chronic immunosuppressive therapy, especially in organ transplant recipients (iatrogenic KS), and in children and young/adults of subequatorial Africa (African KS) . In receAll epidemiological forms of KS generally arise in the skin of the extremities as multiple patches, plaques, or nodular lesions. The tumor shows a particularly aggressive behavior when it is associated with HIV infection, with frequent involvement of mucosae and visceral organs . At histPrevious studies indicated that, at least in early stages, KS is a cytokine-mediated disease and that inflammatory cytokines (IC) and angiogenic factors cooperate in its induction . SpecifiThe early-stage lesions have a polyclonal nature and can regress ,15,16,17In this context, the Tat protein of HIV-1 acts as a progression factor in AIDS-KS and increases the frequency and aggressiveness of AIDS-KS as compared to other non-HIV associated epidemiological KS forms . In parttat gene develop dermal lesions resembling the early phase of KS [tat gene, driven by its own promoter (the HIV-1 LTR) (BKV/Tat transgenic mice), develop dermal lesions like the early phases of KS, as well as tumors of different histotype also observed in HIV-positive persons [Interestingly, transgenic mice carrying the se of KS ,34. In p persons .The dermal lesions appear around the third month of life in 40% of male mice and are characterized by squamous cell dermal hyperplasia and hyperkeratosis localized in the back ,35. ThesAt histology, the bed of the ulcers appears to be formed by granulation tissue with a prominent capillary network, infiltration by inflammatory cells , and proliferation of spindle-shaped cells and endothelial cells, simulating the histopathological features of early KS lesions ,35.Of note, spindle cells isolated from ulcerated KS-like skin lesions of BKV/Tat-transgenic mice co-express antigens specific for endothelial, smooth muscle, and antigen-presenting cells, and express a complex mixture of angiogenic factors, including fibroblast growth factor-2 and vascular endothelial growth factor , as obseThus, many pathological findings observed in this animal model, including KS-like skin lesions, resemble the lesions appearing in AIDS patients, suggesting a relevant role for extracellular Tat in the pathogenesis of such lesions during the course of AIDS.Altogether, these data suggest that BKV/Tat transgenic mice represent a good model to investigate the pathogenic role of extracellular Tat in AIDS-KS development as well as anti-AIDS-KS interventions. Here, we used this model to evaluate the effects of IC and anti-Tat antibodies (Abs) treatment on KS-like lesions’ development and progression.β, TNF-α, and IFN-γ) and anti-Tat Abs on KS-like lesions developing in BKV/Tat transgenic mice.To elucidate the role of IC and extracellular Tat in KS lesion development and progression, we evaluated the effects of IC  and were monitored up to 10 weeks as compared with untreated mice. Lesion dynamic was evaluated by grading lesion severity according to the scoring system depicted in To this aim, BKV/Tat mice bearing initial KS-like lesions or no lesion were injected locally with a mixture of IL-1, TNF-In IC-treated mice, lesion severity and course over time were significantly worse as compared to untreated controls A. MoreovIn order to assess the role of extracellular Tat on lesion course, mice were divided into two groups: IC-treated mice bearing lesions with a score of ≤0.5 were defined as early-stage, whereas mice with lesions of ≥1 were defined as advanced-stage or late-stage. Both groups were injected with IC at day 0, 4, and 8 and anti-Tat Abs or control Abs at day 4, 8, and 12. Lesion course was monitored up to week 12.p = 0.0170)  C, while  0.0170) D. Althou 0.0170) A–D.p = 0.0734)  of the mice treated with anti-Tat Abs at an early-stage regressed to a lesion score stably ≤ 0.5 by week 6, while at the same time point only 50% (5/10) of early-stage control mice reached the same degree of regression. This threshold was never reached by 40% (4/10) of control-Ab mice when the whole observation period was considered (lesion score range: 0.8–6). Finally, the lesions of mice treated at an early stage tended to regress more rapidly as compared to the lesions of mice treated in a more advanced stage  E, while  0.0734) F. Of not 0.0734) .Altogether, these results indicate that anti-Tat Ab treatment may be effective against KS-like lesions of BVK/Tat transgenic mice when administered at early-stages of lesion development.We have shown here that treatment with the same IC that are increased in lesions and blood of KS patients or in subjects at risk of disease development enhances the severity and delays the regression of KS-like lesions arising in BKV/Tat transgenic mice.This observation agrees with preclinical and clinical data indicating that IC play an important role in the development of all KS forms ,15. In pOf note, IC or bFGF are required to observe extracellular-Tat activity either in, in vitro or in vivo models of AIDS-KS, including endothelial and KS cell proliferation, migration, invasion, matrix-metalloproteases expression, and angiogenesis ,46,47,48Interestingly, IC-induced integrins also function as receptors for HIV and HHV8 in several cell types, including dendritic cells and activated endothelial cells ,50,51,52Altogether, these data indicate that Tat and IC synergize in promoting KS development and progression and that IC-treated BKV/Tat transgenic mice represent a suitable model to test interventions against AIDS-KS, although it does not allow the evaluation of HHV8-promoted oncogenesis.By using our transgenic model, we found that three doses of anti-Tat Abs reduced KS-like lesion severity developing in IC-treated BKV/Tat transgenic mice when anti-Tat Abs were administered at an early-stage of lesion development. Early anti-Tat Abs injection also accelerated KS-like lesion regression and reduced the rate of severe-grade lesions. This effect was more evident in the first weeks after Ab treatment, suggesting that a longer treatment with anti-Tat Abs might be more effective, particularly if administered just after lesion development.Previous data obtained in, in vitro and in vivo models of KS and angiogenesis indicated that anti-Tat Abs can neutralize the activity