Neuroimaging text mining extracts knowledge from neuroimaging texts and has received widespread attention. Topic learning is an important research focus of neuroimaging text mining. However, current neuroimaging topic learning researches mainly used traditional probability topic models to extract topics from literature and cannot obtain high-quality neuroimaging topics. The existing topic learning methods also cannot meet the requirements of topic learning oriented to full-text neuroimaging literature. In this paper, three types of neuroimaging research topic events are defined to describe the process and result of neuroimaging researches. An event based topic learning pipeline, called neuroimaging Event-BTM, is proposed to realize topic learning from full-text neuroimaging literature. The experimental results on the PLoS One data set show that the accuracy and completeness of the proposed method are significantly better than the existing main topic learning methods. Neuroimaging text mining is to extract knowledge from neuroimaging texts  and has Topic learning can learn the meaningful expression of the text from the document set , and obtHowever, there are still some problems in the above-mentioned topic models, especially poor interpretability and incoherence of topics. Therefore, how to improve topic accuracy is another core issue in current topic learning researches. The above methods combined with deep neural networks can improve semantic coherence of topics by capturing deep semantic representation . BesidesTopic learning is also a core research issue of neuroimaging text mining. Neurosynth  recognizBased on the above observations, this paper proposes a topic learning pipeline called neuroimaging Event-BTM, to learn topics from neuroimaging literature. An event-based topic learning task is designed to obtain neuroimaging research topics with rich semantics. Following the trend of fusing deep learning, domain knowledge and probabilistic topic models, a new topic learning method combining deep learning with the biterm topic model (BTM) is proposed. Details are described in the following sections.In this section, we will introduce a new topic learning method oriented to full-text neuroimaging literature, including the definition of neuroimaging research events and the proposed neuroimaging Event-BTM.In the definition of ACE , “event”As shown in Table Topic events can be constructed by using meta-events. Therefore, this paper mainly focuses on the extraction and representation of meta-events. These meta-events are usually expressed as a trigger + argument structure . For exaBased on the definition of events in the previous section, we design a topic learning pipeline, called neuroimaging Event-BTM, which combines heterogeneous deep neural networks with BTM. Figure Event recognition includes trigger recognition, argument recognition and trigger-type recognition. This paper uses BiLSTM-CNN  to modelText vectorization The text vectorization layer encodes each sentence of literature as a textual vector for event element recognition. For each word in the sentence, the following four types of vectors are constructed:Word vector: it is to map words into real number vectors to obtain as much semantic and grammatical information as possible . This paCase vector: the ten categories of neuroimaging research meta-events and their corresponding arguments involve a large number of domain terms with the capital abbreviation format, such as fMRI , DMN (default mode network). In order to identify these important upper- and lower-case features, this paper constructs the one-hot case vector which has six dimensions: “numeric”, “allLower”, “allUpper”, “initialUpper”, “mainly_numeric”, “contains_digit” and “other”.Terminology dictionary vector: previous study shows that the term dictionary encoding plays an important role in multi-class named entity recognition . TherefoCharacter vector: CNN is used to construct the character vector. Its input is the 25-dimensional character embedding which is generated by looking up the character random table. In this paper, the character random table is initialized with values drawn from an uniform distribution with range .Based on these four types of vectors, a combined word vector can be constructed as follows:Event element recognition The event element recognition is to decode text vectors for recognizing triggers and arguments. The correct recognition of named entities in sentences depends on the context of the word [the word , so it i-softmax  is used The event extraction is to identify argument roles based on the outputs of event recognition. The lexical-level and sentence-level features are combined to construct the role feature vectors.r is the index of the role type in the event-role table.The lexical-level role feature is involved with word embedding, annotated triggers or arguments, and the context structure of event mentions. For each role, its lexical-level feature vector is defined as follows:r is the index of the role type “research object” in the event-role table. Because the argument “Task difficulty” and the trigger “reveals” are next to each other, both Figure W is the linear transformation matrix of the hidden layer and tanh is the activation function.The sentence-level features are obtained by using CNN . Its inpLinking the lexical-level and sentence-level feature vectors, the role feature vector B is the set of event co-occurrence pairs extracted from the full text of literatures, which is an explicit variable, the topic variable Z represents all event biterms, the implicit variables k: draw a topic-event distribution: for each topic sample get topic distribution of the set b in the event biterm set B:draw a topic assignment: draw two events: for each event biterm The joint probability of an event biterm B is:b is calculated by :10\docuIn this paper, LDA, LF-LDA, LF-DMM and MicroASM are selected as baseline methods to verify the effectiveness of the proposed method.Traditional LDA is the most classical probability topic model. In this paper, LDA was performed on both the full text and the abstract to learn literature topics, respectively.LF-LDA and LF-DMM integrate context information by using the word vector to improve the word-topic mapping learnt. In this paper, LF-LDA and LF-DMM were performed on both full texts and abstracts for comparing with the proposed neuroimaging Event-BTM.MicroASM improves the topic quality by using external domain knowledge. In this paper, external domain knowledge of MicroASM, i.e., the seed topic word pairs, was constructed based on the term dictionary “Cognitive Function”. This baseline method was also performed on both full texts and abstracts, respectively.Original data The experimental data set is composed of 126 full texts of literature from the journal PLoS One, which contain any one of “fMRI”, “functional magnetic resonance imaging” and “functional MRI” in abstracts and were published from January 2018 to July 2019. The data set was divided into the training set and the test set according to the ratio of 9:1, and the two sets were further divided into the full-text set and the abstract set.Term dictionary Neuroimaging Event-BTM integrates domain knowledge with probability topic models. Existing various domain terms are used to annotate arguments in train data sets by the distant supervision approach. The proportion distribution of 3081 terms is shown in Fig. Gross brain anatomy (GRO): 519 brain region terms were collected from the whole brain atlasCognitive function (COG): 839 cognitive function terms, such as “attention”, “activation”, “cognitive dissonance”, were collected from “Concepts” terms on cognitive atlas.2Subject (SUB): 100 crowd-related terms, involved with age, gender, identity, occupation, etc., were collected from the Web.Medical problem (MDI): 602 medical problem terms were collected from “diseases”5Sensory stimuli or response (SEN): 53 perception-related terms, such as “visual”, “auditory” and “emotional”, were collected from the Web.Experimental task (TSK): 763 task terms, such as “abstract/concrete task”, “audit diagnostic observation schedule”, were collected from “Tasks” terms on cognitive atlas.6Experimental measurement (MEA): 21 neuroimaging devices-related terms, such as “MEG ”, “MRI (magnetic resonance imaging)”, “DOI ”, were collected from the Web.Analytical tools and methods (TOL): 140 terms about data-mining algorithms and tools were collected from Baidu Encyclopedia.7Analytical results (RLT): 31 brain networks-related termsEvent element annotation In this study, the “BIO” tagging system [g system  was usedRole annotation The role annotation consists of five parts: role type, the beginning position of the trigger, the ending position of the trigger, the beginning position of the argument and the ending position of the argument. As shown in Fig. Experimental parameters In the experiment, the dimension size of the word vector was set at 100. The epoch was set at 50, the convolution width at 3, the CNN output size at 30, the dimensional number of LSTM hidden layer at 200. We set the number of topics k = 2 for all the methods, and set 2 topic words for each topic.Evaluation parameters As stated above, topic accuracy and completeness are two important indexes of topic learning. The former means that the obtained topics should express the document as accurately as possible. The latter means that the obtained topics should summarize the document as comprehensively as possible. This paper chose topic coherence and KL divergence [vergence , 30 to eV is the set of words in a topic, Topic coherence is to measure the semantic similarity of topic words in a topic for topic evaluation . It can P and Q. For topic evaluation, the average KL divergence of topic pairs can be used to measure the difference of topics, i.e., the difference of topic words distribution among different topics. It can be calculated as follows:p is a topic distribution, p(x) is a topic word in p, and q is another topic distribution, q(x) is a topic word in q. The higher score of KL divergence indicates the higher topic discrimination, i.e., better topic completeness.KL divergence is the asymmetry measurement of the difference between the probability distributions In the experiment, the proposed neuroimaging Event-BTM and LDA were trained based on the training set, and then four baseline methods were performed on both the abstract test set and full-text test set. The neuroimaging Event-BTM was only performed on the full-text test set because the abstract lacks enough information