International Economics and Economic Policy (IEEP) tackles mainly macroeconomic, trade and technology aspects. How will COVID-19 affect the economy given these major trends, will it be a temporary downturn or will it have more permanent effects? Will it accelerate change or hinder adjustments? All papers were presented at an on-line conference organized by the by the IEEP and the EIIW on June 11, 2021.The COVID-19 pandemic and associated lock down measures have hit the global economy and affected the core of our socio economic and business models, which were already exposed to a number of challenging developments. Namely, industrialised countries are experiencing a secular decline of interest and growth rates, possibly related to medium term demographic and technology developments, rising demand for save assets and increasing divergence among people, firms and regions. With the demographic transition, pension and health care are putting additional strain on government budgets. These trends are increasingly restricting monetary and fiscal policies to stabilise the economy in the case of adverse shocks. On the technology side, digitalisation of consumption and production requires structural adjustments on various fronts in order to remain competitive and generate new opportunities for displaced jobs. Since a number of years, globalisation is experiencing sizeable headwinds with rising protectionism and increasing scepticism of universal benefits related to ever increasing international trade and financial integration. Focusing on the interactions between these trends and the COVID-19 shock, this special issue of the journal Karimov and Konings analysing the profile of the employment shock, using a detailed Belgian dataset which allows to distinguish between start-ups and incumbent firms (grouped by age classes). The paper finds that the employment losses are mainly located with incumbent firms but predicts a more structural, medium term negative effect associated missing generation of start-ups. This provides a more nuanced assessment of the shape of the recovery in particular it contrasts with US evidence on business creation in 2020.The special issue starts with a paper by Kollmann identifies the nature of the shock through the lens of a new Keynesian macroeconomic model where policy is constrained by an (expectations driven) liquidity trap. The paper finds that the observed output and inflation responses are most consistent with a persistent negative supply shock giving additional support to the view that negative effects might be longer lasting.The next set of papers discusses the constraints which complicate the policy response and the impact this has on the shape of the recession. The paper by Bismut and Ramajo analyse how COVID-19 might affect the future evolution of real interest rates. Based on an econometric analysis of interest rates for 19 OECD countries over the period 1970–2017 they identify major drivers of interest rates, namely potential output, risk effects and budget deficits. Their estimated equation captures well past developments and predicts that under a scenario of continued low productivity it will be unlikely that the current level of interest rates will change significantly. However pressures from deficits and public debt could emerge, but their effect will depend on saving behaviour.Biase and Doughtery introduces a more granular dimension of the fiscal adjustment by considering the constraints and behaviour of subnational governments. Indeed, subnational entities have more limited fiscal space than central governments. Given social expenditure pressures associated with COVID-19 as well as reduced fiscal revenues at all levels, subnational governments are often forced to reduce public investment, thus making fiscal policy design more pro-cyclical than at the central government level. This is why most of the COVID-19 related fiscal spending has been undertaken by central governments.Van Ark, de Vries, and Erumban compare productivity developments between the US, France and the UK using detailed industry data. The paper finds positive productivity effects of the COVID-19 shock resulting from reduced production of low productivity sectors . However, only the US shows positive within industry effects and the US and the UK show stronger effects for digitally intensive industries. This could be related to more favourable institutional and policy settings.Doehring, Hristov, Maier, Roeger and Thum-Thysen use a two sector model distinguishing between a conventional and a digital service sector. The paper emphasises structural differences concerning scalability, entry barriers and skill demands across these two sectors and explores the economic consequences of a permanent shift towards digital (consumer) services. The paper shows how the distribution of economic rents between workers with and without digital skills and platforms are determined by labour supply conditions and entry barriers.de Braga de Macedo, Oliveira Martins and Tovar Jalles provide a global and long term view on the interactions among globalisation, economic convergence and freedoms. They find that, over the past decades, globalisation, freedoms and economic convergence have supported each other, thus creating a systemic virtuous cycle. The links between globalisation and economic convergence appear particularly strong, supporting the view that the previous and the COVID-19 induced set backs on globalisation may contribute to a continued slowdown of economic growth. The latter may have potentially negative effects on the state of political and civil liberties. Only for non OECD countries there is evidence that the relationship between the level of freedoms and economic convergence could be negative for certain stages of development.Finally, Overall, this special issue gathers a rather rich set of insights and policy-relevant analysis. We hope it may contribute to a better understanding on how the COVID-19 shock has affected the economic outlook and may in the future interact with the ongoing major global trends that are reshaping our economies.


This protocol illustrates a pipeline for modeling the nonlinear behavior of intracellular signaling pathways. At fixed spatial points, nonlinear signaling dynamics are described by ordinary differential equations (ODEs). At constant parameters, these ODEs may have multiple attractors, such as multiple steady states or limit cycles. Standard optimization procedures fine-tune the parameters for the system trajectories localized within the basin of attraction of only one attractor, usually a stable steady state. The suggested protocol samples the parameter space and captures the overall dynamic behavior by analyzing the number and stability of steady states and the shapes of the assembly of nullclines, which are determined as projections of quasi-steady-state trajectories into different 2D spaces of system variables. Our pipeline allows identifying main qualitative features of the model behavior, perform bifurcation analysis, and determine the borders separating the different dynamical regimes within the assembly of 2D parametric planes. Partial differential equation (PDE) systems describing the nonlinear spatiotemporal behavior are derived by coupling fixed point dynamics with species diffusion. The described protocol operates with the already established interaction topology of the signaling network under study; that is, for each node i it is known what nodes activate and/or inhibit it. Mathematically, the network topology is determined by the signed incidence matrix of the ODE system,Here, we present a protocol for computational analysis of the nonlinear dynamic behavior of signaling networks and their transitions between different dynamic regimes. The dynamics of spatially homogenous biochemical systems are described by ordinary differential equations (ODE), whereas the spatiotemporal dynamics are described by partial differential equation (PDE) systems. Knowledge about ODE and PDE is needed as a prerequisite for understanding this protocol. Our computational protocol analyzes the systems’ dynamics at fixed spatial points, and it considers the nonlinear spatiotemporal behavior by coupling fixed point dynamics with species diffusion   .e.g., BioNetFit  with the highest abundance. Using the QSS approximation for the other protein activities , derive Based on the specification of the steady states and the analysis of the shapes of nullclines  to the ODE system for each pair.Scan 2D parametric planes and determine the types of bifurcations that can occur in the system.Scan the parameter planes and determine the borders that separate different dynamic regimes (see The analysis of the changes in the number and stability of steady states can reveal local bifurcations, such as the Andronov-Hopf or saddle-node bifurcations . HoweverIf there are time-series data, fine-tune model parameters to reproduce the system trajectories for a biologically relevant regime. This step uses standard software packages for parameter optimization .Derive a PDE system by adding the terms describing mass transfer processes such as diffusion and convection to the ODE system studied (A set of examples that illustrate the analysis of the dynamics of the RhoA-Rac1 signaling network model and python scripts can be found in “examples.zip” archive.


