Caenorhabditis elegans (C. elegans). However, the contribution of additional neural factors, and a broad understanding of the role of the nervous system in regulating aging remains to be established. Here, we examine the impact of the dopamine system in regulating aging in C. elegans.Despite recent progress in understanding the molecular mechanisms regulating aging and lifespan, and the pathways involved being conserved in different species, a full understanding of the aging process has not been reached. In particular, increasing evidence suggests an active role for the nervous system in lifespan regulation, with sensory neurons, as well as serotonin and GABA signaling, having been shown to regulate lifespan in C. elegans. Activation of D2R using aripiprazole, an antipsychotic drug, robustly extended both lifespan and healthspan. Conversely, inhibition of D2R using quetiapine shortened worm lifespan, further supporting the role of dopamine receptors in lifespan regulation. Mechanistically, D2R signaling regulates lifespan through a dietary restriction mechanism mediated by the AAK-2-DAF-16 pathway. The DAG-PKC/PKD pathway links signaling between dopamine receptors and the downstream AAK-2-DAF-16 pathway to transmit longevity signals.We report that mutations of DOP-4, a dopamine D1-like receptor (D1R), and DOP-2, a dopamine D2-like receptor (D2R) oppositely affected lifespan, fast body movement span, reproductive lifespan, and developmental rate in These data demonstrated a novel role of dopamine receptors in lifespan and dietary restriction regulation. The clinically approved antipsychotic aripiprazole holds potential as a novel anti-aging drug.The online version contains supplementary material available at 10.1186/s12915-022-01272-9. C. elegans [The biological mechanisms of aging are still not well-understood, despite several conserved aging-regulatory pathways that have been identified from yeasts to humans . Increas elegans . HoweverC. elegans: DOP-1 and DOP-4 belong to D1-like dopamine receptors (D1R), while DOP-2 and DOP-3 belong to D2-like dopamine receptors (D2R) [cat-2 had a normal lifespan, suggesting that solely reducing dopamine production does not affect longevity [C. elegans, including decision-making, basal slowing, and food response [C. elegans.Whether the dopamine system regulates the aging process and lifespan is unclear. Dopamine is a biogenic amine neurotransmitter, which primarily modulates behavioral outputs in response to environmental conditions , 7. For rs (D2R) –11. A prresponse –14, it iC. elegans is one of the most widely used model organisms to study aging. Despite its simple nervous system, C. elegans possesses a conserved dopamine system to that of the mammalian nervous system, including biosynthetic enzymes responsible for dopamine synthesis, mechanisms for synaptic release, and the expression of dopamine receptors [C. elegans a suitable genetic model system for investigating the role of the dopamine system in longevity.Because of its short lifespan, observable age-related phenotypes, and conserved aging-related biological pathways , 16, C. eceptors . These fC. elegans by activating the dopamine receptor-mediated pathway. The surprise finding of aripiprazole holds promise for further development as a potentially safe, novel anti-aging drug.In the present study, we demonstrate that dopamine D1- and D2-like receptors oppositely regulate worm lifespan through a dietary restriction (DR) mechanism. Using molecular genetics approach and pharmacological tools, we teased out how dopamine receptors send longevity signals through their G-protein-coupled signaling transduction pathways to downstream DR-related pathway. Our findings uncover a novel mechanism of dopamine signaling in DR and lifespan regulation. Notably, aripiprazole, a clinically widely used antipsychotic drug, robustly extend both the lifespan (> 50%) and healthspan (> 80%) of dop-1, dop-2, dop-3, and dop-4, were used for lifespan assay. Intriguingly, the dop-2 mutant showed a significantly reduced lifespan  than N2 animals, whereas the dop-4 mutant was significantly long-lived  compared with N2 animals. The dop-1 and dop-3 mutants also showed changes in lifespan  compared with N2 animals, which were not statistically significant . Worms exposed to 100 μM of aripiprazole reached a maximum lifespan extension of up to 52.6% (P < 0.0001)  and a PKA regulatory subunit (KIN-2) [DOP-2 signals through Gαo pathways , 23. To  protein . As expe protein . The cAM protein –28. acy- elegans . The C.  (KIN-2) –32. The  (KIN-2) . The resC. elegans ortholog of diacylglycerol kinase [tpa-1, pkc-1, pkc-2 and pkc-3) and two PKD homologs (dkf-1 and dkf-2) [pkc-1, pkc-3 and tpa-1 were required for aripiprazole to extend lifespan, whereas pkc-2 were dispensable  on the healthspan of N2 animals   signaling, silent information regulator 2 (SIR2), and AMP-activated kinase (AMPK) may act upstream of DAF-16 . To deteaak-2 mutant, suggesting that aak-2 is required for the effects of aripiprazole on lifespan. We further tested a mutant bearing a mutation in C. elegans liver kinase B1 (LKB1) homolog PAR-4, an upstream kinase of AMPK. The par-4 mutant displayed no lifespan extension upon aripiprazole-treatment  and D2R (dop-2) mutations of C. elegans oppositely affected worm lifespan in that the dop-2 mutant was short-lived, whereas the dop-4 mutant was long-lived. Pharmacological activation of DOP-2 using aripiprazole robustly extended worm lifespan, whereas inhibition of DOP-2 using quetiapine resulted in shortened lifespan, supporting the selective role of specific dopamine receptors in lifespan regulation. Our data demonstrated that DOP-2 is the major subtype of D2R, and DOP-4 was the major subtype of D1R to regulate lifespan since mutations on the other two dopamine receptors failed to show statistically significant impacts on lifespan. These findings were further supported by the fact that aripiprazole-induced lifespan extension required DOP-2 instead of DOP-3. Mechanistically, dopamine receptors regulate lifespan through a DR mechanism mediated by the AAK-2-DAF-16 pathway. The DAG-PKC/PKD pathway served as a link between dopamine receptors and the AAK-2-DAF-16 pathway to transmit longevity signals. Together, these data represent a novel role of the dopamine system in lifespan regulation, a schematic diagram illustrating the possible mechanism of dopamine system extension of lifespan was shown in Fig. In the present study, we showed that D1R (eat-2(ad1116) mutant has a 57% longer lifespan than the wild-type animals, similar to that of aripiprazole-treated worms, but featuring a nearly 90% reduction in pharyngeal pumping rate [eat-18 mutants with 70-80% reduction in pharyngeal pumping rate only display 15% and 38% lifespan extension, respectively [C. elegans lifespan. The significant lifespan extension effect caused by D2R activation or loss of D1R signaling should be due to direct activation of intrinsic DR pathways in C. elegans instead of altered food intake or inhibited growth of feeding bacterial. Whether D1R agonists and antagonists can affect C. elegans lifespan needs further investigation.A previous study reported that several dopamine receptor agonists could extend worm lifespan . The auting rate , 48. Twoectively , 48. TakC. elegans and stimulate the downstream pathways in the absence of endogenous dopamine. However, interestingly, our results showed that aripiprazole failed to extend the lifespan of the cat-2 mutant, suggesting that a certain dopamine level may be required for aripiprazole to extend lifespan. A possible explanation is that instead of being a conventional agonist of DOP-2, aripiprazole may act as an allosteric modulator whose binding results in a conformational change of DOP-2 and subsequently enhanced affinity to dopamine. Further studies are needed to clarify the agonistic action of aripiprazole on DOP-2 in C. elegans. Notably, our results showed that aripiprazole showed a biphasic effect on dop-2 mutant that is not presented on other cases. This is also an interesting question that demands further investigation.As a reasonable extrapolation from the mammalian data that shows the agonistic action of aripiprazole on D2R, aripiprazole should be able to bind DOP-2 in eat-2 mutant but also induced DR phenotypes including prolonged healthspan, reduced brood size, extended reproductive period as well as decreased lipid storage. Further investigation demonstrated that aripiprazole-mediated DR response was dependent on the AAK-2-DAF-16 pathway. DR is subjected to the regulation of multiple genetic pathways, and different DR regimens may elicit different pathways to confer lifespan extension. For example, Greer et al. described that sDR  and DP (dilution of peptone) extend lifespan through the AAK-2-DAF-16 pathway, whereas other DR regimens could activate SKN-1, PHA-4 or HSF-1 [The conclusion that aripiprazole extends lifespan through a DR mechanism is supported by the fact that aripiprazole not only failed to extend the lifespan of the or HSF-1 . In the How dopamine signaling is transmitted to the AAK-2-DAF-16 pathway to regulate lifespan is an interesting question. The activity of AMPK can be regulated by a series of upstream kinases, such as PKA, protein kinase B (AKT), PKC, PKD, and LKB1. LKB1 phosphorylates AMPK at Thr-172 for activation. Conversely, PKA, PKC, and PKD phosphorylate AMPK at Ser-485/491 to inhibit its activity , 37, 50.egl-30 and egl-8, and the Gαo signaling pathway encoded by goa-1 and dgk-1. A previous study found that GABA receptor GBB-1 modulates lifespan through the EGL-30-EGL-8-DAF-16 pathway, suggesting a lifespan regulatory role of Gαq signaling pathway [C. elegans PKC/PKD isoforms. This may be due to the different expression patterns of these isoforms and the tissue-specific effects of aripiprazole.We found that one possible downstream effector of the dopamine receptors is DAG, which is differentially regulated by Gαq signaling pathway encoded by  pathway . HoweverC. elegans [Dopamine receptors are widely expressed in the nervous system . From mu elegans . HoweverC. elegans [C. elegans through a mechanism other than the Akt pathway, but related to dopamine receptor-mediated DR responses. Since DR has been reported to delay the development of C. elegans [Previous studies based on liquid media have reported that several antidepressants, including mianserin, could extend worm lifespan, mainly by blocking serotonin receptors –55. .A r elegans . Others  elegans . Neverth elegans , our res elegans , 60. Mor elegans –63. TherC. elegans. The clinically proved good long-term safety of aripiprazole as well as the fact that the dopamine system and its downstream DR-related pathways is highly conserved among species support the further translation of our finding into humans. Moreover, developing interventions targeting the dopamine system may be a new direction for aging research which aims to benefit human health and longevity.Taken together, our findings uncover a novel role of dopamine signaling in lifespan regulation. Genetic inhibition of D1R or pharmacological activation of D2R using aripiprazole could robustly extend both lifespan and healthspan in Caenorhabditis Genetics Center  and maintained at appropriate temperature on solid nematode growth medium (NGM) plates seeded with E. coli OP50. Strains used in this study are described in Additional file All strains were obtained from the Aripiprazole and quetiapine, purchased from Meilunbio , were dissolved in dimethyl sulfoxide (DMSO) as stocks. Drugs were added to the liquid NGM before pouring plates. A final DMSO concentration of 0.1% (v/v) was maintained under all conditions.Worms were cultured for three generations without starvation before lifespan assays. All lifespan assays were performed at 20 °C unless specified. Synchronized late L4 larvae were transferred to lifespan assay plates supplemented with 50 μM of 5-fluoro-2′-deoxyuridine  to prevent self-fertilization  IV was used to monitor the translocation of DAF-16:GFP. In each experiment, age-synchronized L4 larvae were treated with 100 μM of aripiprazole or DMSO for 24 h in the same way as described in the lifespan assays. Then, worms were mounted on glass slides with a drop of 0.1% sodium azide and capped with coverslips. Images of the DAF-16:GFP signal was quickly taken with a Nikon TiE fluorescent microscope. Animals were scored as having cytosolic or nuclear localization when localization was observed throughout the entire body or intermediate localization when a mixed distribution pattern was shown. This assay was repeated at least three times and scored by two different individuals.Strain TJ356 n = 10 or 15) were transferred to fresh NGM plates with bacterial lawn. Each worm was placed in one plate and transferred to a fresh NGM plate every 24 h until egg-laying had ceased. The number of hatched worms on each day was counted after 72 h of incubation at 20 °C. The brood size of each worm was the total number of hatched progenies during the assay. The reproductive lifespan of each worm was the time period that it was capable of laying eggs.Worms , and fixed in 2 × MRWB-PFA. Then, worms were washed and dehydrated in isopropanol. After that, isopropanol was replaced with a new ORO solution. After ORO staining, animals were mounted on slides, and images were taken by a Carl Zeiss Axio Imager 2.t-tests. Error bars were presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 8 .The lifespan assays were analyzed using the Kaplan-Meier method and the log-rank test. Brood size assay, pharyngeal pumping rate assay, and ORO staining were analyzed using unpaired Additional file 1: Table S1. List of C. elegans strains used in this study.Additional file 2: Table S2. Supporting data.