about neuroimaging research events.The result of word-based topic learning Traditional topic learning is based on words. Figure The result of neuroimaging Event-BTM different from the baseline methods, neuroimaging Event-BTM adopts event-based topic learning. Figure Evaluation of results The average topic coherence and average KL divergence of topics that we obtained were calculated. The results are shown in Table For topic coherence, traditional LDA is better than LF-LDA. This shows that LF-LDA can extract more diversified topic words because it integrates contextual information by using word embedding. DMM assumes that each document is generated by a single topic, but this experiment sets the number of topics For KL divergence, LF-LDA is better than traditional LDA. This once again confirms that LF-LDA can extract more diversified topic words. The proposed neuroimaging Event-BTM achieves the highest score. This paper introduces domain knowledge into topic learning. Expert knowledge was formalized as three types of neuroimaging research topic events. Formal domain knowledge, i.e., term ontologies or dictionaries, was used to annotate training data by using the distance supervision method. Guiding by domain knowledge, the extracted neuroimaging research events cover the results and processed of neuroimaging researches. Therefore, the topics learned from neuroimaging research events have a higher KL divergence, i.e., topic completeness, and can give more comprehensive literature descriptions than traditional word-based topic learning methods. MicroASM integrates terms belonging to “Cognitive Function” as domain knowledge for topic modeling. Its topic completeness is also improved and achieves the second highest KL divergence.Furthermore, Table By analyzing the process of neuroimaging research and the information availability of neuroimaging literature, three types of neuroimaging research topic events were identified. Based on them, an event-based topic learning task is designed to obtain rich semantic neuroimaging research topics for improving the interpretability and accuracy of topics.By fusing deep learning and domain knowledge with probability topic models, a new topic learning method is proposed to realize event-based topic learning oriented to full-text neuroimaging literature.Aiming at the two core indexes of topic learning, topic coherence and KL divergence were chosen as evaluation parameters. A group of experiments are completed based on actual data to compare the proposed method with four main topic learning methods.Experimental results on actual data sets show that neuroimaging Event-BTM can significantly improve topic accuracy and completeness for neuroimaging literature mining.Currently, neuroimaging topic learning researches mainly adopt typical probability topic models, such as LDA, to extract topics from literature and cannot obtain high-quality neuroimaging topics. Oriented to the full-text topic learning task, existing topic learning methods also cannot effectively meet the requirements of topic learning from full-text neuroimaging literature. For solving this problem, this paper proposes a neuroimaging topic learning pipeline, called neuroimaging Event-BTM, which takes events as the basic unit and extracts topics from neuroimaging full-text literature by combining the deep learning neural network with the topic probability model. These are the following three main contributions:


Dopamine is an important neurotransmitter that plays a key role in a wide range of both locomotive and cognitive functions in humans. Disturbances on the dopaminergic system cause, among others, psychosis, Parkinson’s disease and Huntington’s disease. Antipsychotics are drugs that interact primarily with the dopamine receptors and are thus important for the control of psychosis and related disorders. These drugs function as agonists or antagonists and are classified as such in the literature. However, there is still much to learn about the underlying mechanism of action of these drugs. The goal of this investigation is to analyze the intrinsic chemical reactivity, more specifically, the electron donor–acceptor capacity of 217 molecules used as dopaminergic substances, particularly focusing on drugs used to treat psychosis. We analyzed 86 molecules categorized as agonists and 131 molecules classified as antagonists, applying Density Functional Theory calculations. Results show that most of the agonists are electron donors, as is dopamine, whereas most of the antagonists are electron acceptors. Therefore, a new characterization based on the electron transfer capacity is proposed in this study. This new classification can guide the clinical decision-making process based on the physiopathological knowledge of the dopaminergic diseases. Currently, there are at least eleven different types of dopaminergic drugs for the control of psychotic symptoms. To date, all drugs with antipsychotic efficacy show some affinity and activity at the D2 subtype of the dopamine receptor36.During the second half of the last century, a movement referred to as the third revolution in psychiatry emerged, directly related to the development of new antipsychotic drugs for the treatment of psychosis. Treatment of psychosis has evolved with the development of antipsychotic drugs. The dopamine hypothesis, which defines the physiological mechanism of schizophrenia (a type of psychosis) postulates that this is derived from a primary imbalance in the dopaminergic systemResearch focusing on new antipsychotics has led to greater knowledge on their biochemical effects; however, the physiological mechanism of action underlying their pharmacological therapy still requires explanation. For the most part, antipsychotics can be classified as antagonists or agonists, according to their functionality. Antagonist drugs are those that bind to receptors, in this case dopamine receptors and block them, while agonist drugs are those that interact with the receptors, thereby activating them. An agonist produces a conformational change in the dopamine receptors (coupled to a G-protein) that turns on the synthesis of a second messenger. Antagonists also produce a conformational change in the receptor but without change in signal transduction.39. In addition to agonist–antagonist classification, antipsychotics have been classified according to having affinity for more than one receptor subtype, leading to first and second-generation of antipsychotics40.Experimentally, drugs are classified as either agonists or antagonists based on complex behavioral analysis, as well as rotational experiments with rats47 have used quantum chemistry calculations to help describe the pharmacodynamics of antipsychotic drugs, relating biological activity to chemical reactivity indices, such as chemical hardness and first ionization energy. There is also a comparative study of 32 oral antipsychotics used for treatment of schizophrenia  recently published48. Authors report specific aspects for the antipsychotics such as efficacy, quality of life and side effects. They conclude that, because so many antipsychotics options are available, this analysis should help to find the most suitable drug for each patient. They also found efficacy differences between molecules, but drugs differ more in their side effects than in the effectiveness. It is clear that more research is needed to explain the psychopharmacodynamic effect these drugs have.Previous reportsIn spite of all existing research on dopaminergic agents, to date, very little empirical and theoretical data exist to elucidate mechanisms of action. Based on the idea that all molecules have chemical properties that can be described in terms of response functions related to chemical reactivity, the principal aim of this investigation is to examine 86 molecules classified as agonists and 131 molecules classified as antagonists Tables  by applyThe hypothesis underlying our investigation is that agonist molecules have electron transfer properties similar to those of dopamine; whereas antagonists of dopamine have a different capacity to transfer charge. At molecular level, this may explain why antagonists bind to the receptors without activating them.− and ω+) of the endogenous neurotransmitter dopamine and the related compounds dopexamine, epinine, etilevodopa, ibopamine, levodopa and melevodopa, as well as dopaminergic ligands and closely related substances (86 agonists and 131 antagonists) in order to analyze their electron transfer properties. Dopamine and related compounds are calculated in order to compare their electron transfer properties with that of the pharmaceuticals studied . They bind to multiple receptors or they are used as antidepressants, or they can act as either agonists and/or antagonists, depending on dosage. In order to analyze these results more carefully, we divided the system into two new families. Family I consists of those dopamine receptor ligands that can be easily characterized as either agonists or antagonists, and mainly bind to the D2 receptor of dopamine. In this family, there are 54 molecules classified as agonists and 88 molecules classified as antagonists. The DAM of Family I is reported in Fig. .Family II comprises 76 molecules that are reported as “partial” or “weak” agonists or antagonists, and some of them present binding affinity for multiple receptors. Regardless of whether they are reported as “weak” or “partial” agonists/antagonists, these molecules were included in the conventional classification of agonists/antagonists with antiparkinsonian or antipsychotic effects. Family II form a group that is heterogeneous, with molecules that have affinity for multiple receptors and they are also weak or partial agonists or antagonists. They do not present selectivity to dopamine receptorsi.e. antagonists have similar electron donor properties to dopamine, whereas agonists have different electron donor properties. It is important to emphasize that previously reported experimental data concerning the reactivity of these molecules is either imprecise or indicates that these molecules bind to multiple receptors. The inverse association found in Family II is difficult to explain, but may be an indication of the complications related to the experimental classification of these drugs. The inherent uncertainty associated with the ex vivo or in vivo experiments is a non-parametric entity that is composed of at least two levels of contributions: the supramolecular and the organellar-cellular. The supramolecular contribution of that uncertainty