Dear Readers,The Journal of Innovations in Cardiac Rhythm Management contains an interesting article titled “Epicardial Termination of Left Atrial Appendage Atrial Tachycardia,” in which Polselli et al. describe the ablation of an atrial tachycardia originating from the left atrial appendage (LAA) using an epicardial approach. This article highlights an important topic, which is the role of the LAA in the genesis of atrial arrhythmias.This issue of 1 In addition, the Effect of Empirical LAA Isolation on Long-term Procedure Outcome in Patients With Persistent or Longstanding Persistent AF Undergoing Catheter Ablation (BELIEF) trial,2 published in 2016, demonstrated an incremental benefit of isolating the LAA over conventional ablation alone. However, isolation of the LAA has still not become a mainstream adjunct to pulmonary vein isolation (PVI) during AF ablation procedures. One possible reason for the lack of widespread adoption of LAA isolation is the difficulty in achieving this task. The atrial wall of the LAA is thick and typically requires longer ablation lesions at higher power, more than what is necessary to isolate the pulmonary veins. Moreover, pacing is often required to delineate the location of the left phrenic nerve to avoid its injury. Acute recurrence is also often seen during the procedure, necessitating more ablation in order to achieve durable isolation. Collectively, these aspects can add significant time and challenges to the procedure.The LAA has been suspected since at least 2010 to be a trigger or driver for atrial fibrillation (AF).Two ongoing randomized clinical trials are expected to shed light on this topic. First, the LAA Ligation Adjunctive to PVI for Persistent or Longstanding Persistent AF (aMAZE) trial , which completed enrollment and will be presented at American Heart Association Scientific meeting in November 2021, will help us understand what population(s) might benefit most from LAA isolation and shed light on the safety of this procedure. Another study, the Posterior Wall and LAA Empiric Electrical Isolation for Nonparoxysmal AF (PLEA)  trial, is still enrolling patients and aims to show the benefit of radiofrequency isolation of the LAA. If these trials confirm the role of the LAA in AF maintenance, technological advancements, including the use of pulsed ablation, have the ability to make the task easier.The Journal of Innovations in Cardiac Rhythm Management.Best regards and I hope that you enjoy reading this issue of Sincerely,md, fhrs, faccMoussa Mansour, Editor in ChiefThe Journal of Innovations in Cardiac Rhythm ManagementMMansour@InnovationsInCRM.comDirector, Atrial Fibrillation ProgramJeremy Ruskin and Dan Starks Endowed Chair in CardiologyMassachusetts General HospitalBoston, MA 02114


B cell lymphoma (BCL) is a heterogeneous group of lymphoid malignancies which comprise the majority of canine lymphomas. Diffuse large B cell lymphoma is the most common lymphoma subtype in dogs but other subtypes  have been described. This review aims to explore the use of flow cytometry to refine the diagnosis of canine BCL. Particular emphasis will be given to the possible identification of peculiar immunotypes, putative prognostic markers, staging and minimal residual disease. Flow cytometry (FC) of fine needle aspirates has been increasingly applied as first-line analysis in cases of suspected lymphoma in dogs. It is mainly used to investigate the immunophenotype of the cells and refine the diagnosis obtained from other morphological diagnostic techniques such as cytology and histopathology. In addition to the cell lineage definition, recent studies investigated flow cytometric parameters with a potential clinical and diagnostic relevance. The present review is aimed to summarize these aspects in B-cell lymphoma (BCL).Current histologic and cytologic classification of lymphomas  , 2 requiA restricted expression of kappa or lambda light chain is consistent with a B-cell clonal expansion and the evaluation of the kappa/lambda ratio is the reference flow cytometric method to diagnose a neoplastic proliferation of B-cells in humans. Unfortunately, the kappa/lambda ratio is very low in the normal canine lymphoid populations  with lamIn dogs, B cells generally represent a minority of the population in non-neoplastic lymph nodes , 10, thuThe evaluation of cell size in the lymph node aspirate may also help to support the diagnosis of lymphoma since lymphocytes are mostly small-sized in both normal and reactive lymph nodes  Figure . Cell siThe most reliable way to confirm a neoplastic expansion is the identification of one discrete population with aberrant or abnormal antigenic pattern  . AberranFC cannot provide a definitive diagnosis of B-cell lymphoma if there is no evidence of an obvious neoplastic population  and in presence of a doubtful phenotype . In these cases, histopathology and immunohistochemistry remain mandatory. Additionally, PCR for Antigen Receptor Rearrangements (PARR) analysis can help in achieving the correct diagnosis and define lineage allocation ; howeverAlthough it may add some useful information to the diagnosis of lymphoma and phenotyping (see below), FC is not currently able to distinguish different types of BCL according to the WHO classification system in dogs. Most BCLs diagnosed in dogs are positive for both CD21 and CD79, while, CD21− CD79+ and CD21+ CD79− lymphomas are less common , 18. ExpFinally, no differences in the immunophenotype of different subtypes of BCL were detected in one study . This liFC plays a leading role in defining two major characteristics of lymphomas with possible impact on prognosis: cell lineage (B vs. T) and staging (see below). Other FC features potentially related to prognosis may derive from the evaluation of cell size, the reactivity to some specific prognostic markers, and the evaluation of the residual non-neoplastic cell population.As previously described FSC values are indicative of small, medium, or large cell lymphomas, although a correlation between the flow cytometric and the morphologic information (cytology and histology) has not been assessed so far. Of greater importance, large flow cytometric cell size has been linked to high-grade lymphoma subtypes  and a woRecently, additional prognostic markers have been investigated by FC. Low levels of MHC II expression predicted a poor outcome in BCLs in two studies , 27. HowOne of the main advantages of FC is the multiparametric approach. Numerous parameters or markers can be evaluated on every single cell at the same time. This feature allows an accurate and sensitive description of different subsets within a population. The residual populations of non-neoplastic T lymphocytes are easily identifiable in BCLs and they appear to play a role in the progression of the neoplasm, thus suggesting an active influence of the immune system on the tumor pathobiology and treatment response. A higher percentage of regulatory T-cells (FoxP3+) has proven to be an independent prognostic factor in BCLs  and a reFC analysis of peripheral blood can be run to calculate the lymphocyte-to-monocyte ratio. This ratio has been reported as an independent prognostic factor and a value <=1.2 at the diagnosis is associated with shorter time to progression and survival in dogs with DLBCL treated with chemoimmunotherapy . SimilarFinally, the multiparametric approach of FC is extremely useful also to identify rarer cases of two coexisting neoplasms, such as T-zone lymphoma and BCL –37. An eFC is an excellent tool for staging lymphoma , in particular in fluid matrices including peripheral blood (PB) and bone marrow (BM). The major advantage is that it allows examination for populations with the same scatter and immunophenotypic properties of the primary lesion. Good analytical performances have been reported in the quantification of PB and BM infiltration in dogs with large BCLs . Given tThe issue of staging BM infiltration is debated. Some oncologists do not consider BM analysis mandatory to accurately define the prognosis. This is probably because of the invasiveness of the procedure and because bone marrow evaluation is not considered necessary in absence of cytopenias, presence of circulating blast cells, or other hematological alterations. However, previous studies on DLBCL demonstrated that PB and BM involvements are not directly correlated and it is not possible to predict BM infiltration out of PB results , 40. FurRegarding other BCL subtypes, a recent paper reported a significant association between PB and BM infiltration and prognosis in dogs affected with nodal MZL. Dogs with <30% PB infiltration showed a significantly longer time to progression while BM infiltration stratified cases in three groups  with different survival times .The major role of FC in the follow-up of lymphoma patients is related to the identification of the minimal residual disease (MRD) in PB, BM, and lymph node at the end of the chemotherapeutic protocol. In humans, MRD evaluation plays a key role in the treatment plans of hematologic malignancies and allows individualized tailoring of therapies, in particular for leukemic patients . SimilarA study evaluating MRD in lymph nodes, PB, and BM found only a moderate agreement between FC and PARR at the end of chemotherapy in