Objective: In livestock production, antimicrobial resistance (AMR) is considered an externality as it is the undesired result of preventive and curative antimicrobial use. To address this biosocial issue, our objective is to present an approach based on interdisciplinary research to develop strategies and policies that aim to contain AMR.Method: To do so, we addressed three fundamental questions on which control policies and strategies for agricultural pollution problems are centered in the light of AMR. To ensure the technical, economic, behavioral and political feasibility of the developed measures, we demonstrated the usefulness of systemic approaches to define who, what and how to target by considering the complexity in which the ultimate decision-maker is embedded. We then define how voluntary or compulsory behavioral change can be achieved via five routes, introducing a clear taxonomy for AMR Interventions. Finally, we present three criteria for ex-ante analysis and ex-post evaluation of policies and strategies.Conclusion: Interdisciplinary systemic approaches enable the development of AMR policies and strategies that are technically, politically, economically and, last but not least, behaviorally feasible by allowing the identification of (a) all actors influencing AMU in livestock production, (b) power relations between these actors, (c) adequate regulatory and intervention bases, (d) what behavioral change strategy to use, (e) whom should implement this, as well as the cost-effective assessment of combinations of interventions. Unfortunately, AMR policies and strategies are often investigated within different disciplines and not in a holistic and systemic way, which is why we advocate for more interdisciplinary work and discuss opportunities for further research. For the two past decades, concerns regarding antimicrobial use in farm animals grew considerably due to the growing prevalence of antimicrobial resistance (AMR) and the way this affects human health. AMR is a natural process that results from the ability of microorganisms to quickly adapt to changing conditions. Indeed, the appearance of rare and advantageous mutations that neutralize the effects of antimicrobials is inevitable in large and dense microbial communities and the rapid generation times allow these mutations to quickly become prevalent in growing communities . AdditioBut while AMR is a natural phenomenon, its increasing prevalence is most certainly not. In fact, it is fueled by anthropogenic factors such as the intensive clinical and agricultural use of antimicrobials worldwide, the growth of the world's human population, changes in human lifestyle , and misconceptions and malpractices regarding antimicrobial use (AMU) . Over tiTo contain this serious threat to global health and wealth, the 194 member states of the World Health Organization (WHO) endorsed a global action plan (GAP) in 2015 and committed to establishing national action plans (NAPs) based on the “One Health” approach, which recognizes the interaction between human health, animal health and the environment , 7, woulIn the European Union, this political will to contain AMR has led to a European strengthening of the response to AMR with the development of an EU One Health action plan against AMR and new EU regulations on veterinary medicines [Regulation (EU) 2019/6] and medicated feed [Regulation (EU) 2019/4] . In pracTo address the challenges posed by antimicrobial resistance, countries have been advised to invest in AMR containment  through In this respect, our objective is to add to an interdisciplinary research agenda by providing a perspective on strategies for reducing the dependence on AMU and the threat of AMR from a social science and economic point of view. This perspective was inspired by how social scientists and economists contributed to environmental policies . We discwho among those who play a role in the production of an externality should be targeted. The second question aims to determine the basis for measuring effectiveness or, in other words, what variable(s) control policies and strategies wish to change. Finally, the third question is how to target, i.e., by what mechanism(s) the intended actors and bases should be targeted.Since AMR in livestock and agricultural pollution are both externalities, the design of policies and control strategies for the latter might also be useful for the former. We therefore used three fundamental questions on which the design of policies and control strategies for agricultural environmental pollution is centered  to develTo reduce antimicrobial use and dependence in livestock production, it is necessary that farmers, as ultimate users, and veterinarians, as antibiotics prescriber, change their behavior. It is thus only logical that policies and strategies target them. This is the case in Denmark, where veterinarians do not have the right to sell veterinary drugs  and pig However, the solution to the question “who to target” does not need to be limited to the actual users of the antimicrobials. Indeed, when dealing with externalities, it has been suggested that, in addition to the actual source, others could be targeted . This idTo take a systemic look at antimicrobial use in livestock production, When considering the value chain, it becomes more clear that besides farmers and veterinarians, other actors of the value chain could be targeted. In the upstream part of the value chain, input suppliers such as feed mills could be subject to policies, e.g., by banning or further regulating the production of certain inputs like non-medicated feed to avoid cross-contamination with antimicrobial residues , 45. TarWhen looking at the societal system in which a value chain is integrated, the actors it comprises may also influence actors and practices in the value chain. In Such external actors could also be targeted by policies by for example subsidizing control agencies or farmers' organizations to develop communication campaigns to raise awareness. Universities could be expected to improve courses on AMR in the curriculum of veterinarians or farmers  or couldIn this section, we will explore different options that may provide an optimal basis for measuring impact, or in other words, a variable that policies and strategies are intended to change. To serve as an optimal base to formulate a regulation or strategy and to measure compliance with that regulation/strategy, any elements in the input/technology-production and AMR relationship can be used as long as they are (a) correlated with AMR; (b) enforceable; and (c) targetable in space and time .To identify several bases that could be used, we systemically analyzed the production segment of a livestock value chain. This exercise may of course be expanded to other segments of a value chain or layers of a system, as done by the UK government who produced AMR systems map to provide an overview of the factors influencing the development of antimicrobial resistance and the interactions between them in a one health context . The decStaphylococcus aureus (MRSA) in pig farms in Norway  since different animal species metabolize drugs differently or the amount of output as consuming less animals could also reduce the use of antimicrobials . HoweverFinally, we should keep in mind that AMR can also be introduced in a farm through interactions with the environment or the ‘outside world’. Examples of such interactions include mutual use of farm workers or veterinary practitioners that can introduce resistant microorganisms , deliverWith regard to interactions between farms and the ‘outside world’, it might be more complicated to find compliance bases. In such cases, AMR proxies such as monitored AMR trends could be used. Such options have a higher correlation with AMR but are less attributable to a producer than input/technology and production bases. Moreover, the surveillance of AMR trends in the European animal production is currently limited and results are published with a 2-year delay. To compensate, national surveillance systems have been put in place, each with their own sampling, testing and reporting modalities, yielding results that cannot be compared .Once it is defined who and what should be targeted, the next step is to determine the mechanisms through which the intended actor(s) and variable(s) can be targeted. This entails the choice for a policy instrument, an advisory approach, a structural intervention and other types of options. To change behavior, several intervention frameworks have been developed for different contexts such as policy making and retail . While tAccording to Van Woerkum's framework, a first way to achieve behavioral change involves regulation . This roTo induce a voluntary change, the second route includes provisions and tools, which are instruments that are implemented to change the external material circumstances so that people become motivated to change their behavior. In some cases, the provisions can be restrictive (by making the ‘bad’ behavior less straightforward) and behavioral change is then coerced. However, in the case of AMR and AMU, most provisions and tools are rather enabling and give the intended person external motivation to voluntarily change by making it easier and more achievable to reduce and improve the use of antimicrobials. Examples of tools include coaching sessions to develop and implement farm health plans  and alteAnother way to change external circumstances involves the use of economic and financial incentives. Here, the attempt is to create external circumstances that change the financial conditions in such a way that behavioral change is favored. Typical examples include subsidies for the ‘good’ behavior or taxes (fines) for the “bad” behavior. In the European Union, several countries have levied a tax on the sale of antibiotics , 76. PriIn addition, a third and more direct method to change external motivation of decision makers relates to group pressure and social norms. This mechanism attempts to induce behavioral change by making use of the typical desire of people to comply with the group norm. It is the attempt to use existing social norms, and make people aware of these norms. In some cases, new social norms need to be formed prior to this, if the social norms within the target group does not support the intended behavioral change. An example for the use of group pressure is the benchmarking of farmers and/or veterinarians based on AMU in Belgium, Denmark and the Netherlands .Finally, the last route to voluntary behavioral change goes through communication and education. Through these mechanisms, change agents attempt to change the internal motivation of decision makers so that they become convinced that behavioral change is the best decision. Newly developed tools or economic studies that demonstrate the cost-effectiveness of measure to reduces AMU, such as improved management strategies   can be uThe design of policies and strategies to reduce antimicrobials resistance is essentially guided by three criteria, being effectiveness, efficiency, and fairness (equity). These criteria should be used when evaluating, both ex-ante and ex-post, the performance of policies and strategies. Effectiveness refers to the question whether the implemented policy or strategy achieves its goal, i.e., a reduction in AMR. Policies or strategies that are not effective should not be further considered. In a world with unlimited resources, effectiveness would in fact be the only criterion of importance. However, this is not the case, especially for financial resources and time.All policies and strategies within each of the five behavioral routes involve the allocation of resources. At farm level, policies and strategies usually aim to change AMU, biosecurity and production practices in general and/or to stimulate the adoption of alternative disease management measures. All this involves costs and benefits of which the end result may be negative or positive. Producers of antimicrobials may incur financial losses if the use of antimicrobials is drastically reduced. Policies and strategies such as the use of social norms, communication and education require the investment of financial resources and time by extension agents, researchers and other organizations. As resources are limited, the allocation of resources to one type of policy or strategy may come at the expense of another. Hence, resource allocation of animal health control in general and the reduction of antimicrobial resistance in particular, has to be informed by structured analyses , 