of extracellular Tat ,46,60. TAlthough a clinical translation specifically addressing the effect of anti-Tat Abs in treating AIDS-KS is not available now, observational and interventional studies by our and other groups (extensively reviewed in ), indicatat transgene by polymerase chain reaction (PCR). Briefly, the tail sample was incubated at 50 °C for 14–16 h in lysis buffer (50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)) pH8, 100 mM Ethylene Diaminetetraacetic Acid (EDTA), 100 mM sodium chloride, 1% Sodium Dodecyl Sulfate, 500 ug/mL proteinase K), DNA extracted by phenol-chloroform and analyzed (200–300 ng) by PCR reaction (50 uL) using Tat-specific primers  (1 mM each), 50 uM dNTPs, TaqI DNA polymerase (1 U) and 1X Taq I buffer . Samples were then controlled by gel electrophoresis.The generation of BDF1 transgenic mice carrying BKV/tat sequences, crossing with outbred CD1 mice and subsequent breeding were previously described ,35,36. ATransgenic mice were monitored twice/week for physical and behavioral changes, and examined for KS-like lesion development, which typically appears on the lower dorsal area around 3/4-months age in 40% of male mice ,35.KS-like lesion severity  was gradβ, IFN-γ, TNF-α) at days 0, 4, and 8. Inflammatory cytokines (murine IL-1β (0.2 µg/mouse), IFN-γ (0.5 µg/mouse), TNF-α (0.5 µg/mouse), Roche Diagnostics, S.p. A., Monza, MB, Italy) were combined in a volume of 225 µL, mixed with an equal volume of matrigel  and administered subcutaneously in the lower back of the animals. Untreated mice with initial lesions (score ≤ 0.5) were used as control. All mice were observed for lesion score twice a week up to week 10.To assess the effect of cytokines on development/regression of the lesions, mice with early-stage lesions (score ≤ 0.5) were treated with a cocktail of IC  and mice with late-stage lesions (score ≥1). Both groups were treated with IC at days 0, 4, and 8, and then with anti-Tat Abs or with a control rabbit serum (control groups) at days 4, 8, and 12. Rabbit anti-Tat polyclonal antibodies  or control rabbit polyclonal IgG  were inoculated subcutaneously in the lesion (100 µL). All mice were observed for lesion score twice a week up to week 12.The weekly lesion-score was calculated as the mean of 2-weekly lesion-score evaluation. ANOVA for repeated measures was performed in order to compare the lesion score between groups.® .Kaplan–Meier method was used to assess time to regression, intended as a score consistently ≤ 0.5, and time to progression, intended as the first time of a score ≥ 3.0 and groups were compared by the Log-Rank test. Statistical analyses were carried out using SAS


Establishing synthesis methods to produce a CuBi2O4 photocathode with sufficient cocatalyst sites would be highly beneficial for water splitting. Here, the platinum-enriched porous CuBi2O4 nanofiber (CuBi2O4/Pt) with uniform coverage and high surface area was prepared as a photocathode through an electrospinning and electrodeposition process for water splitting. The prepared photocathode material was composed of a CuBi2O4 nanofiber array, which has a freestanding porous structure, and the Pt nanoparticle is firmly embedded on the rough surface. The highly porous nanofiber structures allow the cocatalyst (Pt) better alignment on the surface of CuBi2O4, which can effectively suppress the electron–hole recombination at the electrolyte interface. The as-fabricated CuBi2O4 nanofiber has a tetragonal crystal structure, and its band gap was determined to be 1.8 eV. The self-supporting porous structure and electrocatalytic activity of Pt can effectively promote the separation of electron–hole pairs, thus obtaining high photocurrent density (0.21 mA/cm2 at 0.6 V vs. RHE) and incident photon-to-current conversion efficiency . This work shows a new view for integrating an amount of Pt nanoparticles with CuBi2O4 nanofibers and demonstrates the synergistic effect of cocatalysts for future solar water splitting.While the CuBi Phototability ,11. Ther2O4, a multinary p-type metal oxide semiconductor that alloys Cu2O with Bi oxide, and its ternary alloy structure allows the photogenerated electrons to be directed toward redox stable metal orbitals [2O4 possesses a sufficiently narrow direct bandgap and exceptionally positive photocurrent onset potential (>1.0 V vs. RHE), thus improving solar energy utilization [2O4 photocatalyst can be realized by hypoxic calcination [2O4 has various desirable properties, it has not achieved a high photoelectric conversion efficiency. Photocathode corrosion often occurs during oxygen reduction due to the poor transport property of the carrier (~1.2 × 10−3 cm2/Vs) [Substantial studies have revealed CuBiorbitals . Such Culization ,14,15,16cination , hydrothcination , and thecination . Althoug2O4 with different noble metal decorative materials has attracted widespread attention due to its synergistic effect, which can increase photoelectrochemical activity [2O4 film can be realized by hydrothermal synthesis, chemical bath deposition [2O4 photocathodes with high photoelectrochemical activity through coupling p-type doping with Au and gradient Cu-vacancy doping [2O4 film decorated with Pt nanoparticles using atomic layer deposition and indicated an attractive p-type material in water splitting without concern for the corrosion problem in aqueous electrolytes [2O4 film coated with Pt layers for water splitting and showed more than double the photoactivity compared to the corresponding monolayer photocathode [2O4 photocathode system has a high application efficiency, its application scope is limited, especially in dense thin films, which often suffer from the recombination of electron–hole pairs [2O4 photoelectrode with sufficient active sites will be very conducive to improving water splitting.Recently, the coupling of film-type CuBiactivity . Such Cuposition , and a tposition . For exay doping ,24. Cao trolytes . Park etocathode . The phoocathode ,27,28,29le pairs . Therefo4 nanotubes [2O4 nanofibers. Here, the novel platinum-enriched porous CuBi2O4 nanofiber (CuBi2O4/Pt) with uniform coverage and high surface area was prepared as a photocathode through an electrospinning and electrodeposition process for efficient water splitting. The prepared photocathode