is related to the lack of abstraction, or “isolation”, of the modeled system being studied . The organellar-cellular contribution of this uncertainty is a “background-noise-like" factor, related to variation in the post-translational modifications of proteins, assimilation of the response signals by several cellular components, termination of these signals by natural mechanisms, among others.The DAM of Family II is included in Fig. + values below or equal to 1.5 are electron donors and those with ω+ values greater than 1.5 are electron acceptors. This new information generated the DAM reported in Fig. + is arbitrary, but this number emerges when we consider experimental information related to the characterization of agonists and antagonists. Within this range, experimental information concurs with theoretical values because all adequately characterized agonists present ω+ values that are less or equal to 1.5, and all adequately characterized antagonists manifest values that exceed a ω+ value of 1.5. This enabled us to classify the molecules with reference to reported experimental and theoretical information.Importantly, behavioral experiments undertaken with rats manifest a degree of ambiguity, inherent to the complexity of biological systems and also to the evaluation and interpretation of data. This degree of ambiguity is not present in quantum chemistry calculations. The hypothesis here is that drugs with electron-transfer properties similar to neurotransmitters will also manifest similar action mechanisms. We thus report new information about the electron donor–acceptor properties of the molecules. This new information is presented in Tables One purpose of antipsychotic treatment is to minimize schizophrenia symptoms, which are caused by a deep imbalance in the dopaminergic system. Reported physiological mechanisms of schizophrenia demonstrate an excess of dopamine activity (direct or indirect) in certain regions of the brain, and little dopamine activity in other regions. We use our information to postulate that electron donors could be useful for modulating schizophrenia symptoms related to little dopamine activity as well as Parkinson’s disease and electron acceptors may be useful for controlling psychosis associated with an excess of dopamine activity as well as Huntington’s disease. Our findings indicate that electron acceptors bind to dopamine receptors and block or inactivate them. Contrarily, agonists interact and donate electrons, thus activating the receptor in a similar way to dopamine.The drugs reported here were classified in the literature as agonists or antagonists. Additionally, electrochemical signaling in cells is an essential process in humans, indicating that electron transfer may be related to the functionality of the molecules that control psychosis. Our results agree with this theory and thus, it is in accordance with the currently believed molecular action mechanism of these drugs. Therefore, we corroborate previously reported postulations with quantum chemistry calculations, and also propose new information for this group of antipsychotic drugs.The main idea of this investigation was to compare intrinsic properties (electron donor–acceptor) between the drugs and neurotransmitters. These intrinsic properties of the molecules are not always in agreement with the conventional classification of agonists and antagonists, specifically for those molecules of Family II that are classified experimentally as “partial” or “weak” agonists/antagonists. The new information reported in this study permits us to define these molecules as "similar to" or "different from" the neurotransmitters.The design of drugs for specific treatments is very demanding. After chemical synthesis and all characterizations have been accomplished, it is necessary to carry out biological tests on the drugs to determine their efficacy, and also in this specific case to define whether they are conventional agonists or antagonists of dopamine or other neurotransmitters. There are many dopaminergic agents available, which vary in terms of effectiveness and side effects, and no single treatment works for all patients. When it is necessary to change medications for specific patients, it is no easy task to decide which medication will help control symptoms. The perception that emerges from this dilemma is that along with the experimental determinations and biological tests, it is possible to do quantum chemical calculations on the molecules in order to obtain more information about their inherent reactivity and susceptibility for binding to receptors. All this information together, including the comparison of these intrinsic chemical properties, should help medical doctors define the most suitable medication for each individual patient.45 it was reported that drugs are like light bulbs and receptors (GPCR proteins) resemble the sockets of a light bulb. Certain light bulb characteristics are independent of the sockets ; in the same way that electron transfer properties of dopaminergic agents are independent of the receptors. This analogy is helpful in explaining the relevance of this information. All of these dopaminergic agents, ordered according to this new information, are reported in Tables Notably, in this analysis we do not include dopamine receptors in the form of G-Protein-Coupled Receptors (GPCRs). This is because the principal aim of this investigation was to report information of the dopaminergic agents based on theoretical Density Functional Theory response functions, related to the electron transfer process. PreviouslyIn this study, new information of 217 antipsychotics is presented based on the theoretical response functions related to the electron transfer process. In order to bind to dopamine receptors and inactivate them, molecules should be electron acceptors. Contrarily, agonists donate electrons and activate them, as dopamine does.As reported previously, clinical use of these drugs is based on their classification as agonists or antagonists, and many times these classifications  is not precise and is insufficient. For this reason, we hope that this new and more rational information will be functional as a guide in the clinical use of the drugs, improving treatment of psychosis, Parkinson’s disease and Huntington’s disease. This research provides new information concerning intrinsic properties of dopaminergic agents, which may be apt for their classification, once affinities for other receptors and biological effects have been taken into account.50, DrugBank 5.051, Guide to Pharmacology52 and Inxight: Drugs53 pharmaceuticals with dopamine receptor affinity used as antipsychotics were selected for this study, particularly focusing on drugs used to treat psychosis. In total 217 (86 molecules categorized as agonists and 131 molecules classified as antagonists) compounds  level of theory59, while applying the continuum solvation model density (SMD) with water, in order to mimic a polar environment60. M06 is one of the hybrid exchange correlation functional designed for main group thermochemistry. This functional has 27% of exact exchange; for the systems studied in this investigation higher percent is not required. Since negative ions are calculated, a triple-ζ basis set was used with diffuse and polarized functions. Harmonic analyses were calculated to verify local minima (zero imaginary frequencies). We considered protonated states of all drugs following the available experimental evidence. All molecular data of the optimized structures are available on request.Gaussian09 was used for all electronic calculations−) and electro-accepting (ω+) powers, previously reported by Gázquez et al.62. These authors defined the propensity to donate charge or ω− (1) as follows:+ (2) is defined asThe response functions that we used in this investigation are the electro-donating (ω− the ionization energy has a higher weight in the equation and in ω+ electron affinity, which is in accordance with chemical intuition. Lower values of ω− imply greater capacity for donating charge. Higher values of ω+ imply greater capacity for accepting charge. In contrast to I and A, ω− and ω+ refer to charge transfers, not necessarily from one electron. This definition is based on a simple charge transfer model expressed in terms of chemical potential and hardness. The Donor–Acceptor Map previously defined49 is a useful graphical tool that has been used successfully in many different chemical systems65. We have plotted ω− and ω+ (Fig. I and A are vertical ionization energy and vertical electron affinity, respectively. Note that in ωSupplementary Information.


Dopamine transporter knockout (DATk) mice are known to demonstrate profound hyperactivity concurrent with elevated (5-fold) extracellular dopamine in the basal ganglia. At the same time, heterozygous DAT mice (DATh) demonstrate a 2-fold increase in dopamine levels yet only a marginal elevation in locomotor activity level. Another model of dopaminergic hyperactivity is the D3 dopamine receptor knockout (D3k) mice, which present only a modest hyperactivity phenotype, predominately manifested as stereotypical behaviors. In the D3k mice, the hyperactivity is also correlated with elevated extracellular dopamine levels (2-fold) in the basal ganglia. Cross-breeding was used to evaluate the functional consequences of the deletion of both genes. In the heterozygous DAT mice, inactivation of the D3R gene (DATh/D3k) resulted in significant hyperactivity and further elevation of striatal extracellular dopamine above levels observed in respective single mutant mice. The decreased weight of DATk mice was evident regardless of the D3 dopamine receptor genotype. In contrast, measures of thermoregulation revealed that the marked hypothermia of DATk mice (−2 °C) was reversed in double knockout mice. Thus, the extracellular dopamine levels elevated by prolonging uptake could be elevated even further by eliminating the D3 receptor. These data also suggest that the hypothermia observed in DATk mice may be mediated through D3 receptors. The dopamine system is involved in the regulation of many vital physiological functions, including locomotor activity, hormonal status, and thermoregulation . ExtraceOn the cellular level, the persistent hyperdopaminergia in DATk mice results in changes in dopamine receptor function. It has been shown that the gene expression of both D1 and D2 dopamine receptors is decreased, whereas the expression of the D3 dopamine receptor gene in the striatum is increased in DATk mice . D3 dopaTo study the function of