84. TwoThe economic efficiency of a policy intervention is the greatest when the social benefits net of social costs are maximized, regardless of how these may be distributed . The twoOne shortcoming of cost-effectiveness analysis or CBA is that they do not consider the distribution of costs and benefits across all actors involved. Important criteria related to this are equity and fairness. While both concepts are related, they are not identical. Equity refers to the mere distribution of costs and benefits of different policies and strategies. Typically, these are not equally distributed over society, especially with an issue like AMR. Whereas the benefits are usually for society at large, through a decreased human health burden, costs often accrue to one or more specific groups, such as farmers, veterinarians or pharmaceutical companies. Nonetheless, the distribution of costs and/or benefits between different regions or groups may be a considerable measure when choosing between different policy options. Likewise, unequal distribution of costs and benefit can give rise to substantial opposition against new policies or strategies and may be the source of considerable lobby group efforts to weaken more severe regulation.A problem is that equity considerations have been very resistant to rigorous analytical treatment. One of the reasons is the many competing notions of equity, which makes that the concept is analytically slippery. This becomes more clear when the notion of equity is replaced by the related concept of fairness. Whereas equity considers the mere distribution of costs and benefits across society, the concept of fairness refers to whether that distribution is socially just and acceptable. Fairness is a concept with which analysts are not comfortable, because it is open to subjective evaluation. One principle to overcome some of this difficulty is the Polluter Pays Principle (PPP), an environmental policy principle which requires that the costs of pollution be borne by those who cause it. In its original emergence, the PPP determined that the costs of pollution prevention and control must be allocated to the polluter. Its immediate goal is that of internalizing the environmental externalities of economic activities, so that the prices of goods and services fully reflect the costs of production .Another concept related to this is the political feasibility of options if dealing with options that have to be decided upon and set by the government, such as public standards and taxes . Even ifIn our introduction, we discussed the global political will to contain AMR through surveillance and optimal antimicrobial use and prescription. For the latter two, strategies are focused on reducing use and dependence, often without taking the behavioral character of AMU and AMR into consideration. To better visualize the interconnection between human decisions concerning AMU and AMR in livestock production, we looked at it from a systems perspective and adapted “the fix that fails” system archetype to represent the relationship between AMR and AMU. System archetypes are causal loop diagrams—or visual representations of balancing (B) and reinforcing (R) processes in a system—that seem to recur in many different life settings. The “fixes that fail” archetype involves the quick implementation of a solution to alleviate symptoms . The relThis simple representation of the interrelationships between animal morbidity, solutions and consequences allows to easily visualize the imbalance that has been created by repeatedly reinforcing the same feedback loop as well as the unintended consequence that results from it. In order to solve this, global efforts are being made to contain AMR, mainly by institutionalizing AMU and by trying to reduce the dependence on AMU, the former acting on the first balancing loop (B1) in Addressing AMR in a sustainable manner is therefore based on the development of strategies and measures that aim to achieve an equilibrium where animal morbidity is reduced through solutions that enhance/preserve animal health, antimicrobials and their alternatives without compromising the effectiveness of the latter two. The effectiveness of these strategies and policies relies on whether these are technically, economically, behaviorally, and politically feasible in a certain temporal and spatial context.In order to develop such strategies and policies, we addressed three fundamental questions on which control policies and strategies for agricultural pollution problems are centered in the light of AMR . We demovia participatory approaches, which could be considered by some as a shift from interdisciplinarity to transdisciplinarity  approaches in order to develop solutions that are adapted to the context in which decision-makers are embedded.Economic analyses in order to identify cost-effective combinations of interventions from different behavioral change routes at system level.The writing was mainly performed by FB and EW. All authors contributed equally to the conceptual thinking and editing of this paper.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.


Objective: This study aimed to explore topics and sentiments using tweets from Ontario, Canada, during the second wave of the COVID-19 pandemic.Methods: Tweets were collected from December 5, 2020, to March 6, 2021, excluding non-individual accounts. Dates of vaccine-related events and policy changes were collected from public health units in Ontario. The daily number of COVID-19 cases was retrieved from the Ontario provincial government’s public health database. Latent Dirichlet Allocation was used for unsupervised topic modelling. VADER was used to calculate daily and average sentiment compound scores for topics identified.Results: Vaccine, pandemic, business, lockdown, mask, and Ontario were six topics identified from the unsupervised topic modelling. The average sentiment compound score for each topic appeared to be slightly positive, yet the daily sentiment compound scores varied greatly between positive and negative emotions for each topic.Conclusion: Our study results have shown a slightly positive sentiment on average during the second wave of the COVID-19 pandemic in Ontario, along with six topics. Our research has also demonstrated a social listening approach to identify what the public sentiments and opinions are in a timely manner. The Coronavirus disease COVID-19) pandemic has persisted for more than a year and resulted in over 141 million infections with over 3 million deaths worldwide 9 pandemi. During Machine learning (ML) techniques have been applied to such research in topic modelling and sentiment analysis. For instance, Latent Dirichlet Allocation (LDA) is commonly applied to topic modelling, and Valence Aware Dictionary and sEntiment Reasoner (VADER) is widely used for sentiment analysis –6. LDA iSocial media and Internet query data have also been used to predict the number of COVID-19 cases –13, espeAlthough recent literature has demonstrated the utility of social media data for various research, unsupervised ML approaches have not been studied as extensively as supervised ML techniques. In other words, data collection in current literature is conducted with pre-identified keywords or hashtags of research interest, and these pre-defined keywords have also been used as criteria for topic classifications –6, 10–18While LDA and VADER have been commonly used for topic modelling and sentiment analysis, respectively, deep learning techniques, such as Long Short-Term Memory (LSTM) and Bidirectional Encoder Representations from Transformers (BERT), have become more successful and have been adopted in more recent literature for sentiment analysis –24. For On the other hand, most studies conducted in the first wave analyzed data with a relatively shorter time frame, several weeks or 1 month, for example –6. Furthvia the Twitter Developer API [English tweets originated in Ontario, Canada, were collected between December 5, 2020, and March 6, 2021, without being filtered by COVID-19 related keywords or hashtags. This is because our study aimed to apply an unsupervised ML approach to identify possible patterns beyond those explicitly mentioning COVID-19 related keywords or hashtags. Instead, major metropolitan areas in Ontario, namely geolocation tags from Toronto and Ottawa, were identified to limit the scope of data collection to a level allowed by available resources. With these query parameters limited to two cities and the timeframe, 569,467 tweets from Toronto and 141,469 tweets from Ottawa were returned from December 5, 2020, to March 6, 2021, oper API .Data were collected on Ontario public health policy changes by each public health unit as a means of comparison against daily case changes and Twitter sentiment from December 5, 2020, to March 6, 2021. Public health policy changes include major vaccine approvals, lockdown announcements, school closures and other public health related enforcement of policies regarding COVID-19 by reviewing each public health unit’s COVID-19 information page within the applicable regions.The daily case counts of all public health units in Ontario from December 5, 2020, to March 6, 2021 were retrieved from the Ontario provincial government’s public health database. The extracted data were organized by active cases, resolved cases, and deaths as defined by the Government of Ontario’s health unit.VADER is used to identify sentiment as it calculates the sentiment attached to individual tokens in text and is adjusted to understand specific tokens commonly used in English social media text . For eac1 and +1 . The com1 and +1 .To determine the accuracy of the unsupervised LDA topic modelling and sentiment analysis. Three researchers—S-FT, HC, and ZB—conducted inter-rater manual validations for 3% of random tweets from each topic and their sentiment. S-FT was the primary rater, HC and ZB served as secondary raters. The 3% random sample is calculated according to Krippendorff’s sampling method with 10% probability of the rarest relevant instances and 95% desired significance level of the answers to our research question . Inter-rThe numbers of active COVID-19 cases and deaths between December 5, 2020, and March 6, 2021, in Toronto and Ottawa, Ontario, are shown in The average sentiment compound score for the topic “lockdown” (in Supplement B The average sentiment compound score for the topic “mask” (in Supplement B The average sentiment compound score for the topic “Ontario” (in Supplement B The average sentiment compound score for the topic “pandemic” (in Supplement B The average sentiment compound score for the topic “vaccine” (in Supplement B Like the “vaccine” topic, the average sentiment compound score for the topic “business” (in Supplement B To our surprise but understandable, there was no obvious correlation between sentiments and cases or key events on average when the pandemic had lasted over 6 months when Ontario, Canada declared its second provincial emergency and lockdown. Sentiments were more related to holidays as positivity was higher from Christmas 2020 to New Year Day, 2021, although the second provincial shutdown kicked in on December 26, 2020. As Positive sentiments across topics are mainly associated with holidays and support for the recommended public health practices such as wearing masks and vaccinations. In contrast, negative sentiment across topics largely reflected frustrations and blame on incompetent political leadership. It is also interesting to observe that Greg Abbott, Texas Governor in the United States, was negatively discussed on Twitter among Canadian users when he lifted the mask mandate in Texas on March 2, 2021 . He was We did not expect the unsupervised LDA approach for topic modelling to show very limited interpretability in our study. In Most existing studies used tweets no later than August 2020, when the first wave of the COVID-19 pandemic was over. In addition, most countries had several lockdowns during the first wave –6. HowevIn addition, our study collected and analyzed tweets over 3 months, which is very different from existing research that collected and analyzed data rarely more than a month . AlthougHowever, our study has several limitations. First of all, due to Twitter’s geolocation query methods, only a subset of tweets has geotags identified. It has been estimated that only about 2.31% of tweets with locations are attached to the tweets, and an even smaller number of those have precise locations . As suchFurthermore, our manual quality validation identified that although small, there is a chance that VADER can misclassify sentiments. For instance, a tweet with sarcasm was regarded as positive but in fact, it should have been considered negative. On the other hand, a tweet with a “surprised” mood was regarded as negative because the word “shocking” was repeated many times. Therefore, it is possible that our sentiment analysis is not perfectly accurate as it should have been. Moreover, tweets classified in each topic were not exclusive to other topics. If a user mentioned several topics within a tweet, it would be assigned to multiple topics, such as the case of Greg Abbott that showed up in both “Ontario” and “business” topics. However, we decided to leave them as they were because it would be inappropriate to assign only one topic to tweets with multiple topics as they naturally occurred. This observation actually showed that some topics could be highly correlated. Accordingly, how to properly choose keywords and their synonyms as filters remains an important challenge to be addressed.In conclusion, our results have shown that Ontarians in Toronto and Ottawa have remained a slightly positive sentiment during the second wave of the COVID-19 pandemic regardless of topics. We also identified six topics that emerged from data over time, and these topics have been highly correlated with the ongoing pandemic, although the average positive sentiment could be driven by the Christmas-New Year holiday break amid the second wave rather than by the public health interventions. Compared with prior studies conducted during the first wave [


