Unsupervised statistical analysis of unstructured data has gained wide acceptance especially in natural language processing and text mining domains. Topic modelling with Latent Dirichlet Allocation is one such statistical tool that has been successfully applied to synthesize collections of legal, biomedical documents and journalistic topics. We applied a novel two-stage topic modelling approach and illustrated the methodology with data from a collection of published abstracts from the University of Nairobi, Kenya. In the first stage, topic modelling with Latent Dirichlet Allocation was applied to derive the per-document topic probabilities. To more succinctly present the topics, in the second stage, hierarchical clustering with Hellinger distance was applied to derive the final clusters of topics. The analysis showed that dominant research themes in the university include: HIV and malaria research, research on agricultural and veterinary services as well as cross-cutting themes in humanities and social sciences. Further, the use of hierarchical clustering in the second stage reduces the discovered latent topics to clusters of homogeneous topics. Natural language processing (NLP) is the ability of machines to read, understand and interpret human language. Over the past decade, NLP has gained popularity especially in artificial intelligence  and human language machine translation. The NLP syntax and semantic analysis methods are based on statistical, probabilistic and machine learning methods , therebyIn recent past, topic modelling has emerged as an effective tool for discovering useful structure in digital text database resources with significant impact on stimulating research culture in academic research –4. TopicGiven that a text corpus contains non-negative entries, Non-Negative Matrix Factorization (NMF) can be applied to decompose the high-dimensional corpus into two lower-dimensional matrices: one for the documents grouped by topic (document-topic distribution), while the second contains words comprising the topics (word-topic distribution) . While sWhile these models assume independence between topics, extensions to allow for direct correlation between topics such as Correlated Topic Models , or indiThe basic idea behind LDA is that a document is generated from a finite mixture of topics distribution where each topic is a distribution over words from the vocabulary. By learning the distributions, the most probable words for each topic can then be used to potentially provide human readable topics. Because of its superiority in analysis of large-scale document collections, better results have been obtained in both academia and non-academia areas such as in –19.Attempts at extracting dominant themes from text copora using LDA-based models are not new. For instance, similar to the analysis presented in this manuscript –22, applHowever, in Africa, there is a paucity of studies that utilize statistical methodology for unstructured data on collections of documents such as digital repositories . The worTwitter or in university e-repositories. Thus, we apply text mining techniques to gather information about scientific publications from the University of Nairobi’s e-repository into a convenient data format and further process the data with open-source R statistical software [The contribution of this manuscript is twofold: first, we showcase some statistical tools for processing unstructured data which is largely encountered in developing countries. For instance, except for donor-funded projects in HIV and Tuberculosis, there are often no proper electronic databases setup for medical records and paper-based records are predominant –29. Furtsoftware  and assoThe second objective is to perform an unsupervised classification  of the UOften, for large text copora, LDA results in a large number of latent topics, say 100 latent topics. Subsequently, succinct presentation and/or visualization of the topic distribution is not trivial. To this end, we propose a two-stage analysis of the case study presented in this manuscript. In the first stage, classical LDA as described in the methodology section (and mostly applied in the related references) is applied to the text copora. For ease of visualization of the original LDA topic composition, we propose simple animation of the topics’ word cloud. Subsequently, the discovered topics are further reduced into clusters of related topics by use of a classical hierarchical clustering algorithm with appropriate topic distance metric. To the best of our knowledge, no previous studies have combined LDA with hierarchical clustering in this manner. The novelty of this manuscript therefore lies in the sequential application of LDA and hierarchical clustering to text copora.The manuscript is organized as follows. First, we describe the case study data and necessary pre-processing steps performed on it. Thereafter, we provide an explanation of the methodology applied in both determining a reasonable number of topics in the documents to model and the final topic modelling using LDA. The results of model fitting are then provided in addition to a concluding section. Additional relevant output is cited in the manuscript and presented separately as supplementary materials to the manuscript.R function was used to scrape the publications metadata into an R dataframe. Key R packages used for this step include: rvest [stringi [tidyverse [foreach [doParallel [Publications available on the University of Nairobi’s “Journal Articles” section of the e-repository (from 1884 to 2019) were downloaded for each of the six colleges separately. A user-defined e: rvest , stringi[stringi , and tididyverse  for webs[foreach  and doPaParallel  for paraM = 10791 documents described in the Data section of this manuscript comprising of a vocabulary of V = 9246 unique words. A document is then a collection of N words W =  where wn is the nth word in the document. Note that the order of occurrence of the words in the original text does not matter (hence the “bag of words” assumption). Subsequently, the final text corpus is a collection of M documents denoted by D = {W1, W2, …, WM}.Consider the collection of W ∈ M:N ∼ Poisson(ξ): denoting the document length—the number of words in the document.Choose θ ∼ Dir(α): the topic distribution for document W .Choose N words wn:(a)zn ∼ Multinomial(θ).Choose a topic (b)wn from p: conditional distribution of the word given a topic and words comprising the topic.Choose a word For each of the LDA considers documents as a finite mixture of latent topics with each topic being defined by a distribution of words from the vocabulary. More formally, to generate a document ξ and α are hyperparameters for the corresponding distributions while β denotes the distribution of words across the topics. For ease of computation, the number of topics is fixed a priori. Optimization of the quantities of interest  can utilize Bayesian Monte Carlo simulation with Gibbs sampling, Laplace approximation, convexity-based variational inferences amongst others. Readers interested in the technical details of LDA inference are referred to [Note that erred to , 40, wheβ) and the posterior document topic distribution (often denoted as γ).For each topic, a word distribution is derived. Subsequently, for each document, a posterior topic probability is computed . The final output from LDA is the posterior word distribution across the topics . This is the principal advantage of LDA in the context of document classification (as is the cases study in this manuscript), since it is expected that although a scientific publication may be based on one dominant topic, several latent sub-topics may be present.On the other hand, the latent topics from LDA are assumed to be independent of each other, which may not be accurate in case of co-occurring topics. For instance, a topic on smoking is more likely to occur together with a topic on lung cancer and less likely to co-occur with a topic on movie reviews. Due to topic independence assumption, LDA would treat smoking and lung cancer themes as independent of each other.To cluster similar topics (based on their document-topic probability). While this is not a replacement for CTM, it may prove useful for identifying related topics without the computational constraints imposed by CTM.To further reduce the dimensions of the discovered topics, thereby, providing concise visualization and synthesis of the results.An alternative approach to identifying related topics is to perform Correlated Topics Modelling (CTM)  rather tTo this end, a hierarchichal clustering model with agglomerative clustering  is perfoa priori. The user has to specify the number of topics to model with LDA [To perform topic modelling, one of the critical tuning parameters is the number of topics to model, which is assumed to be known and fixed with LDA , 44, whiwith LDA . Howeverldatuning::FindTopicsNumber function [ldatuning package to the data and explored T = 2, 3, …, 300 topics with [ldatuning::FindTopicsNumber calls topicmodels::LDA [ldatuning::FindTopicsNumber implements a cross-validation approach which may otherwise result in a pitfall of evaluating a model on the same data it was trained on [Alternatively, when no informative prior knowledge is available on the approximate number of topics in the documents, function  can be aics with , 44, 47 s::LDA  behind tained on .K-fold cross-validation by splitting the data into K partitions, training an LDA model on the K − 1 partitions and using the K-th partition as a test data set. The model perplexity [An alternative approach to determining the number of topics to model is based on rplexity  is then rplexity .ldatuning:FindTopicsNumber and topicmodels::LDA functions require the documents to be processed as a document-term matrix [Corpus object using the DataframeSource function. A document-term matrix was then created by converting the Corpus text to lower case, removing standard English stop words, punctuation, numbers and stripping extra white spaces. Further, only words of at least 3 characters, occurring in at least 5 documents and at most 20% of the documents were retained. The final document-term matrix comprised of 10790 documents, 9246 terms and had a sparsity of 99%.Note that both m matrix , 40. Thetopicmodels::LDA was called with iter = 1000, burnin = 100 and seed = 2019. The analysis was performed in an Amazon elastic compute cloud optimized instance (c5.4xlarge) [R version 3.5.1 and RStudio version 1.1.456 [Finally, note that the approaches described in this section are computer intensive. Therefore, we implemented a parallel computing setup when determining the number of topics. In all cases, 4xlarge)  with 16  1.1.456 .ldatuning::FindTopicsNumber LDA model, the results of which formed the basis for the number of LDA topics to model, is shown in Output of the An LDA model was fitted on the document, term matrix with 100 topics. While not large compared to the dimensions of the document-term matrix, visualization and synthesis of the results is not trivial. Therefore, we first illustrate the key output of the model fitting with the first 25 topics, a pragmatic choice to balance between readability and diversity of output, and provide the complete output of the corresponding complete 100 topics in the supplementary materials for this manuscript.LDA defines a topic as a collection of co-occurring words and further, a document as a collection of topics. For each topic, the posterior probability of a document given a topic is computed. In To link the