In this work, the interactions between hyaluronic acid and bovine serum albumin were investigated. The film-forming properties of the mixture were proven, and the mechanical and surface properties of the films were measured. The results showed the interactions between hyaluronic acid and albumin, mainly by hydrogen bonds. Molecular docking was used for the visualization of the interactions. The films obtained from the mixture of hyaluronic acid possessed different properties to films obtained from the single component. The addition of bovine serum albumin to hyaluronic acid led to a decrease in the mechanical properties, and to an increase in the surface roughness of the film. The new materials that have been obtained by blending can form a new group of materials for biomedicine and cosmetology. Synthetic and natural polymers are composed of repeating fragments called mers . SynthetThe properties of HA can be modified by blending with other polymers and/or biopolymers. In recent years, blends of hyaluronic acid with other polymers have been studied for new material development . New matBiopolymer blend preparation by dissolution in the same solvent allows for avoiding protein denaturation. The specific interaction between the blend components is often called miscibility. The most common interactions in the blends are hydrogen bonding, ionic and dipole, and charge-transfer complexes. The interactions between HA and BSA can determine the properties of the blend. Moreover, the interactions between hyaluronic acid and albumin can be important from a biomedical point of view because the mixture of hyaluronic acid and albumin can mimic the synovial fluid, and may influence the tribological properties of cartilage ,17,18. HIn this work, polymer thin films composed of pure hyaluronic acid of three different molecular weights and their mixtures with BSA have been researched. The interactions between two macromolecules have been studied experimentally and molecular docking has been used for visualization of these interactions. The mechanical and physicochemical properties of the obtained polymeric films were investigated and compared. To the best of our knowledge, the interactions between HA with different molecular weight and BSA have not been studied yet.IR spectra were recorded to determine the interaction between hyaluronic acid and albumin. Individual spectra of pure compounds and mixtures are presented in the figures below . The posThe IR spectra for the three hyaluronic acids are almost identical, regardless of their molecular weight. Minor differences in the wavenumber may be caused by a slight change in conditions when performing spectrometric measurements. The most remarkable difference can be seen between the high- and low-molecular-weight HA spectra and the ultralow-molecular HA spectrum. This may be due to the significant difference in molecular weight and, therefore, the peaks in the ultralow-molecular HA spectrum are much smaller. The obtained IR spectra confirm the presence of amide, hydroxyl, and carboxyl groups in the tested compounds.−1 and 1541 cm−1. In the IR spectra of proteins, there are Amide A, Amide I, and Amide II bands. The Amide A band in BSA is located at 3290 cm−1, whereas Amide I and II are located at 1650 cm−1 and 1541 cm−1, respectively.There are characteristic N-H and C-N bonds in albumin, the presence of which was confirmed in the IR spectrum. The wavenumbers corresponding to these bonds are, respectively, 1650 cm−1. There was a decrease in the peaks corresponding to O-H and C-H bonds and an increase in the COC binding peak. The spectra of HA–bovine albumin mixtures show a peak corresponding to COH binding, which is not present in albumin. Moreover, a shift in the band in the region of OH/Amide A after the mixing of the two biopolymers has been observed. There is also a difference in the position of the bands at 1407 cm−1 in HA; after the addition of BSA, these bands have been shifted to lower wavenumbers. The abovementioned changes in the IR spectra indicate an interaction between albumin and hyaluronic acid. The interaction occurs mainly via hydrogen bonds.There are no significant differences between the spectra of HA–bovine serum albumin mixtures. One can see the difference between the spectra of pure albumin and the mixture with hyaluronic acid. The spectrum of albumin does not show an intense peak corresponding to approximately 590 cmThe mechanical properties of membranes made of pure hyaluronic acid of three different molecular weights and membranes made by mixing hyaluronic acid with bovine serum albumin have been measured. The obtained results are presented in the tables below . No filmmod) was obtained for the high-molecular-weight HA. The highest value of elongation at break was obtained for the low-molecular-weight HA.The mechanical properties of the obtained polymeric films vary depending on the molecular weight of the hyaluronic acid. The highest value of Young’s modulus  was purchased from Merck . The molecular weight of HA was proven by viscometry techniques and was as follows:Hyaluronic acid (HA) of high molecular weight, HA of low molecular weight, and HA of ultralow molecular weight were purchased from the cosmetic company zrobsobiekrem.pl . ProbuminSolutions of pure biopolymers were prepared by dissolution in water with the following concentrations: BSA with 1% high-molecular-weight HA, with 1.5% low-molecular-weight HA, and with 1.5% ultralow-molecular-weight HA.Each of the three hyaluronic acid solutions was mixed with a 1% BSA solution. To do this, 15 g of albumin solution was added to 15 g of HA solution, followed by stirring for 15 min on a magnetic stirrer. Then, 25 g of the obtained mixtures and pure solutions of the two types of HA were poured onto plastic plates (dimension 10 cm × 10 cm). It took a week for the solvent to evaporate and form the polymer film.−1 with 64 scans and scored in the range of 400–4000 cm−1. The spectra were processed using Omnic Spectra 2009.Infrared spectra were recorded on a Nicolet iS10 FTIR spectrophotometer equipped with a diamond crystal ATR device . The IR spectra were registered for membranes obtained from hyaluronic acid and its mixture with albumin. Spectra were recorded with a resolution of 4 cmFrom the obtained polymer films, shapes with the following dimensions were cut: width 1 cm and length 5 cm. Mechanical properties such as Young’s modulus [GPa], tensile strength [MPa], and elongation at break [%] were tested on a Zwick & Roell Z.0.5 testing machine. . Seven to 10 trials were performed for each film. The initial parameters of the test program were as follows: preload 0.1 MPa, test speed 50 mm/min, preload speed 5 mm/min. Data were collected in testXpert II 2017. Statistical analysis was performed using the Q-Dixon test in MS Excel.The surface structure of the polymer films was analyzed based on atomic force microscope images obtained with the MultiMode Scanning Probe Microscope Nanoscale IIIa  operating in trigger mode, in an air atmosphere at room temperature. The roughness parameters were calculated from the 5 µm × 5 µm scan area using the Nanoscope software .To obtain molecular details on HA deposition at BSA (PDB code: 3 v 03), we performed molecular docking of BSA–HA complexes using the VINA method , with deHyaluronic acid can be mixed efficiently with BSA, but the film-forming properties depend on the molecular weight of HA. The interactions between the components of the blend are mainly achieved by hydrogen bonds. The molecular docking experiment shows the most likely binding sites, as well as the fact that hydrogen bonds form mainly with charged amino acids and hydroxyl groups in HA. The addition of bovine albumin causes a deterioration in the mechanical properties of hyaluronic acid. The surface roughness is greater in the blends than in hyaluronic acid films. The obtained biomaterials can be used in the medical and cosmetic industries as film-forming substances and biomimetic adhesives. Moreover, the HA–BSA blends can find potential applications in tissue engineering and regeneration, as injectable hydrogels and as components of cosmetic formulations. The conventional clinical protocol with HA can be modified by the proper addition of BSA.