The last two years, despite the very serious COronaVIrus Disease-2019 (COVID-19) pandemic, have been quite productive in the field of molecular endocrinology and metabolism and our journal section has contributed extensively on that. Several publications were about steroid hormones, such as estrogens and progesterone . Other aOsteoporosis has also been a favorite topic of investigation, even with its connections to vitamin D deficiency and allergy ,7; in thThis extensive interest over the last two years on the molecular aspects of diabetes, obesity, and their cardiometabolic consequences is not surprising, since a higher number of cardiovascular complications have been reported during the COVID-19 era . In addiThis emphasizes the importance of investigating the molecular mechanisms of diabetes and other metabolic alterations, such as obesity, that can increase cardiovascular risk; new Special Issues in the section “Molecular Endocrinology and Metabolism” of the International Journal of Molecular Sciences are currently accepting contributions dealing with the mechanisms of insulin resistance and adipose tissue dysfunction ; obesityIn summary, the section “Molecular Endocrinology and Metabolism” of the International Journal of Molecular Sciences is committed to continue to publish high-quality articles in the field, in order to make a significant contribution to the international scientific and medical community. There are many fields that are still awaiting to be covered, such as: (a) novel insights into physiology, pathophysiology, and therapeutics; (b) cellular interactions and factors involved; (c) cross-disciplinary and integrative studies; and (d) comparative aspects of endocrinology. We can, therefore, use this difficult COVID-19 pandemic time as an opportunity to provide the best updated scientific information and valuable novel data.


KMT2D or KDM6A. It is characterized by infantile hypotonia, developmental delay and/or intellectual disability, long palpebral fissures with everted lateral third of the lower eyelids and typical facial features. Intracranial anomalies occur infrequently in patients with KS and holoprosencephaly has only been recently described. Additionally, though congenital heart diseases are common in patients with KS, to our knowledge truncus arteriosus has never been reported in a patient with KS. We present an unusual case of KS in an infant with holoprosencephaly and truncus arteriosus with partial anomalous pulmonary venous return. Duo whole exome sequencing in our patient identified a pathogenic nonsense variant in exon 10 of KMT2D  establishing the diagnosis. This report further expands the phenotypic spectrum of patients with Kabuki syndrome and emphasizes the utility of performing large scale sequencing in neonates with multiple congenital anomalies.Kabuki syndrome is a rare multiple anomalies syndrome associated with mutations in  KMT2D) (Chr12q13.12) (55–80%) and lysine demethylase 6A (KDM6A) (Chr Xp11.2) (9–14%) were later identified to be causative for KS , skeletal anomalies, dermatoglyphic abnormalities, persistent fingertip pads and intellectual disability (ID); however, many of the characteristic facial features are not clearly evident in early infancy (Kabuki syndrome (KS) [OMIM# 147920 (KS1) and 300867 (KS2)] is a rare pleiotropic, monogenic syndrome that was first identified in 1981 in 10 Japanese patients . Variante for KS . KS is a infancy .Several structural brain anomalies have been recently reported in patients with KS. In 2019, 3 cases of KS1 with holoprosencephaly (HPE) were reported for the first time. Although congenital heart defects (CHDs) have been reported in 70% of patients with KS1, only one of the three cases of KS1 with HPE had right ventricular dilatation and hypertrophy and none of them were reported to have structural heart defects . AdditioThe patient was a female infant born at 32 weeks of gestation. The mother was 19 and the father was 25 at the time of birth. The mother previously had one healthy daughter with a different partner. Prenatal history was significant for late and limited prenatal care in second trimester and lack of folic acid supplementation during the first trimester. The mother denied history of drugs, tobacco or alcohol use. There was no history of diabetes mellitus. Per mother, the facial features of the fetus were not well-visualized during any of the prenatal ultrasounds. The patient was born via precipitous vaginal delivery. Her birthweight was 1.68 kg (<1st percentile) with a head circumference of 27.7 cm (22nd percentile) and length 41 cm (46th percentile). She was initially placed on non-invasive ventilation. Her oxygen saturation remained below 90% despite 100% oxygen. This prompted an echocardiogram which revealed a large ventricular septal defect (VSD), a small atrial septal defect (ASD), probable mitral atresia and probable D-transposition of great arteries (TGA) with adequate ventricular function. Chest X-ray at the outside hospital showed a cystic lesion in the right upper lobe which was concerning for a cystic-adenomatoid malformation (CCAM) pushing the heart inferiorly. She was intubated and transferred to our neonatal intensive care unit (NICU) on day of life two for higher level of care.On admission, her examination was significant for midline cleft lip and palate, hypotelorism, up-slanting palpebral fissures, absent nasal septum, large, prominent ears, clinodactyly, a single flexion crease of the right fifth digit, a single transverse palmar crease on the left, rocker-bottom feet with talipes equinovalgus, anterior anus, sacral dimple, possible craniosynostosis and a single umbilical artery . X-ray oDue to multiple, complex congenital anomalies, the parents decided to withdraw life-sustaining measures. The infant died at 8 days of age.In addition to the above findings, autopsy further showed absence of olfactory tracts and bulbs, diffuse cortical dysplasia, absence of the nasal bone, small unilobular right lung with type II congenital pulmonary airway malformation (CPAM) and abnormal right diaphragm with partial liver herniation .https://genescout.omim.org). The ROH on chromosome 2 contained KYNU, which is associated with vertebral, cardiac, renal and limb defects syndrome 2 , which includes some of the features seen in our patient but not all. We also investigated the homozygous regions for syndromes of uniparental disomy (UPD). Of the chromosomes with ROH, imprinting disorders have been associated with UPD of chromosomes 6, 11, 15 and 16   which revealed a normal female genomic profile with multiple regions of homozygosity (ROHs) . We sear5 and 16 . Materna5 and 16 . While tKMT2D (NM_003482.3) c.2782C > T (p.Gln928Ter) was identified in exon 10, which leads to replacement of the glutamine at amino acid position 928 with a termination codon. This variant was classified as pathogenic for KS1 based on the American College of Medical Genetics (ACMG) guidelines. This classification was supported using the following criteria: PVS1 , PM2 , a half point for PM1  and another half point for PS2  to be de novo). This KMT2D variant has been reported once in literature in a patient with Kabuki syndrome who did not have HPE (KYNU were identified by WES.Duo whole exome sequencing (WES)  was performed on patient’s and mother’s blood. We were unable to obtain a paternal sample. The following phenotypic terms were applied for analysis: ASD, VSD, dextro-transposition of the great arteries, hypoplastic left heart, PAPVR, pulmonary arterial hypoplasia, possible pneumomediastinum, pneumothorax, cystic abnormality of upper lobe of right lung, possible congenital cystic adenomatoid malformation or congenital pulmonary airway malformation, alobar holoprosencephaly, microcephaly, craniosynostosis, ectopic pelvic right kidney, sacral dimple, thoracic vertebral abnormalities, malformation of S2 vertebra, anteriorly displaced anus, hypotelorism, upslanting palpebral fissures, vascular flat marking on forehead, cleft lip and palate, hypoplastic nasal bone and low-set ears. 99.37% of target bases in disease causative genes were fully covered. The average coverage per target base was 135.54. A complete list of genes analyzed is provided in have HPE . No pathKMT2D  or KDM6A (X-linked dominant) and to date over 500 cases of genetically confirmed KS have been reported in literature. The 2019 international consensus criteria for definitive diagnosis of KS, require a history of infantile hypotonia, developmental delay and/or ID with pathogenic/likely pathogenic variant in KMT2D or KDM6A and/or typical dysmorphic features   . Howeverip pads) . Our patKMT2D and KDM6A are interacting members of the histone methyl transferase family that participate in chromatin remodeling, thereby impacting the transcription of a variety of downstream genes . TA has never been reported to occur in a patient with Kabuki syndrome. Of the 32 patients with molecularly confirmed KS who had structural brain anomalies (including the present case), 53% were found to have CHD  have been reported to occur in up to 80% of patients with KS-septal defects are the most common followed by coarctation of the aorta and bicuspid aortic valve . Conotruhave CHD .KMT2D mutation associated with TA. The KMT2D histone methyltransferase has been shown to control neural crest cell (NCC) differentiation and facial morphology (To our knowledge, this is the first case of rphology . NCCs anrphology .KMT2D/KDM6A pathogenic mutations with HPE and/or TA and other conotruncal abnormalities will be identified. This case further illustrates the clinical utility of WES, providing an answer that would not have been attainable given the absence of important, defining clinical features early in life. Establishing the diagnosis helped to provide closure for the family and informed recurrence risk for future pregnancies. In general, WES and WGS also provide important information regarding prognosis and surveillance measures and inform clinical management decisions (Although we cannot absolutely determine whether HPE and TA are part of the KS phenotypic spectrum or if they occur coincidentally, we did not identify any other genetic/environmental cause of HPE or TA in this patient, and patients with Kabuki syndrome have presented with features that fall within either the HPE or conotruncal spectrum. Unlike neural tube defects, folic acid supplementation has not been shown to protect against HPE . It is pecisions .As many of the classical features of KS are not apparent until later in life, neonates with KS with critical congenital anomalies may remain undiagnosed clinically. KS should be included in the initial differential diagnosis of all neonates with HPE and/or conotruncal heart defects, as the association with KS might be higher than previously recognized. Early WES or WGS in the neonatal period may help identify more cases of KS, further expand the neonatal phenotype of this rare syndrome and help the medical team to appropriately counsel families.