LDA-derived topics to the natural “College” hierarchy in the data, data was obtained from six  colleges from the University of Nairobi, we coloured the documents per topic by the college they were derived from.A clearer association between the topics is now evident upon adding the document source label. For instance, while from One feature of LDA is that each document is collection of topics whereby the probability of a document contributing to each topic is computed. In γ using the Ward.D2 agglomerative clustering algorithm [R hclust.As highlighted in the methodology section, we performed hierarchical clustering in order to reduce the number of topics into a smaller set using hierarchical clustering. We performed hierarchical clustering of the LDA document-topic posterior probability distribution matrix lgorithm  in R hclThe resulting dendrogram using the Cosine distance between documents in a topic is presented in In To further evaluate the results of hierarchical clustering with Hellinger distance, which collapses the 100 LDA topics to 10 thematic areas, we present the top 10 terms from the topics comprising each cluster in These clusters signify a higher-level summary of the dominant research themes in the university. For instance, from Further, from We applied natural language processing statistical tools to abstracts from six different colleges in the University of Nairobi. Topic modelling has proven to be a powerful tool for synthesis of large text Copora. In particular, LDA, being an unsupervised algorithm, summarizes the document text into fewer latent topics.We have shown that while the tools are mature enough to be used with reasonable expertise, the synthesis of the results in a more succinct manner is not straightforward. Visualizations are powerful tools used to communicate results especially to non-domain experts and proper strategies to summarise results may be necessary. To this end, we applied a mix of graphical and video visualizations.LDA, as implemented in this manuscript, assumes independence between the latent topics. On the other hand, CTM imposes a correlation between the latent topics and could potentially be applicable in the case study presented in this manuscript. However, CTM is computationally intensive and our attempts to fit a CTM model were futile due to limitations on the computing power.As an alternative, we proposed to apply classical hierarchical clustering to document-topic probabilities matrix. As a measure of dissimilarity, appropriate distance metrics such as Hellinger and Cosine were applied. To the best of our knowledge, no previous studies have combined LDA with hierarchical clustering in this manner. In most cases, the two methods are treated independently and results compared. For instance , appliedFrom the LDA model fitting results, it was evident that some topics predominantly comprised of documents from specific colleges hence very domain specific. On the other hand, documents across different colleges contributed to several other topics. This can be attributed to use of terms in the abstracts that are not domain-specific. Further, we note that for five of the six colleges, at least two topics could be almost exclusively attributed to these colleges. This was not the case for the College of Health Sciences (CHS) whose documents were almost entirely equally distributed across the topics.A potential explanation for this could be that research in CHS is multi-disciplinary and hence the documents text contribute to several  topics. In fact, upon clustering the 100 topics into more homogeneous clusters, we note that CHS dominates clusters 3 and 9, but jointly contributes to cluster 5 with College of Agriculture and Veterinary Services (CAVS) and to cluster 8 with both CAVS and College of Physical and Biological Sciences (CPBS).Based on the hierarchical clustering of LDA topics, it was evident that the different colleges are individually involved in at least one domain-specific research. Moreover, cross-cutting research themes were discovered especially in the case of human and animal health-related topics. As expected, most of the human health research centres around HIV and malaria (clusters 3 and 8), while agriculture (cluster 10), which is a mainstay economic activity also attract a lot of research interest.The use of open source tools for data wrangling and analysis has been illustrated with a case study comprising of a two-stage analysis of an unstructured text corpus. The hierarchical clustering approach proposed for the second stage still poses a challenge in terms of visualizing the topic clusters in relation to the LDA input documents. This is particularly the case because, while hierarchical clustering generates clusters of the original LDA topics, in the LDA step, each document may contribute to several topics. For visualization of each document within a hierarchical cluster, we used the maximum posterior topic probability across the respective topics. Thus, if hierarchical cluster one comprises of five LDA topics, the maximum of the posterior topic probability for each document is assigned as the respective posterior cluster probability.This choice may however mask the fact that all five posterior topic probabilities for a document contributed to the formation of that hierarchical cluster. An alternative approach may be to compute a joint topic probability for each document within a hierarchical cluster (the product of the posterior topic probability for the five topics for each document). This choice has not been fully investigated and will be a subject of future improvements.The methods described in this manuscript have many use cases in developing countries. For instance, the analysis of open-ended replies to survey questions, unstructured medical and legal documents, government social media handles and potentially for evaluating the dynamics of trending topics in the national discourse. While often written formally (in English or Swahili), more often than not, non-official social media communication- that may have relevant information in terms of trending topics- in Kenya uses slang. The methods we proposed here may potentially be used to analyse the dynamics of the language, say between urban and rural setting, across different generations or demographics or more generally, the evolution of specific text. In fact, the methodology presented in this manuscript may be applied to daily press releases by the Kenyan Ministry of Health regarding the SARS-CoV-2 outbreak. The ultimate goal would then be to evaluate the dynamic trend of the government messaging with the evolution of the pandemic.In general, the adoption of natural language processing methods in developing countries can be hampered by the computational power required. In part, this problem can be solved by accessing cloud computing services such as Amazon Web Services, as long as a decent internet connection is available since the cloud services are web-based. However, there is still the research financing hurdle for cloud computing to be widely adopted in such countries.S1 FigEach row denotes a document with the row×column cell representing the corresponding document probability for each topic.(PDF)Click here for additional data file.S2 FigHighest posterior probability of the top 10 terms comprising of clusters of topics based on hierarchical clustering with Cosine distance.(PDF)Click here for additional data file.S3 FigPosterior topic probability for the topic clusters. X-axis: maximum posterior probability per document for the topics comprising a cluster.(PDF)Click here for additional data file.S1 VideoTop 100 terms comprising each topic. The size of the text corresponds with each term’s frequency.(MPEG)Click here for additional data file.S1 GifTop 100 terms comprising each topic. The size of the text corresponds with each term’s frequency.(GIF)Click here for additional data file.S1 Data(CSV)Click here for additional data file.


R) of 115%, and an ion exchange capacity (IEC) of 2.70 meq/g. It was used for the adsorptive removal of methyl orange (MO) from an aqueous solution via batch processing. The effect of several operating factors, including contact time, membrane dosage, initial concentration of aqueous dye solution, and temperature on the percentage discharge of MO and adsorption capacity, was evaluated. Experimental data for adsorption of MO onto the DMEA-grafted AEM was analyzed with two parameter and three parameter nonlinear adsorption isotherm models but fitted best using a nonlinear Freundlich isotherm. Adsorption kinetics were studied by using several models, and attained results showed that experimental data fitted well to pseudo-second-order kinetics. A thermodynamic study showed that adsorption of MO onto the prepared DMEA-grafted AEM was an endothermic process. Moreover, it was a feasible and spontaneous process.This manuscript describes the synthesis of dimethylethanolamine (DMEA)-grafted anion exchange membrane (AEM) by incorporating dimethylethanolamine as ion-exchange content into the polymer matrix via the solution casting method. The synthesis of the DMEA-grafted AEM was demonstrated by Fourier transform infrared (FTIR) spectroscopy. The prepared DMEA-grafted AEM exhibited higher thermal stability, homogeneous morphology, water uptake (W Around 10% to 15% of them are expelled as drainage into water resources without any pretreatment. It is vigorously polluting the environment and poignant aquatic organisms as well as human life. There are more than 10,000 kinds of commercially available dyes, most of which are recognized as toxic for the environment. Moreover, several dye molecules can endure for a long time in the environment, and, resultantly, it is essential to discharge them from industrial drainage before their dumping in hydrological systems [Several industries, including the plastics, textile, pharmaceutical, paper, and cosmetics industries, etc., widely utilize dyes and pigments to color their final products . A cruci systems ,3,4,5. To date, a lot of methods, including ion exchange membrane (IEM) , reverseIn the past, researchers investigated the appropriateness of employing a lot of adsorbent with varying cost and efficiency such as waste apricot , coconutIn this article, the synthesis of dimethylethanolamine (DMEA)-grafted AEM was carried out via the solution casting method by introducing DMEA as an ion-exchange moiety into the polymer matrix. The synthesis of DMEA-grafted AEM was accomplished by using Fourier transform infrared spectroscopy. The synthesized AEM was characterized in terms of water uptake, thermal stability, ion exchange capacity, and morphology. The prepared DMEA-grafted AEM was used for the adsorptive discharge of MO from an aqueous solution at room temperature. The effect of operational parameters on the percentage discharge of MO from aqueous solution and adsorption capacity was explored. Adsorption equilibrium, kinetics, and thermodynamics were also studied.2SO4), dimethylethanolamine (DMEA), ethanol, potassium chromate (K2CrO4), chloroform, 2,2′-Azo-bis-isobutyro nitrile (AIBN), silver nitrate (AgNO3), and N-bromo-succinimide (NBS), were kindly supplied by Sinopharm Chemical reagent Co. Ltd, Shanghai, China and used as obtained. Throughout this research, deionized (DI) water was utilized. Poly  (PPO) was kindly supplied by Sigma-Aldrich chemicals . Methyl orange, chlorobenzene, sodium chloride (NaCl), N-methyl-2-pyrrolidone (NMP), sodium sulfate  (PPO) into brominated PPO (BPPO) was performed as described  Figure  See Sec. We employed