dogs with DLBCL. Flow cytometric cutoff was set at 1%. PARR was more sensitive than FC in predicting time to relapse but only the combined interpretation of the two methods was predictive of the overall survival . One of The development of new specific antibodies suitable for FC and targeting more B-cell antigens may be useful to improve the accuracy of the diagnosis of BCL and improve prognostic abilities. In particular, specific antibodies recognizing plasma cells (CD38) or immature B cells  labeled with different fluorochromes are needed to be included in the diagnostic panels.The wide range of possible applications of FC makes it an important tool for future developments in the field of canine lymphoma. FC can contribute to the discovery of new therapeutic targets and identification of new drugs with the ultimate goal of more individualized therapies. Some examples include studies focusing on specific markers such as PD-1 / PD-L1 , 48, c-kThe diagnostic usefulness of flow cytometric analysis in dogs affected by lymphoma is increasing. In this review, we summarized recently reported flow cytometric indexes and features with clinical significance for canine B-cell lymphomas. To confirm and validate the diagnostic and prognostic role of these parameters, larger multicentric prospective studies are warranted. Collectively, they represent important milestones in the exploration of the full power of flow cytometry in veterinary oncology. They lay the foundation to understand why similar lymphomas behave differently and to improve the ability to differentiate prognosis among B-cell neoplasms and within a single lymphoma subtype. Currently, flow cytometry has the highest diagnostic value if combined with other morphologic techniques such as cytology and/or histology. Its potential will be expanded in the future but we believe the right way is to aim for the result of fully integrated multidisciplinary  lymphoma diagnosis.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Correlation of in vivo imaging to histomorphological pathology in animal models requires comparative interdisciplinary expertise of different fields of medicine. From the morphological point of view, there is an urgent need to improve histopathological evaluation in animal model-based research to expedite translation into clinical applications. While different other fields of translational science were standardized over the last years, little was done to improve the pipeline of experimental pathology to ensure reproducibility based on pathological expertise in experimental animal models with respect to defined guidelines and classifications. Additionally, longitudinal analyses of preclinical models often use a variety of imaging methods and much more attention should be drawn to enable for proper co-registration of in vivo imaging methods with the ex vivo morphological read-outs. Here we present the development of the Comparative Experimental Pathology (CEP) unit embedded in the Institute of Pathology of the Technical University of Munich during the Collaborative Research Center 824 (CRC824) funding period together with selected approaches of histomorphological techniques for correlation of in vivo imaging to morphomolecular pathology. Characterization of experimental animal models in translational research ranges from in vivo longitudinal imaging methods to ex vivo histopathological evaluation and downstream molecular analysis. The applicability of results achieved in animal models in general can be problematic and is not always successful .Because of a variety of new technologies during the last years , 3, whicExperimental animal model-based pathology investigates animal models specifically generated to mimic human diseases [diseases Comparative pathology of spontaneous diseases in domestic and laboratory animals detects similarities and differences of sick animals to corresponding human diseases to advance diagnostics and therapies in humans [n humans Comparative veterinary pathology observes and compares diseases of different species which can be biologically highly variable (including humans) [ humans) .The term “comparative pathology” is defined as pathology of analogous diseases in different species . It can In translational research, comparative pathology is usually used synonymously to experimental animal-based pathology.Several authors pointed out the looming need of pathologists focusing on comparative aspects for handling the increased use of genetically engineered mice –12. For The main reasons for the poor transferability of results in animal model systems to clinical applications with respect to pathology are: 1. missing or incomplete pathologic description/evaluation, 2. lack of current comparative classifications of animal models, and 3. lack in pathologists with appropriate training in exercise of comparative issues . With reThe focus of CRC824 research groups is on oncology. Translational oncological research uses different systems of animal models resulting in different levels of complexity which influence comparability and reproducibility . Table 1Given the fact that there is a lack of surgical pathologists with background knowledge of animal model pathology and in reverse only very few veterinary pathologists are focusing on translational and imaging issues, it became clear that there is an urgent need bringing the two disciplines together. Besides the appropriate planning and evaluation of animal model-based experiments, a major task of comparative facilities lies in the correct and effective training of pathologists. Additionally, with respect to animal protection and ethics, it must be the aim to decrease animal numbers in research. By planning and evaluating experiments in a scientifically sound way, repetitions and false conclusions can be prevented. The ARRIVE  guidelines as well as the 3R  principles have to be considered. The majority of animal-based research to date is performed with mice due to their fast reproduction rate and their high availability for genetic engineering methods. There are two main aspects that have to be considered when choosing the mouse as a model organism: 1. characterizing the animal on a morphological and molecular level to ensure that the model used is suitable for the question and 2. correct planning including necropsy and slide preparation as well as consultation of pathologists with expertise in animal model pathology to ensure reproducibility , 15.These circumstances resulted in the establishment of the CEP in 2016, a veterinary pathology unit embedded in the Institute of Pathology at the Technical University of Munich (TUM) consisting of a technical and a scientific part. The CRC824 was the first funding where the CEP unit was involved as a central project (Z-project), meaning a scientific service unit for the research groups included in the CRC824 and focusing on correlation of in vivo imaging techniques to histomorphological evaluation. The CEP rapidly developed and increased within the setting of the CRC824 and became one of the largest comparative pathology facilities in Europe. Started with a basic equipment, it now expanded to a fully equipped and automated histopathological laboratory which enables for processing of the high amount of requests in an appropriate time and way. Its unique feature is the embedding at the Institute of Pathology at the TUM together with a complex network of a broad field of translational research groups focusing on in vivo multimodal molecular imaging. Over the last years, several additional consortial fundings have been added.Our performance monitoring for the period of 2018 to 2020 showed a total number of processed projects between 500 and 600 per year. Thereof, most projects were mouse studies which accounted for ~ 50% every year . Translational projects aiming at comparative evaluation of human tissue specimen also made up a major part of our work: around 40% of projects per year requested comparative human evaluation . Rat tissue requests had a lower percentage ranging from 1.7% to 3.2% in the given period. The remaining specimen (around 5% every year) originated from various other species. A detailed analysis of the project requests in 2020 (despite a slight drop due to the Coronavirus pandemic) is shown in Fig. For scientific requests, we provided a well-trained team of veterinary pathologists, veterinary pathologists in training and digital pathology expertise. From the surgical pathology site, 9 pathologists  were involved in CEP-projects.As a central project in the CRC824, technical and scientific support of the CEP mainly focused on correlating in vivo imaging to histomorphology.Basically, co-registrations of in vivo imaging techniques with histomorphological features include exact embedding in the right plane of interest and appropriate labeling of the relevant sites. Besides, it is obligate to ensure appropriate necropsy and fixation of samples. For this reason, pathologists should be involved before starting in vivo imaging or at least before planning of animal necropsy so that the workflow and processing of samples can be adapted in an appropriate way. Table Basic co-localization (cell line based xenograft model): solid growing subcutaneous tumors:For example, basic co-localization was