Brucellosis is widespread globally and one of the most important zoonotic diseases. Therefore, to fully comprehend the disease and discover ways of prevention and treatment, researchers have conducted some research in this field. Hence, this study will focus on the topic trend of scientific publications of brucellosis.  This study is an applied research using text mining techniques with an analytical approach. The statistical population of the present research is all global publications related to brucellosis. For data extraction, the Scopus citation database was used in the period from 1900 to 2020. The main keywords for search strategy design have been extracted from consultation with thematic specialists and using MESH. Python programming language has been applied to analyze data and implement text mining algorithms.  According to results, eight main topics of “Prevention,” “Clinical symptoms,” “Diagnosis,” “Control,” “Treatment,” “Immunology,” “Structural Features,” and “Pathogenicity” have been identified for brucellosis publications. Moreover, the topics “Prevention” and “Pathogenicity” had the highest and lowest prevalence in the field of brucellosis over time, respectively.  This study has revealed the topics published in the global publications of brucellosis; the findings can be useful for research centers and universities in determining research priorities in the field of brucellosis. Brucellosis is one of the zoonotic infections between humans and animals, known as undulant fever in humans and brucellosis in animals. The disease is globally widespread due to the dissemination of the infection among domestic and wild animals . The conDue to the importance of the disease, researchers have conducted many studies in this field and published their findings to reach a broad understanding of the disease and discover methods of prevention and treatment. In recent years, due to the increasing rate of online article submission in the information databases apace with the annual publication trend of research, manual review of this research is impossible or extremely time-consuming . TherefoInvestigating the publication trend of articles in various scientific fields can provide a proper insight into the efforts of researchers in various fields of knowledge, especially in life sciences. Reviewing the published articles in the field of biomedical sciences makes it possible to identify alterations in the field of knowledge, intelligent classification of information, and identification of different subject areas of knowledge. Therefore, it is necessary to use the methods and techniques of automatic and intelligent investigation of scientific publications.Furthermore, investigating the publication trend of each scientific field in a specific period represents how much its main issues and subtopics have changed over this time and what subfields each scientific field includes. Moreover, the review of research topics in each scientific field indicates that the highest and lowest research popularities during different periods are assigned to what topics ; these rIn this regard, text mining is one of the techniques capable of intelligent data and knowledge extraction from a large volume of scientific publications in various fields of medical sciences, and its purpose is to extract hidden knowledge from texts . As one In this regard, various studies in different fields of medical sciences have applied text mining and topic modeling techniques to discover hidden knowledge and identify topics in scientific publications, for example, studies in the subject areas of anesthesia , drug saAccordingly, the analysis of texts published in citation databases and identifying research trends in various scientific fields is one of the most important applications of text mining. Therefore, discovering and analyzing the structure of topics and identifying the topic evolution of scientific publications of brucellosis over a specific time are the main issues addressed in the present investigation.This study is applied research that has been carried out by applying text mining techniques and an analytical approach. The statistical population includes all scientific publications of the subject area of brucellosis in the Scopus citation database, which is considered a suitable database for scientometric research due to its comprehensiveness for various fields of science and indexing a significant number of articles –19. Medi(TITLE (brucellosis) OR TITLE  OR TITLE  OR TITLE (rock AND fever) OR TITLE (cyprus AND fever) OR TITLE (brucella AND infection) OR TITLE (brucella AND infections) OR TITLE (undulant AND fever) OR TITLE (brucellosis) OR TITLE (bangs AND disease) OR TITLE (bang AND disease) OR TITLE-ABS-KEY (brucella) OR TITLE (pulmonary AND brucellosis) OR TITLE (mediterranean AND fever)).Titles, abstracts, and keywords of retrieved publications were merged to perform the text mining process. Afterward, preprocessing and data cleansing operations were applied to the studied data for increasing data quality and validity of patterns and extracted relationships. It is noteworthy that data cleansing only maintains the required and relevant textual data .Eliminating unimportant graphemes, including extra empty spaces, text formatting tags, and removing nonalphabetic graphemes such as deleting punctuation marks or numbers from the text; these operations lead to focusing on the words and phrases in the text .Tokenization of the text components into words and phrases, which is one of the essential processes of text preprocessing in text mining .Converting capital letters to lowercase letters for the purpose of text uniformity; since capital and lowercase words are stored in a separate and different computer language, converting all capitals to lowercase letters makes all words uniform .The uniforming of synonymous words was performed manually. The lemmatization method was also applied to make different forms of uniform words. Lemmatization is replacing words or their basic dictionary forms with the conjugated forms of words . LemmatiRemoving stop words and some words that are not valuable for retrieving or analyzing documents , such asThe following steps were implemented to perform data cleansing operations:Topic modeling is a statistical and textual analysis method that evaluates documents to identify their themes or topics. The results of this algorithm can be used to analyze how topics relate to each other and how they evolve over time . Topic mIn the present research, a topic modeling algorithm named the latent Dirichlet allocation (LDA) has been applied for topic modeling operations. LDA is one of the most important methods of implementing topic modeling and one of the best and most extensively used algorithms, which significantly identifies relevant semantic issues in scientific texts .Latent Dirichlet allocation (LDA) is a probabilistic model that can extract latent topics from a collection of documents. According to Blei et al., topic modeling assumes that each document of a corpus is structured by a set of hidden topics . By applMany social scientists have used topic modeling, especially latent Dirichlet allocation (LDA), which is the most popular method of topic modeling , 29, 30.Since the LDA method cannot determine the number of appropriate topics, the coefficient of variation (Cv) coherence measure was applied. An important index, called Cv coherence, is employed for measuring the co-occurrence of the words extracted using the topic model. The model's great performance is interpreted if the words from similar topics co-occur frequently, otherwise stated, in the case of high Cv coherence .It was indicated in previous studies that the performance of this criterion is excellent in determining the number of topics and is closely related to human judgments about the interpretation of topics , 33.In this article, at the outset, the number of main topics was obtained for all data of brucellosis publications, and then, the determination of the number of topics and topic modeling was performed for publications related to each of the obtained topics; hence, the subtopics dependent on each main topic were also identified.Furthermore, after the words were identified, the publications related to each topic were labeled for that topic by consultation with a topical expert.Python programming language and text mining-related libraries, including Gensim, NLTK, and SpaCy, have been applied to implement these steps . Python A total of 25,846 publications related to brucellosis were extracted from the Scopus database. The LDA topic modeling has been used to select the number of topics. The results obtained from topic modeling are shown in the word cloud presented in Furthermore, It is indicated in In the topic “Prevention,” the highest publication rate is dedicated to the subtopic “Vaccine” with 49.96%, and the subtopic “Bioterrorism” has the lowest publication rate with 2.80%.In the topic “Diagnosis,” the highest publication rate belongs to the subtopic “Elisa” with 33.70%, and the lowest publication rate belongs to the subtopic “Brucellacapt” with 1.40%.In the topic “Clinical symptoms,” the subtopics “disease” with 25.25% and “Fever” with 1.24% have the highest and lowest publication rates, respectively.In the topic “Immunology,” the subtopic “macrophage” with 35.98% has the highest publication rate, and the subtopic “lysosomal” with 0.12% has the lowest publication rate.Brucella” with 36.67% have the highest and lowest publication rates, respectively.In the topic “Pathogenicity,” the subtopics “pathogens” with 63.33% and “In the topic “control,” the subtopic “vaccine” with 51.66% and the subtopic “patient” with 48.34% have the highest and lowest publications rates, respectively.In the topic “treatment,” the subtopic “melitensis” with 51.95% has the highest publication rate, and the subtopic “Bacteria” with 8.59% has the lowest publication rate.According to Brucella is a Gram-negative, optional intracellular, nonmotile, non-spore-forming coccobacillus causing brucellosis [cellosis . Undulancellosis . Human bcellosis , 37. Acccellosis . TherefoAccording to the results of this study, eight main topics have been identified for the scientific publications of brucellosis by investigating the scientific publications of brucellosis. “Prevention,” “Clinical symptoms,” “Diagnosis,” “Control,” “Treatment,” “Immunology,” “Structural features,” and “Pathogenicity” are in the respective order in terms of highest to lowest publication rates for these eight main topics.The topic “prevention” has the highest publication rate in the field of brucellosis. This topic includes the subtopics “Milk,” “zoonotic,” “livestock,” “bioterrorism,” and “vaccine.” It was also found that most publications on this topic have been dedicated to the subtopic “Vaccine,” which indicates the important role of vaccine use in preventing brucellosis. It also means that research in the field of “vaccine” is in a progressive trend, and today's human need research in the field of brucellosis is focused on the vaccines. Nowadays, there are several types of vaccines available; some of them are suitable for human use and some others for animals. These vaccines are in various types, including “Live,” “Inactive cell lysate,” “Subunit,” “DNA,” “Synthetic peptide,” and “Live vectored” .“Clinical symptoms” was the second subject area of publications with high frequency, and “attack,” “child,” “lesion,” “disease,” acute, “fever,” “amyloidosis,” and “chronic” were its subtopics. The results demonstrated that the subtopic “Disease” had the most application in publications in this field. The children's involvement is also a major challenge in the field of health and, in this regard, has dedicated a large part of the literature on the topic “clinical symptoms.” The disease is known as the “Disease of mistakes” due to its nonspecific clinical symptoms. It was shown in extensive studies in this field that fever is the most obvious clinical sign of the disease and also the highest rate of infection in children and adults occurs in males, the reason of which is that men and boys, compared with women and girls, are more responsible for doing affairs related to livestock such as shepherding and milking .Brucella and other immunoglobulins [The subject area “Diagnosis” is in the third rank of publication rate of the field of brucellosis, which includes the subtopics “Brucellacapt,” “ELISA,” “detect,” “organism,” “positive,” “PCR,” “culture,” and “laboratory.” Rapid and precise diagnosis of brucellosis is important due to the multisystem involvement in the body. Culture and microbiological identification, serological tests including rose Bengal slide agglutination test, serum agglutination test (SAT), microagglutination test , indireclobulins . Accordi“Control” is another topic in brucellosis publications, which included the subtopics “Patient” and “vaccine,” and the “vaccine” has been the most studied subtopic in this topic. The term “vaccine” in the “prevention” set is also considered the most common topic in the field, which indicates its significance.melitensis,” and “antibiotic.” In this topic, the highest number of phrases has been dedicated to “melitensis.” Since Brucella melitensis has been the most aggressive among Brucella strains and constitutes the most human infections [“Treatment” is the next topic in brucellosis publications, which included the subtopics “Culture,” “bacteria,” “fections , it is oBrucella in the human body depends on its viability and proliferation in macrophage cells, the term macrophage has been the most investigated topic in the field of immunology. “Superoxide dismutase,” “Type IV Secretion System (T4SS),” “Cyclic β-1-2-glucans (CβG),” “Cytochrome oxidase,” “Nitric oxide reductase (NorD),” and “Brucella virulence factor A (BvfA)” are among the factors of Brucella virulence to fight against cells of the human immune system, especially macrophages [“Immunology” is the subsequent topic in brucellosis publications, which included the subtopics “Intracellular,” “expression,” “macrophage,” “pathway,” “caspase,” “neutrophil,” “response,” “nitric_oxide,” “apoptosis,” “lysosomal,” and “cytokine.” Since the pathogenesis of rophages . The nexBrucella” and “pathogen,” and the term pathogen has been the most considered term in this field.The results also revealed that the topic “protein” has the highest publication rate in articles related to the subject area “Structural features.” Moreover, “Pathogenicity” is the topic with the lowest publication rate in the field of brucellosis, which included the subtopics “In general, the high publication rates in the topics “Prevention,” “Clinical symptoms,” “Diagnosis,” and “Control” can be related to the global publication of articles, easier and faster access to research resources of basic medical sciences, modern scientific tools for studying these topics, and finally nowadays humans' need to prevention, recognition of clinical symptoms, and diagnosis and control of brucellosis. Besides, the low publication rates of the topics “Pathogenicity,” “Structural features,” and “Immunology” can indicate that the research life of these topics with current science and scientific tools is now coming to an end, and a complete scientific knowledge has been developed in these areas. If a new scientific research method or tool is discovered or invented in the world of experimental science, there would be an advancement possibility in scientific research and publications in these areas.In this context, similar studies tried to identify the topics of scientific publications of other microbial diseases. Danesh et al. have identified eight topics for the global spreading of coronavirus as follows: “structure and proteomics,” “Cell signaling and immune response,” “clinical presentation and detection,” “Gene sequence and genomics,” “Diagnosis tests,” “vaccine and immune response and outbreak,” “Epidemiology and Transmission,” and “gastrointestinal tissue” . DastaniScientific research in the field of clinical microbiology, in chronological order, and in terms of the evolution of scientific research primarily focuses on recognizing the nature of the pathogenic agent, clinical symptoms, diagnosis and treatment, and finally control and prevention of the pathogenic agent, which means that without recognizing the structural features of a pathogenic agent or its pathogenesis, the logical trend of scientific research will never be to seek treatment and control the pathogenic agent. In other words, each of the mentioned eight topical phrases has a specific research life, and the researchers' focus on the subject areas will change with increasing the scientific research on the subject area of brucellosis. However, some factors such as today's scientific need, advances in specific technologies , and researchers' hidden purposes  affect the logical trend of scientific research. According to the results of the present investigation, the publication of basic topics in the field of brucellosis has been on the rise with a constant logical trend, which can be justified by the advancement of technology and scientific tools. Moreover, some factors, such as the incidence of a pandemic, can also lead to a specific subject area being of high interest. In this regard, Danesh et al. have indicated that the publication trend in the field of coronavirus has been associated with a significant growth by the incidence and prevalence of each of MERS, SARS, and COVID-19 .The findings of this scientific investigation, as the first text mining study in the field of brucellosis publications, have shown the subject trend of brucellosis publications of the last 120 years. The text mining and investigation of subject areas of scientific topics in clinical microbiology assist in understanding the subject areas and today's scientific need of the world, planning and policy-making for the scientific activity, and publication of articles on the topics related to clinical microbiology. The results of this study are of great importance for the publication databases of scientific articles and can be significantly effective in their future orientation in publishing articles related to brucellosis. These results can also be used by the research centers and universities for the purpose of replanning the proposals related to brucellosis. For future research, it is suggested that the publication trends of global articles and each country's domestic publications are studied and compared, which would be important in the collaboration of global research centers to meet today's scientific need in the field of brucellosis disease.