the solution casting method to synthesize the homogeneous DMEA-grafted AEM, as reported in our previous work ,43,44,451H NMR (DMX 300 NMR spectrometer working at 300 MHZ), FTIR spectrometer , Field emission scanning electron microscope , and the Shimadzu TGA-50H analyzer  were used WR=Ww− form. It was then washed with deionized water to discharge excessive NaCl. After that, it was soaked in 0.5 M Na2SO4 solutions for 48 h. The quantity of Cl− ions released was calculated by titration method by using 0.05 M AgNO3 as titrant and K2CrO4 as an indicator. It was estimated by the following equationm, V, and C depict the dry mass of the DMEA-grafted AEM, titer volume, and the concentration of AgNO3, respectively, used during titration.The classical Mohr’s method was employed to determine the ion exchange capacity of the DMEA-grafted AEM. Initially, the dried DMEA-grafted membrane was soaked in 1.0 M NaCl solution for 48 h to convert charge sites into the Clmax. = 464 nm for MO. The concentration of MO was determined from the calibration curve. The adsorption capacity and the percentage removal were determined by the below equations0C and tC denote MO concentrations at the intital state at time t, respectively. Similarly, V indicates the volume of MO solution, and W is the weight of the prepared DMEA-grafted AEM, respectively.Herein, a batch adsorption procedure was used as described previously ,35,36,47Several nonlinear adsorption isotherms were used to explain the adsorption of MO onto the DMEA-grafted AEM See .Adsorption kinetics was studied by using several kinetic models for MO adsorption onto the DMEA-grafted AEM See . R, ∆Go, Kc, ∆So, and ∆Ho are the general gas constant, change in Gibb’s free energy (KJ/mol), equilibrium constant, change in entropy (J/mol·K), and change in enthalpy (KJ/mol), respectively.Adsorption thermo dynamics were evaluated using the equations below1H NMR spectroscopy was used, and the attained 1H NMR spectrum is denoted in The bromination of poly containing DMEA to the polymer architecture [membrane . It was membrane ,50. The itecture . It authThe thermal stability of the DMEA-grafted AEM was illustrated by thermogravimetric analysis (TGA). The structure of ion exchange membranes also has a significant effect on the removal of dyes from an aqueous solution. It was investigated by scanning electron microscopy (SEM). Water uptake and ion exchange capacity indicate the hydrophilicity of the ion exchange membranes. These parameters are established on the amount of ion-exchange contents into a polymers architecture. For the prepared DMEA-grafted AEM, water uptake was found to be 115% at room temperature. Herein, ion exchange was estimated by employing classical Mohr’s method, and the obtained IEC value was found to be 2.70 meq/g. The theoretical IEC of the DMEA-grafted AEM was 2.85 meq/g. Water uptake and ion exchange capacity have a crucial effect on the adsorption efficiency of a prepared membrane. From this, we concluded that the synthesized membrane was highly hydrophilic and suitable for the adsorption of MO from an aqueous solution. Adsorption of dyes is dependent on operating endowments. In this manuscript, we investigated the influence of four operating parameters such as contact time, membrane dosage , initial concentration of dye solution, and temperature on the percentage discharge of MO from aqueous solution and adsorption capacity. The effect of the initial concentration of dye solution onto the adsorption capacity of DMEA-grafted AEM and the percentage discharge of MO from aqueous solution is represented in Two parameter nonlinear isotherms ), and three parameter nonlinear isotherms (Redlich-Peterson (R-P), Hill, and SIPS) were also used to explain adsorption of MO onto the prepared DMEA-grafted AEM. All isotherm parameters were calculated by nonlinear regression by employing Igor Pro WaveMatrices 6.2.1 software . The staf is the adsorption capacity of the prepared DMEA-grafted AEM. The attained value of mean adsorption energy (12.50 kJ/mol) from the D-R isotherm model demonstrated that adsorption of MO onto the DMEA-grafted AEM was a chemical ion-exchange adsorption process.1 and qe are given in 2) was 0.836. There were also large differences between experimental adsorption capacity (qe and exp.) and calculated adsorption capacity values , indicating that a pseudo-first-order model can not explain the rate process. The plot of the pseudo-second-order model is shown in 2 > 0.999) was close to unity, demonstrating that the model fit the experiment data well. Similarly, the plots of the Elovich model and the modified Freundlich equation are shown in 2) were 0.724 and 0.708 for the Elovich model and the modified Freundlich equation, respectively, show that these models were not suitable for explaining the experimental data. Moreover, the plot of the Bangham equation is represented in c for the adsorption of MO onto the prepared DMEA-grafted AEM, and the attained results of the thermodynamic study are given in oG) were negative, demonstrating the spontaneity and feasibility of the MO adsorption process onto the DMEA-grafted AEM under the utilized experimental conditions. The positive value of enthalpy (ΔoH) confirmed that adsorption of MO onto the DMEA-grafted AEM was an endothermic process. Moreover, the positive value of entropy (ΔoS) exhibited the enhancement in randomness during adsorption of MO onto the DMEA-grafted AEM at the dye-membrane interface.Thermodynamical evaluation is crucial for the investigation of heat change, spontaneity, and feasibility of the adsorption process onto the surface of adsorbent and adsorption mechanism. In summary, the DMEA-grafted AEM was successfully synthesized. The FTIR spectroscopy demonstrated the synthesis of the DMEA-grafted AEM. The synthesized AEM exhibited a homogeneous structure and excellent thermal stability. From an aqueous solution, the percentage MO removed was increased with membrane dosage, contact time, and temperature, whereas it declined by increasing the initial concentration of MO. Similarly, the adsorption capacity was increased with contact time, initial concentration of dye, and temperature, whereas it decreased with membrane dosage. The kinetic study represented that the adsorption of MO onto the DMEA-grafted AEM fit using a pseudo-second-order model. Isotherms evaluation indicated that adsorption of MO onto the grafted AEM was fitted well to the nonlinear Freundlich isotherm model. The thermodynamic adsorption study showed that adsorption of MO onto the DMEA-grafted membrane was a spontaneous and endothermic process. Hence, the synthesized DMEA-grafted AEM could be employed as an extraordinary candidate for the removal of MO from aqueous solution at an ambient temperature.


While the interference effect between transport channels is manifested through our results, we also differentiate the contributions towards transmission from the bulk states and the edge states, by understanding the difference in the Green’s functions obtained from direct integration method and iterative method, separately.In this study we look into the interference effect in multi-thread molecular junctions in between carbon-nanotube (CNT) electrodes of assorted edges. From the tube end into the tube bulk of selected CNTs, we investigate surface Green’s function and layer-by-layer local density of states (LDOS), and find that both the cross-cut and the angled-cut armchair CNTs exhibit 3-layer-cycled LDOS oscillations. Moreover, the angled-cut armchair CNTs, which possess a zigzag rim at the cut, exhibit not only the oscillations, but also edge state component that decays into the tube bulk. In the case of cross-cut zigzag CNTs, the LDOS shows no sign of oscillations, but prominent singularity feature due to edge states. With these cut CNTs as leads, we study the single-polyene and two-polyene molecular junctions via both  Such a trend has been enhanced further as graphene was rediscovered in 2004, which brought even more attention toward all different materials based on the honeycomb carbon structure. Later as the theoretical research turned to look at the boundaries of these materials, such as edges of graphene, graphite or nano ribbons11, or finite-sized CNTs of different tips15, experiments kept up as well. Scanning tunneling microscopy studies20 on LDOS of different graphite edges have been performed, and the edge states of graphite are confirmed. However, similar experimental efforts on CNTs boundaries22 are even more challenging and yet to be solidly realized.Since the discovery of carbon nanotubes (CNTs) in 199123 using CNTs is an alternative and interesting choice, compared with the largely studied single-molecule junctions with metal-molecule linkages26. The CNTs, proposed to serve as the junction connecting the electrodes, or the electrodes themselves, or forming a heterostructured junction, give rise to peculiar properties in transport through these devices30.Due to the relatively well-defined lead-molecule covalent bonding and the finite transport channel involved, prospected nano-devices31, while in others the linkage morphology, or the question of whether the linkage is formed by multiple molecules32, becomes important and therefore requires further confirmation. As the idea of a single-molecule transistor is gradually realized in these systems, the properties of the CNT leads and the details of the molecule-CNT contact are both crucial for further pursuit. Interference effect35, among all the above, should accompany the formation of multi-molecule junctions in between CNT leads, and is an example that illustrates the interplay between the two roles.In recent years, molecular junctions employing CNT electrodes have been fabricated and measured by much more controlled and developed strategies. In some cases a single protein can be site-specifically attached to the leadsab initio calculations. With these tools we examine the surface Green’s function, whose projection onto contact sites helps to explain the transmission behavior of molecular junctions. We also look into the LDOS, the by-product of the surface Green’s function. For different cuts of CNTs we carry out the LDOS comparison that shows the dependence of edge state contribution on the type of cut. The transmission in double-threaded molecular junctions shows clearly the interference effect, namely, how the pick of contact sites, where the molecules attach to the CNT leads, affact the transport. This is an effect that has been studied for the cross-cut armchair CNTs, and now for the angled-cut armchair CNTs and cross-cut zigzag CNTs. Again, we realize that the interference is a consequence of superposition from the properly chosen even and odd channels.In this study, we aim at understanding the assorted cross-cut or angled-cut semi-infinite CNTs and the transport through molecular junctions bridging such leads. On the one hand we use a one-parameter  tight-binding (TB) model that describes the 36 to optimize the CNT bulk structure, with a force criterion of 0.02 eV/Å, an energy cutoff of 200 Ryd, the Double-Å × 20 Å × a is used, where Å, which allows enough vacuum to pass the bond length convergence test.We use the geometry relaxation process implemented in the SIESTA packageÅ × 20 Å × D Å, where In a similar way, the structures of the junctions are then relaxed. For each junction we use a rectangular supercell of 20 ab initio transport calculations, which are performed by the Nanodcal package37 with the density functional LDA_PZ81 and the DZP basis. Non-equilibrium Green’s function (NEGF) method38 is adopted in this ab initio process, where the leads’ self-consistent field (SCF) calculation uses the bulk CNT structure, and is followed by the SCF and transmission calculation for the open system of CNT-junction-CNT. Having done the transmission convergence test, we choose a 3-primitive-unit-cell buffer layer to sit at both sides of the scattering region, to form the linkages between the junction and the leads. Note that the Brillouin zone is sampled by a 1 × 1 × 200 Monkhorst-Pack grid, while a supercell of 30 Å × 30 Å × D Å and a corresponding real-space grid of 450 × 450 × 15 D are used to ensure enough vacuum and accurate transmission calculations performed by Nanodcal.The relaxed junction structures are then put into the Theoretical results shown in this article are derived from a single-parameter model, where a uniform ((i)The iterative method: All CNT leads considered here have the semi-infinite symmetry. Based on such a symmetry, and this symmetry alone, one gets the self-consistent formula:(ii)The integration method: Out of the electronic states of the infinite 2-D graphene, one finds the allowed 3. By doing so one obtains the r, r′) projection of the surface Green’s function isλ, λ′ and λ″ to label the eigen-states of the bulk tube. The real space position is labeled by layer k and −k, composing the complete orthonormal basis that satisfies the angled-cut boundary conditions. Obviously the boundary conditions of the angled-cut armchair CNTs’ rim are more complicated than those of the cross-cut tubes, in the sense that there are We calculate the surface Green’s function of the electrodes with the TB Hamiltonian described above, via two different paths:mentclass2pt{minimWith this integration method, the Green’s function thereby derived comes from the bulk’s Bloch states only. In other words, evanescent waves are excluded in the integration method, while the iteration method includes everything. Our inspection via the integration method is therefore valuable in the sense that it helps to separate the contributions from the edge states and the bulk states.The surface LDOS of selected CNTs are shown in Fig. EF. The peak of these EF states is wider in all cases of angled-cut Moreover, for cases of cross-cut Next we investigate the layer-by-layer LDOS, for energy at 17H19, the 17-carbon polyene molecule(s), and perform the ab initio calculations for transmission through all non-equivalent one-polyene junctions  channel is the even (odd) combination of the real-space two parallel polyenes. These two channels diagonalize the effective two-dimensional subspace characterizing the junction of two threads. In Fig. ab initio transmission data on top of the iterative method data. For all cases it appears that the TB polyene molecular level spacings are smaller than those in the ab initio version.Note that the hopping energy ab initio calculation for polyene C17H19 in vacuum, via the SIESTA package with the same standard we use for the junctions. The HOMO and LUMO of C17H19 suggest an intra-molecular hopping that is 1.3 times the CNT hopping. As the polyenes act as the bridge in the junction, the average molecular C-C bond length increases by only 0.5%, while the standard deviation shrinks to under 22% of the vacuum value. Although the increased average and the shrunken standard deviation of bond length should bring smaller molecular level spacings, all C-C bonds of the polyene in the junction are shorter than the bulk CNT’s two different bond lengths (1.412 Å and 1.415 Å). This means, the intra-molecular hopping is still larger than the CNT hopping, even in the junction form, which explains the disagreement between the TB and the ab initio results on energy spacings of the molecular level resonance.To see this, we do the We use the two cross-cut zigzag CNTs, ument}$$1,0$$\end{8H10, the 8-carbon polyene(s) as the junction, and investigate the one-polyene case and the two-polyene cases, as shown in Fig. With the detailed energy dependence of the surface Green’s function matrix elements readily calculated, we then consider CIn Fig. We present in this article a further study for the interference effect in multi-thread molecular junctions in between CNT leads of various cuts. To this purpose, we first show our calculations concerning the surface Green’s functions of cross-cut and angled-cut ab initio results with the iterative method results. In the two-thread cases, the agreement between both approaches displays the effect of interference between the even and the odd channels. The discrepancy between the two approaches mainly results from overlooking the difference between the intra-molecular hopping and intra-CNT hopping, and is explained by our observation on the molecular bond lengths.In the follow-up calculations for transmissions through molecular junctions in between angled-cut 8H10 junction in between cross-cut F.We also present a transmission study on one- and two-CAs the fabrication of CNT-junction-CNT systems is becoming more developed and controlled, theoretical understanding of electronic structures at CNT edges, contact and molecule properties, and the interplay between the previous two aspects, are practically needed. Our study reveals the importance of the interference effect, through the discussion of the LDOS of CNT edges, and the interplay between the molecule and the leads via the contact selections.


Antimicrobial resistance (AMR) has the potential to threaten tens of millions of lives and poses major global economic and development challenges. As the AMR threat grows, it is increasingly important to strengthen the scientific evidence base on AMR policy interventions, to learn from existing policies and programmes, and to integrate scientific evidence into the global AMR response.While rigorous evaluations of AMR policy interventions are the ideal, they are far from the current reality. To strengthen this evidence base, we describe a framework for planning, conducting and disseminating research on AMR policy interventions. The framework identifies challenges in AMR research, areas for enhanced coordination and cooperation with decision-makers, and best practices in the design of impact evaluations for AMR policies.This framework offers a path forward, enabling increased local and global cooperation, and overcoming common limitations in existing research on AMR policy interventions. Antimicrobial resistance (AMR) — the process by which microbes acquire resistance to antimicrobial medicines — is widely recognised as a serious threat to global public health. The likelihood of drug resistance increases when microbes are exposed to antimicrobials and, unlike previous generations, we can no longer count on the development of new drugs to overcome this threat –3. The dAMR is politically, economically and microbially difficult to tackle from a policy perspective. Efforts to evaluate AMR interventions would be significantly improved by increasing investments in monitoring and surveillance for antimicrobial resistance. Controlling AMR will require a suite of effective antimicrobial stewardship and conservation strategies to ensure the appropriate use of antimicrobials , in addiAs the threat posed by AMR grows, it is increasingly important to strengthen the scientific evidence base on AMR policy interventions, to learn from existing policies and programmes, and integrate scientific evidence into the global AMR response . The goaAMR researchers need to prioritise the study of ‘what’ works, ‘when’ it works, ‘why’ it works, and ‘what’ elements are necessary for its success.AMR needs better mechanisms for prioritising important research questions that can shape effective action. There has already been substantial research on the root social and microbial causes of AMR –33 and wMore formally, research prioritisation can be improved by undertaking structured prioritisation and consensus processes in collaboration with stakeholder groups, including policy-makers at different levels of government, civil society, health professionals and research funders. Research funders can support this work through opportunities for strategically funded research to address AMR rather than relying on researcher-led operating grants. For example, the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) recently funded an international workshop and formal consensus process to develop 10 research priorities for behavioural approaches to develop more impactful hospital antimicrobial stewardship programmes . The JamResearchers need to use rigorous systematic reviews to inform research prioritisation and to summarise the effectiveness of policy interventions.Rigorous systematic reviews and evidence gap maps can support the planning of policy interventions by ensuring that they are adequately supported by evidence. However, to be useful, these reviews must be high quality and regularly updated. Health Systems Evidence has appraised and catalogued more than 50 systematic reviews related to health systems and antimicrobial use dating back to the year 2000, and this database shows that the rigour and quality of these reviews is mixed .Conducting systematic reviews to summarise what we currently know is an essential input for research prioritisation. Reviews can collate empirical evidence to answer a specific research question  but theySystematic reviews can also ensure that research efforts are not wasted on questions that have already been definitively answered , 42. AltResearchers need to work with stakeholders to ensure that rigorous evaluations of all new AMR programmes are the norm.Researchers can actively advance progress on AMR by ensuring that evaluations of policies become the norm. While rigorous evaluations of all AMR interventions would be ideal, at present, we are very far from this reality . Policy Without a culture of evaluation, we risk implementing, maintaining and even spreading ineffective or inefficient AMR policies, at great financial and opportunity costs. Additionally, though policy-makers do not always see their added value, there are political advantages in conducting good impact evaluations; evaluation puts policy-makers in the politically attractive position of continuous policy improvement, enables them to ensure that research assessing their initiative is appropriate, and reduces political risk because they can acknowledge that they are operating with imperfect information . Where pAMR research needs to be planned to address policy-makers’ questions about effectiveness, implementation, costs and equity.Moving from evidence to policy inevitably involves consideration of pragmatic and ideological factors beyond evidence of effectiveness . ResearcOne simple and intuitive tool for enabling policy-maker engagement in research planning is to use the APEASE criteria  (Box 2).Researchers need to use theory, frameworks and logic models to design more coherent AMR policy interventions.Unfortunately, to date, the approach to designing AMR policy interventions has been ad hoc and seems to be guided by the ‘it seemed like a good idea at the time’ principle, rather than by an explicit process that considers the determinants of the problem, relevant theory and available empirical evidence , 21. ThiResearchers can improve the design of AMR policy interventions by employing and advocating for the inclusion of theory, frameworks and logic models in the early stages of intervention design to describe how and why an intervention is expected to work. These steps can substantially address the common tendency in