dopamine D3 receptors, mice lacking these receptors were created ,11. The In this work, we created double knockout mice deficient in both DAT and D3 receptors to further study the interaction of these two mechanisms of extracellular dopamine regulation and their influence on neurochemistry, behavior, and major physiological parameters.As DAT and D3 genes are located in different chromosomes ,2,16, mip < 0.001). Double knockout mice DATk/D3k also had lower body weight (19.6 ± 0.7 g) compared to WT and D3k animals (p < 0.001). Other genotypes did not show any growth abnormalities   .p < 0.001). Interestingly, the elimination of the D3 receptor together with DAT protein restored body temperature to WT level (body temperature for DATk/D3k 36.4 ± 0.3 °C) . DATk had a significantly lower body temperature 34.6 ± 0.3 °C compared to WT animals ( 0.3 °C) .p < 0.05).Spontaneous locomotor activity was measured using an automated monitor system in all animals of all genotypes except for DATk/D3h and DATk/D3k groups. D3k animals had only a slight increase in locomotor activity A. Meanwhp < 0.05). In heterozygous animals for both genes (DATh/D3h), locomotor activity was elevated compared to DATh animals (p < 0.05) but did not exceed the activity of D3h animals. Most interestingly, the activity of DATh/D3k animals was higher compared to both D3k and DATh animals (p < 0.05), indicating additive effects of both mutations at the locomotor activity level. Stereotypy score was higher for all mutants compared to WT animals (p < 0.05) without significant differences between mutant genotypes  were used: WT (n = 9), DATh (n = 5), DATk (n = 4), D3h (n = 5), D3k (n = 7), DATh/D3h (n = 6), and DATh/D3k (n = 4). The extracellular level of dopamine in the dorsal striatum was measured using a quantitative “low perfusion rate” microdialysis that allows the measurement of “true” extracellular levels of a neurotransmitter  Figure . The strIn this study, we used double mutant mice with a disruption in either or both DAT and dopamine D3 receptor genes to evaluate the relative contribution of these extracellular dopamine regulatory mechanisms in physiological functions. Furthermore, we aimed to investigate the specific role of the D3 dopamine receptor under the condition of hyperdopaminergia. Dopamine is involved in the regulation of many functions, including movement, motivation, reward, and temperature control . Thus, tC. elegance [The deletion of the DAT gene results in anterior pituitary hypoplasia and dwarfism in mice . DAT elielegance . Howeverelegance , so theielegance . In contOne of the major neurotransmitters involved in body thermoregulation is dopamine. However, which dopamine receptor is most critically involved in the mediation of the effects on body temperature is still a matter of debate ,22. StudSeveral mechanisms regulate extracellular dopamine levels. The major one is reuptake of dopamine by DAT ,3,4,16. In agreement with previous studies, we observed elevated quantitative microdialysis measures of extracellular dopamine levels in both D3 and DAT mutant mice ,37. LackDopamine is critically involved in the control of locomotion. It is well established that an increased level of extracellular dopamine leads to enhanced locomotion ,3,4,5,16D3h mice are known to have a greatly reduced D3 binding, but D3 mRNA can still be detected in D3h mice . In our The excessive grooming resulting in self-injuring was observed in DATk/D3h and, to a lesser extent, in DATk/D3k animals. Increased grooming was previously reported in D3k mice suggesting that it could be related to increased extracellular DA level . SimultaDouble mutant animals were generated by crossing DATk and D3k mutant lines . Parental DATk and D3k strains of mice of mixed C57Bl6 × 129SvJ background were described earlier ,16. AnimFor measurements of locomotor activity and stereotypy, mice were placed in Plexiglas boxes (size 20 × 20 cm) and activity was registered for 30 min. Measurements were performed during a light period between 14:00 and 18:00 with an Omnitech monitor  equipped with infrared photobeam detectors. The horizontal activity was measured as total distance traveled. Stereotypy was determined as the detection of repetitive movement with an interval of less than 1 s. The vertical activity was measured as beam breaks of the vertical sensors placed 7 cm from the floor of the cage .Rectal body temperatures were determined by a digital thermometer . The probe was inserted into the rectum of unanesthetized animals in home cages and maintained until the temperature reading had stabilized . Measurements were performed once for each tested animal.+ 150; K+ 3.0; Ca2+ 1.4; PO43− 1.0; Cl− 155; ESA, Bedford, MA, USA) with 0.25 mM ascorbate, pH 7.3. To measure the ”true” extracellular concentration of dopamine, a quantitative “low perfusion rate” microdialysis was performed [4 every 90 min. Perfusate samples were assayed for dopamine using HPLC-ECD. Monoamines and metabolites were separated on a microbore reverse-phase column  with a mobile phase consisting of 0.03 M citrate-phosphate buffer with 2.1 mM octyl sodium sulfate, 0.1 mM EDTA, 10 mM NaCl, and 17% methanol (pH 3.6) at a flow rate of 90 μL/min and detected by a 3-mm glass carbon electrode  set at +0.8 V. The volume of injection was 5 μL [Mice were anesthetized with chloral hydrate  and placed in a stereotaxic frame. Dialysis probes  with CMA-11 guide cannulae were implanted into the right striatum. The stereotaxic coordinates for implantation of microdialysis probes were (in mm): AP 0.0, DV 4.4, ML 2.5 relative to bregma. The placement of the probe was verified by histological examination after the experiments. Following surgery, animals were returned to their home cages with free access to food and water. Twenty-four hours after surgery, the dialysis probe was connected to a syringe pump and perfused at flow rate 1 μL/min with artificial cerebrospinal fluid . Significance at the A comparative analysis of DAT and D3 single mutants and DAT/D3 double mutants revealed some new functions of D3 receptors under conditions of hyperdopaminergia. The deficiency of DAT or D3 results in an elevated level of extracellular dopamine. The deficiency of both of these proteins results in an even higher elevated striatal extracellular dopamine level in DATh/D3k animals. This leads to an increase in the parameters of locomotor activity in double knockouts resulting in self-injuring behavior likely due to an increased level of stereotypy. For the development of dwarfism, the lack of DAT was crucial and the absence of D3 did not influence body weight either in normal animals or DATk mice. The decrease in body temperature observed in DAT knockout mice was abolished when D3 receptors were also removed indicating a specific role of D3 in hyperdopaminergia-related hypothermia in DATk mice.


The mechanical and chemical properties of natural plant fibers are determined by many different factors, both intrinsic and extrinsic to the plant, during growth but also after harvest. A better understanding of how all these factors exert their effect and how they interact is necessary to be able to optimize fiber quality for use in different industries. One important factor is the post-harvest process known as retting, representing the first step in the extraction of bast fibers from the stem of species such as flax and hemp. During this process microorganisms colonize the stem and produce hydrolytic enzymes that target cell wall polymers thereby facilitating the progressive destruction of the stem and fiber bundles. Recent advances in sequencing technology have allowed researchers to implement targeted metagenomics leading to a much better characterization of the microbial communities involved in retting, as well as an improved understanding of microbial dynamics. In this paper we review how our current knowledge of the microbiology of retting has been improved by targeted metagenomics and discuss how related ‘-omics’ approaches might be used to fully characterize the functional capability of the retting microbiome. Natural fibers from different plant species have long been used by man to make textiles and are now being increasingly exploited as a viable replacement for synthetic fibers in composite materials. From a biological point of view the fibers used in textiles and composites are single cells, much longer than they are wide and characterized by the presence of a thick secondary cell wall. Such structures can be found in the xylem (wood fibers) and/or associated with the phloem in the outer tissues of the stem of non-woody plants , as well as in the walls of the fruits or the leaves of some species . In bast fiber species, the individual fiber cells (elementary fibers) are grouped together to form so-called fiber bundles.The ‘quality’ of the fabricated textiles and natural fiber composites (NFCs) is determined by both the mechanical and chemical properties of the isolated fibers/fiber bundles and the subsequent industrial transformation process , and res1. The sequences of other fiber plants followed a few years later, most likely as a result of the size and/or complexity of the genome with cotton, jute and ramie being sequenced, respectively in 2015 , cell wall composition  and organization . At the biological level, these aspects are determined by the combined and coordinated spatio-temporal expression of several hundreds of genes, and a major challenge is therefore to identify which ones are involved in this process and to understand their role(s). As a result, genomic and genetic studies aimed at improving fiber plants have so far mainly focused on genome sequencing and the identification of genes/traits associated with various agronomic traits  or cell wall construction . Flax was the second fiber plant to have its genome sequenced  after he in 2015 , 2016  to gene expression profiles in field-grown flax is an extremely challenging task as these profiles are continually modified during plant growth according to developmental and environmental cues . Both mixysporum . In bothThe situation is further complicated by the fact that flax fibers are extracted from the stem by mechanical defibering that may alter their mechanical and chemical properties especially at the level of so-called ‘technical fibers’, corresponding to whole or fragmented fiber bundles. Furthermore, mechanical extraction itself is generally preceded by a retting step that can also modify fiber properties if not managed properly .In field-/dew-retting, plants are pulled (uprooted) and left on the ground for