Background: The Hajj is an annual religious mass gathering event held in Makkah, Saudi Arabia. With millions of participants from across the globe attending the Hajj, the risk of importation, transmission, and global spread of infectious diseases is high. The emergence of antimicrobial resistant (AMR) bacteria is of worldwide concern and the Hajj poses a serious risk to its dissemination. This review aims to synthesize published literature on AMR bacteria acquisition and transmission associated with the Hajj. Methods: We searched electronic databases to identify literature published between January 1990 and December 2021. The search strategy included medical subject headings and keyword terms related to AMR bacteria and the Hajj. Results: After screening 2214 search results, 51 studies were included in the analysis. The review found 6455 AMR bacteria transmissions related to the Hajj. Thirty predominantly enteric or respiratory disease-causing AMR bacterial species were reported with isolates identified in cases on five continents. Most were male, aged above 50 years and were diagnosed in Makkah. Most cases were identified through hospital-based research; few cases were detected in community or primary health care settings. Conclusions: This review provides a contemporary account of knowledge related to AMR transmission at the Hajj. It emphasizes the need for the enhancement of surveillance for AMR bacteria globally. Antimicrobial resistance has emerged as a global threat to public health and health systems . AntimicMycobacterium tuberculosis (TB), where around 29% of deaths among TB patients have been attributable to AMR TB ) were identified in Makkah, the city in which the Hajj takes place and from where most pilgrims come ) reported in nine studies [Acinetobacter spp. (73.89% [95% CI: 69.92%–77.85%]) were isolated within hospital settings [Pseudomonas aeruginosa were a total of 466 out of 984 (47.36% [95% CI: 44.24%–50.48%]) Pseudomonas aeruginosa isolates. All were reported within six studies, and all were within hospital settings ) within their studies  of the tested enteric infection-causing bacteria were resistant to beta-lactams. This was similar to what other studies have found. For instance, Santos et al. (2020) found that phenotypically 74% and genetically 94% of tested enteric samples were resistant to at least one beta-lactam antibiotic . ResistaOur findings show that the most common (68.58% [95% CI: 66.87%–70.29%]) AMR expressed in respiratory infection-causing bacteria was beta-lactams and that this rate was significantly higher among cases detected in Makkah. The higher detected rate of beta-lactam resistance in Makkah compared to outside Makkah could be due to the high rate of respiratory infections among the Hajj pilgrims  or simplAcinetobacter spp. with about 88% of isolates collected resistant to at least one beta-lactam antibiotic. This is similar to a similar study’s findings  of sampled bacteria collected in hospital settings in Makkah were resistant to at least one antibiotic; this is in line with the range reported elsewhere (27% to 82%) . Moreovefindings . Infectiveloping . TherefoSixty-five percent of the studies included in our review reported AMR detection through hospital-based research. Both routine and incidental hospital and community-based surveillance are crucial to assess, predict, and follow up on the development and transmission of AMR bacteria within the Hajj . MoreoveThe prevalence of AMR transmission at the Hajj should be emphasised on prevention, early detection, and rapid response. Measures that should be considered include the development of policies that require mandatory vaccination  and prophylaxis before coming to Hajj; strategies to strengthen hospitals’ infection controls; the development of Hajj-specific guidelines for AMR prevention, detection and control; enhancement of hospital- and community-based surveillance for AMR; streamlining testing processes; establishment of governance arrangements that support the adoption of novel (or improved) methods of tackling AMR risk; and investing in social, clinical, and operational research to better understand transmission dynamics.This study has several limitations. First, most of the included studies provided limited information about participants’ demographics. Demographic data (such as age and gender) are important, as some diseases have high occurrence rates associated with specific ages. Second, we relied on the information provided in the reviewed articles and could not interrogate or validate their findings. Third, some studies were excluded as they were not available in English, which may have resulted in relevant information being missed. Forth, most studies were based on data collected as part of the research conducted in Makkah and hence, AMR detection associated with Makkah is likely overrepresented. Finally, presence of bias was not universally discussed in the manuscripts review. Existence of bias in the literature reviewed may impact the validity and generalizability of our results.This scoping review provides a point-in-time collation of available scientific information related to AMR transmission associated with the Hajj. It provides an accessible resource for researchers and practitioners seeking insights into AMR-associated incidence and burden and identifies knowledge gaps that may be filled. Finally, the work offers generalizable advice that those responsible for the management of AMR at mass gatherings may refer to inform their efforts.


The context of the COVID-19 pandemic has brought to light the infodemic phenomenon and the problem of misinformation. Agencies involved in managing COVID-19 immunization programs are also looking for ways to combat this problem, demanding analytical tools specialized in identifying patterns of misinformation and understanding how they have evolved in time and space to demonstrate their effects on public trust. The aim of this article is to present the results of a study applying topic analysis in space and time with respect to public opinion on the Brazilian COVID-19 immunization program. The analytical process involves applying topic discovery to tweets with geoinformation extracted from the COVID-19 vaccination theme. After extracting the topics, they were submitted to manual annotation, whereby the polarity labels pro, anti, and neutral were applied based on the support and trust in the COVID-19 vaccination. A space and time analysis was carried out using the topic and polarity distributions, making it possible to understand moments during which the most significant quantities of posts occurred and the cities that generated the most tweets. The analytical process describes a framework capable of meeting the needs of agencies for tools, providing indications of how misinformation has evolved and where its dissemination focuses, in addition to defining the granularity of this information according to what managers define as adequate. The following research outcomes can be highlighted. (1) We identified a specific date containing a peak that stands out among the other dates, indicating an event that mobilized public opinion about COVID-19 vaccination. (2) We extracted 23 topics, enabling the manual polarity annotation of each topic and an understanding of which polarities were associated with tweets. (3) Based on the association between polarities, topics, and tweets, it was possible to identify the Brazilian cities that produced the majority of tweets for each polarity and the amount distribution of tweets relative to cities populations. The pandemic has significantly impacted people’s daily lives worldwide, affecting work, education, and social interactions and leading to the enforcement of distancing measures worldwide ,2,3. PhyThe opinions and sentiments expressed by people about aspects of the pandemic have triggered worldwide research seeking to analyze the associated patterns and generate informational inputs to support actions in favor of combating the social effects of COVID-19 ,12,13,14In this context, Cotfas et al.  analyzedAbdulaziz et al.  also focMelton et al.  applied Shi et al.  developeIn a study with a medical infoveillance orientation, Mackey et al.  applied Daradkeh  presentek-means clustering algorithm, obtaining as results keywords related to the public information about vaccination, a co-keywords network, and an “information needs framework” based on the results of both methods, elucidating, for instance, the information needs of various social groups.Wang et al.  analyzedZhu et al.  developeXie et al.  reviewedAnother interesting study related to the pandemic was developed by Zhou et al.  in ChinaThe vaccination campaign against COVID-19 in Brazil was marked by the division of public opinion, with discussions and confrontations between pro- and anti-vaccination groups on social networks and the notable dissemination of misinformation about vaccines ,25. PoliThe dissemination of misinformation about COVID-19 vaccination in Brazil and the world brought to light the need for immunization program management agencies to properly analyze the infodemic phenomenon . It inclSome recent studies have indicated paths to be followed to address this need. We highlight those that are most associated with the research we carry out: applying methods to discover patterns in comments and opinions indicating the commenter’s behavior ; systemaThe present article reports a new study expanding the findings of de Carvalho et al. , who appThis study attempts to address the needs identified above by applying a methodological approach that public agencies managing the COVID-19 immunization program can use to combat the spread of misinformation, considering natural language processing tools, text mining, and machine learning to provide structured and helpful information.(1)What topics were discussed during 2020 and 2021 on COVID-19 immunization in Brazil?(2)What can be understood/interpreted about the extracted topics?(3)How did topics evolve over time?