material was composed of a CuBi2O4 nanofiber array, which has a freestanding porous structure, and the Pt nanoparticle was firmly embedded on the rough surface. The porous nanofiber structure makes the cocatalyst (Pt) better arranged on the CuBi2O4 surface, which effectively prevents the electron–hole pair recombination at the electrolyte interface. The as-fabricated CuBi2O4 nanofiber has a tetragonal crystal structure, and its band gap was determined to be 1.8 eV. The self-supporting porous structure and cocatalytic activity of Pt can effectively promote the separation of electron–hole pairs, resulting in high photocurrent density (0.21 mA/cm2 at 0.6 V vs. RHE) and IPCE (4% at 380 nm). This study provides a new idea for the integration of Pt nanoparticles and CuBi2O4 nanofibers and provides a synergistic catalyst for future solar water splitting.Electrospinning provides a simple and scalable synthesis method to fabricate one-dimensional nanomaterial  and has anotubes  for sola2O4/Pt nanofiber film was synthesized via a three-step process: electrospinning, annealing, and deposition, which is shown in The CuBi2PtCl6·6H2O) were from Aladdin, Shanghai, China. The bismuth nitrate pentahydrate (Bi(NO3)3·5H2O), cupric nitrate (Cu(NO3)2·3H2O), N,N-Dimethylformamide (DMF), and acetic acid (CH3COOH) were obtained from J&K Chemical Ltd., B, Beijing, China. All materials were of analytical grade without further purification.The polyvinylpyrrolidone  and chloroplatinic acid (H3)3·5H2O and Cu(NO3)2·3H2O were added to a mixture of acetic acid and DMF and stirred 1 h to ensure dissolution; then the PVP was added to the above mixture and stirred 10 h to form the homogeneous precursor (2) was pasted on the counter plate to collect nanofibers. The electrospinning was performed at a distance of 20 cm between the tip of the steel needle and the plate collector, at a high voltage of 20 kV, at an injection rate of 0.1 mL/h, and with an air humidity of 40%. After electrospinning for 25 min, the films collected on the FTO glass , an Ag/AgCl reference electrode, and a platinum counter electrode. The electrolyte was 0.1 mM H2PtCl6•6H2O in 0.1 M potassium borate buffer (pH = 7.0). The electrodeposition was carried out using an electrochemical workstation (Zahner Zennium) at −0.20 V versus Ag/AgCl for 1 min.The Pt nanoparticles were loaded onto the CuBi2O4 nanofiber was measured by the Brunauer–Emmett–Teller . The UV-visible diffuse reflectance spectrum was characterized by a UV-vis spectrophotometer (PE lambda 750) with an integrated sphere attachment.In order to investigate the nanofiber, its morphology was measured by a scanning electron microscope , an energy-dispersive X-ray spectroscope (EDS), and a transmission electron microscope . Its chemical element and crystallinity were characterized by X-ray photoelectron spectroscopy  and X-ray diffraction , respectively. The surface area of the CuBi2 was carried out using the CEL-PAEM-D8 photocatalytic activity evaluation system, which consisted of a gas chromatograph (AgilentTechnologies GC-7890B) and a 300 W Xe lamp . The circulation water of 25 °C was applied to maintain the reaction temperature of the solution. The photoelectrochemical experiments on the CuBi2O4/Pt nanofiber photocathode were performed on the Zahner electrochemical workstation in a three-electrode cell . For photocurrent measurements, the electrolyte was 0.2 M PBS (pH 7.0). The Xe lamp  was used as the illumination source. The light intensity was adjusted by a calibrated photodetector. For incident photon-to-current efficiency (IPCE) measurements, the CIMPS TLS03 model (Zahner tunable light source system) was employed for monochromatic light excitation. The chopped photocurrent–voltammetry measurement was carried out with a scan speed of 10 mV/s and a chopped light time of 8 s.The photocatalytic H2O4 photocathode. The morphology of the CuBi2O4 nanofibers was evaluated by TEM and SEM measurements. As shown in 2O4 tetragonal phase (JCPDS 01-080-0996). 2O4 nanofiber after depositing Pt. It can be seen that the Bi, Cu, and O elements were uniformly distributed inside the nanofiber. In addition, the Pt element was uniformly distributed on the surface of the nanofiber.As indicated in 2O4, respectively (JCPDS 01-080-0996). The SnO2 phase of FTO glass was also marked on the same figure with an orange line. No other diffraction peak was found in the XRD pattern, which confirmed the high crystalline and phase purity of CuBi2O4 after annealing. Detailed information about the surface element composition as well as the chemical state can be obtained by XPS. The survey scan spectrum  bonds, respectively. Together, both the XRD and XPS results confirmed that these nanofibers were highly crystallized.As shown in spectrum b revealespectrum c–e, two O1s peak e ranging2O4 nanofibers exhibited strong UV-vis absorbance in both the ultraviolet and visible light regions, and their absorption cutoff wavelength was about 650 nm. By employing the linear part of (αhv)2 vs. hv, the band gap was calculated to be 1.8 eV, which is similar to that reported in other studies (1.74 eV) [2O4 nanofibers  of nanoparticles reported in other research [As indicated in 1.74 eV) . From thnofibers b, the spresearch .2O4 photocathode, the photocurrent–voltammetry measurement under AM 1.5 was performed. As shown in 2O4/Pt nanofibers showed little cathodic transient spikes, which presumably were caused by surface recombination. 