Antimicrobial-resistant bacteria and their antimicrobial resistance (AMR) genes can spread by hitchhiking in human guts. International travel can exacerbate this public health threat when travelers acquire AMR genes endemic to their destinations and bring them back to their home countries. Prior studies have demonstrated travel-related acquisition of specific opportunistic pathogens and AMR genes, but the extent and magnitude of travel’s effects on the gut resistome remain largely unknown.Using whole metagenomic shotgun sequencing, functional metagenomics, and Dirichlet multinomial mixture models, we investigated the abundance, diversity, function, resistome architecture, and context of AMR genes in the fecal microbiomes of 190 Dutch individuals, before and after travel to diverse international locations.qnr fluoroquinolone resistance genes, blaCTX-M family extended-spectrum β-lactamases, and the plasmid-borne mcr-1 colistin resistance gene.Travel markedly increased the abundance and α-diversity of AMR genes in the travelers’ gut resistome, and we determined that 56 unique AMR genes showed significant acquisition following international travel. These acquisition events were biased towards AMR genes with efflux, inactivation, and target replacement resistance mechanisms. Travel-induced shaping of the gut resistome had distinct correlations with geographical destination, so individuals returning to The Netherlands from the same destination country were more likely to have similar resistome features. Finally, we identified and detailed specific acquisition events of high-risk, mobile genetic element-associated AMR genes including Our results show that travel shapes the architecture of the human gut resistome and results in AMR gene acquisition against a variety of antimicrobial drug classes. These broad acquisitions highlight the putative risks that international travel poses to public health by gut resistome perturbation and the global spread of locally endemic AMR genes.The online version contains supplementary material available at 10.1186/s13073-021-00893-z. Antimicrobial resistance (AMR) is a major global public health threat with a high mortality cost –6. AMR bAMR is rising worldwide, but there are large geographic differences in the prevalence and type of resistant bacteria and their AMR genes , 8. Low-qnr, and mcr-1 are often associated with mobile genetic elements like plasmids and are of particularly high concern due to their ease of spread )  between samples decreased (p<2e−16 [Fisher’s exact test])  Fig. a. Each mn=3) or post-travel (n=3) time point  between the pre-travel and post-travel samples , suggesting that having the same travel destination makes traveler resistomes more similar , the post-travel metaresistomes had a strong regional bias (p<2e−16 [Fisher’s exact test])  but not before travel (p=0.7021 [permanova]). This demonstrates that individuals traveling to the same destination are far more likely to have their post-travel samples cluster in the same metaresistome than their pre-travel samples.To interrogate these region-specific effects, we rebuilt Dirichlet multinomial mixture models after separating the pre-travel and post-travel samples. Separating the time points removes possible random effects due to subject identity allowing a narrower focus on destination. This analysis yielded 9 metaresistomes (4 in the pre-travel samples and 5 in the post-travel samples). Though the pre-travel metaresistomes did not show significant bias by destination   for AMR genes in our Dutch traveler cohort , inactivation (p=2.76e−4), and target protection (p=7.87e−8) all had significant positive correlations, and the trend for antibiotic target alteration was also positive. In contrast, the trend for antibiotic target replacement is nearly flat, showing that target replacement genes we detected in gut resistomes do not have a corresponding increase in abundance when they are prevalent in more samples.We found a positive correlation between prevalence and abundance p<2e−6 for AMRp=1.1e−9), efflux (p=3.4e−3), and inactivation (p=8.0e−8) of antibiotics all significantly increased after travel. This indicates that at the level of AMR mechanisms, there is a significant effect of travel in structuring the gut resistome. By further classifying the AMR genes families into gene classes defined by CARD ontology, we observed that 11 of 20 detected classes had increased abundance in the post-travel samples compared to the pre-travel samples  and for antibiotic target replacement (p=0.0295 [permutation test]) were both highly enriched in the significantly acquired set of genes. In contrast, AMR genes for antibiotic target protection (p=8.29e−6 [permutation test]) were completely absent in the significantly acquired genes (far less than predicted under a null model).We detected 56 AMR genes with significant evidence of acquisition after travel, compared to only 4 showing significant loss following travel Fig. : Fig. S8tetX, an antibiotic-inactivating monooxygenase which confers resistance against all clinically relevant tetracyclines, including last-resort antibiotics like tigecycline, eravacycline, and omadacycline [tetX variants , while the variant of tetX discovered in our functional selections was significantly acquired during travel .The diversity of AMR genes with significantly increased abundance and acquisition post-travel demonstrates the importance of expanding AMR analysis beyond ESBLs to the entire gut resistome. For example, we detected two variants of acycline , 85. TetcatA, a chloramphenicol acetyltransferase  and those visiting Northern Africa having the lowest AMR gene acquisition . Six of the 56 significantly acquired AMR genes identified in Fig. dfrA1 variants, which confer resistance against trimethoprim. Each dfrA1 variant had a fold change increase between 3.62 and 3.92 in prevalence in post-travel samples. Increases we saw in trimethoprim-sulfamethoxazole resistance genes   as presented in Additional file tetM, tetQ, ermB, and mefAE) were present in all of the samples, and two genes (qnrA and mcr-2) were not present in any samples. We conducted acquisition analysis and destination bias analysis for the remaining 10 genes.Concurrent with our comprehensive metagenomic resistome analysis, we specifically targeted 16 clinically important AMR genes in our samples by qPCR. Of these 16, four genes , and blaCTX-M-9 was predominantly acquired in Southeastern Asia (82.1%). Uniquely, mcr-1 was only acquired by travelers to Southeastern Asia  [E. coli from a traveler returning from Vietnam also harbored mcr-1 on an IncI2 type plasmid.To further investigate the ive Fig. . Plasmidatabase) , 90. A fatabase)  of the 6atabase)  demonstrmcr-1 containing contig, and the top hit (99.9% identity with 100% query coverage) was to a plasmid from gastroenteritis-causing E. coli . In both plasmids . T4SS have known involvement in horizontal gene transfer [mcr-1 colocalization with T4SS in plasmids [mcr-1 was on different plasmid incompatibility types than IncI2.We searched NCBI for the best BLAST match of subject S032’s transfer –97. Therplasmids , 98, thoGlobal AMR spread threatens decades of success in treating bacterial infections with antibiotics , 99–101.A study by Langelier et al. in 2019 reported on the resistome in 10 travelers to Asia or Africa . Eight otetW and catA [tetW and catA were also more likely to be found in our pre-travel than post-travel samples. In contrast, the antibiotic-treated ICU patient resistomes in the Buelow et al. study were enriched for AMR genes such as erm and an aac(6’) family gene, both of which were also acquired and increased in our post-travel samples. With antibiotic perturbation, the effects on the gut resistome can vary based on the spectrum of the antibiotic [An individual’s gut resistome response to travel perturbation may parallel the response from other non-travel gut perturbations, including hospitalization and antimicrobial treatment , 105. Intibiotic , 42, 81.tibiotic . Even ifqnr endemicity in Vietnam with 100% carriage of qnr by qPCR of fecal samples from 93 Vietnamese residents of the Ha Nam province [blaCTX-M-1 (38%) and blaCTX-M-9 (61%). These results correspond well with both the high acquisition rate we saw for these genes in individuals returning from Southeastern Asia and the blaCTX-M-9 predominance we saw in travelers returning from Southeastern Asia.The high-risk gene acquisitions we observed are concordant with qPCR-based research of endemic antibiotic resistance in our cohort’s travel destinations. In 2019, Bich et al. demonstrated province . This samcr-1 [E. coli isolates from all 2001 participants (540 to Southeastern Asia) in the COMBAT study detected mcr-1 in 6 E. coli isolates, indicating higher detection sensitivity for mcr-1 using qPCR directly from the stool. These results are also comparable to another culture-based isolate study [mcr-harboring E. coli.Our cohort’s mcr-1 Southeast Asian geographic acquisition bias is also consistent with Bich et al. where 88% of tested Vietnamese residents carried mcr-1 . In compmcr-1  by Arcilte study  where 20mcr-1-containing plasmids across a number of different bacterial isolates from around the globe [mcr-1 plasmids, which corresponds to our detection of mcr-1 in travelers to Southeastern Asia. The authors found that mcr-1 initially mobilized to plasmids through an ISApl1 transposon. This is consistent with the reference plasmid in Fig. In 2018, Wang et al. analyzed he globe . China amcr-1 results advocate for a combined approach of AMR gene detection. Short-read shotgun metagenomic sequencing provided us with excellent data for understanding gut resistome composition changes, diversity changes, and AMR gene acquisitions due to travel, but only 1 of 18 (5.6%) mcr-1 qPCR-positive stool samples we assembled yielded an mcr-1 contig. However, we show that AMR gene contig assembly yields an important genomic context surrounding resistance genes that could have implications for understanding and modeling AMR gene spread. Contig assembly using short-read shotgun metagenomic sequencing may differ by AMR gene. For example, we successfully assembled tetX in 56 of 143 (39.2%) ShortBRED-positive samples. Future studies may employ chromosome conformation capture or long-read sequencing in concert with short-read sequencing to improve metagenomic assembly and give even more detailed genomic context to resistance gene detection directly from stool [Our om stool .Our study design was optimized to understand the acquisition within travelers, and we do not have samples from travelers’ contacts while abroad. Pre-travel samples from our Dutch cohort also contain resistant bacteria and AMR genes, but our study is only equipped to show unidirectional gene transfer from the destination to the travelers; it is also possible that travelers could deposit AMR genes in their travel destinations. Future investigation into travelers’ contacts at home and abroad may resolve AMR gene transmission networks. We also observed that grouping samples by subregion better explains the sample composition than grouping by continent. It is possible that we are missing even more granular effects that would be found at the country or even city level , 108.CTX-M and mcr-1) and functionally discovered AMR genes with no known homologs in the current databases. We also show AMR gene acquisition and carriage in the gut resistome is travel destination-specific with compositional signatures lasting at least until the traveler returns home. Interventions to reduce AMR burden in low- and middle-income countries with current high endemic AMR burdens may reduce traveler AMR gene acquisitions. Developments in risk stratification for AMR genes could help target such efforts [We provide new data regarding the effect of international travel to low- and middle-income regions on the gut resistome of travelers from a high-income country. We show that such travelers acquire AMR genes abroad and carry these AMR genes back to their countries of origin. These AMR genes include both known clinically relevant AMR genes that are common in pathogens , supplementary table legends (S1-S9), and supplementary figures (S1-S15).Additional file 2: Supplementary tables (S1-S9).Additional file 3: Source data for all figure panels.Additional file 4: Scripts that takes in community data matrix and outputs Dirichlet Multinomial model object.Additional file 5: Takes in community data matrix and directory with Dirichlet Multinomial model objects and outputs data from the best fit model.Additional file 6: Outputs a data table for AIC values of DMN models and makes aggregated AIC and Laplace plots.