(4)How are they distributed in Brazilian territory, and which places have generated the most tweets?Four research questions can be defined regarding our research problem:polarity → topic → tweet, it was possible to identify the Brazilian cities that produced the most tweets according to each polarity, and the distribution of the number of tweets according to the cities’ population.Among the research findings, we highlight the most interesting: (1) the highest peak in the number of tweets for most of the topics and all polarities on 17 January 2021, the date of the first COVID-19 vaccine applied in Brazil; (2) twenty-three topics were extracted and, after manual annotation, fifteen of them received the pro-vaccination label, five the neutral label, and three the anti-vaccination label, indicating a predominance of topics supporting vaccination in Brazil within the period studied, according to the annotators’ judgments; (3) through the association (1)Texts collection from Twitter(2)Texts preprocessing(3)Feature selection(4)Topic modeling and discovery(5)Spatio-temporal topic analysisThe methodological approach applied in our research was based on a social web mining framework  with theThe texts were collected using Twitter API 2.0 through a scraping script developed in Python, applying functions from the “requests” library. Larger corpora were constructed, as reported in de Carvalho et al. , but a s(1)text field)Tweet text ((2)place_name field)Place name ((3)country field)Country name ((4)point field)Geographic coordinates ((5)created_at field)Creation date  file with the following fields:latitude and longitude, and it was later decomposed into specific vectors for each part of the coordinate. The dates were transformed into a readable format by the temporal analysis scripts.The geographic coordinates field consists of A set of preprocessing functions was applied to minimize textual noise effects: case folding; duplicities exclusion; tasks for eliminating punctuation, emojis/emoticons, numbers, stop words, links/URLs; and tokenization. The preprocessing was developed through another Python script using mainly the Spacy library , which iAfter preprocessing, the texts were vectorized using simple term counting (defining the frequency of the term) and the term frequency-inverse document frequency (TF-IDF). Both vectorization processes were applied using the respective functions available through the Scikit-Learn framework .Each vectorization method received the lemmatized tokens extracted from the tweets. So, for the subsequent topic modeling and analysis phases, the words (treated as uni-grams) were the selected features.Topic modeling methods consist of converting numeric (document × term) vectors representing documents to (topic × term) and (document × topic) vectors . The topN denotes a sequence of words w = , θ is a k-dimensional Dirichlet random variable that can assume values in the (k—1)-simplex vector. In our study, we used Scikit-Learn’s LDA implementation considering the online variational Bayes method [Algorithm 1 Latent Dirichlet Allocation Generative Process1.w in a corpus D:For each document 1.1Choose1.2Choose 1.3N words nw:For each of the 1.3.1Choose a topic 1.3.2nw from nzChoose a word The model was proposed and described by Blei et al.  and can s method , using mA hyperparameter tuning using Scikit-Learn’s Grid Search function was performed, considering learning decays of 0.5, 0.7, and 0.9 for between 3 and 30 topics. The learning decays are parameters that control the learning rate when using the LDA online variational Bayes method and can vary from 0.3 to 1.0.The Grid Search can deliver test scores such as the model perplexity and the log-likelihood  intertopic distance mapping, (2) the most salient terms evidencing, and (3) topic segregation. For the intertopic distance mapping and the most salient terms, we used a Python library called pyLDAvis ,46, autoIn the intertopic distance map, the extracted topics are represented by circles, and their area corresponds to each topic’s term prevalence. The center of each topic is defined by calculating the distance between the topics, using a multidimensional scaling process to represent the distances on a two-dimensional plane . The prow is for determining the generating topic T, versus a randomly-selected term w’. Equation (3) describes how to calculate the salience measure, considering that given the number of words, the list of the most probable terms contains more generic words than the list of specific terms [According to Chuang et al. , Equatioic terms .t-distributed stochastic neighbor embedding (t-SNE) implemented in Scikit-Learn and the Bokeh library [t-SNE was designed to alleviate the crowding problem in the traditional stochastic neighbor embedding (SNE) using a symmetric version of the SNE cost function [ijp described by Equations (4) and (5) are defined to measure similarities between objects ix and jx, symmetrizing two conditional probabilities, where iσ represents the bandwidth of Gaussian kernels defined so that the perplexity of the conditional distribution iP is equal to a predefined perplexity u [For topic segregation, we used the  library  for plotfunction . Joint plexity u ,51.(4)p(1)(1)Supportive topic (pro), with positive relation, denoting trust about the COVID-19 vaccination.(2)Negationist or unsupportive topic (anti), with negative relation, denoting mistrust about vaccination.(3)Neutral topic , not associated with the types above.Manual topic polarity labeling, dedicated to annotating the polarity trend associated with each topic into three types:(2)Geographical Topic Analysis is dedicated to visualizing and understanding the topics’ distribution in a geographic area. There are two possible ways to perform this analysis : (1) dis(3)Time Topic Analysis is dedicated to visualizing and understanding the topics’ distribution over time. One example of this application can be seen in the study by Martin et al. .The final part of the methodologic workflow is the space and time topic analysis. Using the results of the previous phase, we can perform the following tasks:In our research, the manual topic polarity annotation was performed by an initial group of three scholars, from different research areas, with different backgrounds: Informatics, Economics, and Life/Health Sciences. This manual labeling becomes necessary to ensure an alignment based on human knowledge and the consequent reliability of the topics concerning their positioning on the investigated theme. The use of three annotators is a way to ensure that, if there is a disagreement between the labels of two of them, the labeling of a third one can serve as a basis to define which polarity label will be defined . If therThe Brazilian geographic space is considered for the analysis performed in this study, and the geographical topic analysis is aligned with the idea of discovering topics of interest across the Brazilian geographic regions. Using data collected from June 2020 to October 2021, we could perform the time topic analysis, demonstrating the evolution of the discussions. Graphs were developed using the Python Matplotlib library , and theThe results will be presented following, divided into (1) the general findings, considering initial data about the corpus constructed for the spatio-temporal analysis; (2) the topic modeling results according to the Grid Search applied; and (3) topic analysis findings, considering the manual labeling, and the spatio-temporal analysis.Considering the geocoded tweets extracted from one of the corpora presented by de Carvalho et al. , the iniThe number of tweets started to increase in mid-December 2020; in addition, there was a peak with 1224 tweets on 17 January 2021, identified by analyzing the collected data. Both situations were also noted in the study by de Carvalho et al. . Figure There are twenty-five cities with at least 300 tweets registered in the corpus; among them, twenty cities are state capitals, including Brasília , and the others are major urban centers in their respective states. (1)For the words vector obtained with the count method: a topic model with only three components (topics), using a learning decay equals 0.7. This result is corroborated by the best log-likelihood of −840,183.6751 and perplexity of 1479.7684.(2)For the words vector obtained with TF-IDF method, a topic model with only three components, and in this case, using a leaning decay equals 0.5. This result is corroborated by the log-likelihood of −281,057.5610 and perplexity of 4225.0856.The topic modeling process considered two word-vectors, one obtained using the count vectorizer and the other the TF-IDF vectorizer. The main results for the Grid Searches using these vectors were:However, the results of this test require a sensitivity analysis that can be applied using the learning decay’s performance graphs. This analysis can help define a more significant number of topics within the determined limits. Although the increase in the number of topics for each learning decay may cause a decrease in the log probability  regardless of the applied vectorization, there will be a better margin of topics for analysis based on the diversification of the sub-themes involved. The graph grid in Graphs A1 and B1 in Learning decay curves for the words vector obtained using the count vectorizer presented the definitive overcoming of the learning decay curve of 0.7 between 22 and 22.5 for the number of topics. The curve initially containing the best result was surpassed by the learning decay curve of 0.9, as presented in Graph A2 . So, forFor the learning decay curves related to the words’ vectors obtained using the TF-IDF vectorizer, there were several points where the curves of 0.5 and 0.7 crossed each other. For instance, the first time was between 3.10 and 3.15 topics, where the learning curve of 0.5  was overcome by the curve of 0.7. However, as recorded in Graph B2 , the leaBased on this sensitivity analysis, we opted to use a model with 23 topics according to the words’ vector obtained through the count method.The initial output of the topic modeling and extraction is the overall topic structure. There are several forms to present this structure. vaccineyes → vaccine yesvaccineforeverybody → vaccination for everybodyvaccinenow → vaccine nowoutbolsonaro → out Bolsonaro (the family name of the Brazilian president at the time this study was carried out)Sistema Unico de Saúde, the Brazilian Public Health System)hailthesus → hail the SUS  presented label ties as each of the three annotators applied a different label. A fourth annotator (an Engineering researcher) was invited to support resolving the indecision in the two topics. The results of the polarity label annotation are shown in After resolving the ties, the labels’ distribution defined fifteen pro-vaccination topics, five neutral, and only three anti-vaccination. These labels enable the visualization of opinion distributions associated with the topics in space and time, helping to complete the analysis of the support and trust orientation regarding the COVID-19 immunization program in Brazil. After applying these labels to the corpus, the tweets’ counts were: 41,868 pro-vaccination, 9725 neutral, and 4165 anti-vaccination.The topic extraction process allowed the determination of the timelines for each of the 23 topics according to the number of tweets posted over the 17 months. As we obtained the topic → tweet association, it was possible to compute the totals of tweets collected according to each day for each topic.x-axis, the dates, and the y-axis, the tweets count). Each topic showed different peaks in the number of tweets on specific dates, with the highest peak in Topic 3 between January and February 2021, specifically on January 17, which is in line with the peak in the overall timeline of tweets . The map in Topic 13, about “Vaccine saving lives concerning COVID-19” also with pro-vaccination polarity, has the second most significant number of tweets (n = 4815). From the topic polarity annotation, in addition to analyzing the evolution of these polarities over 17 months, it is possible to visualize the geographic distribution of tweets according to the labels applied. The grid in It was also possible to extract the ten cities with the highest number of tweets on each map according to polarities. The data in The percentual distribution scores represent interesting information to understand the opinions’ penetration according to each city’s population size. In cities with large populations, such as São Paulo and Rio de Janeiro, which also had some of the highest numbers of tweets, it is noted lower percentual distributions of tweets, a fact that may indicate that the extension of opinion effects is smaller than in cities with smaller populations and large amounts of tweets.For example, looking at the pro-vaccination polarity, the city with the smallest population, São Luís , had a higher percentual distribution than São Paulo and Rio de Janeiro, indicating that the opinions may have had better penetration. Regarding opinions supporting vaccination, it is potentially desirable for the agencies managing the programs.On the other