2O4 nanofibers rose slowly when decreasing the potential, and yielded −0.12 mA/cm2 at 0.6 V vs. RHE. On the contrary, after depositing the Pt nanoparticles, their photocurrent rose quickly when decreasing the potential and yielded −0.21 mA/cm2 at 0.6 V vs. RHE. Remarkably, the photocurrent of CuBi2O4/Pt nanofibers was about 75% higher than that of the pristine nanofibers. Then, the IPCE measurement by the tunable light source TLS03 model at 0.6 V vs. RHE was performed. As indicated in 2O4/Pt nanofibers showed a higher IPCE reaching up to 4% at 380 nm. As shown in 2O4 nanofilm (0.15 mA/cm2 at 0.6 V vs. RHE) [2 at 0.6 V vs. RHE) showed higher photoelectrochemical performance than that of the as-prepared CuBi2O4/Pt nanofiber due to its internal electric field promoting charge separation. [To investigate the photoelectrochemical activity of the CuBiaration. .2O4/Pt nanofibers photocathode decreased obviously with the increase in time at 0.6 V versus RHE and decreased by nearly 40% after 25 min illumination. Nevertheless, the photostability of the CuBi2O4/Pt nanofibers photocathode was better than that of the pure Cu2O [2O4 photocathode consisting of open windows and struts [2 production of the fabricated nanofibers was measured by the gas chromatography-mass spectrometer (GC-7890B). As shown in 2O4/Pt nanofibers exhibited higher photocatalytic performance (380 μmol/(g·h)) compared to the CuBi2O4 nanofibers (290 μmol/(g·h)). Nonetheless, its photocatalytic performance was much lower than that of Pt/TiO2 nanosheet with exposed (001) facet (8500 μmol/(g·h), [−3 cm2/Vs, [2O4 nanofibers presents a major challenge for solar water splitting, and protection layers using atomic layer deposition were essential in order to use them as a practical photocathode.As indicated in ure Cu2O  and CuO ure Cu2O ) photocad struts . The phol/(g·h), ). The ma cm2/Vs, ). In add2O4 nanofibers could absorb photons and excite the valence electron to the conduction band. Then the photogenerated electron moved to the interface between the photocathode and the electrolyte due to the downward band bending and injected into the electrolyte to take part in the reduction reaction of hydrogen, which was similar to the charge transfer of BiVO4 anode [2O4/Pt nanofibers show better photocatalytic activity. The main reasons are as follows: Firstly, nanofibers have a large specific surface area and porous structure, which maintain good contact with electrolytes and enrich the active sites. Moreover, the nanofibers, possessing a one-dimensional structure, can also shorten the length of hole diffusing to the FTO substrate and decrease the recombination of the electron–hole pair. In addition, the Pt cocatalyst can also efficiently extract a photogenerated electron from the space charge layer. Therefore, more electrons can be transferred into electrolytes to take part in the reduction reaction of hydrogen, and the electron–hole recombination is significantly hindered. In summary, the main reasons for the enhanced photoelectrochemical performance of CuBi2O4/Pt nanofiber photocathode are porous nanofibers and cocatalysts.Based on the above analysis of experimental data, the transfer process of the electron–hole pair in the photocathode was shown in O4 anode . As show2O4 nanofibers (CuBi2O4/Pt) with uniform coverage and high surface area were prepared as a photocathode through an electrospinning and electrodeposition process for improving the photoelectrochemical water splitting. The CuBi2O4 nanofibers showed an average diameter of 225 nanometers, and lengths up to several dozens of micrometers. The porous nanofiber structures allow the cocatalyst (Pt) to have better alignment on the surface of CuBi2O4, which can effectively hinder the electron–hole recombination at the electrolyte interface. These nanofibers have a tetragonal crystal structure, and their band gap was determined to be 1.8 eV. After depositing Pt nanoparticles, their photocurrent density was 0.21 mA/cm2 at 0.6 V vs. RHE under AM 1.5 illumination, and the IPCE was 4% at 380 nm. The enhanced photoelectrochemical ability was mainly attributed to the porous nanofibers, large specific surface area, and the cocatalytic activity of Pt nanoparticles. This work shows a new view for integrating an amount of Pt nanoparticles with CuBi2O4 nanofibers, indicating the synergistic effect of cocatalysts for efficient storage of solar energy into hydrogen.In summary, the platinum-enriched porous CuBi


Clot–lysis was assessed using our plasma-based in-house assay. Platelet count, activated partial thromboplastin time (aPTT), international normalised ratio (INR), fibrinogen, fibrin D-dimer, antithrombin, thrombin generation, circulating fibrinolysis markers and organ dysfunction markers were analysed. Disseminated intravascular coagulation score, Sequential Organ Failure Assessment (SOFA) score and 30-day mortality were registered. Results: Three distinct clot–lysis profiles emerged in the patients: (1) severely decreased fibrin formation (flat clot–lysis curve), (2) normal fibrin formation and lysis and (3) pronounced lysis resistance. Patients with abnormal curves had lower platelet counts (p = 0.05), more prolonged aPTT (p = 0.04), higher lactate (p < 0.01) and a tendency towards higher SOFA scores (p = 0.09) than patients with normal clot–lysis curves. Fibrinogen and fibrin D-dimer were not associated with clot–lysis profile (p ≥ 0.37). Conclusion: Septic shock patients showed distinct and abnormal clot–lysis profiles that were associated with markers of coagulation and organ dysfunction. Our results provide important new insights into sepsis-related fibrinolysis disturbances and support the importance of assessing fibrinolytic capacity in septic shock.Background: Septic shock patients are prone to altered fibrinolysis, which contributes to microthrombus formation, organ failure and mortality. However, characterisation of the individual patient’s fibrinolytic capacity remains a challenge due to a lack of global fibrinolysis biomarkers. We aimed to assess fibrinolysis in septic shock patients using a plasma-based fibrin clot formation and lysis (clot–lysis) assay and investigate the association between clot–lysis parameters and other haemostatic markers, organ dysfunction and mortality. Methods: This was a prospective cohort study including adult septic shock patients ( Septic shock patients are at high risk of developing coagulation disturbances, which, in their most severe form, may manifest as disseminated intravascular coagulation (DIC), a life-threatening condition ,2. AlterSeveral methods have been developed to assess fibrin formation and lysis . The fiThe present study aimed to investigate fibrin formation and lysis in septic shock patients and its association with other markers of coagulation and fibrinolysis as well as DIC, organ dysfunction severity and 30-day mortality, using our in-house clot–lysis assay .The present study was a post-hoc study to a prospective cohort study including adult septic shock patients admitted to the intensive care unit (ICU) . CitrateThree distinct clot–lysis profiles emerged in our patient