several weeks during which they are colonized by microorganisms  that produce cell wall degrading enzymes contributing to the progressive de-solidification of fiber bundles from the other stem tissues and partial dissociation of the fiber bundles . Fibers Until recently, our knowledge of the different microorganisms present during retting, the dynamics of colonization, and the complexity of the cell wall degrading enzyme arsenal has been severely limited by our incapacity to cultivate and study the great majority of these species. Furthermore, we know hardly anything about how assembly, activities  of the retting microbial community and interactions among members are affected by environmental conditions or by plant genotype . The facCurrently, rapid advances in high-throughput sequencing (HTS) technologies are allowing us to exploit the rapidly expanding field of metagenomics to improve our understanding of retting in flax and other fiber plants . In thisOne of the first definitions of retting is as follows: ‘Retting refers to the operation that textile plants are subjected to in order to free the fibers of the liber from the gum-resinous substance which binds them and keeps them attached to the woody stem of the plant’ . The ‘arGenerally, two main types of retting are used: “water retting” and “field-/dew-retting.” For water retting, flax stems are harvested, grouped in bundles and submerged in water. Historically, this was done in natural basins  for 5–7 days, followed by a period where stems were dried directly on the ground for one to 2 weeks . More reMore recently, various alternative methods using commercial enzyme cocktails mainly containing pectinases and xylanases have been investigated in an attempt to provide better control of the process and therefore improve fiber yield and quality . HoweverIndividual flax fiber cells (elementary fibers) are grouped together to form fiber bundles that are found in the stem outer tissues surrounding the xylem and pith that constitute the inner tissues . The bunRhizomucor pusillus (Mucoromycota formerly Zygomycota) and Fusarium lateritium (Ascomycota) have shown a capacity for decohesion of fibers, in particular thanks to a high level of pectinolytic activity  are known to produce pectin lyases , who concluded that the bacteria  pectins . The secelsineum . A few ycohesion . This stLater, Bacillus licheniformis and Bacillus subtilis were the most dominant during the first and second phase, corresponding to the growth phase and the rapid pectinolytic phase (between 10–40 h) of water-retting. Clostridium acetobutylicurn and Clostridium felsineum appear during the last phase known as the slow pectinolytic phase  followed the evolution of anaerobic bacteria during water-retting, both at an industrial and laboratory scale. ic phase . All theClostridium genus and the aerobic strains to the Bacillus or Paenibacillus genera. Anaerobic colonies with significant polygalacturonase activity belonged to two phylogenetic clusters assigned to the Clostridium acetobutylicum/Clostridium felsineum and Clostridium saccharobutylicum species. For aerobic bacteria, colonies with significant polygalacturonase activity belonged to two phylogenetic clusters assigned to the Bacillus subtilis species  ARDRA technique. All of the anaerobic strains were assigned to the  species . All thein vitro retting tests was carried out by Fila and coworkers but no new fungal genus was identified  phyla . In the entified .Fusarium lateritium and more particularly Epicoccum nigrum, recognized as being a primary saprophyte in retting , Bacteroidetes (8 vs. 11%) and Firmicute (1% or less) but differed for Actinobacteria (2.3 vs. 11%).There are currently very few studies on the holistic interactions between microorganisms and the flax plant during its growth and their implication on the quality of fibers. The only interactions studied for the moment are those taking place during retting, and that concern the capacity of microorganisms to degrade the plant cell wall and therefore ture use . The firture use . The autture use . More reture use . Howeverture use . This stBacillus cereus HDYM-02, previously characterized in their laboratory as being very effective in the decohesion of fibers. Interestingly, this species had been identified previously as an efficient retting agent that did adversely affect fiber mechanical properties  making up the community. Furthermore, metabarcoding cannot determine which functional genes are expressed. Nevertheless, recent advances in sequencing technologies and computational capabilities offer a unique way for different meta-omic approaches  to fill the gap from taxonomic information to functions by generating information on the functional gene composition of the retting microbial community.To our knowledge, while there are currently no publications reporting the use of these meta-omics approaches to study retting, recent studies have been performed on biologically similar  processes such as litter degradation. This latter phenomenon is indeed commonly described as a breakdown of dead plant material, which is composed of cellulose, hemicellulose, lignin, pectin but also proteins and can therefore be compared to the retting process. In the following sections we present some examples of how different meta-omic approaches have been used to look at plant biomass breakdown and discuss how these approaches could be exploited to provide functional information on retting.Shotgun metagenomics is a high-throughput, culture-independent genomic analysis of all microbes present in a sample. It starts with extraction of total DNA from a sample followed by library preparation and sequencing. Shotgun metagenomics is thus an untargeted global sequencing approach compared to the 16S/ITS/18S rRNA sequencing used for metabarcoding (targeted metagenomics) and which is sometimes erroneously referred to as metagenomics. In the former approach, the aim is to sequence all the genomes present, whereas, in the metabarcoding strategy only the rRNA amplicons are sequenced. Coupled with functional profiling, shotgun metagenomics enables the description of the total genetic content of the microbial community. The functional annotation of the obtained sequences requires the use of several databases, including well-known broad databases such as NCBI nt, ortholog databases such as Clusters of Orthologous Groups (COG) and Evolutionary genealogy of genes: Non-supervised Orthologous Groups (EggNOG) for inference of functional categories, pathway databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG)  for metaWith regard to retting, no shotgun metagenomic studies have been conducted to date, and insights into genes involved in fiber degradation are still only obtained via culture-dependent genomic studies . NeverthIn a study of Amazonian rainforest and rainforest-to-pasture converted soils, A MAG approach can also be applied to explore the functional diversity among members of the same phylum . In thisThe study of the effect of agricultural management practices on crop soil microbial communities is also a growing field for the use of shotgun metagenomics and aims to correlate these practices to microbial community taxonomic diversity and the pool of functions . In theiNasutitermes corniger. They then combined these data with metabolic data collected in the literature, resulting in a set of 2,988 metabolic reactions, that they used to construct a species-wide lignocellulose network. They identified 15 key bacterial species, all exclusively found in one gut compartment, producing a variety of enzymes that can breakdown lignocellulose including endoglucanases, exoglucanases, endo-1,4-β-xylanase, β-glucosidase, β-xylosidase, and cellobiohydrolase. In turn, the degradation products fed two other key bacterial species involved in fermentation.Another interesting example of the use of shotgun metagenomics to understand microbial degradation of plant cell wall polymers concerns the recent work by Shotgun metagenomics offers a unique view of the functional potential  of the microbial community and how it is affected (or not) by its environment. It does not, however, provide any information about the functions that are actually realized  by each member of the community. A first step in obtaining this information can be gained from gene expression studies through RNA sequencing. When applied to complex microbial communities this is called metatranscriptomics. Metatranscriptomics provides a snapshot of gene expression and constitutes a first step in going from ‘what the microbial community can do’ to ‘what the microbial community does.’ By analyzing gene expression at the community level, metatranscriptomics allows scientists to determine which metabolic pathways are active as well as their relative importance, thereby enabling predictions to be made about the functional role of the community. In addition, by accumulating metatranscriptomic data over the time course of a biological process it becomes possible to obtain a more accurate picture of the functional dynamics. The principle of metatranscriptomics is similar to the one described above for shotgun metagenomics except that total RNA (and not DNA) from a sample is extracted. The RNA then has to be processed for mRNA enrichment and/or rRNA depletion since rRNA are highly abundant and will compete for sequencing, resulting in poor representation of mRNA. As in all expression studies, particular precautions have to be taken to avoid RNA degradation during sample transport and preparation which can be challenging for field studies. A functional annotation of quality also relies on the use of several databases (including annotated metagenomes) to produce the most comprehensive transcript function prediction possible .As for shotgun metagenomics, metatranscriptomics has not yet been used in the context of retting. Nevertheless, some studies on biologically related environments have been done . In theiThanks to advances in sequencing technologies and analysis tools in the last decade, more and more shotgun metagenomics and metatranscriptomics data can be obtained and analyzed thereby enabling in depth descriptions of microbial community structure and functions. The complexity of some environments such as soil that was previously a major drawback for analysis can now be alleviated by using other techniques to experimentally reduce the complexity of the community . Despite15N-labeled plant-derived organic matter in a soil microbial community provided novel information on community structure and microorganism