done for a murine xenograft model of human myeloid sarcoma aiming at monitoring of human central memory T-cells by Immuno-Positron Emission Tomography (Immuno-PET) imaging . PositroComplex co-localization : tissue-specific tumors:For tissue-/organ-specific tumors, we developed a workflow for identification of tumor imaging parameters of mouse pancreatic ductal adenocarcinomas (PDACs) and analysis of their applicability for translation to human settings. The study was performed using genetically engineered mouse models that develop an endogenous pancreatic ductal adenocarcinoma . NeoplasFor complex co-localization of endogenous or orthotopic tumors, we additionally developed different other approaches with respect to the specific research question:During the CRC824 funding period, several in vivo–ex vivo co-localization approaches were successfully applied to different issues depending on the complexity of the research question. Basic and quite simple co-localization can be done for easily accessible tumors, like subcutaneous neoplasias, whereas tissue-specific endogenous tumors, like endogenous abdominal neoplasias, are more challenging and require a more complex and difficult workflow for satisfying co-registration results.For co-localization of detection and monitoring of esophageal cancer severity with capsule optoacoustic endoscopy (COE) in a porcine model, the ex vivo samples were cut along the lumen immediately after imaging to open the esophagus flat and to ensure the precise dissection of the area of interest . VasculaCo-localization of the thyroid gland proved to be quite easy to handle when removing the thyroid gland together with the trachea . TogetheNeuroendocrine tumors of the pituitary and the adrenal gland are quite challenging. Exact co-localization of the pituitary gland is only possible if the pituitary remains in the skull. Thus, decalcification is necessary for histomorphological analysis which means that the sample is no longer available for additional methods. Co-localization of adrenals requires the removal of the adrenal with the adjacent kidney, leading to a loss of additional landmarks.For co-localization of lymphomas, one should keep in mind that, in contrast with humans, lymphoma of the mouse occurs multicentrically in lymphnodes .13C pyruvate magnetic resonance spectroscopy [Co-localization projects of hepatocellular carcinoma (HCC) nicely showed the difference of levels of complexity between subcutaneous and tissue-specific tumors. Subcutaneous tumors were easily accessible and easy to process for histology after hyperpolarized troscopy . Processtroscopy , 24.Research requests aiming at correlation of human in vivo imaging with histomorphology of resection specimen included projects working on prostate cancer (unpublished data) and pancreatic ductal adenocarcinoma as well , 25.Light Sheet Fluorescence Microscopy (LSFM):From the pathologists point of view, for all co-localization issues which include histomorphology and immunohistochemistry, processing of FFPE tissue should always be preferred to cryo sectioning. In cryo sections, cell morphology is not so well maintained and cryo artifacts can interfere with co-localization results. If cryo sectioning is necessary due to a local setup , the cryo tissue should be stored unprocessed in liquid nitrogen or embedded in cryo cassettes with an appropriate embedding material and be stored at − 80 °C.LSFM allows a three-dimensional fluorescence based visualization of whole tissue samples. Two thin counterpropagating sheets of laser light illuminate a thin layer of the sample . TherefoClearing of samples was done using the 3DISCO (3D imaging of solvent-cleared organs) protocol . After iThe last decades have shown that genetically engineered animal models are highly adaptable to a great variety of research questions but bring along the difficulty to translate their findings to the human setting. Therefore, the field of comparative pathology together with correlated in vivo imaging became more important. Meanwhile, several authors emphasized the need of pathologists with comparative knowledge focusing on translational research and animal model pathology , 10, 32.


Sex steroid hormones (SSH) are cholesterol-derived molecules. They are secreted into saliva and enter the oral cavity, triggering physiological responses from oral tissues, with possible clinical implications, such as gingival inflammation and bleeding. SSH and hormonal changes affect not only oral host cells but also oral microorganisms.Historically, most research has focused on the effect of hormonal changes on specific bacteria and yeasts. Recently a broader effect of SSH on oral microorganisms was suggested. In order to assess the role of SSH in host-microbe interactions in the oral cavity, this review focuses on how and up to what extent SSH can influence the composition and behavior of the oral microbiome. The available literature was reviewed and a comprehensive hypothesis about the role of SSH in host-microbiome interactions is presented. The limited research available indicates that SSH may influence the balance between the host and its microbes in the oral cavity. Sex steroid hormones (SSH) are regulatory molecules synthesized or converted by several different living organisms ranging from mammals to plants, and from parasites to bacteria . In mammDuring pregnancy, the surge in hormonal levels triggers responses by oral tissues, with 30-100% of women undergoing gingival changes such as inflammation and increased bleeding . SimilarResearchers have tried to explain why oral clinical changes take place, which mechanisms are involved, and the possible implications of both for the host. It has been proposed that SSH are capable of affecting periodontal tissues and their surrounding environment through four different mechanisms: (1) by locally influencing proliferation of fibroblasts and epithelial cells; (2) by increasing vascular permeability; (3) by elevating levels of immune cells in the periodontium; and (4) by inducing changes in certain oral microorganisms . HoweverStudies in females and males on the effects of SSH on oral bacteria and fungi have been inconclusive. Moreover, the study of the oral microbiome, understood as the microorganisms present in the oral cavity, their genetic information and the environment in which they interact  has gainin vitro and clinical studies provided the basis for a hypothesis about host-microbiome interactions mediated by SSH. Possible implications for the host’s oral health and future perspectives are discussed.The purpose of this review was to assess the role of SSH in host-microbe interactions in the oral cavity, and the extent to which SSH can influence the composition and behavior of the oral microbiome. Our review of in vitro using estradiol, progesterone and testosterone on different oral microorganisms, especially periodontal bacteria. The specific focus on periodontal bacteria derives from earlier studies showing that SSH can compensate for the absence of vitamin K  the interaction between oral microorganisms and SSH; and (2) the response of host oral cells to the presence of both hormone metabolizing microorganisms and SSH. These direct and indirect influences of SSH on oral microbiota are discussed below.To date, Candida species have been reported to respond to the presence of SSH. For the purpose of this review, SSH-responsive microorganisms have been classified into four groups based on their putative role in oral health and disease and in line with the classification used in the majority of the reviewed literature. These are: (1) periodontal-disease-associated bacteria; (2) caries-associated bacteria; (3) oral-health-associated bacteria; and (4) oral fungi. Their characteristics and known response to SSH are summarized in Several oral bacteria and Bacteroides melaninogenicus subsp. intermedius (now Prevotella intermedia), and Bacteroides melaninogenicus subsp. melaninogenicus (now Prevotella melaninogenica) – used estradiol and progesterone as growth factors in the absence of vitamin K  and P. intermedia were able to nearly quintuplicate their SSH uptake when fumarate was added to the medium, significantly enhancing their metabolism.In the early 1980s, Kornman and Loesche conducted the first study which showed that, in the absence of vitamin K, two periodontal bacteria – itamin K . They alPrevotella intermedia group. Estradiol positively increases protein levels and formation of polysaccharides in biofilms as well as enhancing its coaggregation with Fusobacterium nucleatum. This could have an influence in biofilm formation (Prevotella intermedia group bacteria express enzymes that act as virulence factors. One of these enzymes is the protease dipeptidyl peptidase IV (DPPIV) which is involved in the destruction of periodontal tissue. The activity of this protease is increased by the presence of estradiol, which thus enhances its virulence  or 4-androstenedione  and progesterone  binding sites  synthesis of surface capsular proteins; (2) persistence and dissemination in their human host; (3) co- and inter-aggregation and; (4) production of energy er chain . It has er chain .Candida. The different responses triggered by SSH not only in disease-associated bacteria but also health-associated