AMR to re-invent the ‘square’ wheel rather than build on existing evidence from behavioural and implementation science. When planned without the use of theory, interventions are more likely to be unclear about the behaviours and outcomes targeted, and the means by which the intervention will achieve its intended effect . The proResearchers need to ensure that AMR policy interventions are evaluated using the most rigorous designs feasible in the given circumstances.When promoting a culture of AMR policy evaluation, improving and strengthening the design of evaluations should also be considered. One challenge in AMR research has been a lack of differentiation between programme evaluation  and research evaluation . While programme evaluation is important, policy-making needs to be guided by robust evidence generated using rigorous study designs. AMR policy intervention research has been plagued by poor quality and inappropriate evaluation methods. In a recent systematic review, 30 of 69 included studies used uncontrolled before–after designs and simple descriptive methods that cannot control for important design concerns, including bias, confounding and secular trends, and are generally uninterpretable , 53.Overall, interventions should be evaluated using the most rigorous study designs feasible in the given circumstances in order to minimise bias and maximise the generalisability of findings. The choice of evaluation design will be guided by numerous factors, such as whether it is feasible to randomise intervention sites, whether it is necessary to introduce the intervention at all sites simultaneously, the acceptability of having a no-intervention control group, and the number of available intervention sites Fig. .Fig. 2CThe focus on education, attitudes and behaviour change in AMR research, and the associated high likelihood of contamination, will likely preclude the use of randomised controlled trials (RCTs) with individuals  as the units of randomisation in much of AMR policy intervention research. In most cases, a cluster RCT should be considered the gold standard for AMR policy intervention evaluations . ClusterWhere random allocation is not feasible  interrupted time series (ITS) methods are the strongest study design for AMR research . HoweverUncontrolled before–after study designs, which historically have been common in AMR research, should be avoided if at all possible. The apparent effects of an intervention using these designs are completely confounded by secular trends and concurrent events. Even controlled before–after studies, which mitigate this threat of confounding to a limited extent, should be avoided unless there are at least two intervention sites and two control sites; however, given more sites, other more rigorous study designs may be feasible and preferable. Controlled before–after studies and non-randomised trials are an option when randomisation is not possible and there is an insufficient number of time intervals to conduct an ITS; however, these designs should only be considered as hypothesis generating. Figure AMR policy research needs to include head-to-head comparisons of different intervention variations and promote radical incrementalism to enhance the effectiveness of extant policies.Researchers can advocate for the use of more rigorous study designs and partner with policy-makers to ensure that evaluations are appropriately conducted. To move beyond effectiveness, research evaluations need to incorporate opportunities for addressing the other important AMR policy questions described earlier, namely why interventions work and what elements are necessary for their success. Once the initial effectiveness of an AMR policy intervention has been shown, there is an opportunity to conduct controlled head-to-head comparisons of different intervention iterations in the interest of optimisation, either through sequential trials comparing variations of the intervention, or using factorial designs to evaluate whether the addition of co-interventions meaningfully changes the intervention’s effectiveness. Evaluations can be facilitated by the development of implementation laboratories, which involve close collaboration between research teams and health systems delivering implementation strategies at scale . Trials Researchers need to define a set of core outcome measures for AMR research that address appropriate antimicrobial prescribing, development of antimicrobial resistance, and cost-effectiveness of policy interventions.AMR lacks both an agreed-upon metric for evaluating progress and a common system for measuring the scope of the problem. Systematic reviews of interventions to reduce antimicrobial use have shown that researchers use a wide range of prescribing and dispensing-focused metrics , 17. TheA harmonised set of measures for conducting and evaluating interventions serves three purposes. First, it creates consistency between evaluations that facilitates systematic reviews to inform evidence-informed policy-making. Lack of a shared outcome measure is a common challenge in public health systematic reviews and one which limits the amount of evidence that can be rigorously synthesised . Second,While there are substantial political and economic barriers to improving data collection and evaluation, from a metrics viewpoint, there are numerous successful initiatives that have adopted research governance principles to improve data collection and comparability of research studies, including the Core Outcome Measures in Effectiveness Trials (COMET) group  and ESSEResearchers need to register research protocols and evaluation trials and use reporting guidelines and checklists to improve reporting quality.An effective and coherent global response to AMR requires full and transparent reporting of all aspects of AMR intervention studies. Unfortunately, public health research as a whole, and AMR research specifically, faces challenges in this domain. Inaccessible research, partial reporting, and publication bias are all common in AMR research , 67 and Researchers should register protocols, primary evaluations of policy interventions (both RCTs and quasi-experiments) and systematic reviews in trial registers. Pre-registration of an evaluation helps address the tendency to avoid publishing the results of research with neutral or negative findings, which is common to much of scientific research, and may be particularly common in policy research where government partners may feel that they lose credibility if a policy intervention is found to be less effective than expected. The fields of clinical trials and systematic reviewing — both of which use protocol registration to reduce publication bias and to enable other researchers to see whether a research study on a particular topic is ongoing — have benefited from the support of research funders, journals and research ethics boards to promote these efforts to improve research reporting . Along wResearchers should also embrace the use of reporting guidelines and checklists to overcome common reporting challenges such as not describing the intervention specifically enough to allow replication , 50, usiResearchers need to embrace open data and open access opportunities to widely disseminate AMR research findings.The principle of shared learning is familiar to researchers and embedded in much of health research. A key question facing the field is how best to promote this ideal when working with stakeholders to develop effective and efficient AMR policy interventions. However, as many governments embrace nudge units, innovation hubs and radical incrementalism , there iAlthough the threat posed by AMR has been well-known for many decades, recent escalation in multidrug-resistant and extensively drug-resistant bacterial infections has elevated AMR to a more prominent position on the international political agenda. Substantial work is ongoing at the national level to address domestic AMR concerns , and theThese national and international efforts require substantially more evidence for the effectiveness and feasibility of AMR policy interventions than is currently available. However, this gap provides an opportunity for researchers to engage in a meaningful conversation about the importance of evidence-informed policy-making for AMR. Mitigating the threat posed by AMR will also require substantial collaboration among researchers and policy-makers. Table As we have highlighted, it is increasingly important to strengthen the scientific evidence base on AMR policy interventions, to learn from existing policies and programmes, and integrate scientific evidence into the global AMR response . This fr


Mycobacterium tuberculosis complex (MTBC) have been recorded in captive white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros. More recently, cases have been documented in free-ranging populations of both species in bovine tuberculosis (bTB) endemic areas of South Africa. There is limited information on risk factors and transmission patterns for MTBC infections in African rhinoceros, however, extrapolation from literature on MTBC infections in other species and multi-host systems provides a foundation for understanding TB epidemiology in rhinoceros species. Current diagnostic tests include blood-based immunoassays but distinguishing between subclinical and active infections remains challenging due to the lack of diagnostic techniques. In other species, demographic risk factors for MTBC infection include sex and age, where males and adults are generally at higher risk than females and younger individuals. Limited available historical information reflects similar age- and sex-associated patterns for TB in captive black and white rhinoceros, with more reports of MTBC-associated disease in black rhinoceros than in white rhinoceros. The degree of MTBC exposure in susceptible wildlife depends on their level of interaction, either directly with other infected individuals or indirectly through MTBC contaminated environments, which is dependent on the presence and abundance of infected reservoir hosts and the amount of MTBC shed in their excreta. Captive African rhinoceros have shown evidence of MTBC shedding, and although infection levels are low in free-ranging rhinoceros, there is a risk for intraspecies transmission. Free-ranging rhinoceros in bTB endemic areas may be exposed to MTBC from other infected host species, such as the African buffalo (Syncerus caffer) and greater kudu (Tragelaphus strepsiceros), through shared environmental niches, and resource co-utilization. This review describes current knowledge and information gaps regarding the epidemiology of TB in African rhinoceros.Cases of tuberculosis (TB) resulting from infection with  Mycobacterium tuberculosis complex (MTBC) (Mycobacterium bovis (M. bovis), which is known to infect livestock as well as captive and free-ranging wildlife species (Tuberculosis (TB) is a chronic infectious disease that affects a broad range of host species . It is cx (MTBC) . Bovine  species , 3. For M. tuberculosis and M. bovis have been recorded in captive, semi-captive (maintained in private reserves and more intensively managed), and free-ranging rhinoceros worldwide  and white (Ceratotherium simum) rhinoceros in South Africa. As of 2016, HiP housed ~1,500 white rhinoceros and 360 black rhinoceros (M. bovis–infected wildlife (over 20 species in KNP), including important bTB maintenance hosts such as African buffaloes (Syncerus caffer) (Tragelaphus strepsiceros)  and Hluhluwe–iMfolozi Park (HiP) are home to large populations of free-ranging black  –27. The siceros) , 19.M. bovis and M. tuberculosis have not been considered an immediate threat to the world's African rhinoceros populations, the potential impact of these pathogens on their health and conservation is largely unknown. Because bTB is a World Organization for Animal Health (OIE) and nationally notifiable disease  and MTBC antigen stimulated cytokine assays  , 55. Mycpositive . In humapositive , 58. Thipositive , as wellpositive . Regardlpositive , 60.M. bovis isolates may be useful not just for diagnosis, but also for tracing the origin of MTBC infection in multi-host systems (Trichosurus volpecula)  genotyping, or whole genome sequencing of  systems , 61–64.  