hand, Porto Alegre, the city with the second highest population in the southern Brazilian region and the highest number of anti-vaccination tweets, had the third highest percentual distribution, demonstrating a greater tendency for penetration of negationist opinions. Still within the anti-vaccination polarity, the city of Vila Velha, despite having the smallest population among the ten cities listed, has the highest percentual distribution score, indicating that there may be a potential domain of negationist opinions shared by Twitter users.Considering the ten cities with the highest number of tweets for each polarity, we can see that all cities are state capitals for the pro-vaccination polarity. In the neutral polarity, there is a predominance of capitals, except for two cities: Campinas and Uberlândia. Campinas has the third-largest population in the state of São Paulo, and Uberlândia has the second-largest population in the state of Minas Gerais. Finally, for the anti-vaccination polarity, there is also a predominance of the capital, except for two cities: Uberlândia and Vila Velha. Vila Velha is the second largest city in terms of population of the state of Espirito Santo, even ahead of the state capital, Vitória, which does not appear in From a theoretical point of view, our study provides a methodological approach to analyze topics in space and time, using data obtained from Twitter about the Brazilian COVID-19 immunization program. However, the methodology was designed to be independent of research themes, contributing to the development of other research to understand how public opinion on the subject of interest evolves in space and time. There are other methods specifically for topic modeling and extraction; however, we rely on the literature when using LDA  are responsible for creating and spreading misinformation also becomes possible, delimiting the analysis to more particular levels. Based on these last comments, the infodemic opening space for cybercrimes or generating situations of civil danger is an issue to be explored by further studies ,62.polarity → topic → tweet.In this context, while in our previous study we dealt only with the opinion classification based on a textual training dataset containing tweets with pro, anti-vaccination, or neutral orientation, manually searching for correlated events on social web and news search engines, in the current work we sought to deepen the investigation with new elements. We identified the main topics discussed so that researchers from different areas can apply manual annotation of polarity labels, no longer using machine classification, but now classification based on human judgment, for later automation of the hierarchical association From the previous work, we only used two sets of informational elements that we consider fundamental: (1) a part of one of the textual bases that contained geocoded tweets so that we could expand our developments with the distribution of topics in the Brazilian geographic space; and (2) the three-category classification scheme, with the pro, anti, and neutral labels.t-SNE, enabling a more detailed view of the distribution of topics and polarities, according to the number of tweets, in time and Brazilian geographic space. In contrast, our previous work applied only classification models allowing the polarities evolution over time. The use of topic modeling was fundamental to increasing the specificity of the results obtained concerning the previous study.The framework in the current study used different models than the previous one, although they were machine learning models in both cases. The present study applied topic modeling, using the Latent Dirichlet Allocation, and consequently clustering for visualization, with the One last implication to be mentioned is the development of new epidemiologic analyses of the COVID-19 outbreak : infodemAlthough our methodological approach is based on techniques and tools implemented through secure and robust software, there is a need for subsequent refinements, especially in the text preprocessing and the definition and extraction of the features used in the process. There are several difficulties involved, listing some of them: we need to deal with texts outside the formal standard of the Portuguese language, which generates several noises such as abbreviations, the use of emojis/emoticons, and even the expansion of the stop words lists to include also associated abbreviations, and grammatical errors .In this way, the construction of preprocessing scripts becomes challenging , even thAnother difficulty is related to the geocoding of tweets. We extracted geographic information as described in the methodological section, but we need to complete the information about the coordinates through points with latitude and longitude using Google’s geographic API. The geographic information we could extract directly via the Twitter API brought us polygonal coordinates when our objective was the points referring to cities. As we also obtained the names of cities through Twitter API, we developed a process that retrieves the geographic points referring to these cities via Google API. Furthermore, the number of tweets obtained with geocoding was much lower than the number of tweets we parameterized to be searched and retrieved, as we reported in our previous work , implyinThus, a critical limitation was the low number of geocoded tweets, with information from specific points to determine which cities generated the posts. It is crucial to retrieve more extensive amounts of geocoded tweets, to ensure statistical significance to the corpus to train topic extraction methods and consequently to ensure reliable analyses and results. Using tweets containing geographic information allows new studies to apply methods for analyzing correlations between variables identified through the texts with the variables traditionally used by management agencies, as in the case of vaccination programs. For example, the influence that pro- and anti-vaccination texts can exert on the flow of people receiving vaccinations can be studied using methods such as those applied by Huang et al. .Even though the LDA fixes existing problems in methods prior to it , there aAnother issue that still needs further development concerns the psychological effects of COVID-19 and related issues on people and the way they manifest on the social web. In this study, although we are considering people’s opinions, we have not developed any analysis of psychological effects in parallel with the study of opinions. Several studies have demonstrated the need to investigate the effects of the pandemic on people’s mental health, evaluating, for example, effects such as anxiety and the causes of hesitation to receive the vaccine  about vaccination on social networks in the infodemic context, which can help guide efforts to combat potentially harmful information  to society and vaccination programs.The temporal evolution analysis allowed the visualization of the moment when Brazilian posts started intensifying on the explored theme: from the launch of the national vaccination campaign in December 2020. It was also possible to detect when the highest peak of posts occurred: on 17 January 2021, when the first vaccine was applied in Brazil. There was a tendency for most of the topics to present peak on the same date.Regarding the evolution of tweets for polarities, the peak on 17 December 2021, was more marked in the pro-vaccination orientation. However, they are also perceived in the anti-vaccination and neutral polarities on minor scales.In the analysis of the distribution in the Brazilian geographic space, a greater concentration of tweets was identified in large cities, especially in the two largest cities in the country: São Paulo and Rio de Janeiro. However, based on the data collected, these two cities also had the lowest tweet percentual distributions according to their populations. The spatial analysis of the hierarchical association polarity → topic → tweet allowed the observation of the potential of some cities concerning the penetration of each opinion polarity in their populations.It is worth mentioning that any other immunization program for other diseases can use the methodological approach presented. If we further expand this vision, the agencies involved in managing any crisis in any critical public sector are potential methodology users.As directions for further research, we highlight: (1) improvement of the technical data scraping process to extract more significant amounts of geocoded tweets about the desired topic; (2) development of an improved process for manual annotation on the opinion polarities, also considering labels referring to emotions such as happiness, anger, anguish, anxiety, fear, sadness, surprise, etc. Additionally, we highlight  for new


Asexual reproduction is an additional unique reproduction mode of plants relative to traditional sexual reproduction. The innovation of breeding and propagation technology is the key to and an imperative segment of the competition in the crop breeding industry. The asexual reproduction approach is an important direction in the development of modern cell engineering and biotechnology breeding and has great development potential for crop artificial seed and plant factories. However, at present, the regulation principle and molecular mechanism of asexual reproduction and propagation are not clear, and the related knowledge and biotechnology are insufficient. In this Research Topic, we aimed to gather new research, hypotheses, and reviews that would help to better understand the progress and prospects of crop asexual reproduction to promote theoretical and technological innovation in this field. In this way, a series of articles provides an overview of the discoveries in the field of asexual reproduction and its potential applications in crops.Plants display a remarkable capacity for somatic cell plasticity for asexual reproduction. This notable example of totipotency is demonstrated by plant cells that can develop into embryos and then complete plants  transgenes from S. italica, each a member of a different phylogenetic BBM clade. All SiBBM transgenes induced various levels of parthenogenetic embryo development, resulting in viable haploid T1 seedlings in rice.Embryogenesis and reproduction are not strictly dependent on fertilization in plants, which can produce embryos in seeds asexually, a process known as apomixis. Investigations on the molecular basis and identification of functional genes involved in apomixis visual labeling system is a powerful and useful tool for in vitro cell manipulation as well as genetic transformation when used in combination with modern molecular biotechnology in plants. Hu et al. report an optimized protein fluorescence reporting system for somatic embryogenesis regeneration screening and visual labeling of functional genes in the cotton genetic transformation process. It provides a visual marker for labeling transgenic somatic embryos using the fluorescence reporting system based on the screening of 11 fluorescent proteins. The optimized fluorescence labeling system in this study offers the potential for accelerating somatic cell regeneration efficiency and the in vivo monitoring of diverse cellular processes. In addition, the study by Nie et al. successfully induced calli from two diploid cotton species, Gossypium. raimondii and Gossypium. sturtianum, and transcriptome sequencing analysis was performed on the calli to reveal new genes responding to salt stress, which will lay the foundation for plant