and commensal microorganisms hint a versatile influence of SSH in an oral microcosm.Since the beginning of the exploration of the effect of SSH on oral bacteria and fungi, it has repeatedly been observed that a number of sub- and supragingival bacteria are capable of changing their behavior in the presence of these molecules. This has also been observed for the fungus Changes in the metabolism of fibroblasts have also been documented. This has shown that SSH are not only capable of affecting the microorganisms in the environment, but may also as a result, change the response of the host.In vitro studies have set the fundamentals to question the role of the oral microbiome during periods of changing hormone levels, giving enough reason to presume that not only inflammation and changes in the immune response of the host are responsible for the observable clinical changes. As explained above, there are unfortunately no in vitro studies that have tested the response of an oral microcosm to the presence of SSH. The hypothesis of changes in a polymicrobial setting have only been investigated in vivo and these results will be discussed below.Since gingival alterations during pregnancy were linked to hormonal changes  differenThe contribution of female SSH to the alteration of periodontal bacteria has been thoroughly reviewed  and it wThis, paired with differences in study design and microbiological analysis methods have made interpretation of the results a challenging task. However, since the publication of the aforementioned review, several new studies have been published. Recent studies have focused on endogenous fluctuations  as well as exogenous administration of hormones. Species affected during these changes are listed on Physiological and physical changes taking place during lifetime are regulated by various biological processes, including important fluctuations in SSH. These fluctuations occur differently depending on the sex of the individual. While females experience marked increases or decreases of SSH -especially during pregnancy- males face progressive and steady changes . This coThe menstrual cycle is a periodic shift in hormone production paired with physical changes of the uterus and ovaries in women of reproductive age . These cCampylobacter, Haemophilus, Prevotella and Oribacterium) and a higher diversity of species during the luteal phase. These results support the hypothesis that periodic hormonal shifts can influence the oral microbiome, but more studies are needed to confirm this and assess the possible clinical implications.A limited number of studies have investigated the effects of the menstrual cycle on both gingival and oral microbiological changes, without reaching definite consensus . This coth – 7th week of pregnancy after which the production is taken over by the placenta. Estrogen increases steadily until delivery, rising from 0.1 ng/mL during the follicular phase of a normal menstrual cycle to 6-30 ng/mL at the end of the third trimester of pregnancy. Progesterone rises from less than 1 ng/mL during the follicular phase to 100-300 ng/mL at term . Two studies have analyzed the microbiome in depth by means of sequencing techniques.Recent studies have mostly targeted specific species known for their affinity for SSH by means of selective media, PCR, qPCR and Fluorescence In vitro studies have not reported any effect of either estrogen or progesterone on Aggregatibacter actinomycetemcomitans. However, this bacterium appears to be more prevalent during the second and third trimester of pregnancy according to Prevotella intermedia paired with increased gingival inflammation during the 2nd and 3rd trimester of pregnancy  but not to a significant level. This could be related to the small sample size and a high drop-out rate. In vitro, P. nigrescens shows enhanced biofilm formation at estradiol concentrations present during the second trimester of pregnancy and enhanced planktonic growth at estradiol concentrations corresponding to the third trimester of pregnancy  using 16S-pyrotag sequencing, to investigate the synergistic effects of smoking during pregnancy . A signiA second cross-sectional study aimed to present a snapshot of the oral microbiome during all trimesters of pregnancy and post-partum using 16S rRNA gene sequencing . DiversiOnly one longitudinal study has used sequencing methods to assess the oral microbiome of pregnant women . Unlike Prevotella, Treponema, Cardiobacterium, Aggregatibacter, Leptotrichia among others. Even though the study sample was small, a shift could be observed, suggesting that hormonal changes during pregnancy could push the biofilm towards an unbalanced or dysbiotic state.It was reported that several genera are responsible for the clustering according to the pregnancy trimester and that different genera were positively correlated with the surge in SSH, including Two cross-sectional studies have explored the microbial composition of post-menopausal women . Even thF. nucleatum, P. gingivalis, P. intermedia, S. oralis and T. forsythia were significantly higher in individuals presenting PCOS and gingivitis, compared to controls. Also, serum antibody levels were measured using ELISA. Individuals with PCOS and gingivitis showed an increased antibody-mediated immunological response to P. gingivalis, and periodontally healthy individuals with PCOS, showed an increased response to P. intermedia and S. oralis.Polycystic ovary syndrome (PCOS) is an endocrine, reproductive and metabolic condition affecting women of reproductive age, with a prevalence between 15-20% . RecentlOne more study used PCR to evaluate the presence of nine different periodontal bacteria in young individuals with and without PCOS . In thisActinobacteria without further significant variation in community composition and diversity between the studied groups. This reduction could favor an environment suitable for disease-associated bacteria, increasing the risk of dysbiosis. A small sample size and the absence of more severe forms of PCOS could have influenced the obtained results.To date, only one study has used next generation sequencing to investigate possible changes in the oral microbiome of individuals with PCOS . This stExisting research remains contradictory regarding the role of PCOS in the composition of the oral microbiome and its contribution to inflammation and periodontal health/disease.Hormonal contraceptive methods consist of synthetic forms of progestagens and/or estrogens at levels aimed to suppress ovulation, similar to the ones achieved during pregnancy. These come in different dosages and formulations and can be administered orally , in the form of injections, implants, intravaginal, topical patches and intra uterine devices .Previous studies have investigated the possible influence of oral contraceptives (OC) on the composition of the subgingival microbiome . These sP. gingivalis, P. intermedia and A. actinomycetemcomitans have been observed in women using oral contraceptives together with an increased risk of developing periodontitis  or combined (estrogen-progesterone) preparations. Administration can be oral, transdermal, percutaneous, intramuscular, intranasal or local with dosing and timing depending on the case .P. gingivalis, P. intermedia and T. forsythia – after 2 years of HRT use. Even though these results seem optimistic and could indicate a positive influence of HRT on periodontal health, the role of HRT on clinical attachment loss (CAL) remains debatable and inconclusive used by individuals looking forward to gaining muscular mass, leading to different complications .Studies evaluating oral health and the use of androgens are scarce. The reason behind this could be that androgens do not experiment extreme fluctuations like female SSH do. This results in a more stable concentration throughout the life of an individual .A. actinomycetemcomitans, P. intermedia, P. gingivalis and Candida species associated to a diseased oral state, compared to a control group of anabolic steroids non-users.It can be expected that androgens have an effect in the oral cavity as androgen receptors have been described in diverse oral tissues such as gingiva , oral muTaken together, the above-described studies indicate that it is likely that SSH affect the oral cavity, however more studies are needed to understand how endogenous and exogenous androgens can affect the balance between the host and the oral microbiome.in vitro and in vivo studies have aimed to clarify the direct and indirect effects of SSH on the oral microbiota and host tissues. It is important to note that both these approaches have advantages and limitations. In vitro studies provide a controlled environment and can explain the mechanisms involved in microbial responses to SSH. Nonetheless, they provide only a simplified interpretation of reality and lack important factors that play a role in the complex host-microbiome interaction. And while in vivo studies offer real-life conditions whose results can be directly applicable to the clinical field, there is a high degree of subject variability and, more importantly, a real risk of bias. Both in vitro and in vivo studies have mostly targeted specific periodontal pathogens and failed to look at the oral microbiome in its