group: (1) severely decreased fibrin formation (flat curve), (2) normal fibrin formation and lysis  and (3) pronounced lysis resistance (lysis-resistant curve); see On day 1, 10 of the 34 patients (30%) demonstrated flat curves, with no or little fibrin formation and area under curve = 0. Another 13 (38%) patients showed normal curves, with normal or increased fibrin formation and full lysis of the clot. Nine of these 13 patients had an increased area under curve when using our local reference intervals  as cut-oOn days 2 and 3, 29 and 23 patients remained included in the cohort, with plasma available for analysis. On both days, the three distinct curve patterns remained . On day 9/L, normal curves 247 (175–299) × 109/L, lysis-resistant curves 152 (111–245) × 109/L, p = 0.05 ; see p = 0.02), but not between patients with normal curves and lysis-resistant curves (p = 0.17). Median (IQR) for aPTT between the three patient groups were: flat curves 41 (37–50) seconds, normal curves 32 (27–40) seconds, lysis-resistant curves 33 (27–39) seconds, p = 0.04 . Fibrinogen, fibrin D-dimer, international normalised ratio (INR) and antithrombin did not differ between patients with different clot–lysis curves (p = 0.79).In general, patients with abnormal clot–lysis curves (flat or lysis-resistant curves) had lower platelet counts and more prolonged activated partial thromboplastin time (aPTT) B than pas curves B. Fibrin® (CAT). As previously described [n = 10) displayed flat thrombin generation curves with endogenous thrombin potential (ETP) being 0, indicating severely impaired thrombin generation. There was a considerable overlap between patients with flat thrombin generation curves and those with flat clot–lysis curves, as 8 out of 10 patients with flat clot–lysis curves also had flat thrombin generation curves . Peak thrombin concentration tended to be higher in patients with lysis-resistant curves than patients with normal clot–lysis curves (mean 216 (SD 77) nmol/L vs. 151 (SD 77) nmol/L, p = 0.06), but ETP did not differ between patients with normal clot–lysis curves and those with lysis-resistant curves (data not shown).Ex vivo thrombin generation was analysed using Calibrated Automated Thrombogramescribed , approxip > 0.001) and moderately with peak thrombin concentration . Net fibrin formation (area under curve) correlated moderately with plasma fibrinogen  but not with peak thrombin concentration  or with ETP .Peak fibrin formation (peak absorbance) correlated strongly with plasma fibrinogen  466 (202–700) µg/L vs. 79 (57–173) µg/L, p < 0.01). Similar results were found on day 2, where median PAI-1 was 168 (IQR: 57–365) µg/L in lysis-resistant patients (n = 6) vs. 38 (IQR: 19–49) µg/L in patients with normal curves (n = 7). Levels of thrombin-activatable fibrinolysis inhibitor (TAFI) and tissue plasminogen activator (tPA) did not differ between patients with normal and lysis-resistant curves on day 1. In patients with normal curves (n = 13), where 50% lysis time could be calculated, PAI-1 correlated positively and significantly with 50% clot–lysis time on day 1 . We found only weak and non-significant correlations between 50% clot–lysis time and plasminogen, tPA, TAFI or fibrin D-dimer on day 1 .As displayed in p = 0.09 . The difference between patients with flat and normal curves reached statistical significance (p = 0.03 (Mann–Whitney test)). Median (IQR) lactate in the three patient groups were: flat curves 2.5 (1.6–6.0) mmol/L, normal curves 1.2 (1.0–1.4) mmol/L, lysis-resistant curves 2.8 (1.4–4.7) mmol/L, p < 0.01 . Patients with flat or lysis-resistant curves appeared to have higher DIC scores than patients with normal curves, but this was not statistically significant  in accordance with local institutional guidelines, we cannot exclude that this could influence fibrin formation capacity and contribute to the flat curves in some of the patients. However, all patients received LMWH during their ICU stay and it was dosed in the evening, while blood sampling was performed in the morning. Unfortunately, we could not measure anti-Xa activity due to lack of material.The presence of three distinct clot–lysis patterns remained on days 2 and 3. Furthermore, we found that 75% of patients exhibited the same pattern on day 2 as on day 1, while on day 3, this number had decreased to 61%. The change from day 1 to day 3 probably reflects that sepsis-related coagulopathy and altered fibrinolysis are dynamic conditions that may fluctuate during the course of disease. However, the high concordance between day 1 and day 2 supports the validity of our results. It should be noted, however, that some patients dropped out of the study between day 1 and 2 or day 2 and 3 due to discharge from ICU, transfer to different hospitals or passing, and we do not have access to data from these patients; thus, these concordance rates may be subject to bias and should be interpreted with caution.A detailed assessment of fibrin formation and lysis capacity in critically ill patients can support the early diagnosis of microthrombus formation, but it may also benefit the bleeding patient. Septic shock patients are at high risk of bleeding due to low platelet counts, consumption coagulopathy and higher need for invasive procedures such as lumbar puncture, surgical exploration and vascular access for, e.g., haemodialysis or central venous catheters. Currently, haemostatic support with tranexamic acid is considered contraindicated in sepsis, especially when DIC is suspected . HoweverFour recent studies assessed fibrin clot properties in sepsis with clot–lysis assays similar to ours ,20,21,22The clot–lysis assay appears well suited for characterisation of the fibrin formation and fibrinolytic capacity in septic shock patients and holds potential as a research assay, as it is sensitive to both hyper- and hypofibrinolysis, can be run in batch and can be modified according to different clinical research questions. However, turnover time, lack of standardisation or harmonisation between laboratories  and lackSome limitations to the present study should be considered. It was a post-hoc study with a small sample size. The study was not powered to investigate clinical endpoints such as bleeding, thrombosis and mortality. We did not have access to