regeneration and protoplast fusion.The development of a Chen et al. report that uniconazole augments abscisic acid in promoting somatic embryogenesis in Gossypium hirsutum. The study suggests that uniconazole could modulate callus proliferation and callus differentiation rate by regulating the endogenous levels of IAA and ABA.Dynamic balance , ethylene for preglobular embryos, GA for globular embryos, and ABA for mature embryos) and interactions with hormones play a pivotal role in switching cell fate during somatic embryogenesis developmental plasticity of asexual reproduction. They can be considered vital regulators of developmental switching during the totipotency of somatic plant cells. While emphasizing the endogenous signal of the switch, exogenous plant growth regulators are also fundamental. Wang et al. demonstrate that GhLBDs (LATERAL ORGAN BOUNDARIES DOMAIN transcription factors in Gossypium hirsutum) promote callus initiation and act as selectable markers to increase transformation efficiency. This study provides new insights into callus initiation regulatory mechanisms and strategies for improving transformation efficiency in crops.Moreover, the Leafy cotyledon (LEC), Wuschel (WUS), Baby boom (BBM), Monooxygenase, and somatic embryogenesis receptor kinase (SERK) identified may serve as markers to distinguish embryogenic cells, enabling an early diagnosis of embryogenic potential. Joshi et al. review the promotion role and molecular mechanism of AGAMOUS-Like15 (AGL15) in somatic embryogenesis, focusing on the MADS-domain transcription factor AGL15, and show a positive correlation between accumulation levels and the capacity for somatic embryogenesis. The review relates AGL15 function to other transcription factors, hormones, and epigenetic modifiers involved in somatic embryo development. Yin et al. review the options for engineering apomixis in plants. This review summarizes the current understanding of apomixis and highlights the successful introduction of apomixis methods into sexual crops. They also summarize three schemes to achieve engineered apomixis, which will offer more opportunities for the realization of apomictic reproduction.The Research Topic also features Review Articles focusing on asexual reproduction. in vitro, somatic embryogenesis, and apomixis in vivo. As the basis for plant cell engineering and biotechnology applications, asexual reproduction is of great practical importance in plant factories and modern breeding. It is imperative to unravel the regulatory mechanisms of asexual reproduction and developmental flexibility for potential applications in crops, but a long and difficult road is ahead.Taken together, these articles provide an overview of the molecular basis and regulation of asexual reproduction, including embryonic differentiation All authors listed have made a substantial and intellectual contribution to the work and approved it for publication.National Key Research and Development Program (2021YFF1001203), the Agricultural Seed Project of Shandong Province (2020LZGC002), and the National Natural Science Foundation of China (32001589).We would like to thank all authors for their contributions to this Research Topic. We are also grateful to all reviewers for their evaluation of the manuscripts submitted.The authors declare that the research was conducted in the absence of commercial or financial relationships as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Antimicrobial resistance (AMR) is a growing global health concern for both animal and public health, and collaborative strategies are needed to combat the threat. The level of awareness and funding for policies focused on reducing AMR varies between countries. The aim of this study was to compare the integrated surveillance systems for AMR in high and low–middle economies of the Asia-Pacific Economic Cooperation and determine whether there was any improvement from 2015 to 2018. We conducted a survey with a group of 21 countries at different development levels. Associations between the economic development level and the questions of AMR awareness and funding were established using Fisher’s exact test. Improvements were identified where countries established public policies for integrated surveillance of AMR. High economies showed greater advancement in several topics related to AMR than low–middle economies. The survey revealed that there is a better understanding surrounding the implications of the emergence of AMR in human medicine than in veterinary medicine, agriculture, and food production. Our results show that countries enhanced overall AMR surveillance over the 4-year-period; however, more research is needed concerning these advances, especially in low–middle economies and the food production sector. Antimicrobial resistance (AMR) is a global issue that threatens a century of progress in both animal and public health  and the Given the increasing relevance of AMR, the Food and Agriculture Organization of the United Nations (FAO), the World Organization for Animal Health (WOAH), and the World Health Organization (WHO) Tripartite High-Level Coordination Forum set AMR as one of five priority issues and as a flagship topic for One Health, establishing an agreement to develop a joint action plan for AMR in 2014 . In 2022Using the official recommendations of WHO, FAO and WOAH, many high economies have established surveillance programs or systems to monitor AMR for antibiotic stewardship, while most low–middle economies have little to no programs to address this issue  due to lIn 2015 and 2018, our research group surveyed two Asia-Pacific Economic Cooperation (APEC) projects, both focusing on AMR control strategies in humans and food production animals, to evaluate the status of AMR integrated surveillance in various APEC economies at different development levels [A total of 14 (66.7%) APEC participant responses were received. The first group, called “High economies”, included Australia, Canada, the United States, Hong Kong, Japan, and New Zealand. The second group, named “Low–middle economies”, included Chile, Indonesia, Malaysia, Mexico, Peru, Philippines, Thailand, and Vietnam. These classifications resemble the Organization for Economic Co-operation and Development (OECD) classification terminology . All resp-value (≤0.05) (The funding and reports of respondent surveillance systems can be observed in  (≤0.05) , represeEven when AMR in humans is recognized as a major global emergency problem , it is wTo better understand the awareness of different economies, regardless of the level of development, we conducted two APEC projects surveying participating economies. Our results show that since the first survey in 2015, there has been an improvement in all economies with respect to AMR surveillance in both human health and animal production. This could be attributed to the economies’ participation in the first APEC project and the subsequent development and improvement of surveillance systems. The least amount of improvement and greatest differences between high and low–middle economies were in the animal production sector. The shortfall occurs due to differences in resources, laboratories, legal frameworks, and human capacity. In low–middle economies, some areas of AMR research have been neglected ,25,26.The main concerns regarding AMR varied between high economies and low–middle economies. High economies emphasized the lack of alternatives to antibiotics and the lack of audits of veterinary farms and clinics. Low–middle economies highlighted the use of critically important antimicrobials in agroindustry and their lack of resources to act on this issue. Up-to-date knowledge on optimal antibiotic use was found to be higher in high economies for both human and veterinary use; meanwhile, in low–middle economies, it was only found for human antimicrobial use. This gap could be the result of limited animal health guidelines. In some cases, when these guidelines exist, they are not tailored to the end users or are only applicable to large-scale animal production. More research concerning the different characteristics of animal production in each economy is needed to develop customized guidelines that really address this problem.All high economies surveyed received funding for both human and food chain surveillance, while only some low–middle economies received funding for human and food chain AMR surveillance. The same difference is observed in the proportion of reports about advances in surveillance in humans and in the food chain. Surveillance programs should integrate data originating from humans, food production animals, and retail meats . AMR is Results indicate that by 2018, there was a better understanding of the implications of AMR occurrence in human medicine than in veterinary medicine, agriculture, and/or food production. Most economies have conducted AMR surveillance systems focused on human health, and only in the high economies have surveillance systems been developed for animal health. This is similar to that reported by Founou et al. , who poiIt is known that AMR can only be controlled through a concerted global effort, led by heads of state and global institutions, and through coordinated action by the human and animal health sectors, agriculture, in collaboration with the food industry, and community organizations under the concept of One Health . Our resAll economies have made progress since 2015 in the development and implementation of national action plans for AMR under a One Health approach. However, a new survey is needed since the COVID-19 emergency deprioritized planned activities and diverted human and financial resources. Furthermore, antimicrobial use in humans increased in 2020, fueled by COVID-19 prevention or treatment of bacterial coinfections . All theIn 2018, a follow-up survey was sent to the 21 APEC economies previously surveyed by Lapierre et al. , a multiAPEC economies are very different and have different degrees of development. Therefore, they were divided into two groups, “High economies” and “Low–middle economies”, according to the classification of APEC economies considered “travel-eligible” . This APMultiple analyses were performed for each question with the response data “Yes”, “No”, and “Not known” and the economic level category “High”, “Medium”, “Low”, “None”, and “Not known” using Fisher’s exact test . Only thThis pilot survey and its results from different years of reporting contribute to the global objective of tackling AMR in both high and low–middle economies. Cooperation between economies is a key principle in decreasing the gaps between groups and ensuring every economy can implement a viable national plan to control AMR. This model could be expanded to other countries or international entities to encourage the flow of information about AMR surveillance and control strategies. It is very important that high economies, which have surveillance systems in place , can share their knowledge and experiences with low–middle economies, making implementation more effective and efficient since they can communicate not only good results obtained but also their failures. AMR must be looked at as a global public threat, so every country has a shared responsibility and should collaborate to try to decrease AMR and help prioritize areas where these actions are most critical, such as food production surveillance and its integration with human AMR surveillance. Finally, it is necessary for a third survey assessment in the APEC economies to measure the impact of their advances due to the health, economic, and social effects of COVID-19.