systems , includilpecula) , badgerslpecula) , 66, deelpecula)  or Africlpecula)  and cattlpecula) –68. TherAs a result of the popularity of rhinoceros for zoological exhibition, they have historically been globally distributed through importation. Reports of TB in captive rhinoceros in zoological gardens worldwide date back to the late 1800's. Historical cases of bTB and TB in black and white rhinoceros are summarized in According to limited information, most TB cases have been recorded in black rhinoceros, with an apparent paucity of cases identified in white rhinoceros –19. ThisM. tuberculosis, with only a few caused by M. bovis. This is hypothesized to be a result of a high level of exposure of captive animals to M. tuberculosis through direct or indirect interactions with humans , while in captivity or during exportation  black rhinoceros, euthanized due to loss of condition, had small non-encapsulated pulmonary granulomas associated with nfection . Similar changes . Interesears old . In addiears old , 15, 18.There is limited literature that characterizes risk factors of MTBC exposure and transmission patterns in free-ranging black and white rhinoceros populations, likely a result of the logistical and technical difficulties associated with disease surveillance and sporadic cases in these animals. Therefore, extrapolation from literature on TB in other species and multi-host systems may aid in understanding the epidemiology of MTBC in African rhinoceros populations. Demographic risk factors for MTBC infection, such as sex and age, have been described in humans, mice, cattle, and limited species of wildlife , 73–82. In many TB-susceptible species, sex is considered a risk factor for infection , 73–78. Both testosterone and progesterone are immunosuppressive. These hormones impair macrophage activation and may increase TB susceptibility , 86. In Loxodonta africana) tested in KNP, overall TB seroprevalence was higher in males than in females (Meles meles) than in females (Odocoileus virginianus) in Michigan also reported a higher odds of being bTB test-positive in males compared to females  and African elephants (>25 years old) in bTB endemic regions also showed a higher seroprevalence than their younger counterparts  . Human sterparts , 49. Incterparts .While most studies in wildlife show increasing bTB prevalence with age, studies conducted in the Iberian Peninsula report higher prevalence in juvenile wild boar than adults in high-prevalence multi-host systems , 95. ThiT. volpecula) in New Zealand (Sus scrofa), red deer and fallow deer (Dama dama) in Spain (O. virginianus) in the USA (Cervus canadensis) (Bison bison) (M. meles) in the United Kingdom   and Amern bison)  in Canadsmission , 104–107uffaloes .M. bovis transmission to herbivores is largely unknown but has been attributed to indirect interactions through contamination of pastures, feed, or browse with MTBC shed by infected hosts. Various studies have demonstrated that infected hosts shed M. bovis into the environment  are implicated as bTB maintenance hosts where spillover into wildlife occurs , 111. Hoironment , 112. Thironment . Since t2  may be an important mode of bTB transmission to black rhinoceros. Buffaloes, greater kudus, black and white rhinoceros share water pans, which may also increase the frequency of interactions within and between these species , and with greater soil moisture content (~50%) in spring, compared to higher air and soil temperatures  and lower soil moisture content (~2%) in summer in this region (M. bovis (measured by PCR and culture) in contaminated environmental substrates, which varied between 4 weeks and 6 months, was shortened by exposure to high ambient temperatures, increased intensity of solar radiation, and higher loss of substrate moisture through evapotranspiration  can disseminate M. bovis from contaminated animal feces to the surrounding soil through casting egestion  with these species. Exposure to M. tuberculosis is less likely in free-ranging populations than in captive rhinoceros, as they have little to no interaction with humans, which are the most affected by this pathogen.While an hosts . This anan hosts . Free-raM. tuberculosis or M. bovis exposure. Disease surveillance and reporting biases also exist because not all institutions that house rhinoceros conduct post-mortem TB surveillance or have equal diagnostic capabilities, not all cases of TB in rhinoceros are recorded in the literature, and non-cases are often not reported . Bas. BasM. tM. bovis is expected to increase with age, due to more intense, and consistent exposure of adults in their smaller, more “settled” home ranges compared to that of the more nomadic young, and the accumulation of repeat exposures over their lifetime. The prevalence of M. bovis may also be higher in older animals, reflecting the chronic nature of disease in these long-lived species. The impact of increasing age on the overall TB risk of these populations is still unknown, and requires more investigation.In free-ranging populations of African rhinoceros in SA, incidence of M. bovis in this species; this is expected to occur as a result of their sharing of this habitat with the suspected bTB maintenance host, the greater kudu, as well as the longer persistence of M. bovis in shady (vs. irradiated) conditions. Conversely, the more frequent association of white rhinoceros with soil-associated bTB reservoirs and African buffaloes (a prominent bTB maintenance host) with whom they share their environment and food source, is likely to increase their M. bovis exposure. Additionally, because the white rhinoceros is considered a more social species than the black rhinoceros, it is hypothesized that M. bovis risk resulting from intra-species interactions will be comparatively higher in white rhinoceros. Overall, because of the higher population of buffaloes (the maintenance host with which white rhinoceros is expected to more frequently associate) than greater kudus (with which the black rhinoceros is expected to more frequently associate), as well as the expected occurrence of more intra-species interactions in white rhinoceros, it is hypothesized that there will be a higher risk M. bovis exposure in white rhinoceros populations than in black rhinoceros populations in KNP and HiP.The characteristic closer association of black rhinoceros with closed, shady environments than white rhinoceros may increase exposure to M. bovis infection risk. During hot or dry periods, the M. bovis exposure of rhinoceros (and other species) may increase due to increased aggregation of M. bovis-infected and susceptible hosts at water sources. During hotter periods, specifically, the aggregation of infected and susceptible hosts in shady areas and/or mud wallows may be associated with increased M. bovis exposure. Seasonal fluctuations in soil-associated bTB reservoirs such as free -living M. bovis in the soil, as well as earthworms and amoeba, may also be associated with seasonal changes in incidence M. bovis infection in African rhinoceros and other animals.In free-ranging African rhinoceros populations in South Africa, it is hypothesized that there is an association between seasonal fluctuations in environmental conditions, rhinoceros spatial patterns, and The most pressing concern related to TB in African rhinoceros is the acquisition of knowledge and the development of tools to inform surveillance and control strategies for the disease, as well as conservation plans.The development of sophisticated diagnostic tools may allow for early detection of infection; this would enable earlier interventions that could improve the prognosis of infected individuals and mitigate the spread of the infection in captive and free-ranging systems. Of particular consideration is logistical feasibility and fitness-for-purpose of a test. Capture and immobilization of rhinoceros, especially in free-ranging populations, is extremely costly and is a source of stress for the animal. Therefore, the use of a test like the TST, in addition to being unreliable in this species, would be ill-advised, as it involves immobilization for both administration and interpretation of the test on separate occasions, increasing the cost and the stress for the rhinoceros undergoing testing. Development of blood-based cytokine release assays for bTB in African rhinoceros is currently ongoing; these tests require a single capture and immobilization, and once validated, may be reliable, cost effective diagnostic methods for bTB in rhinoceros.M. bovis infection and disease progression in African rhinoceros. This would involve ante-mortem monitoring for MTBC infection, as well as thorough post-mortem exams that include histopathology and ancillary diagnostic tests. Because zoological facilities tend to keep curated medical records, retrospective, longitudinal data may already be available to address these knowledge gaps. Careful, standardized data curation across institutions could inform and enumerate a study population and be used to identify cases and non-cases. In any such study, attention to confounding factors such as differences in exposure to MTBC based on animal origin, movement history, and TB prevalence in human populations should be considered.Coordinated studies in captive populations may help to clarify demographic factors  as risks for In South Africa, population-based epidemiological studies of bTB in free-ranging African rhinoceros populations are currently ongoing. Findings from such studies could help identify major drivers of bTB infection in free-ranging populations as well as identify low risk individuals, which would have immediate benefit to current conservation and translocation efforts. This knowledge could be applied to inform management decisions for these populations, e.g., to minimize the probability of moving a false-negative infected animal out of bTB endemic areas and inadvertently spreading bTB to other areas. Such studies may also aid in identifying important bTB risk mitigation opportunities aimed to decrease continued spread of bTB in black and white rhinoceros living in these fragile ecosystems.M. bovis and M. tuberculosis) to African rhinoceros species. It has also drawn attention to major knowledge gaps pertaining to TB in rhinoceros. By identifying and systematically addressing each of these knowledge gaps, advances will inform management decisions for conservation of African rhinoceros, and South African biodiversity.This review has focused on available literature that could help to characterize the risk posed by MTBC (including RD, MM, WG, and CW reviewed available literature and wrote and edited the manuscript. PB edited the manuscript. RD and MM generated 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. The reviewer BB declared a past co-authorship with one of the authors PB to the handling editor.