arterial lactate levels at the time of ICU admission but only at study enrolment and thus were not able to take arterial lactate at admission into account. The use of different TF and tPA concentrations challenges the comparison of our results with those of others. Our clot–lysis protocol uses relatively high concentrations of tPA compared with other laboratories ,20. EvenThe present study was a post-hoc study of a prospective cohort study  including at 20 °C for 25 min within 1 h of sampling and stored at −80 °C until analysis within 12 months. Repeated freeze–thawing was avoided.Blood was drawn from an arterial cannula placed in the radial or femoral artery. Citrated plasma for clot formation and lysis assay, thrombin generation assay, plasma fibrinolysis markers and other coagulation analyses was obtained as previously described , centrifg. Final concentrations in the wells were as follows: Recombinant TF , diluted 1:5000; phospholipid suspension containing phosphatidylserine, phosphatidylcholine and sphingomyelin , 4 µM; tPA , 116 µg/L; CaCl2, 26.7 mM. Normal pooled plasma  was used as internal control and added to all plates. After addition of CaCl2 as the final step, the plate was shaken for 10 s. Absorbance was read continuously at 405 nm for 90 min . The analyses were performed in duplicate, and the mean value of the duplicate readings was used for statistics. The raw data (absorbance units (AU) with corresponding time points) were exported from the reader and processed using the 2030 WorkOut and WorkOut 2.5 software . The following parameters were derived from the raw data: lag phase (seconds), peak absorbance (AU), area under curve (AU*seconds), 50% clot–lysis time (seconds) calculated as time from peak absorbance until 50% lysis (The fibrin clot formation and lysis assay (clot–lysis assay) was performed at the Thrombosis and Haemostasis Research Unit, Department of Clinical Biochemistry, Aarhus University Hospital according to our local protocol . Briefly0% lysis .Laboratory analyses and quality assessment were performed by experienced laboratory technicians blinded to the patients’ clinical and laboratory information. Individual patient samples were re-analysed if the intra-individual (between duplicates) coefficient of variation of any parameter was >15%. The plate was re-analysed if the intra-assay (between controls) coefficient of variation of any parameter was >15%.Our previously published reference intervals for the method were used for comparison of septic shock patients with healthy individuals .®, Technoclone, Vienna, Austria), PAI-1 antigen  and TAFI antigen . Plasminogen was analysed employing a Sysmex CS2100i .The following proteins were analysed using commercial ELISA kits according to the manufacturers’ instructions: tPA antigen (Technozyme® (CAT)) was analysed at the Thrombosis and Haemostasis Research Unit, Department of Clinical Biochemistry, Aarhus University Hospital as previously described [Ex vivo thrombin generation , fibrin D-dimer, antithrombin , CRP, creatinine, alanine transaminase, bilirubin and arterial blood lactate.® 14 . Graphs were created using GraphPad® Prism 8 . Normal distribution was assessed visually with quantile–quantile (QQ) plots. Unpaired t-tests or one-way analysis of variance (ANOVA) were used to test for difference between groups if data followed a normal distribution, and Mann–Whitney’s test or Kruskal–Wallis’ test if they did not. For correlation analysis, Pearson’s r was used for normally distributed data and Spearman’s rho for non-normally distributed data. As the present study was a post-hoc study with a fixed population size, we did not perform a sample size calculation.Statistical analyses were performed using StataSeptic shock patients showed distinct fibrin clot formation and lysis profiles detected with our assay, and these profiles correlated with markers of coagulation and organ dysfunction. Our results stress the importance of employing dynamic assays to assess fibrinolytic capacity, as these assays may provide valuable information to support and guide the treatment of sepsis-related coagulopathy.


Ocypode spp.) are known to build sand structures near the entrance to their burrows. It has always been assumed that the structure played a role in sexual selection and mate attraction. We hypothesized that the larger males would make relatively larger pyramids in order to enhance fitness. We studied Red Sea ghost crabs at Eilat, Israel, and found that the larger crabs actually did not put much effort into building pyramids, and it was the medium-sized crabs that invested the most in building the pyramids.Male ghost crabs , in which males build display pyramids to attract females. The study was conducted at the beach in Eilat, Israel. At each session, we measured the height (in cm) of all pyramids and the dimensions  of the burrow entrance. We assumed that the size of the entrance represented the relative size of the carapace width of the occupant. The mean (± SE) entrance volume was 230.8 ± 11.7 cm, and the height of the pyramid was 11.8 ± 0.49 cm (n = 54). The results of our study did not support our hypothesis because we had expected to find a linear correlation between body size and pyramid height, i.e., the larger the male, the larger the pyramid. However, our results show that the largest males in the population either built small pyramids or not at all, and the cut-off of the larger crab’s body size appears to be around 350 cm3. We discovered a step-wise function in the data in that crabs with the smallest body size of ca. 250 cm3 constructed the highest pyramids, with a declining tendency between 250–350 cm3 and extremely low pyramids beyond 350 cm3. However, our findings need to be further studied with a stress on the ambiance and elucidate whether the habitats differ in temperature, humidity, prey-base, etc., before concluding as to why the larger males desist from building pyramids. This study underwrites the importance of studying the mating systems of the macro-fauna of the beaches that are fast disappearing owing to anthropogenic development.Display, wherein males attempt to maximize fitness by attracting sexually mature females to mate, is known to drive speciation by Sexual Selection. We researched the Red Sea Ghost Crab ( Since the theory of speciation and natural selection were co-proposed ,2, the sDisplay, wherein males attempt to maximize fitness by attracting reproductive females to mate, is known to drive SSS . The subTrying to impress females to choose themselves in a lek mating system requires males to outdo their competition by being either more vocal or colorful, executing a more elaborate dance and/or having exemplary ornamentation . AlthougOcypode, Red Sea Ghost Crab , in which males build display pyramids to attract females [According to this, we researched a little-studied species of the genus  females ,21. In s females . We reasOcypode and Holpocypode spp.) are considered to be reliable bioindicators of beach and environmental quality [Ocypode species include air-borne sounds and substrate vibrations by either rapping or stridulating; visual courtship cues were male waving or chela-forward display; or petrified display-signals that males make from sand excavated from their burrows [Ghost crabs  high and the base circumference averaged 28 cm  wide [3  of sand excavated from within the burrow and required about 80 min  to construct. He also suggested that the pyramids appear to be the sign-stimulus for attracting females, intruding males are fought off by the owner, and the nearest-neighbor-distance was 134 cm. In another study, it was reported that a male will guard his pyramid for 4–8 days, during which it does not feed [It was found that the pyramids are on average 16.5  wide . For thenot feed . Only whnot feed ,27. It wnot feed ,26.The study was conducted on the northeast beach in Eilat, Israel . Eilat is situated in the northwestern corner of the eastern arm of the Red Sea. Owing to shore-development projects by the local municipality wherein gravel was used to layer the sandy shores for the sake of beach tourism, a short section of ca. 400 m still remains in its original, sandy substrate. This stretch is further divided into two sections—ca. 100 m of sandy beach for unregulated tourism and a further 300 m that extends eastwards into a military fortification and the international border with Aqaba, Jordan.We conducted our study on the tourist section of the beach. At each session, we located all sand pyramids that were visible on the beach and measured their height (in cm), and the dimensions  of the burrow entrance. We assumed that the size of the entrance represented the relative size of the individual living in it, i.e., the carapace width ,29,30,31We analyzed relationships between entrance volume  and the heights of the pyramids as linear and/or quadratic tendency. In the first step, both variables were standardized, and the mean value was used as a reference point:x—not standardized variable;µ—mean value calculated from population.Thus, the standardized variable means how much of the entrance volume/height of pyramids deviates from the average of the whole population. We then described the relationship of both variables using the equation of a linear and a quadratic function, where the standardized height of the pyramid was used as the dependent variable. Next, we performed Mandell’s test to check which model better described our relationship. We also calculated an extreme of quadratic functions according to the equation: R 4.0.3 .n = 54. We found a negative significant correlation between these variables for both raw and standardized variables: r = −0.60, p < 0.001, n = 54; r = −0.60, p < 0.001, n = 54. The beta-coefficient (± SE) for linear relationships was −2.64 ± 4.82 and was different from 0 , and R2 for this relationship was 0.36. For quadratic function, the beta-coefficient (± SE) was −1.65 ± 6.06 and also was different from 0 , but in this case, R2 was higher than for linear regression and was 0.42. Mandel’s test to check which model is better suited to the data was significant , which suggested that the quadratic function better describes our relationship than the linear model. The extreme of the entrance volume was 169.01 mm, and after crossing this point of burrow entrance volume, the height of the pyramid decreased (The mean (± SE) entrance volume and height of the pyramid were respectively 230.8 ± 11.7 cm and 11.8 ± 0.49 cm, resp., ecreased .3. We discovered a step-wise function in the data in those crabs with the smallest body size of ca. 250 cm3 constructed the highest pyramids, with a declining tendency between 250–350 cm3, and extremely low pyramids beyond 350 cm3  are attracted to males with larger larders, and in extreme conditions, even choose to be polygynous over monogamy, which is the norm in the species, as the mating system for the season [The results are in contrast to the SSS and the Handicap Principle, wherein it is always assumed that the larger, greater or more excessive the demonstration by the male, the better attraction of females and increased fitness. For example, female Southern Grey Shrikes (e season ,34.In the past, the extreme dimorphism between the sexes and elaborate and diverse decorations of males even lead to mistakenly identifying them as separate species . As prevUca spp. was classified as resource-defense polygyny in some species and as resource-free and promiscuous in others [The mating system in n others . In our n others .Early researchers concluded that in field conditions, the negative effects of burdening offspring with handicap genes outweighed the advantages of mating with a fitter male and, thus, disproved the Handicap Principle ,42. In oIn conclusion, we present the results of our study on display in Red Sea Ghost Crabs wherein the degree of investment in display-related sand pyramids is related to body size, but against the intuitive assumption that larger males build larger pyramids, we found that the smaller the male is the larger the pyramid it needs to build to try and obtain the attention of the vying females. However, our findings need to be further studied with a stress on the ambience and to elucidate whether the habitats differ in temperature, humidity, prey-base, etc., before concluding as to why the larger males desist from building pyramids. This study underwrites the importance of studying the mating systems of the macro-fauna of the beaches that are disappearing due to anthropogenic recreational activities and development.