An accurate timescale of evolutionary history is essential to testing hypotheses about the influence of historical events and processes, and the timescale for evolution is increasingly derived from analysis of DNA sequences. But variation in the rate of molecular evolution complicates the inference of time from DNA. Evidence is growing for numerous factors, such as life history and habitat, that are linked both to the molecular processes of mutation and fixation and to rates of macroevolutionary diversification. However, the most widely used methods rely on idealised models of rate variation, such as the uncorrelated and autocorrelated clocks, and molecular dating methods are rarely tested against complex models of rate change. One relationship that is not accounted for in molecular dating is the potential for interaction between molecular substitution rates and speciation, a relationship that has been supported by empirical studies in a growing number of taxa. If these relationships are as widespread as current evidence suggests, they may have a significant influence on molecular dates.We simulate phylogenies and molecular sequences under three different realistic rate variation models—one in which speciation rates and substitution rates both vary but are unlinked, one in which they covary continuously and one punctuated model in which molecular change is concentrated in speciation events, using empirical case studies to parameterise realistic simulations. We test three commonly used “relaxed clock” molecular dating methods against these realistic simulations to explore the degree of error in molecular dates under each model. We find average divergence time inference errors ranging from 12% of node age for the unlinked model when reconstructed under an uncorrelated rate prior using BEAST 2, to up to 91% when sequences evolved under the punctuated model are reconstructed under an autocorrelated prior using PAML.We demonstrate the potential for substantial errors in molecular dates when both speciation rates and substitution rates vary between lineages. This study highlights the need for tests of molecular dating methods against realistic models of rate variation generated from empirical parameters and known relationships.The online version contains supplementary material available at 10.1186/s12862-022-02015-8. Understanding the timescale of evolution is critical to researching the processes that generate and shape the diversity of life on Earth. Analysis of DNA sequences offers the possibility of investigating the tempo and mode of evolution of all extant lineages, not just those that have a detailed fossil record. The earliest molecular dating analyses concluded that the average rate of molecular evolution was relatively constant over time in some proteins because of an apparently linear accumulation of amino acid differences when compared between species , 2. HoweA wide variety of models have been developed to account for different patterns of among-lineage molecular rate variation –16. In tTesting over the last two decades has generally supported the robustness of divergence time estimates under the Bayesian methodology to the choice of rate prior, provided that enough separate rate parameters are allowed and sufficient calibrating information is provided –33; but Currently used models of rate variation are generally either uncorrelated, so that each lineage-specific rate is drawn independently from a common distribution, or autocorrelated so that lineage-specific rates evolve from the rates of their parent lineages according to a stochastic, random walk. However, the factors shaping variation in rate of molecular evolution are complex: in addition to random variation in rates between lineages, there is clearly also systematic variation influenced by many factors including species traits, population dynamics, environment and evolutionary history . EvolutiIn addition to covarying with other evolving traits, there is evidence that variation in molecular rates can be associated with macroevolutionary patterns. Relationships between the relative size of sister clades, representing their net diversification rates, and their molecular rates have been detected in several studies, include in reptiles , 57, birThe mechanism behind observed correlations between diversification and molecular evolution remains a matter of debate. One line of argument proposes that higher molecular rates could promote speciation by speeding the development of reproductive isolation . In thisAn association between substitution rates and diversification rates could affect the performance of existing divergence time estimation methods (e.g. ). In parHere, we investigate the potential influence of speciation-related rates of molecular evolution on the reliability of molecular dating studies. Rather than assuming only one specific model of the association between diversification rates and rates of molecular evolution, we model a range of possible causal links. We include three alternative models of the association between rates of molecular evolution and speciation rates. In the first, both speciation rates and substitution rates vary over the phylogeny, but neither influences the other. In the second, we model continuous variation in a linked manner, which might represent either direct influence of one on the other  or an inIn this study, we simulate the evolution of molecular sequences under all three models of the link between speciation and rate of molecular evolution—unlinked, continuous coevolution and punctuated models—and observe the average effects on errors in Bayesian divergence time estimates. Most simulation studies use arbitrarily chosen values to parameterize the simulations. In order to make our test have real-world significance, we base our simulations, as far as possible, on empirically determined relationships between rates of molecular evolution and diversification rate. As a convenient well-studied case study, we parameterize our models using values taken from empirical inferences on bird data , 80, or In order to ensure our simulations are as close as possible to real-world data, we chose to parameterize the simulations using empirical estimates of parameters from bird data, because birds are one of the most well-studied groups for both substitution rate variation and diversification rate. While these parameters might not exactly capture processes in other taxa, and different values could alter results, we believe that basing the simulations on a real case study lends a degree of realism that would be lacking if we arbitrarily chose convenient parameter values. We checked that the simulation procedure produced trees with average characteristics similar to the avian clades used to parameterize the study , Continuous (speciation rates and molecular rates covary), and Punctuated (molecular change occurs in bursts associated with speciation events as well as continuously within lineages) Table . We usedNode dates were then inferred using three different reconstruction methods: BEAST 2 , 84 usinFirst, we examined the median absolute percentage error (MAPE) in the ages of all nodes within each tree whose topology was correctly reconstructed Fig. . For treSecond, for trees reconstructed using BEAST 2 (UCLN), we also investigated the degree of topological error via the Robinson-Foulds distance between the true and estimated tree . This meThird, we calculated the uncertainty of node age reconstruction as reported by the method via the width of 95% highest posterior density intervals as a proportion of node age. Division by node age is required because the infinite-sites model of molecular evolution predicts that the relationship between posterior mean node age estimates and 95% credible intervals should approach a straight line with sufficient molecular data , 77. UsiIf the difference in gamma values between reconstructed and true trees is negative, the reconstructed tree has nodes distributed more towards the root, while if it is positive the reconstructed tree has nodes distributed more towards the tips than the true tree. Using BEAST 2 (UCLN), gamma values for the reconstructed Unlinked trees were distributed evenly around the gamma value of the simulated tree with a mean shift of + 0.11  and PAML . Clearly, these methods and models do not represent all possible approaches to inferring molecular dates using a rate-variable method, but they were chosen because they are widely used and differ in some key respects, and therefore present two useful test cases for the accuracy of “relaxed clock” methods in the face of systematic rate variation.The Unlinked model describes the error that can be expected from independent variation in speciation and molecular rates. Inference on trees simulated under this model produced median absolute errors in node age inference of about 12% on average using BEAST 2 (UCLN), and high values of 66% using the PAML (ACLN) and 77% using PAML (UCLN) Fig. . The souThe causes of the extremely high errors of the PAML(ACLN) and PAML (UCLN) phylogenetic inference for the Unlinked trees (and throughout the study for the other models) are unclear. Examination of the output trees suggests that the most extreme errors are in trees with shallower calibrations, and that the ages of large uncalibrated clades are sometimes overestimated severely. The inferences from PAML and BEAST 2 are not directly comparable due to numerous technical differences: for example, tree topology is fixed in PAML but estimated in BEAST 2; and BEAST 2 has prior distributions on speciation and extinction parameters, which are fixed in PAML. Nevertheless it is surprising that the performance of the ACLN model in PAML was worse than the UCLN model in BEAST 2 even though the underlying rate variation in these simulations is indeed autocorrelated. The fact that even higher errors and greater HPD widths are found when using PAML (UCLN) than using PAML (ACLN) Figs. , 3 suggeThe Continuous model has substitution rates and speciation rates that coevolve continuously rather than varying independently. Notably, we chose the degree of covariance that reproduced two well-established empirical phenomena – the correlation of clade size and branch length contrasts detected in a sister pairs analysis by Lanfear et al. , and theLevels of absolute error were highest under BEAST 2 (UCLN) when speciation events were associated with large punctuational bursts of molecular evolution , the Punctuated model had the lowest degree of topological error  an an107]) Our results here show the value of testing molecular phylogenetic and dating methods against complex simulation models informed by empirically determined patterns of rate variation. We have demonstrated that commonly applied Bayesian divergence time estimation methods can experience average errors of 12% in node dates under a model in which speciation and molecular rates vary independently, 14% when substitution rates are linked to speciation rates and 20% under a punctuational model when analysed using an uncorrelated rates prior in BEAST (UCLN). Errors were much higher under all models when trees were analysed with an autocorrelated rate prior in PAML (ACLN), and still higher with the uncorrelated lognormal rate prior in PAML (UCLN). As demonstrated by the negative skewness of gamma statistics, for trees with links between speciation and molecular evolution these errors could lead to systematic overestimation of node ages. Regardless of the mechanism generating the association between speciation rates and substitution rates, we show that the potential for divergence time estimation error associated with known empirical relationships between molecular evolution (manifest in substitution rate) and macroevolution (manifest in the topology and branch lengths of phylogenetic trees) can be significant and should be taken into account. Future work should see a broader range of sophisticated divergence time estimation methods be tested against a wider variety of more empirically realistic simulated data, including geographic and environmental biases, concerted changes in population size, and directional changes in life history characteristics. Ultimately the goal should be to produce new models that relax the assumption of independence between priors on divergence times and priors on substitution rates. These will lead to greater confidence in applications that depend on robust estimates from molecular dating procedures.We refer throughout to simulated phylogenies, which are known without error, as the ‘simulated trees’, and to the phylogenies inferred from analyzing the sequences evolved along each of these simulated phylogenies as the ‘reconstructed trees’. Comparison of the reconstructed trees to the simulated trees allows us to evaluate the degree to which phylogenetic inference methods correctly infer the known evolutionary history of the simulated data.Our overall design is summarised in Fig. We simulate sets of 50 phylogenetic trees and nucleotide sequence alignments under each of three different models describing the association between speciation rates and rates of molecular evolution: one “Unlinked” model in which molecular and diversification rates each vary independently, as well as two alternative mechanisms for an association between rates of molecular evolution and speciation rates , as implemented in the software package ‘BEAST 2’ , in which rates for each branch are independent draws from a common lognormal distribution;the autocorrelated lognormal rate prior (ACLN), implemented in the ‘mcmctreere suite , in whicFor each reconstructed tree, we calculated accuracy in the estimation of node times using the median absolute percentage error (MAPE) of inferred node times for clades that are present in both true and reconstructed trees. The median was chosen rather than the mean to reduce the impact of extremely high values for the estimates of shallow nodes. Since these are only calculated for correctly reconstructed branches and do not capture errors in reconstructing topology, we also calculate the Robinson-Foulds distance or number of non-shared bipartitions, which only reflects topological error .Model misspecification in Bayesian molecular dating methods can sometimes be associated with higher uncertainty in the reconstructed node ages, as measured by the width of Bayesian credible intervals. This is undesirable because the reconstructed history is less useful as an explanatory tool and less informative in downstream analyses, but it can also be a simple way to diagnose problems with the specified model. We examined the uncertainty with which node ages were reconstructed by the two Bayesian molecular dating methods. We scored uncertainty by averaging the width of the reconstructed 95% highest posterior density intervals as a proportion of reconstructed node height across each tree (HPD width), with a larger score indicating less precise estimates.We examine the direction of error by calculating the gamma statistic for each tree and comparing to the true tree . The gamWe checked to see if we can use the reconstructed branch lengths, which are the median of posterior branch lengths calculated by BEAST 2 (UCLN), PAML (ACLN) or PAML (UCLN), to detect the correlation between branch lengths and clade sizes found by sister pair analysis on the simulated trees (see Additional file Additional file 1: Supplementary methods and results.