Acyrthosiphon pisum has been used as a biological model for studies on phenotypic regulation for adaptation, and its alternative phenotypes are typically and physiologically based on maternal effects. We have observed an artificially induced and host-related maternal effect that may be a new aspect to consider in maternal regulation studies using A. pisum. Marked phenotypic changes in the cuticular melanization of daughter A. pisum were detected via tyrosine hydroxylase knockdown in the mothers during their period of host plants alternations. This phenotypic change was found to be both remarkable and repeatable. We performed several studies to understand its regulation and concluded that it may be controlled via the dopamine pathway. The downregulation and phenotypes observed were verified and described in detail. Additionally, based on histological and immunofluorescence analyses, the phenotypic changes caused by cuticular dysplasia were physiologically detected. Furthermore, we found that this abnormal development could not be reversed after birth. Transcriptome sequencing confirmed that this abnormal development represents a systemic developmental failure with numerous transcriptional changes, and chemical interventions suggested that transgenerational signals were not transferred through the nervous system. Our data show that transgenerational regulation  was responsible for the melanization failure. The developmental signals were received by the embryos from the mother aphids and were retained after birth. APTH RNAi disrupted the phenotypic determination process. We demonstrate that non-neuronal dopamine regulation plays a crucial role in the transgenerational phenotypic regulation of A. pisum. These results enhance our understanding of phenotyping via maternal regulation in aphids.Maternal phenotypic regulations between different generations of aphid species help aphids to adapt to environmental challenges. The pea aphid  Acyrthosiphon pisum (Harris) is utilized as a biological model of insect–plant interactions, phenotypic plasticity, and in symbiosis studies –wingless (apterae) and sexual–asexual morphs, are determined by the mothers  in the epidermal cells. Thereafter, L-DOPA decarboxylase (DDC) converts L-DOPA into dopamine. Acyldopamines, N-acetyldopamine (NADA) and N-β-alanyldopamine (NBAD), are synthesized from dopamine via the actions of the arylalkylamine-N-acetyltransferase (aaNAT) and NBAD synthase (ebony), respectively, and are transported to the extracellular tissues. Laccases  catalyze melanization (pigmentation) and sclerotization in the cuticles and other tissues (TH RNA interference (RNAi) treatments were shown to exhibit a pale body color , which can be summarized as follows. In the cuticle, TH converts tyrosine into  tissues . Numeroudy color . L-DOPA L-DOPA, which is functional upstream in animal melanization regulatory and nervous systems, is also found in plants, including the broad bean Vicia faba, the hosts of A. pisum. Pea aphids attack a variety of legume crops, including V. faba, white clover (Trifolium repens), and alfalfa (Medicago sativa)  . V. fabaf L-DOPA , which if L-DOPA  and alsof L-DOPA . Howevernization . The L-DA. pisum, under hosts alternations. The phenomenon has been observed in response to A. pisum TH (APTH) knockdowns in the nymphs of RNAi-treated mothers during the period of transition between host plants (with high L-DOPA to low L-DOPA content). In response to this treatment, perturbations of the cuticle melanization and nymph development were observed. RNAi is widely used in gene function studies . We also performed detailed analyses of the phenotypic and biological changes in the nymphs, to compare the induced phenotypic changes and normal maternal phenotypic regulations. Histological examinations, immunofluorescence detection, transcriptomic analyses, and chemical interventions were also performed to understand the regulatory mechanisms underlying these transgenerational phenotypic changes.To elucidate the mechanisms underlying the unexpected phenotypic changes in A. pisum were collected from M. sativa plants in Lanzhou, Gansu Province, China  and reared on V. faba in Shaanxi, China, for approximately 5 years. Prior to the experiments, the aphids were cultured in low densities on V. faba and T. repens under long-day conditions  for more than 30 generations at the Key Laboratory of Applied Entomology, Northwest A&F University, Yangling, Shaanxi, China.Red V. faba  during host plant alternations and RNAi from V. faba. However, given the impracticality of working with the much smaller M. sativa leaves, we selected T. repens (fit-size leaves for cells of 24-well plate) for the further experiments.Only wingless aphids were used in the experiments. They were replenished by rearing all the insects at densities in excess of 30 aphids/plant, for more than three generations. The aphids during the host transition period were used for the RNAi experiments. The newborn aphids  on the APTH was prepared for downregulation investigations, to modify the biosynthesis of L-DOPA. The dsRNA of the lymphotoxin-alpha gene  of Mus musculus was used as a control  are shown in The dsRNA of  control . The syn control  of the d control , and theT. repens for 72 h after treatment and subsequently collected for LC/MS analysis. Newborn nymphs (within 30 min after birth) were collected from mothers between 72 and 96 h after the dsRNA treatments and prepared for LC/MS analysis. This methodology is detailed in S1.2  and a SDPTOP-SZN71 microscope system .The following biological parameters of the treated mothers (ds-TH and ds-lta) and their daughters were analyzed: proportion of abnormal producers (mothers); life span, survival rate, and duration of development for each stage (nymphs). This is further detailed in S1.3 .Newborn (first instar) and new molted (second instar) nymphs were prepared for video recording  staining, and five samples of the cross-sections of the T2 segments of the third-instar nymphs from each treatment were selected for immunofluorescence assays. Transverse longitudinal sections, from the head to the tail, of the treated mother aphids were also collected 48 h after the dsRNA injection for immunofluorescence experiments. The method is detailed in S1.3  of software for comparison.Histological sectioning, staining (HE) and immunofluorescence were performed for abnormal  for transcriptome sequencing and preliminary analysis  and dopamine receptor antagonists  were injected into A. pisum to investigate the transition signals of the nervous system underlying this transgenerational phenomenon. The method is detailed in S1.4 . Values of the chemical amounts, body lengths, and developmental times were subjected to one-way analysis of variance. Differences among means were calculated using Duncan’s test at a significance level of P < 0.05. Quantitative analyses of the L-DOPA and dopamine and the proportions of the abnormal nymph producers were subjected to a Student’s t-test where P < 0.05 indicated significance. The survival data were analyzed using an χ2 test where P < 0.05 also indicated significance. All data were processed using SPSS v. 22 . Immunofluorescence images were obtained and analyzed using CaseViewer . Charts and diagrams were constructed with Microsoft® Excel 2016 MSO  and GraphPad Prism .A. pisum, a significant reduction in the expression of APTH-RNAi was observed in the head on the second day . In contrast, no significant transcriptional differences were detected in the abdomen  or the whole body . Additional experiments with more replicates revealed that the abdominal APTH expression was unstable with the ds-TH1 and ds-TH2 treatments .After the injection of dsRNAs  into APTH between the different developmental stages are shown in DDC under APTH-RNAi are shown in For more information and physiological background, the transcriptional differences in the expression of APTH in the head was relatively stable, and therefore, we analyzed the duration of APTH repression in the head. No significant reduction in APTH expression was detected in the heads of the dsRNA-treated aphids on the first day . The dsRNA treatment reduced APTH expression on days 2 and 3 . Thereafter, APTH expression was indistinguishable from that of the control individuals . Additionally, the ds-TH1 treatment was slightly more effective than the ds-TH2 treatment.The downregulation of L-DOPA content significantly decreased in aphids with APTH downregulation .Differences in immunofluorescence could also be observed in the embryos . All embAPTH knockdown did not differ in appearance. However, phenotypic alterations were observed in their daughters. The melanization levels in the heads, antennae, corniculi, and legs of the nymphs were substantially lower than those of their mothers . A reduction in melanization was also detected by monitoring the early tanning after nymph molting. Nymphs subjected to different treatments and molting simultaneously exhibited very different tanning rates. Control aphids tanned faster than those treated with ds-TH . In addition, abnormal nymphs developed by day 7 after the TH dsRNA injections (ds-TH1 and ds-TH2) showed that most of the abnormal aphids were low-tanning individuals. However, two control nymphs with abnormal tanning were also observed on days 3 and 6  did not significantly change over 3 days after the dsRNAs treatments .L-DOPA and dopamine levels between the nymphs laid by the mothers from the ds-TH and the ds-lta treatments  than those of the control nymphs in the first and second instars  and of the control nymphs (ds-lta). This phenomenon was observed at all three developmental stages examined  consisted of thinner layers  and healthy individuals. The cuticle protein genes (CPs) were either upregulated or downregulated in response to the ds-TH treatment . The tranchanged . Only a nchanged .t = −3.168, df = 4, P = 0.034; XM_029488103.1, t = −3.887, df = 4, P = 0.018; t = 0.002, df = 4, P = 0.999; NM_001163201.1, t = 1.354, df = 4, P = 0.247; XM_003247688.4, t = 11.416, df = 4, P = 0.201; NM_001326676.1, t = 5.681, df = 4, P = 0.809; XM_001944914.5, t = 0.807, df = 4, P = 0.465; Transcriptome sequencing.rar).Transcriptional verification by qRT-PCR of the selected candidate genes that exhibited relatively large exchange rate changes. The transcriptional results (qRT-PCR) showed different results from those of the transcriptomic data. Some results were similar, such as changes to XM_001949658.5 and XM_029488103.1 .To investigate whether dopamine-related pathways of the nervous system were involved in this transgenerational control system, we treated individual specimens with an inhibitor of dopamine biosynthesis or with receptor antagonists. Only individuals treated with the dopamine biosynthesis inhibitor (metirosine) gave birth to abnormal nymphs. We found that 7 of the 32 mothers were abnormal nymph producers, and phenotypic changes were detected in their daughters . All indA. pisum are maternally determined and can lead to phenotypic changes in their daughters. In this study, artificially induced (RNAi) and host-related (under host transition period) transgenerational phenotypic