Cholangiocarcinomas (CCAs) are heterogeneous biliary tract malignancies with dismal prognosis, mainly due to tumor aggressiveness, late diagnosis, and poor response to current therapeutic options. High-throughput technologies have been used as a fundamental tool in unveiling CCA molecular landscape, and several molecular classifications have been proposed, leading to various targeted therapy trials. In this review, we aim to analyze the critical issues concerning the status of precision medicine in CCA, discussing molecular signatures and clusters, related to both anatomical classification and different etiopathogenesis, and the latest therapeutic strategies. Furthermore, we propose an integrated approach comprising the CCA molecular mechanism, pathobiology, clinical and histological findings, and treatment perspectives for the ultimate purpose of improving the methods of patient allocations in clinical trials and the response to personalized therapies. O. viverrini and C. sinensis infections in some Eastern countries. In Western countries, the incidence of CCA, taken together, is estimated to be <four cases/100,000 people/year, while it is significantly higher in Asian countries, with peaks of 85 cases/100,000 people/year observed in Northern Thailand [Cholangiocarcinoma is a complex nosological entity, including a heterogeneous group of biliary tract malignancies, which represents the second most common primary liver cancer and accounts for 3% of all gastrointestinal neoplasms worldwide . CCA incThailand . Over thThailand . The debThailand , is worrThailand , althougThailand ,10,11 haThailand ,13, reveThailand ,15, defiThailand , also knThailand , where pThailand , and, whThailand . ConversThailand . In our Molecular profiling studies have better delineated the genomic and transcriptomic landscape of CCA. Several studies on diverse cohorts of patients have defined different mutational profiles in iCCA and p/dCCA and have identified molecular subgroups, associated with driver gene combinations and patient prognosis ,22.Nakamura et al.  performeThe role of the risk factors, such as viral hepatitis and cirrhosis, was investigated with regard to molecular characterization by Sia et al. , who defA different clustering method, when compared to the ones previously cited, was adopted by Nepal et al. , who anaAs far as PSC is concerned, genomic and transcriptomic studies included a low percentage of patients with this condition, although it represents a predominant risk factor for CCA in Western world. To overcome this limitation, 186 PSC-BTCs tissue specimens from 11 centers in Europe and U.S. were recently analyzed by Goeppert et al. , who estThe hypothesis of chronic inflammation driving specific molecular alterations has been supported by Jusakul et al. , who carCCA is a group of malignancies characterized by dismal prognosis and very high intertumoral variability. The previously described genetic and epigenetic alterations, the underlying etiology and the complex tumor microenvironment determine this variability, presumably together with different cells of origin .It is therefore crucial to consider all these variables to identify the prognosis and treatment response and propose the most suitable targeted molecular agent. Unfortunately, several targeted therapies tested so far have shown only little or no overall survival benefits , but queHereinafter, we explore the main pathways that have been targeted in clinical trials, and we analyze the criteria of patient enrollment. Furthermore, we summarize the possibility of targeted therapies, both already studied and yet to be established, with respect to molecular clusters and driver gene mutations identified in the previous sections .As described above ,29, IDH1As previously mentioned, almost 15–28% of iCCA exhibit gene alterations in FGFR2, demonstrating its great potential in the role as a molecular target in this setting of patients. FGFR2 inhibitors, such as pemigatinib, derazatinib (NCT03230318) and infigratinib (BGJ394), have shown promising results. In particular, pemigatinib, in an umbrella multicenter, open-label, phase II clinical trial (FIGHT-202), was administered in three groups of patients with cholangiocarcinoma, who had FGFR2 fusions or rearrangements, other FGFR2 alterations and a FGFR2 wild type. In this study, the patients belonging to the first group showed an overall response rate (ORR) of 35.5%, with a median duration of response (mDOR) of 7.5 months . On the Due to this promising evidence , its safThe overexpression of vascular endothelial growth factor receptor (VEGFR) has been demonstrated in 31.4–75.6% of iCCA and p/dCCA, and the presence of EGFR and VEGFR family members in BTC is linked to a negative prognostic role . The inhOn these bases, the efficacy of targeting VEGF with bevacizumab has been studied in a multicenter phase II trial in combination with gemcitabine and capecitabine . The resRegorafenib has shown promising result in patients with advanced BTC, with progression on the standard first-line therapy with a PFS of 15.6 weeks and OS of 31.8 weeks in this poor prognosis population, and confirmatory phase III studies are warranted .To date, EGFR inhibitors did not show encouraging results when administered in CCA clinical trials. Several trials have explored the efficacy of combination regimens of monoclonal antibodies with chemotherapy, with mixed results.A phase II clinical trial evaluated the efficacy of cetuximab in combination with GEMOX vs. GEMOX alone in patients with advanced BTC . The patThe combination of panitumumab, a monoclonal anti-EGFR1 antibody, with gemcitabine and irinotecan showed good tolerability and encouraging results, with a median PFS of 9.7 months and median OS of 12.9 months . These eOther studies in the past years did not show any clear benefits in terms of the OS from a combination of chemotherapy regimens with lapatinib  or erlotSeveral solid malignancies, such as breast and gastroesophageal cancers , have knThe PI3k/AKT/mTOR pathway is implicated in the metabolism, cell cycle and survival. Alterations of this pathway have been observed in several solid tumors, including iCCA and p/dCCA ,27,29. NBortezomib is a proteasome inhibitor already approved for multiple myeloma and other hematological diseases. Despite that a phase II trial did not observe a confirmed response in unstratified patients , leadingCCA is characterized by the notable presence of desmoplastic stroma that is implicated in the reduced efficacy of the immune response and in chemoresistance ,64. ReceCurrently, there is no recommendation in favor of performing molecular profiling or specific gene alterations analyses as routine in CCA patients management . A retroThe CCA molecular mechanism has aroused growing interest over the years, and it has been studied on several cohorts through different high-throughput techniques and clustering methods. Multi-omics datasets have provided a large amount of information about CCA pathobiology, although not fully elucidated yet. Nonetheless, this approach is challenging because of the wide dimension and high heterogeneity of datasets, the limited number of patients due to CCA generally low incidence and different data-merging methods . Thus, hThe de facto poor translatability of preclinical studies and lack of patient stratification in clinical trials have contributed to the disappointing results of several therapeutic strategies so far tested in CCA. While, in various clinical trials, patients have been treated with targeted therapies without a previous molecular characterization, the risk of a molecular-centered approach is, conversely, to neglect the tumor pathobiology and clinical background, overestimating the prognostic value of molecular signatures.Hence, we propose an integrated strategy based on the correlation between the etiological, clinical, histological, radiological and molecular findings. Analyzing the clinical–epidemiological and histological features, correlating them to the underlying molecular mechanisms and reproducing the interactions between them in experimental models may be the keys to develop more efficient diagnostic and therapeutic tools. The identification of molecular alterations associated with specific risk factors/underlining liver diseases, clinical presentation and histological and radiological patterns would allow a targeted molecular analysis, instead of time-consuming and expensive high-throughput profiling method and, ultimately, proper patient allocations in clinical trials.Acknowledging the complexity and heterogeneity of this disease and bearing it in mind when designing both preclinical and clinical studies represent the first step towards effective precision medicine in CCAs, which seems increasingly probable in the near future.