Klinefelter syndrome (KS) is the most common aneuploidy in men and has long-term sequelae on health and wellbeing. KS is a chronic, lifelong condition and adolescents/young adults (AYAs) with KS face challenges in transitioning from pediatric to adult-oriented services. Discontinuity of care contributes to poor outcomes for health and wellbeing and transition programs for KS are lacking. We aimed to develop and test a mobile health tool (KS Transition Passport) to educate patients about KS, encourage self-management and support successful transition to adult-oriented care. First, we conducted a retrospective chart review and patient survey to examine KS transition at a university hospital. Second, we conducted a systematic scoping review of the literature on AYAs with KS. Last, we developed a mobile health transition passport and evaluated it with patient support groups. Participants evaluated the tool using the System Usability Scale and Patient Education Materials Assessment Tool (PEMAT). Chart review identified 21 AYAs diagnosed between 3.9-16.8 years-old (median 10.2 years). The survey revealed only 4/10 (40%) were on testosterone therapy and fewer  had regular medical care. The scoping review identified 21 relevant articles highlighting key aspects of care for AYAs with KS. An interprofessional team developed the mobile-health KS transition passport using an iterative process. Support group members (n=35) rated passport usability as ‘ok’ to ‘good’ . Of PEMAT dimensions, 5/6 were deemed ‘high quality’ (86-90/100) and participants knew what to do with the information (actionability = 83/100). In conclusion, many patients with KS appear to have gaps in transition to adult-oriented care. Iterative development of a KS transition passport produced a mobile health tool that was usable, understandable and had high ratings for actionability. Klinefelter syndrome KS, 47, XXY) is the most common chromosomal disorder in males occurring in approximately 150 per 100,000 males (1:660)  and subs7, XXY isKS can have wide-ranging effects on health and wellbeing , 7. The To address the knowledge gap related to KS transition, we used a three-tiered approach. First, we conducted a retrospective chart review to examine the discontinuity in transitional care for patients with KS. Second, we conducted a systematic scoping review to evaluate the available evidence and recommendations for adolescents and young adults (AYAs) with KS during the transition to adult-oriented care. Third, we developed a novel mobile health  ‘transition passport’ to support and guide patients in self-management during transition and to ensure information transfer. Finally, we sought to validate the transition passport with KS patient-support groups by examining usability, understandability, and actionability of the transition passport.This study was conducted in accordance with the Declaration of Helsinki and was approved by local ethics committees . All participants provided informed consent prior to study activities. The study was comprised of three parts. First, we conducted a chart review of the Lausanne University Children’s Hospital (Switzerland) and surveyed patients about their experiences during transition to adult-oriented care. The results of part I pointed to gaps in care and challenges during transition that spurred us to develop a mobile-health tool to support transition for AYAs with KS. The second part of the study involved a systematic scoping review examining the literature on transitional care for patients with KS. This was used to gather the best available evidence for creating the mobile-health tool. Last, we created a transition passport for patients with KS and validated it with patient support organizations.To better understand the needs of patients with KS during transition we conducted a retrospective, descriptive follow-up study of AYAs with KS (>16 yrs.) seen in the pediatric endocrine service of the Lausanne University Children’s Hospital. Patients were identified by searching the hospital database of patients (2000-2014). Patients after 2014 were not included - as a structured transition program  was launched in 2014 . IndividTo review the data and evidence base related to transitional care for AYAs with KS, we conducted a scoping review  according to the five-stage Arksey and O’Malley framework . FindingAn interprofessional team including pediatric/adult endocrinology, urology, and nursing engaged in an iterative process to develop content and design a mobile, electronic health  tool  to support AYAs transition from pediatric to adult-oriented care. Broadly, the goal was to create an interactive tool (PDF) organized into easily navigable sections enabling patients to link/jump to preferred topics by clicking on icons - without having to read through the entire document . The KS transition passport serves as a psychoeducational tool enabling AYAs to learn about KS as well as a means to document and record information essential to their medical management. The interactive PDF format was chosen for reasons of simplicity as the format can be used with standard mobile electronic devices and independent of web connectivity and/or linkage to electronic health records. Language editing, word choice, layout, and design elements were modified and refined in an iterative development process. The final version (V.20) was translated from French to English. We assessed readability using algorithms that calculate the number of difficult words/sentences to provide an estimated age range and grade reading level. There is no accepted “gold standard” algorithm to assess readability so we utilized several validated instruments: Automated Readability Index, Linsear Write Formula, Flesch-Kincaid Grade Level, The Coleman-Liau Index, Flesch Reading Ease, and Gunning Fog Index and reported the calculated consensus reading level as previously reported .To evaluate the transition passport, we partnered with patient organizations and used a web-based approach to reach geographically dispersed patients . Adult pTo assess usability of the transition passport, participants completed the System Usability Scale (SUS) . Users rp value <0.05 was considered statistically significant.Data are reported using descriptive statistics. Student’s T-tests and Mann-Whitney rank sum tests were used (as appropriate) to compare SUS ratings between patients and parents/guardians. PEMAT scores ≥80% on a given parameter were deemed be ‘high-quality’ . Chi squThe retrospective search of the Lausanne University Hospital (CHUV) pediatric endocrine database identified 21 White non-Hispanic patients with KS (2000-2014). Age at initial consultation ranged from 3.9-16.8 years-old (median 10.2 yrs.). Of those responding to the survey, more than half of patients  received testosterone therapy in the pediatric setting. The duration of care in pediatrics ranged from 1-11 yrs. . A survey was sent to patients and 10/14 (71%) completed the survey. Half of patients  reported being clinically followed in adult endocrinology and 3/10 (30%) reported that they did not seek regular medical care. Only 4/10 (40%) had ongoing testosterone treatment. We inquired about the preferred timing of transition and the majority of patients preferred transition between 16-19 yrs. . In terms of the transition process, results were divided. Half of patients (5/10) preferred to see the adult provider alone while the other half preferred to have parents included in medical visits.To explore the survey findings suggesting gaps in care and challenges with adherence to treatment, we conducted a systematic scoping study of the available literature on AYAs with KS 1987-2017). From the initial review of 134 articles, 22 were included for full review and data extraction. The publications were from Australia, Belgium, France, Netherlands, Switzerland, and the United States. Data were extracted from articles on AYAs with KS including 11 review articles 7-2017. F–33, two Spurred by the findings of the chart review and drawing on scoping review findings, an interprofessional team  developed a KS transition passport to support effective transition to adult-oriented care. A series of face-to-face meetings charted the structure of the passport  was rated 83/100. Patients with KS and parents/guardians exhibited similar PEMAT scores (p=0.9) – yet this observation should be interpreted with caution given the limited number of parent/guardian participants.To evaluate the KS transition passport, we recruited subjects through several KS patient support organizations. In total, 35 participants completed the evaluation  with KS. First, our retrospective chart review indicated that a significant portion of young men with KS struggled with transitioning from pediatric to adult-oriented care resulting in gaps in treatment and care. Second, the systematic review of the literature and scoping study highlighted a lack of consensus regarding transitional care of AYAs with KS. The paucity of high-quality data limits the development of evidence-based guidelines for practice. We developed a KS transition passport using an iterative process with an interprofessional team incorporating findings form the scoping review. Evaluating the KS transition passport with patient groups showed that the passport is usable, understandable, and actionable – and thus, may represent an important avenue for extending the reach of care for AYAs with KS.Given the broad phenotypic spectrum of KS, some AYAs with KS may not differ significantly from their peers. Overt KS phenotype is often evident only after puberty or when fertility is pursued , 44. SubTo support AYAs during transition , an interprofessional team developed the “KS transition passport”. The m-health