feeding strategies  in a given habitat . Recent As is the case for metagenomics and metatranscriptomics, there are actually very few descriptions of a metaproteomics approach being applied to flax retting. Nevertheless, some general conclusions and useful information can be obtained from the studies on biologically similar systems. In a recent metaproteomics study of lodgepole pine decay, a diverse array of carbohydrate-active enzymes (CAZymes) were identified, representing a total of 132 families or subfamilies among which 672 glycoside hydrolases (GHs) including highly expressed cellulases or hemicellulases . The autCladosporium and Cryptococcus  in the microbial community. This approach is of course much more informative than metabarcoding but requires a much greater sequencing depth and therefore a much higher cost. Likewise, the need to assemble large-scale sequencing data requires skills and adequate computer resources. Extensive reviews of best-practices can be found in the literature and provide useful guidelines, from study design to data analysis, and address common pitfalls of such an approach .If shotgun metagenomics can answer the questions “who is there” and “what enzymatic potential” is present, it cannot, however, provide any information about the functions that are really present . Such information can be obtained by metatranscriptomics. Despite the fact that the concept of metatranscriptomics has been around for several years , there iIf metatranscriptomics provides data on gene expression in microbial communities only metaproteomics can provide information about the different proteins responsible for the observed biological activity of the community . WhateveAnother issue concerns the size and the complexity of multi-organism protein sequence databases that are likely to contain many highly similar orthologs . There iIn conclusion, by using integrative meta-omic approaches such as meta-genomics, metatranscriptomics and metaproteomics, a much better characterization and understanding of the dynamics of the retting microbial community and its molecular interactions can be obtained. However, for all methods, the same ‘bottlenecks’ remain: standardization of appropriate experimental designs to deal with the tremendous heterogeneity of samples, the use of state-of-the-art methodologies coupled with optimal genomic sequence information, pertinent database selection, and appropriate bioinformatic tools to analyze, integrate and visualize comprehensive global data sets . As indiA multitude of intrinsic and extrinsic factors, both during plant growth and after harvest, all contribute to establishing the final phenotype of plant fibers reaching the factory . Since tSG and SH revised the manuscript. All authors were involved in the conceptualization of the manuscript and wrote the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neisseria gonorrhoeae in high prevalence populations have been shown to result in extremely high levels of antibiotic consumption. No randomized controlled trials have been conducted to validate this strategy and an important concern of this approach is that it may induce antimicrobial resistance. To contribute to this debate, we assessed if mass treatment in the related species, Neisseria meningitidis, was associated with the emergence of antimicrobial resistance. To this end, we conducted a historical review of the effect of mass meningococcal treatment programmes on the prevalence of N. meningitidis and the emergence of antimicrobial resistance. We found evidence that mass treatment programmes were associated with the emergence of antimicrobial resistance. In the absence of a vaccine, current antibiotic-dependent efforts to reduce the prevalence of  Neisseria (N.) gonorrhoeae by 90% by 2030 faces two growing challenges—antimicrobial resistance and rising rather than falling incidence of N. gonorrhoeae in many key populations [N. gonorrhoeae and other organisms [The World Health Organization’s plan to reduce the incidence of ulations ,2. A numulations . A concerganisms . Neisseria, N. meningitidis. There have been few mass treatment trials of N. gonorrhoeae. These studies have typically found that mass treatment has no effect [To assist in the evaluation of this concern, we undertook a historical review of the effect of mass antimicrobial treatments on antimicrobial susceptibility of the related o effect ,6,7, or o effect ,9. Only o effect ,9. N. meningitidis. These mass treatment studies involved the widespread administration of antibiotic therapy (chemoprophylaxis) to a community with either excess cases of meningococcal disease or a raised prevalence of asymptomatic N. meningitidis [Considerably more mass treatment studies have been conducted for ngitidis ,11,12,13N. meningitidis and N. gonorrhoeae, there are also considerable similarities [Neisseria genus that are classified as strict human pathogens, the majority of both infections are asymptomatic and resolve spontaneously. Both infections cluster in particular population groups. In the case of meningococcus, and in keeping with its respiratory transmission, epidemics and high carriage rates are predominantly associated with young adults living in crowded conditions [N. gonorrhoeae is sexually transmitted and thus a high prevalence has been linked to factors such as high rates of sexual partner turnover which generate dense sexual networks and high equilibrium prevalences of N. gonorrhoeae [N. gonorrhoeae in these populations—typically around 10% [Neisseria via well-developed systems of transformation [Neisseria species has been established as a key way that both the gono- and meningococcus have acquired antimicrobial resistance [N. gonorrhoeae has been noted to be more susceptible to the emergence of AMR than N. meningitidis [N. meningitidis is associated with the emergence of AMR, this would provide a cautionary warning for using antibiotic based strategies to reduce the prevalence of N. gonorrhoeae in high prevalence settings such as PrEP cohorts.Although there are important differences in the mode of transmission, preferred site of colonization, clinical presentation and host immune response between larities ,15. Despnditions ,12,13. Norrhoeae ,17,18,19ound 10% ,20. Crucormation ,22,23. Tsistance ,22,23. Nngitidis . These cN. meningitidis on the prevalence of the bacteria and the emergence of AMR in over 33 studies [A recent review paper by MacNamara et al. evaluated the effect of mass treatment of  studies . The aut studies . This re studies . Although the effect on AMR was not assessed in all studies, when it was assessed, AMR emerged fairly frequently. Resistance to rifampicin was particularly evident and found in all three community studies where this was assessed ,25,27,28N. meningitidis carriage found that penicillin, rifampicin, minocycline, ciprofloxacin and ceftriaxone were effective at eradicating carriage for up to four weeks [A systematic review of the efficacy of various antibiotics for the eradication of ur weeks . Eleven ur weeks . The useWe could not find any systematic reviews on this topic, but there was broad consensus in the literature we reviewed that overcrowding (particularly for young adults) played a crucial role in outbreaks of meningococcal disease and increases in prevalence ,13,26,32N. meningitidis but this effect did not appear to persist beyond six months. Mass treatments appeared to result in the emergence of AMR to rifampicin and sulphadiazine. There was little or no data for other classes of antibiotics. Mass treatment was fairly effective in the short term in reducing the prevalence of N. gonorrhoeae (azithromycin/ceftriaxone) was not assessed. There are also important biological differences between N. meningitidis and N. gonorrhoeae, as well as differences between the mass administration of antibiotics during a meningococcal outbreak and the sustained high levels of antibiotic exposure in a PrEP cohort.The utility of these findings is limited by the fact that the effect of mass treatment with the antibiotics currently mostly used to treat N. meningitidis does provide additional motivation to be alert for the emergence of gonococcal AMR in PrEP and other high antibiotic exposure populations. There is increasing evidence that horizontal gene transfer plays an important role in the genesis of AMR in N. meningitidis and even more so in N. gonorrhoeae. For example, it has been established that transformation from commensal pharyngeal Neisseria spp. played an important role in N. gonorrhoeae’s acquisition of resistance to extended spectrum cephalosporins [N. gonorrhoeae. These resistance genes can then be taken up months later by incoming gono- and meningococci [Neisseria species and, thus, we were unable to evaluate this effect. Unsurprisingly, however, studies have found a link between antibiotic susceptibility of commensal Neisseria and antibiotic consumption [Neisseria subflava to have high miminum inhibitory concentrations for penicillin, cefixime, ciprofloxacin and tetracycline [Neisseria to be related to recent cephalosporin consumption [Despite these important reservations, the fact that AMR can emerge so rapidly in the related osporins ,34,35. Tngococci ,22,23. Wsumption . A studyacycline . This waacycline ,37. Moresumption .Two trials have been conducted to assess if doxycycline pre- and post-exposure could reduce the incidence of bacterial STIs including gonorrhoea ,40. BothNeisseria species, any of which can become a reservoir for AMR upon repeated exposure to antibiotic treatment of the host [Neisseria could serve as an AMR early warning sign and it may be prudent to monitor the antibiotic susceptibilities of these commensal Neisseria species in high gonococcal prevalence and high antibiotic consumption populations, such as those on PrEP [Most people are persistently colonised during their lifetime with a variety of commensal the host . As a rethe host . As a re on PrEP ,43. N. gonorrhoeae in this group [N. meningiditis [N. meningitidis group B outer membrane vaccine), appears to only have limited efficacy and for a short period [N. gonorrhoeae have focused on strategies relying on antibiotics. The evidence reviewed here suggests that extensive use of antibiotics to control N. meningitidis prevalence runs the risk of inducing AMR. These findings provide further justification to reconsider antibiotic based strategies to reduce gonococcal prevalence—such as three-monthly screening for gohorrhoea/chlamydia in PrEP cohorts. They also provide further motivation for enhanced surveillance of AMR in all Neisseria species in high prevalence, high antibiotic consumption populations.A further relevant parallel between gono- and meningococci is how the prevalence of both infections is strongly influenced by underlying dense contact networks—sexual networks and spatial networks, respectively ,18,19. Iis group ,20. Thisngiditis ,47,48,49t period ,51,52. I