regulations in A. pisum were observed. These findings are particularly useful for furthering our understanding of the mechanisms underlying the phenotypic determination in aphids. We detected strong, predictable, repeatable, and consistent phenotypic changes in the daughter aphids of the treated individuals. Low and high levels of cuticular melanization and sclerotization were detected in the nymphs. The periods of host transition, from feeding on V. faba (high L-DOPA content) to the T. repens (low L-DOPA content), and simultaneous treatments with APTH (converts L-tyrosine to L-DOPA) dsRNAs were both essential for this phenomenon. We suggested that the maternal effects played an important role, and a transgenerational system based on dopamine/L-DOPA signals between the mother and embryos could exist. This transgenerational regulation may be related to the phenotypic plasticity of the pea aphid but requires further investigation to improve our understanding.Many of the phenotypic features of TH, the target gene silenced by RNAi in this study, is a key regulatory enzyme upstream of melanization pathway . This severe reduction in L-DOPA assimilation could promote a reorganization of the internal biochemical environment. The reduction in L-DOPA content may have triggered endogenous L-DOPA biosynthesis and maintained its downstream reactions. The dopamine pathway is moderately susceptible to imbalances, and even weak interference from exogenous dsRNA could disrupt the transmission of molecular signals and cause irregular and disordered expression feedback of APTH in A. pisum. pathway . IrregulTH confirmed that APTH was downregulated in the insect cuticle and explained the irregular transcriptional feedback in the abdomen. Distribution differences of TH were detected in the developing cuticle tissues of the embryos. These results are consistent with the findings from a study using microarrays and A. pisum . Further experiments with the nymphs, including transcriptional analysis, l effect . Winged l effect . These ds aphids .APTH-RNAi was an atypical maternal effect in aphids. The fact that densely stimulated  mothers can produce winged nymphs (irreversible phenotypic changes), whereas nutritional or photoperiod stimulations may induce mothers to generate sexual nymphs (irreversible phenotypic changes), reflect their adaptations to environmental changes  a certain proportion of the nymphs showed phenotypic changes ; (2) the changes .TH) upstream of melanization regulation. In contrast, several genes downstream from APTH, including laccase-like, yellow-like, and dopamine N-acetyltransferase-like  and fully functional enzymes (including TH). Therefore, abnormal melanization may account for the observed phenotypic changes. The presence of the TH protein negated the RNA intergenerational interference and the irregular distribution of the TH protein provided evidence for the collapse of the cuticle structure. A malfunctioning cuticle layer would not support melanization, regardless of whether the substrates and key enzymes were sufficient and functional, respectively.The malfunctioning cuticular layer observed with the transcriptional changes directly explained the physiological causes of these phenotypes. Cuticular dysplasia with microstructural collapse in pitation . MalformL-DOPA, the reaction product of TH and one of the key chemicals that differ among A. pisum individuals, plays an important role in trans-generational regulation. In the present study, we observed the phenotypic changes in response to the changing levels of L-DOPA intake  and L-DOPA self-synthesis modifications (APTH-RNAi). Our results suggest that L-DOPA and dopamine (the neurotransmitter derived from L-DOPA) might participate in the intergenerational signal transmission system. An L-DOPA/dopamine-based developmental signal is transmitted from the mothers to the embryos and determines the future developmental patterns of the nymphs. In this study, a disordered signal induced by APTH dsRNA was received by the embryos and caused pathological phenotypic changes thereafter.We hypothesized that L-DOPA intake considerably reduced), L-DOPA/dopamine reductions must be the key factor, but dopamine receptor-based neurological regulation is not involved in this transgenerational phenotypic regulation. We suggest the presence of another downstream pathway, which is regulated by dopamine/L-DOPA content, that acts as a transgenerational signal between the mothers and embryos. Further research is required to fully understand this non-neuronal dopamine regulation.The transgenerational signal might not be transmitted via the nervous system (dopamine and its receptors). Studies have shown that physical contact induces wing dimorphism in aphid offspring , and thiA. pisum. Variable RNAi efficiencies in aphids have previously been reported (We utilized RNAi and found that it could induce strong phenotypic changes in reported . This inA. pisum can be affected in certain conditions, based on the transcriptional modifications. Mother aphids determine the developmental patterns of their daughters, and the dopamine pathway regulation is likely to be involved in cuticular development in daughter aphids via a pathway that does not depend on dopamine receptor-based neurological regulation. L-DOPA, the key chemical in this process, is present in both plants and aphids and functions in the physiological regulations of this process. However, how mother aphids transmit signals to their embryos and how the embryos receive them remains poorly understood. Future studies should focus on determining the genes that may be involved in this process.The aphid phenotypes observed were indicative of the complexity and flexibility of the transgenerational regulations. The present study shows that maternal phenotypic regulation systems of All datasets generated for this study are included in the article/YZ and T-XL designed the research. X-XW performed the research. Z-JF, Z-SC, and Z-FZ provided the assistance. YZ and X-XW analyzed the data. and YZ, H-GT, X-XW, and T-XL wrote 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.


P = .022). Testing for additional actionable gene alterations such as TERT promoter mutations or RET and NTRK gene rearrangements further increased the diagnostic yield to 78%‐85% and up to 95% when including the ThyraMIR Thyroid miRNA Classifier. These data support the role of molecular cytopathology in surgical and therapeutic decision‐making and warrant additional studies.Risk assessment is critical to determine the timing of elective surgeries and preserve valuable resources in time of pandemic. This study was undertaken to better understand the potential value of molecular testing to risk‐stratify thyroid nodules with malignant cytology (Bethesda VI). Systematic review of the literature contributed 21 studies representing 2036 preoperative specimens. The BRAF p.V600E substitution was detected in 46% to 90% of cases with a pooled positivity rate of 70% . None of the studies used comprehensive oncogene panels. Retrospective analysis of 531 clinical specimens evaluated with the next‐generation sequencing ThyGeNEXT Thyroid Oncogene Panel identified a total of 436 gene alterations. BRAF mutation rate was 64% in specimens tested as part of standard clinical care and 75% in specimens from cross‐sectional research studies ( Studies limited to RNA biomarkers were not included in the analysis. Retrospective review of cases previously tested as part of standard clinical care in Interpace Biosciences' laboratory or as part of clinical research studies was performed exclusively on deidentified molecular data. The analyses did not involve any clinical data or protected health information that could be linked to individual subjects and did not constitute human subjects research as defined in 45 CFR 46.102. Pooled rates and I2 heterogeneity tests were calculated using fixed or random effect models and the Cochran Q statistic. Proportions were compared using Pearson chi‐square tests.The biomedical literature database from the National Center for Biotechnology Information (32 = 46%) for a fixed effect model and 70%  for a random effect model . The exact nucleotide variations are described in Supplemental Table Review of cases submitted for molecular testing as part of standard clinical care between April 2015 and June 2020 yielded 381 unique Bethesda VI specimens with molecular data . The majority of positive cases harbored a BRAF p.V600E substitution in both cohorts , 64% for CLIA specimens and 75% for research specimens (P = .022). The second most frequent gene alteration was TERT promoter mutations (26/241 or 11%), highlighting the limitation of molecular strategies that only interrogate exome variants. For example, the Afirma Xpression Atlas, based on transcriptome RNA sequencing, has a reported false negative rate of 26% relative to targeted DNA sequencing and cannot assess variants outside the fraction of exonic sequences that are actually transcribed into mRNA.In our clinical experience analysis, a total of 436 gene alterations were identified in 414 of 531 preoperative specimens with malignant cytology. There were significant differences between the two cohorts and their respective collection schemes. The clinical research cohort was a representative cross‐sectional sampling of thyroid nodules at the time of FNAB diagnosis while the CLIA cohort consisted of more challenging cases that required molecular testing as part of their routine clinical management. Predictably, the positivity rate was lower among CLIA specimens relative to research specimens . The use of different molecular methods, reagents and protocols probably contributed to the broad range of reported rates (46% to 90%). Methods with poor analytical sensitivity, for example, Sanger sequencing, or with clinical cutoffs set at high percent variant, are expected to have a lower detection rate. In one of the reviewed studies, Lee et alThe high frequency of oncogenic BRAF mutations in preoperative thyroid FNAB has important clinical implications. Multiple studies have shown that BRAF p.V600E correlates with aggressive features of thyroid carcinoma such as extrathyroidal extensions, vascular invasion, larger nodule size, advanced staging, lymph node metastasis and recurrence.In summary, our systematic review of the literature indicates that 70% to 75% of FNAB with malignant/Bethesda VI cytology are expected to be positive for the oncogenic BRAF p.V600E substitution. The majority of these studies (71%) assessed only BRAF mutational status. Our analysis of 531 representative clinical specimens is the first to report the potential value of a comprehensive oncogene panel combined with a miRNA expression classifier. Additional work is required to fully assess the role of molecular testing for the preoperative risk stratification of malignant FNAB, clinical decision‐making, timing of surgery and optimal utilization of valuable healthcare resources.E.L. is a consultant for Interpace Biosciences Inc.Table S1 Distribution of DNA mutations in n = 270 CLIA specimens positive with the ThyGeNEXT Thyroid Oncogene Panel.Click here for additional data file.