tool was intended to serve as a psychoeducational support for therapeutic education to help patients better understand KS, as well as a resource promoting patient activation  enabling patients to record vital health information during the transfer from pediatric to adult-oriented care. The passport can equally be used as a standardized information transfer tool between health professionals. The idea of using a “passport” to support AYAs during transition is not new. Indeed, the Lausanne University Children’s Hospital has developed and uses a transition passport for type 1 diabetes and Turner Syndrome. Other groups have published findings on similar passports for AYAs with diabetes , inflammRelative strengths of the study include the sequential process . The study has a number of limitations including the relatively small sample size for the retrospective chart review/survey (n=21) and the evaluation with KS patient organizations (n=35). We did not collect data on self-identified race/ethnicity. As such, the findings should not be considered to represent the perspectives of individuals with Klinefelter syndrome from diverse ancestry. Indeed, a recent review has identified noted paucity of data on the experiences patients with Klinefelter syndrome from and Black, Indigenous, People of Color (BIPOC) communities . SimilarThe iterative development process involved an interprofessional team yet did not involve patient/parent stakeholders. This is relevant as involving patient stakeholders in a “design-thinking”  process can improve understandability, actionability and acceptability of co-created patient-facing materials 16. Almost a third  of patient/parents provided free-text comments that were generally positive and expressed appreciation for the work. However, a number of comments also noted a desire for greater detail regarding mosaicism, thrombosis risk, complexity of behavioral issues, how to find a specialist/ask for referrals, and starting an occupation. Indeed, comments from patients/parents requesting information and greater detail on learning difficulties, academic supports, disability/function, and psychiatric diagnoses may reflect that behavioral health specialists  were not involved in the iterative ‘design thinking’ process. In hindsight, inclusion of such perspectives could have strengthened the development of the m-health tool. Notably, patient-facing material need to be regularly reviewed and updated to ensure that the information accurately reflects the current state of the science and standards of care as well as unmet patient needs. Future work should include systematic follow up of AYAs following transition to adult-oriented care. Additionally, establishing rigorous criteria to identify uniform groups of patients with Klinefelter syndrome  could support more targeted and rigorous clinical and fundamental research on Klinefelter syndrome.In conclusion, many patients with KS appear to have gaps in transition to adult-oriented care. Iterative development of a KS transition passport produced a tool that was usable, understandable, and had high ratings for actionability. Such eHealth tools can support a comprehensive, structured approach to transitional care for AYAs with KS. Future work may examine the effect of the KS transition passport on knowledge, patient activation, adherence to treatment, and continuity of care.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Lausanne University Hospital Ethics Committee and Boston College Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.AD made substantial contributions to the study design, acquired and analysed the literature for the scoping review, contributed to the development of the transition passport, collected and analysed the data for the evaluation of the transition passport and drafted the manuscript. VH made substantial contributions to collecting and analysing the chart review and patient survey data, contributed to the development of the transition passport, provided critical feedback on the manuscript and approved the final manuscript. SL made substantial contributions acquiring and analysing the literature for the scoping review, provided critical feedback on the manuscript and approved the final manuscript. LE made substantial contributions to collecting and analysing the data for the evaluation of the transition passport, provided critical feedback on the manuscript and approved the final manuscript. GP, LV, and NP made substantial contributions to developing the transition passport, provided critical feedback on the manuscript and approved the final manuscript. MH made substantial contributions to the study design, led the development of the transition passport, provided critical feedback on the manuscript and approved the final manuscript. All authors contributed to the article and approved the submitted version.This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Open access funding provided by University of Lausanne.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


During the past few decades, several FDA-approved medicines inspired by natural products have reached the clinic. The time is therefore right to publish a special topic dedicated to the topic of natural products and drug discovery. This special topic contains one review article, four perspectives, two research highlights, two research articles and a personal interview. We have invited a collection of recognized experts in the field of natural product chemistry to contribute to this special topic, which provides a broad survey of progress over the past few years.The special topic starts with an interesting and detailed review by David J. Newman, who discusses historically important discoveries regarding the therapeutic use of natural products as anti-infective, antitumor and antimetabolic agents. Some of these are currently widely used as first-line drugs or have provided the starting point for developing much better semi-synthetic analogs for the treatment of infectious diseases, cancer and metabolic diseases.In accordance with the majority of research in this area, the four perspectives presented by Geoffrey A. Cordell, Jian-Min Yue, Hui Ming Ge and Xiaoyu Tang are devoted to describing recent progress in sustainable strategy development, and the isolation, synthesis and biosynthesis of biologically active natural products by using emerging tools and techniques. Geoffrey A. Cordell discusses the topic ‘Evolving paradigms for natural-product drug discovery’ and presents his long-standing desire to integrate the technologies of the Fourth Industrial Revolution (4IR) with sustainability in biologically active natural products to meet global human health-care needs. Bin Zhou and Jian-Min Yue present a perspective entitled ‘Natural products are the treasure pool for antimalarial agents’ and highlight the historical achievements and current trends in antimalarial drug discovery based on natural products. In the perspective contributed by Bo Zhang and Hui Ming Ge, the authors discuss state-of-the-art pericyclases for cycloaddition and outline the issues associated with, and opportunities for, further studies of pericyclases. The perspective entitled ‘Harnessing nature's biosynthetic capacity to facilitate total synthesis’ by Xiaoyu Tang and coworkers highlights representative applications of synthetic biology in the total syntheses of complex molecules by harnessing the power of biosynthetic enzymes.Escherichia coli 50S ribosome. The author focuses on an integrated approach that facilitates the identification of new and potent antibiotics with reduced bacterial resistance. The highlight contributed by Ziyang Zhang, entitled ‘Beyond natural targets: chemical synthesis reprograms the target specificity of rapamycin’, describes a compelling strategy developed by Jun O. Liu and coworkers, which shows that modification of natural products can not only expand the target landscape of natural products, but also impart improved properties such as potency and selectivity to the parent molecules.In the research highlight section, Tuoping Luo discusses the technology developed by Ian B. Seiple and coworkers in which total synthesis is combined with cryo-electron microscopy to resolve complexes of virginiamycin derivatives with the Anisomeles indica (L) Kuntze (Labiatae) leaves, which has been identified as an oral agent against SARS-CoV-2 in mice.This special topic also contains two research articles, by Xiaoguang Lei's group and Zhen Yang's group, on drug discovery based on natural products. Xiaoguang Lei's group designed and synthesized bifunctional chemical probes based on the natural product polycarcin V as site-specific DNA-damaging agents, and uncovered novel DNA–protein interactions. This highlights their potential use in the light-controlled treatment of skin diseases such as psoriasis. Zhen Yang's group reported their efforts to achieve the isolation and semi-synthesis of the natural product (−)-anisomelic acid from in vitro generation of many pluripotent stem cells from patients. Such cells have the potential to be used for cell therapy, drug screening and disease modeling, and are the most important ‘seed cells’ in the field of regenerative medicine.In the final section of the special topic, Hongkui Deng summarizes the efforts of his group to use small-molecule natural products for This special topic provides information on research in the areas of natural products and drug discovery. We hope that this five-part publication, which is devoted to demonstrating that natural products are a primary source of medicines, reflects current state-of-the-art drug discovery, and will become a useful reference both for beginners and veterans in this dynamically developing field.