The hydrophobic property of gelatin nanofibers was also found to be improved with the addition of AEO. The nanofibers incorporated with AEO showed significant antioxidant activity and inhibitory effect against both Gram-negative and Gram-positive bacteria in a concentration dependent manner. Furthermore, the 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay demonstrated that the developed gelatin/AEO nanofibers revealed no cytotoxicity effect. Thus, gelatin nanofibers incorporated with AEO can be used as potential food packaging.The development of food packaging possessing bioactivities which could extend the shelf life of food has gained increased interest in recent years. In this study, gelatin nanofibers with encapsulated angelica essential oil (AEO) were fabricated via electrospinning. The morphology of gelatin/AEO nanofibers was examined by scanning electron microscopy (SEM) and the addition of AEO resulted in the increase of fiber diameter. The proton nuclear magnetic resonance ( Nowadays, petrochemical-based plastics are widely used as food packaging, however they are not environmentally-friendly, recyclable and biodegradable, resulted in serious environmental problems ,2,3. BioGelatin is one such material. It is a natural Generally Recognized as Safe (GRAS) polymer, which can be extracted from collagen, one of the most abundant protein in animals, and has been widely used in food, pharmaceutical, cosmetic industries ,8. HowevAngelica sinensis (Oliv.) herb, known as Dong-gui in Chinese, has been demonstrated to possess outstanding antibacterial and antioxidant activities [Essential oils (EOs), which are extracted from aromatic plants, have been widely studied in the food and pharmaceutical fields not only because they are natural GRAS products but also due to their various bioactive properties such as antioxidant, antimicrobial, anti-tumor, and anti-inflammatory ,16,17. Stivities ,22, whic1H-NMR) and water contact angle (WCA) measurements, respectively. The antioxidant and antibacterial activities were evaluated by 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) radical scavenging assay and the dynamic contact method against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively. Finally, the cytotoxicity of the AEO/gelatin nanofiber was investigated by MTT assay.In our previous study, the gelatin nanofibers encapsulated with peppermint and chamomile essential oil were successfully fabricated, which showed great potential as active food packaging . In thisMw ≈ 60 kDa, G2625, Sigma-Aldrich, Shanghai China), 2,2-Diphenyl-1-picrylhydrazyl  were purchased from Sigma-Aldrich . Angelica essential oil  was from Jiangxi Cedar Natural Medicinal Oil Co., Ltd., Ji’an, China. Glacial acetic acid  was provided from Tianjin Jindong Tianzheng Fine Chemical Reagent Factory, Tianjin, China. Phosphate buffer saline (PBS), dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S) and trypsin were obtained from Thermofisher Scientific . -2, 5-diphenyl tetrazolium bromide  was from Shanghai Yuanju Biotechnology Co., Ltd, Shanghai, China. Muller Hinton (MH) broth and agar nutrient broth were supplied from Guangdong Huankai Microbiology Technology Co., Ltd., Guangzhou, China.Gelatin powder  at 25 °C by immersing the probe into gelatin-AEO solutions. The viscosity of the solutions was measured through a digital display viscosimeter  at 25 °C using spindle L-3 at constant speed of 60 rpm. All measurements were performed in triplicates, and all data were statistically analyzed to be expressed as mean ± standard deviation (SD).The schematic of electrospun nanofiber preparation is shown in The electrospun solution was loaded into a 1 mL syringe and ejected at a pumping speed of 0.3 mL/h. A bias voltage of 15 kV was applied between the stainless steel needle (23-gauge) of the syringe and the collector at a distance of 20 cm. After electrospinning, the sample was dried overnight in a desiccator to remove the residual solvent and set aside for experiments.The morphology of gelatin/AEO nanofibers was directly observed under scanning electron microscope  without gold sputtering at an accelerating voltage of 15 kV. The diameter of nanofibers was measured by analyzing the SEM images with ImagePro Plus 6.0 soft imaging system. At least 150 fibers in each sample were randomly selected and measured for calculating the average diameter. Frequency distribution histogram and Gaussian fitting curve were made to quantify the fiber deposition accuracy. All data were shown as average diameter (AD) ± standard deviation (SD).1H NMR  spectra at 400 MHz [To prove the existence of AEO in nanofibers, 20 mg of nanofiber was dissolved into 500 μL of DMSO-d6 and characterized with  400 MHz . The chet = 0). Each sample was tested at least three times at different locations for averaging.The hydrophilicity of the firous mats was evaluated with an automatic contact angle meter . Briefly, 2 μL of distilled water was dropped on the surface of nanofibrous mat and photographed immediately , and after 24 h fresh colonies were used to prepare bacterial suspensions. The turbidity of bacterial suspension was adjusted to 0.5 McFarland standard (around 1.5 × 108 CFU /mL). Then, 20 mg of each nanofibrous sample was cut and sterilized with UV irradiation for 20 min on each side, and soaked in 3 mL of fresh Muller Hinton broth containing 10 μL of the prepared bacterial suspension (5 × 105 CFU/mL) at 37 °C for 24 h. The bacterial solution without any nanofiber was applied as a control. Finally, the growth of bacterial was indirectly measured by optical density at 625 nm with a UV-vis spectrometer . Then the antibacterial activity of nanofiber can be calculated as follows:Ac is the absorbance of the bacterial solution in control group, while A is the absorbance of the bacterial solution with according nanofiber sample.The antibacterial activity of the gelatin/AEO nanofibers was evaluated with a semi-quantitative shake flask method against two representative microorganisms, . aureus . E. coli2 in a humidified atmosphere incubator. The glass slide (22 × 22 mm) covered with gelatin/AEO nanofibers was sterilized by UV light for 15 min and placed in 6-well plate. Then 105 NIH/3T3 cells were seeded on each nanofiber sample. After culture for 24 h and 48 h, the culture medium in each well was replaced by 2 mL of MTT solution (0.5 mg/mL). After incubation for 4 h, 2 mL DMSO was added to exchange the MTT solution in each well. Then the purple solution in each well was transferred into a 96-well plate and the absorbance was measured using a microplate reader at 570 nm . Experiments were carried out in triplicate.The biocompatibility of gelatin/AEO nanofibers was measured according to our previous reported method by MTT assay . NIH/3T3p < 0.05) were employed.Experiments were performed in triplicate (n = 3) and results were expressed as means ±SD. Statistical analyses were performed using GraphPad Prism 7. To determine the statistical difference, one-way analysis of variance (ANOVA) and Tukey’s post hoc test . This result indicated that gelatin/AEO nanofibers possess outstanding antioxidant property and could protect food from oxidative decay.Antioxidants can serve as effective preservative in food-packaging applications. AEO has been proved to possess excellent antioxidant activity due mainly to the presence of E-3-butylidenephthalide . In ordeE. coli and S. aureus after 24 h of incubation, and even facilitation to the growth of these two bacteria, which is consistent with the previous report [E. coli and S. aureus. The result demonstrates that gelatin/AEO nanofiber membrane could effectively control the development of decay caused by microorganisms and is a promising candidate of active packaging for extending shelf life and maintaining food safety.Microbial infestation is a major cause of food spoilage. It has been reported that AEO has effective and broad-spectrum antibacterial activity, which could be related to ligustilide, one of the main compounds of AEO . The ants report . In contTo further verify the safety of gelatin/AEO nanofibers as active food packaging, the biocompatibility of the nanofibers was examined by MTT assay. As shown in v/v) via electrospinning technique. SEM images revealed all the electrospun membranes possessed homogeneous nanofibrous morphology. 1H-NMR analysis confirmed the existence of AEO in the gelatin/AEO nanofibers. The addition of AEO reduced the wettability and enhanced the water barrier function of gelatin nanofibers. The radical scavenging assay and shake flask method indicated that the gelatin/AEO nanofibers have excellent antioxidant activity and good antibacterial function against both Gram-positive and Gram-negative bacteria. Moreover, MTT assay revealed that the non-cytotoxicity of gelatin/AEO nanofibers. These results indicate that the developed gelatin/AEO nanofibers show great potential as a food packaging material.In this study, we have fabricated gelatin/AEO nanofibers with a certain percentage of AEO (0%, 3%, 6%, 9%