totality. The incorporation of both approaches is therefore most suitable to grasp the whole picture.We set out to review the data available on the effect of SSH in the oral cavity. Various in vivo and in vitro studies makes them difficult to compare. The technologies used to classify microorganisms in early studies were less sensitive and did not document all species correctly.The great variability between the methodologies applied to date in The available evidence makes it evident that hormones can affect the oral microbiome both directly and indirectly. The remaining question is if and to what extent these effects play a role in oral health and disease and what the underlying mechanisms are.Recent studies have approached this question using clinical evaluation paired with current technologies such as sequencing, that focus not only on classic periodontal pathogens but also on the oral microbiome . These aBuilding on the available evidence, we propose a hypothesis that describes the mechanism of this interplay between SSH, host and microbiome.By metabolizing SSH, certain bacteria can directly and indirectly influence SSH concentration in the environment . Their cWe therefore propose a hypothesis to explain how SSH concentrations in saliva can lead to shifts in the oral microbiome and the oral milieu that directly induce a response from the host. These shifts include the proliferation of certain species and the metabolism of SSH into other molecules. Meanwhile, the host responds to these changes and – in turn – metabolizes SSH in the medium, adapting its physiological response to the environmental conditions Figure 2Toxoplasma gondii – a protozoan parasite – can induce the steroidogenesis of testosterone, supporting the hypothesis that microorganisms can also modulate the synthesis of SSH to their own benefit , University of Amsterdam and VU University Amsterdam.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bifidobacterium genus are among the most abundant colonizers of the gut of healthy infants  with its host, and their application as probiotics to improve human and animal health. Here, we summarize some of the scientific highlights of the articles published in this special issue.The present Research Topic entitled “Kelly et al.). Many so-called non-digestible glycans specifically stimulate growth of particular bifidobacterial strains and/or species. Among them, galacto-oligosaccharides (GOS) are widely used as prebiotics in infant nutrition. Ambrogi et al. showed that the enzyme BgaE, from Bifidobacterium longum subsp. longum NCIMB 8809, is suitable for in vitro GOS synthesis using lactose as the starting substrate. This work highlights the potential of bifidobacterial enzymes to be exploited for the development of dietary prebiotics.Bifidobacteria metabolize a variety of complex carbohydrates that through their metabolic end products also provide nutrients to the host. This remarkable metabolic behavior reflects the importance of carbohydrate utilization as part of bifidobacterial gut colonization and persistence. The current knowledge of bifidobacterial carbohydrate metabolism is reviewed, with a focus on plant poly-/oligosaccharide degradation  biosynthesis-associated genes across the genus Bifidobacterium. The authors generated spontaneous riboflavin-overproducing variants of Bifidobacterium longum subsp. infantis ATCC 15697, which were also shown to increase vitamin B2 concentration in a fecal fermentation system, thereby providing promising data for application of this isolate as a functional food ingredient.Another feature of bifidobacterial metabolism with potentially important health implications is the Yuan et al. to evaluate the effect of bifidobacteria on intestinal epithelial layers when damaged by pathogenic Escherichia coli.Commensal bacteria colonize the gut and by doing so offer protection against pathogens. Mechanisms of gastrointestinal protection by probiotic bacteria against infection involve modulation of intestinal epithelial barrier function. The application of trans-epithelial electrical resistance (TEER) has been used by Hickey et al.The production of extracellular layers, such as exopolysaccharide (EPS), by certain bifidobacteria taxa, allows them to overcome gastrointestinal challenges and to persist for longer periods in the gut .Alterations in gut bacteria and their metabolites on several inflammatory and immune processes have been associated with carcinogenesis and tumor etiology . The administration of the bifidobacterial strain resulted in the reduction of gut inflammation in mice without recovering gut microbiome diversity. This positive effect in the gut only occurs when proinflammatory species-induced gut inflammation .Probiotics are believed to become a potent tool to modify the composition of the gut microbiome and to benefit host health in multiple ways. As part of this Research Topic, several papers discuss the use of bifidobacterial strains as probiotics. For example, the use of Horigome et al. in low birth weight infants, the oral administration of Bifidobacterium breve M-16V allowed its gut colonization for several weeks post-administration and appeared to improve gut microbiota formation. In addition, bifidobacteria isolated from dogs were characterized in vitro and in vivo for potential use to support canine health .To exert a probiotic effect, a bacterial strain may need to colonize and persist in the host. In the clinical trial carried out by Bifidobacterium species that efficiently colonize the infant gut. Yan et al. explored the co-occurrence of Bifidobacterium phylotypes in mother–breast milk–infant triads. The authors describe that the groEL gene is an effective target for in depth resolution of Bifidobacterium communities. This approach allowed the assignment of Bifidobacterium phylotypes in mother–infant pairs.The period immediately following birth is crucial for the appropriate development of the gut microbiota and infant development  systems. In this regard, a novel genetic tool for bifidobacterial targeted mutagenesis, based on a synthetic vector (pFREM28) lacking known B. breve R-M motifs, was described and validated by Hoedt et al.. This approach can be applied to design synthetic plasmids to target other genetically inaccessible bifidobacterial species. Additionally, an adapted plasmid expressing mCherry with OVA (pMG-mCherry-OVA) for bifidobacteria was developed to study bifidobacterial-host interactions in both in vitro and in vivo studies .To shed light on probiotic features and gut adaptation of bifidobacteria more mechanistic and functional analysis are needed through embracing different “omics” approaches in combination with novel/existing molecular tools, technologies and models. Members of the genus In summary, the integration of various approaches to determine the presence and functionality of bifidobacteria in the gut are crucial to improve our understanding of the complex interactions between the human (gut) and  bifidobacteria. Future probiotic interventions directed to modulate gut microbiota will lead to “next-generation” probiotic bifidobacterial strains in functional food and nutraceutical industries.MET and DS wrote and edited the manuscript. All authors have made substantial contributions to the article and approved the manuscript for publication.DS and MET are members of APC Microbiome Ireland, which is a research center funded by the Science Foundation Ireland (SFI), through the Irish Government's National Development Plan. The authors and their work were supported by SFI (Grant SFI/12/RC/2273), Irish Research Council Postdoctoral Fellowship (GOIPD/2017/1302) and MSCA-IF (Grant number 898088). LR has received funding from the Spanish State Research Agency RTI2018-095021-J-I00 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction: In recent periods the United Arab Emirates (UAE) has strengthened economic measures in its pharmaceutical policy by promoting local manufacturing and facilitating the use of generic medicines. International examples indicate the importance of quality control elements in the implementation of cost containment policies. Multicriteria Decision Analysis (MCDA) is increasingly used in health care to facilitate health care decision based on multiple objectives. Our objective was to develop a pilot MCDA tool for repeated use to support the value-based purchasing of generic medicines in the UAE.Methods: An international evidence framework was adapted to UAE in a multistakeholder workshop organized by Dubai Health Authority. After validating the relevance of nine criteria in the local jurisdiction, participants decided the ranking and weight of each criterion by anonymous voting.Results: The top four criteria focused on quality elements starting with real-world clinical or economic outcomes (with 19.8% weight), followed by the quality assurance of manufacturing (17.3%), then evidence on the equivalence with the original product (14.8%), and drug formulation and stability (12.3%). The pharmaceutical acquisition cost criteria ranked fifth with 9.4% weight. The bottom four criteria, including reliability of drug supply, macroeconomic benefit, pharmacovigilance and added value services related to the product had similar weights in the range of 5.5–7.7%.Conclusion: Policy-makers in Dubai put high emphasis of value-based health care by incentivizing manufacturers of off-patent pharmaceuticals to generate additional scientific evidence compared to the mandatory minimum and acknowledging efforts to improve quality standards. The MCDA tool is considered suitable to improve the transparency and consistency of decision making in UAE for off-patent pharmaceuticals, and subsequently for other health technologies. The UAE has a comprehensive, government-funded health service and a rapidly developing private health sector that delivers a high standard of health care to the population. Health care in the UAE is funded mainly by the government, however, private health care financing plays a crucial role in outpatient care especially in Dubai and Abu Dhabi .In Dubai healthcare is regulated at both the federal level by the Ministry of Health and local level by the Dubai Health Authority (DHA) . DHA hasIn the United Arab Emirates (UAE), most pharmaceutical products are imported from around the globe under the regulatory framework of the Ministry of Health. In recent periods economic measures in the local pharmaceutical policies have been strengthened by promoting local manufacturing and facilitating the use of generic medicines .External price referencing is applied to interpret the Emirati pharmaceutical prices in comparison with the prices in the country of origin, in Gulf and other countries (such as the United Kingdom) in the reference basket . The finInternal price referencing is applied for chemically and pharmacologically similar products registered in the UAE. Locally produced generic products must be priced 30% lower than the innovator or market leader medicine and imported generics must have at least a 40% price discount .The efficiency of generic drug policies is usually assessed by measuring the level of price erosion and market share of generics . Indeed,In 2019, over 61% of the UAE’s market share in standard units on prescribed OPPs were toward chronic disease; yet, OPPs had significantly higher market share in acute diseases, such as antiinfective and gastrointestinal medicines. On the other hand the utilization of OPPs is still relatively low in specialty areas with high growth rate, such as oncology  or diabeWhilst cost-savings due to price erosion is an important benefit of utilizing generic medicines, drug quality, equivalence with the originator medicine, drug formulation, supply reliability, medical adherence and persistence, real world health outcomes and non-pharmaceutical costs also contribute to the success of generic drug policies . IgnorinIn 2015 a group of international experts selected 22 potential criteria that could be included into multicriteria decision analysis frameworks of OPPs . By consOur objective was to take a step forward in the implementation of value-based health care in UAE by developing a pilot MCDA tool for repeated use to support the value-based purchasing of OPPs (excluding biosimilar medicines) based on the international evidence framework.The process of developing the MCDA tool to facilitate value-based policy decisions for off-patent pharmaceuticals in Dubai is described in The preparatory work between leading pharmaceutical policy experts of Dubai Health Authority (DHA) and international experts contributing to the EFOR development started in January 2020. After concluding on the objectives of DHA, a two-day workshop structure was selected for the development of the MCDA tool by final selection, ranking and weighting of decision criteria. Due to the COVID pandemic, the face-to-face workshop had to be postponed and finally replaced by a one-day virtual workshop. To facilitate the workshop, a special Excel tool was prepared to lead workshop participants through each step.A virtual workshop was hosted by the Dubai Health Authority on October 1, 2020. Local stakeholders were present at the meeting room of DHA by keeping appropriate social distancing. International MCDA experts—with responsibility of facilitating the workshop without any influence on voting—were connected by webstream.The workshop started with an introductory session to introduce the concept of MCDA and the evidence framework for OPPs to the participants and an overview on pharmaceutical policies and market trends in UAE.Preformed questions on the Mentimeter platform allowed the international moderator to implement the anonymous voting remotely. The voting results were presented after all participants submitted their vote to avoid consensus bias . After eAs a first step participants were asked to provide consent their anonymous answers could be aggregated and used in the scientific publication.The first voting question explored the number of criteria included in the MCDA tool. After that, participants voted for the threshold of price differential compared to the cheapest OPP alternative.The next step was weight elicitation in two phases, in which participants first established an order of importance of the criteria by applying the SMART method. After selecting and ranking the most important three criteria, the second most important three criteria were selected and ranked and finally the bottom three criteria were ranked. The final round of voting explored the relative incremental weight of each criterion compared to the previous criterion , then the final weights were calculated by normalizing the sum of weights to 100% .The Excel tool included a test case which allowed participants to understand how the MCDA tool works in practice, to test its reliability and to discuss whether any amendments should be considered.Out of the 21 participants, 19 decision-makers represented the public sector and the remaining two participants represented the private insurance sector. The majority of participants were mainly representing Dubai’s regulatory and policy makers from the Dubai Health Insurance Corporation, the Dubai’s strategy and corporate development sector and Health Regulation Sector. All participants gave consent about publishing the aggregated anonymous answers.At first, participants voted about the number of criteria on the MCDA tool. 57% of the participants voted for not excluding any of the nine proposed criteria, while 19% participants voted for the inclusion of eight criteria, 24% participants voted for the inclusion of only seven criteria. Therefore, with majority voting all nine EFOR criteria were included to the MCDA tool.Subsequently, participants voted for 204% threshold of price differential compared to the cheapest OPP alternative. This means that if the price of the cheapest alternative is 100 AED, a product with 202 AED price would receive 50% of scores for the price criterion, and any medicines above 304 AED price is not eligible for any scores of the price criterion.Finally, participants completed the SMART ranking and swing weighting exercise. Results of the criteria ranking, and weighting are presented in MCDA is increasingly used in health care globally to improve the consistency and transparency of policy decisions . MCDA haThe intention of DHA to facilitate value-based health care is reflected in the choices of the MCDA tool for OPPs. The four most highly rated criteria focus solely on quality elements starting with real-world clinical or economic outcomes, followed by the quality assurance, then the equivalence with the reference product and closing with stability and drug formulation, leaving no more quality elements in the MCDA tool. These criteria would incentivize manufacturers to generate additional scientific evidence for example from observational studies or database analyses compared to the mandatory minimum and acknowledging efforts to improve quality standards. In fact, it is unlikely that many pharmaceutical companies can submit clinical trial or real-world evidence about the incremental benefits or improved stability or drug formulating of their OPPs. Such products can be considered value-added medicines, hence DHA intends to take a pioneer step to acknowledge evolutionary innovation of generic medicines .Surprisingly, the pharmaceutical price criterion ranked only fifth with 9.4% weight. In Kuwait the price criterion in the MCDA tool had 35% weight, and in Indonesia 40% weight . As oppoIt should be highlighted that the number of workshop participants was relatively small and may not be fully representative to all stakeholder groups with important contributions to pharmaceutical policies in Dubai. Hence, the MCDA tool for OPPs can be considered as a starting point for utilizing MCDA in healthcare decision-making in the UAE, which should be tested in pilot settings. The real-world experience during the pilot phase could then provide a basis for necessary adjustments of the MCDA tool in another workshop with a preferably broader representation of different stakeholder groups, including eminent pharmacists and physicians. Subsequently the MCDA tool can be used routinely in pharmaceutical purchasing decisions. An if the tool proves to be useful to facilitate value-based purchasing of generic medicines, further MCDA tools may be developed with similar methodology to support the purchasing decisions of other health technologies, e.g., biosimilar medicines, orphan drugs, vaccines or medical devices.