Commercial poly methyl methacrylate (PMMA)-based cement is currently used in the field of orthopedics. However, it suffers from lack of bioactivity, mechanical weakness, and monomer toxicity. In this study, a PMMA-based cement nanocomposite reinforced with hydroxyapatite (HA) nanofibers and two-dimensional (2D) magnesium phosphate MgP nanosheets was synthesized and optimized in terms of mechanical property and cytocompatibility. The HA nanofibers and the MgP nanosheets were synthesized using a hydrothermal homogeneous precipitation method and tuning the crystallization of the sodium-magnesium-phosphate ternary system, respectively. Compressive strength and MTT assay tests were conducted to evaluate the mechanical property and the cytocompatibility of the PMMA-HA-MgP nanocomposites prepared at different ratios of HA and MgP. To optimize the developed nanocomposites, the standard response surface methodology (RSM) design known as the central composite design (CCD) was employed. Two regression models generated by CCD were analyzed and compared with the experimental results, and good agreement was observed. Statistical analysis revealed the significance of both factors, namely, the HA nanofibers and the MgP nanosheets, in improving the compressive strength and cell viability of the PMMA-MgP-HA nanocomposite. Finally, it was demonstrated that the HA nanofibers of 7.5% wt and the MgP nanosheets of 6.12% wt result in the PMMA-HA-MgP nanocomposite with the optimum compressive strength and cell viability. Over the past fifty years, PMMA has been utilized in odontology and orthopedic applications as the main material for fixation of prosthesis to transfer forces from bone to prosthesis ,2. Altho2 nanotubes [Incorporation of the reinforcement materials into the matrix of the PMMA bone cement is considered as one of the potential approaches for improving its bioactivity and mechanical properties ,18. For anotubes  have beeRecently, 2D nanomaterials have attracted significant interest as promising nanoplatforms for biomedical applications owing to their ultrathin thickness, specific physicochemical properties, high surface-area-to-mass ratio, and 2D morphological feature . MagnesiThe purpose of this study is to investigate the synergistic effect of the HA nanofibers and 2D MgP nanosheets ratios on the mechanical property and the cytocompatibility of the PMMA nanocomposites. Therefore, synthesized the MgP nanosheets and the HA nanofibers were mixed into the PMMA matrix at three different ratios. Central composite design (CCD) as a useful statistical-based experimental design tool was employed for designing the experiment. The simultaneous effect of HA and MgP ratios on the ultimate compressive strength and cell viability of the PMMA-HA-MgP nanocomposites was investigated. The empirical models based on the effective factors and interactions were developed and applied as a prediction tool for optimization. In addition, the synthesized HA nanofibers, the MgP nanosheets, and the PMMA-HA-MgP nanocomposites were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), field emission scanning electron microscope (FESEM) and energy-dispersive X-ray spectroscopy (EDS). 3 (0.5 M) was then added to this solution until its pH value reached 3.50. After stirring for 30 min, the solution was transferred to a 100 mL stainless-steel autoclave and was treated hydrothermally at 160 °C for 6 h. Finally, the products were centrifugally cleaned with deionized water and absolute ethanol and then dried in an oven at 80 °C for 5 h [In a typical procedure, the aqueous solution of calcium nitrate tetrahydrate (0.01 M) as the calcium (Ca) source, diammonium hydrogen phosphate (0.06 M) as the phosphorus (P) source, and urea (1 M) solution were first mixed together. The molar ratio of Ca/P was kept at 1.67. HNO for 5 h .2–H3PO4 was employed to synthesize two dimensional MgP nanosheets. First, the MgOH (1 M) was dissolved with the magnetic stirrer in H3PO4 (1 M) until a fully clear solution was obtained. Thereafter, the NaOH solution was added and standed for 2 h. Finally, the prepared solution was centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. The molar ratios of MgOH, NaOH and H3PO4 were adjusted to 0.18, 0.45, and 0.37, respectively. The solid precipitate was vacuum dried at 70 °C for 24 h.According to the study reported by Laurenti et al., , a ternaThe PMMA-HA-MgP nanocomposites were prepared by mixing different ratios of the HA nanofibers and the MgP nanosheets in PMMA according to the experimental design in X” and dependent variable “Y”. Generally, a low order polynomial is employed in some of the independent variables; however, a polynomial of the higher degree such as the second order model is employed when there is curvature in the system as stated in Equation (1). [i, j, b, k, and e stand for the linear coefficients, the quadratic coefficients, the regression coefficients, the number of experimental factors, and the random error, respectively.Response surface methodology (RSM) is defined as a collection of the statistical and mathematical techniques that can be employed to determine the effect of several factors at different levels and further their effects on each other . This teion (1). :(1)Y=β0 In this study, a standard RSM design known as CCD was employed using Design of Expert software version 10  to investigate the simultaneous effect of independent factors including the HA nanofibers and the MgP nanosheets ratios on the ultimate compressive strength (UCS) and cell viability of PMM-HA-MgP nanocomposite. CCD is suitable for fitting a quadratic model, which usually works well for predication and optimization . Among dAccording to ISO 5833 standard , the cyl2 and 95% humidity. Three disk shape samples of nanocomposites with dimension of 6 × 2 mm (D × W) were placed in a 96-well plate. Cultured cells were then seeded into the plate with the concentration of 10,000 cells per well followed by incubation for 24 h. MTT solution was prepared by diluting MTT in PBS with a concentration of 5 mg/mL. Subsequently, MTT solution was added, and the well-plate was incubated for 4 h. Dimethyl sulfoxide (DMSO) solvent was then added to each well to solubilize formazan salts. At the end, absorption was measured by a microplate reader  at 570 nm wavelength.In this study, cytotoxicity of PMMA-HA-MgP nanocomposites was evaluated using the MTT assay method. First, fibroblasts cells (3T3-J2) were cultured in Dulbecco’s modified eagle medium (DMEM) with addition of 10% fetal bovine serum (FBS) and 1% penicillin streptomycin followed by incubation at 37 °C with 5% COScanning electron microscope (SEM) (Hitachi S-3000N VPSEM) equipped with energy-dispersive X-ray spectroscopy (EDS) and field emission scanning electron microscope (FESEM) (JEOL JSM-6320F) were used to investigate surface morphology and elemental compositions. In addition, the nanostructures of the HA nanofibers and the 2D were observed under scanning transmission electron microscopy (STEM) (JEOL JEM-ARM200CF). The XRD patterns were recorded using a Bruker D8 Discover X-ray diffraction system equipped with a copper sealed X-ray tube source, producing Cu-Kα (λ = 1.5418 Å). The diffractometer was operated at 40.0 kV and 40.0 mA at a 2θ range of 5–60° with a step size of 0.02 and an exposure time of 1 s/step.It was reported that the optimal fiber used as reinforcement in a polymer-based composite should have high aspect ratio so that the stresses developed within them are remarkably larger than the nominal stresses in the composites . Therefo3(PO4)2, Na2HPO4·2H2O, Mg2PO4(OH), Na2Mg5(PO4)4·7H2O compounds. X-ray diffraction patterns of the HA nanofibers and the MgP nanosheets are shown in The normal probability plot of residuals for UCS and cell viability of PMMA-HA-MgP nanocomposite is shown in The normal probability plot of the residuals for UCS and cell viability of the PMMA-HA-MgP nanocomposite is shown in The main effect plot and the interaction plot for UCS are shown in 2, and B2 are significant factors. The “Lack of Fit F-value” of 0.26 implies there are 85.23 chances that it could occur due to noise. The Pred R-Squared of 0.9816% is in reasonable agreement with the Adj R-Squared of 0.9739. This indicates that this regression model can reasonably predict responses for new observations. The ANOVA table for UCS is summarized in 2, and B2 are significant factors because their values of “Prob > F” are less than 0.05. However, AB is a nonsignificant factor because its value is greater than 0.1. In order to improve the model, the nonsignificant terms need to be removed. Therefore, it is essential to apply the model reduction. Adeq Precision measures the signal to noise ratio, and a ratio greater than four is desirable. In this model, the ratio is 33.121, implying an adequate signal. Therefore, this model can be used to navigate the design space. The coefficient of variation (CV), which is a ratio of the standard deviation to the mean, is 4.41%. This amount is lower than 10%, implying that the model is reproducible. The ANOVA table for cell viability is summarized in The “Lack of Fit F-value” of 2.61 implies there is a 10.64% chance that a “Lack of Fit F-value” could occur due to the noise. The Pred R-Squared of 0.8830 has good agreement with the Adj R-Squared of 0.8470. Adeq Precision measures the signal to the noise ratio, and a ratio greater than four is desirable. In this model, the ratio is 13.559 implying an adequate signal. This model can be used to navigate the design space. The CV of this model is 2.04%, which is lower than 10%, implying that this model is also reproducible. Final equations in terms of actual factors for the UCS and cell viability are presented in Equations (2) and (3). The adequacy of these developed models needs to be assessed prior to their use in the optimization process.The adequacy of the developed models for UCS and cell viability was assessed by six confirmation tests. The conditions for the first three confirmation run experiments are among the conducted tests, and the last three confirmation tests are not previously performed but are within the previously defined levels. The residuals between the actual and predicted values for cell viability indicate that the percentage errors are between 0–4.5%. The confirmation tests revealed good correlation between the predicted and the experimental values with less than 10% of error, which is acceptable. Therefore, it can be said that the empirical models derived from RSM are reasonably accurate and, thereby, can be employed in the optimization process. The optimization process determines the combination of the dependent factor levels that simultaneously satisfy the sought requirements . OptimalThe PMMA-HA-MgP with the HA nanofibers ratio of 7.5% wt and the MgP nanosheets ratio of 6.12% wt reveals the maximum UCS and cell viability with the highest level of desirability (91.1%). In this study, the HA nanofibers and the MgP nanosheets as the reinforcement materials were synthesized through hydrothermal homogeneous precipitation and tuning of the crystallization of the sodium–magnesium–phosphate ternary system, respectively. A novel PMMA bone cement nanocomposite was then developed by mixing the HA nanofibers and the MgP nanosheets at different ratios of reinforcement. A standard RSM design known as CCD was employed to investigate the effect of the HA nanofibers and the MgP nanosheets ratios on the ultimate compressive strength and cell viability of the PMM-HA-MgP nanocomposite.The results demonstrated that both the HA nanofibers and the MgP nanosheets are considered as two significant factors to improve UCS. However, the effect of the MgP nanosheets was observed to be more significant. In addition, both reinforcements are significant factors for enhancing the cytocompatibility of the PMMA-based nanocomposite under the influence of the higher amounts of the HA nanofibers. A set of confirmation tests demonstrated that the empirical models derived from RSM can be used to describe the relationship between the independent factors and responses. The optimization results demonstrated that the maximum UCS and cell viability of the PMMA-HA-MgP nanocomposite is obtained with the HA nanofibers ratio of 7.5% wt and the MgP nanosheets ratio of 6.12% wt with the highest level of desirability (91.1%). The current result is based on the selected ratios of reinforcements. Therefore, more experiments need to be conducted at different ranges of ratio. In addition, more research needs to be conducted to investigate the mechanism of this nanocomposite.