The molecular weight of self-emulsifying amphiphilic epoxy sizing emulsions has a big effect on the carbon fibres and interfacial properties of their composites. Novel amphiphilic epoxy sizing emulsions with four different molecular weights  were successfully prepared by a self-emulsifying method and applied to improve interfacial bonding between carbon fibres (CFs) and an epoxy resin (EP). The effect of molecular weight on the quality of emulsions, the sized CFs and the interfacial properties of the CF/EP composite system were studied. The results reveal that these novel sizing emulsions exhibited strong emulsifying ability and high processability. The most favourable wettability and adequate CF surface free energy were obtained by the emulsion with a molecular weight of 7500. Compared with unsized CFs, the monofilament fibre tensile performance was remarkably improved when increasing the shape parameter from 5.08 to 7.20. The interfacial sheer strength (IFSS) of the CF/EP composite was greatly increased by 96% with the emulsion of 7500. The enhanced interfacial adhesion benefits were attributed mainly from the enhanced charge interaction between CFs and the sizing layer as well as the compatibility and the mechanical interlock between the sizing layer and the epoxy matrix. Carbon fibre reinforced plastic composites (CFRPs) are widely used in industry due to the many advantages they offer, including high specific mechanical properties, adequate protection from corrosion and fatigue resistance. The performance of CFRPs, which are composed of different materials, are affected by their components, the mechanical properties of which are dominated by carbon fibres (CFs). Conversely, the mechanical strength of CFs may be reduced due to defects and damage produced by mechanical friction in processing, transportation and use. Furthermore, the chemical inertness on CF surfaces can cause undesirable wetting of the resin matrix resulting in a diminishing effect on the quality of the interface. The interface between CFs and the resin matrix is directly related to the stress transfer efficiency from the resin matrix to CFs and the overall mechanical performance of CFRPs ,2,3,4. SWater-based sizing has drawn increased attention recently due to its environmental friendliness in comparison to solvent-based sizing. As of now, two strategies have been developed to prepare a water-based sizing agent. The first strategy is to incorporate an emulsifier into a hydrophobic polymer to form an emulsion using a mechanical method ,6,7,8,9.Thermoset epoxy (EP) is widely employed as a matrix for CFRPs because of its ideal processability and chemical stability. Epoxy-based sizing agents used for CF/EP composites were investigated in this study. To prepare a self-emulsifying epoxy sizing agent, the hydrophilic groups such as p-aminobenzoic acid (PABA) , diethanA novel self-emulsifying amphiphilic epoxy sizing emulsion with strong emulsifying ability was developed with the purpose of improving the interfacial bonding between the CFs and the epoxy matrix. Amino-terminated polyoxypropylene (PEA) has many ether groups and terminated amino groups in its molecular chain, which implies outstanding hydrophilicity with desirable compatibility and reactivity with an epoxy. It was therefore considered an appropriate epoxy to modify for the purpose of this study. Octadecylamine (ODA) contains a long hydrocarbon chain with amino groups and can be introduced into the molecular segment of an epoxy via the opening reaction of an epoxy ring to enhance the hydrophobic properties of resulting Amphiphilic epoxy and compatibility with epoxy matrix. In this study, amphiphilic epoxy sizing emulsions with four different molecular weights were prepared by modifying an epoxy with PEA and ODA via a self-emulsifying method. The emulsions with nanoscale particle size, even distribution and strong stability were obtained. The influence of molecular weight on the emulsions, the CFs and the interfacial adhesion in the CF/EP system was investigated.® 6240) mixed with an amine curing agent was obtained from Tianjin Hansort Advanced Materials Co. , Ltd. The ODA was obtained from Tianjin Fuchen Chemical Reagents Co. , Ltd. Tianjin Dongfang Chemical Industry Co.  provided acetone, dichloromethane (DCM) and acetic acid. Deionised water was prepared in the laboratory.The CFs (T700-24K) used in this study were provided by Jilin Jinggong Carbon Fiber Co. , Ltd. Bisphenol A epoxy resin (E-1NT) with a molecular weight of 370 and PEA of 1000 were purchased at Jilin Qianren Innovative Materials Co.  The structure of PEA is shown in 2 and maintained at 70 °C to evaporate the acetone until the temperature started to increase. Afterwards, the mixture was heated up to 110 °C for 3 h and was then cooled down to 40 °C. Finally, acetic acid and deionised water were added and a cationic emulsion with a solid content of 40% was formed. The four epoxy sizing emulsions were prepared by the same procedure but with different molecular weights. The quantities of reactants are supplied in n1/n2 is the molar ratio of ODA to PEA and 1n + 2n = 1; polymerM, ODAM, PEAM and E-1NTM are, respectively, the target molecular weight of sizing agent and the molecular weights of ODA, PEA and E-1NT; and m is the degree of repeating units according to the polymerM.A schematic of the synthesising process of sizing emulsions is provided in The sizing emulsions were diluted by deionised water to a concentration of 3% and then put into a tank. The CF tows were passed through sizing treatment and dried immediately in an oven at 110 °C for 1 h to evaporate the water. The four types of sized CFs were prepared by the same procedure.N,N-dimethylformamide (DMF) as the eluent with a flow rate of 1.0 mL/min by a GPC system . The particle size and distribution (PSD) of emulsions was tested by dynamic light scattering (DLS) . The heat resistance of sizing emulsions was examined by thermal gravimetric analysis (TGA) . The range was 25–500 °C under N2 and the heating rate was 10 °C/min.Gel permeation chromatography (GPC) analysis was performed using −1. Firstly, a sample was taken before the sizing agent was acidified and diluted with acetone, and then it was dried under an infrared lamp to obtain a test sample. Secondly, a background scan of the KBr sample was performed. Finally, the test sample was smeared on the KBr sample and scanned to obtain an infrared spectrum.A Fourier transform infrared (FTIR) spectrometer  was used to measure the functional groups in the range of 4000–500 cm2I2) and surface energy were obtained by a DCA meter . The sessile drop method was used to measure the contact angles. The unsized and sized carbon fibre tows were compacted to form a flat surface. Drops of water and CH2I2 were, respectively, dropped on the surface of carbon fibres by a syringe. The contact angle was measured by the instrument.The dynamic contact angles (DCA) of unsized and sized CFs with water and diiodomethane (CHScanning electron microscopy (SEM)  and atomic force microscopy (AFM)  were used to characterise the surface morphology of sized CFs. The tapping mode of AFM with silicon tapping cantilever (OTESPA) was applied to scan the CF surface. The amplitude setpoint was 24 mV. Height images were obtained and flattened to display the CF surface clearly.The monofilament fibre tensile tests were performed by a tensile strength tester with a force range of 100 cN  based on ISO 5079-1995 . The tenm are Weibull parameters and can be deduced by using the equation of Weibull distribution  was mixed with the DCM to form the solution. The microdroplet was dripped on fibre using a needle and cured at 135 °C for 1 h. One end of the frame was placed in the upper clamp of the tester to ensure the resin microdroplet was close to the kerf. The other end was free. The IFSS value was calculated according to Equation (2). One value was obtained by testing at least five effective specimens. F is the peak pullout force, d is the average diameter of the fibre and l is the embedded length of the resin droplet.The tests of pulling a monofilament fibre from cured epoxy droplets were performed to determine the IFSS of CF/EP composite by the tensile strength tester  with a displacement rate of 1 mm/min. Glue was used to fix the CF to the paper frame. The epoxy resin (Hansortn) and average molecular weights (Mw) are also presented in −1 is attributed to the characteristic absorption peaks of the epoxy group, which is not visible for the synthesised emulsions. The sizing emulsions with different molecular weights exhibited similar IR spectra. A new absorption peak was observed at 3400 cm−1, which is assigned as the characteristic stretching vibration of hydroxyl groups. In the synthesis of emulsions, the epoxy groups underwent ring-opening reactions to form hydroxyl groups. This resulted in a decrease of epoxy groups and the increase of hydroxyl groups in the system [−1, which is characteristic of an ether group. The stretching vibration of –CH2 groups was found in the range of 3000–2800 cm−1 and became stronger as the molecular weight was increased. The spectra indicated that the emulsions were synthesised by reactions among epoxy, amino, hydrophobic and hydrophilic groups, suggesting that a successful introduction had occurred.FTIR results of neat epoxy (E-1NT) and all the sizing emulsions are shown in e system . A new w2I2 and the surface energy of unsized carbon fibres were, respectively, 93.06°, 101.86° and 16.87 mN/m. The wettability was improved by sizing procedure. The contact angles with water remained almost constant as the molecular weight changed. The emulsion of 7500 exhibited the smallest contact angle with CH2I2, resulting in the highest surface energy of 34.3 mN/m. The hydrophobic properties of ODA were probably restricted by the entanglement of molecular chains at high molecular weight [2I2 were decreased to 109.2° and 63.1°, respectively, and the corresponding surface energy was altered from 22.3 to 26.8 mN/m, confirming the speculation. DCA results showed that the emulsion of 7500 produced the best wetting effect among the four emulsions.The results of DCA tests are shown in r weight . To verim was increased for the sized CFs. The sizing emulsion of 7500 exhibited the best stability with a maximum value of 7.20, which revealed that sizing emulsions were conducive to bridge the defects and repair the damages on the CF surface in accordance with the surface morphology  were successfully prepared by a self-emulsifying method through introducing hydrophilic and hydrophobic groups into the epoxy molecular structure. The particle size of the obtained emulsions is in the nanometres range. The four emulsions prepared during this study all have strong thermal stability. When they lose 5% of their mass by heating, the required decomposition temperature is above 300 °C. The sized CFs by the emulsion of 7500 exhibited the most favourable wettability. The grooves on the CF surface became shallower when undergoing sizing treatment but the emulsions with high molecular weight  induced irregular bulges.The novel sizing agent emulsion was shown to be effective for improving the mechanical performance of sized CFs and interfacial adhesion. The monofilament fibre tensile strength was enhanced by treating the defects on the CF surface. The sizing agent of low molecular weight (7500) exhibited the best quality with a tensile strength of 3.44 GPa and a maximum shape parameter of 7.20. The IFSS of CF/EP composite was enhanced by 96% with the emulsion of 7500 compared with unsized CF. This resulted from the increased charge interaction between the CF and the sizing layer as well as the compatibility and the mechanical interlock between the sizing layer and the epoxy matrix.