A 30-year-old man with a history of an in-situ melanoma of the forehead was referred for cardiac evaluation because of tachycardia and elevated levels of serum troponin. The transthoracic echocardiogram revealed multiple masses attached to the walls of both ventricles and the right atrium (RA). A large mass was occupying almost one third of the right ventricle (RV), resulting in reduction of the end-diastolic RV volume and tachycardia. A cardiac magnetic resonance imaging confirmed multifocal myocardial infiltration and intracavitary masses and excluded the presence of thrombus in any of the cardiac chambers. Diffuse metastatic involvement in the liver, the spleen, and the brain by computed tomography precluded surgical management. Being BRAF-unmutated, the patient was initially treated with a combination of nivolumab and ipilimumab. One month later, the cardiac metastases in RA and left ventricle were unchanged on echocardiogram, while the tumor in RV was enlarged occupying the majority of the chamber, resulting in further reduction of the cardiac output and tachycardia. The treatment was changed to a combination of dacarbazine and carboplatin, but the patient eventually died two months later. Heart is not a common metastatic site of melanoma and cardiac involvement is usually clinically silent making ante mortem diagnosis difficult. Multimodalidy imaging plays a pivotal role in the diagnostic work up. Cardiac melanoma metastases indicate an advance stage disease with poor prognosis.


By analogy to the conductor in an orchestra managing multiple instrumental sections into a dynamically integrated musical piece, the cross-talk between these signaling cascades places the mitochondria as a major conductor of cellular integrity.Several studies reported that mitochondrial stress induces cytosolic proteostasis. How mitochondrial stress activates proteostasis in the cytosol remains unclear. However, the cross-talk between the mitochondria and cytosolic proteostasis has far reaching implications for treatment of proteopathies including neurodegenerative diseases. This possibility appears within reach since selected drugs have begun to emerge as being able to stimulate mitochondrial-mediated cytosolic proteostasis. In this review, we focus on studies describing how mitochondrial stress activates proteostasis in the cytosol across multiple model organisms. A model is proposed linking mitochondrial-mediated regulation of cytosolic translation, folding capacity, ubiquitination, and proteasome degradation and autophagy as a multi layered control of cytosolic proteostasis that overlaps with the integrated stress response (ISR) and the mitochondrial unfolded protein response (UPR While unique protein quality controls exist for individual organelles, disruption of the homeostasis of one organelle can affect the function of the others . Therefomt). In addition, stress in both the endoplasmic reticulum and the mitochondria have been found to converge on the regulation of translation through phosphorylation and attenuation of the translation elongation factor eIF2a. This response is referred as the integrated stress response (ISR). Subsequently, a clear interconnection between the UPRmt and the ISR was described. Since both of these pathways have been the topic of excellent recent reviews  have been reported to regulate cytosolic proteostasis, but their relation to the ISR and the UPRmt remains unclear.The UPRmt to include a well-orchestrated coordination of all steps leading to balanced and functional proteome.Proteostasis is defined as the process by which a functional and balanced proteome is maintained. In order to be achieved, a balanced proteome requires the coordination between mRNA translation, protein folding and regulated proteasome degradation as well as autophagy . The picThe ISR refers a network of stress-activated kinases that phosphorylates the eukaryotic translation initiation factor 2 (eIF2a) leading to its inactivation and attenuation of general translation. These kinases are protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), heme-regulated eIF2a kinase (HRI), and general control non-repressed kinase 2 (GCN2). An array of different stresses activates these kinases including amino acid starvation, heme deficiencies, viral infection, and stress in the endoplasmic reticulum . The attHowever, while general translation is attenuated, selected mRNAs that contain upstream open reading frames (uORFs) are selectively translated during ISR . The traC. elegans), ROS are necessary for the GCN-2 dependent phosphorylation of eIF2a. GCN-2 is a nutrient sensor and is mainly known to be activated by starvation or amino acid depletion via deacylated tRNA . In agreement with this possibility, they found that deletion of GCN-2 in C. elegans increases the dependency on ATFS-1 for survival  and is considered to be a distinct response to the UPRmt, since the UPRmt is best known to regulate mitochondrial proteases and chaperones.While the latter study demonstrated that defect in mitochondrial proteins import due to inactivation of the Tim23 translocase complex promotes mitochondrial proteostasis, defect in mitochondrial import specifically in the inter-membrane space (IMS) of the mitochondria was shown to affect cytosolic proteostasis. Having two sub-compartments, the IMS and the matrix, importing proteins into the mitochondria requires a precise sorting of proteins destined to these respective sub-compartments. The MIA machinery is responsible for the import of proteins specifically in the IMS . The ChaNuclear factor erythroid derived 2-related factor 1, Nrf1 also named NFE2L1 , a potent transcription factor and leads to the up-regulation of proteasome activity , a key transcription factor for the heat shock response in the cytosol (mt and heat shock response (HSR), they named this response the mitochondria-to-cytosol response (MCSR)  . Of notee (MCSR) .C. elegans. They found that activation of the MCSR reduced the accumulation and toxicity of polyQ protein aggregates in skeletal muscle and improved motility in C. elegans  genes. Nutrient starvation was initially shown to be the mechanism of activation of autophagy . Subsequutophagy . Importautophagy . The linutophagy .mt has also been reported to activate the transcription of several autophagy genes  inhibitor perhexiline (PHX) leads to inhibition of fatty acid oxidation, CPT inhibitors are already used clinically to improve heart function. The Dillin lab showed that by inducing MCSR pathway, CPT inhibition by PHX reduces the accumulation of polyQ protein aggregates . Therefomt in C. elegans but not the HSR and was found to reduce amyloid beta deposits in the SH-SY5Y neuroblastoma cell line  in neurodegenerative diseases especially due to the observation of sex differences that characterize these diseases and because the basis of these differences are largely unknown . Severalmt leading to stimulation of proteasome activity is also a promising avenue to stimulate cytosolic proteostasis. Further, since raloxifene is widely used clinically (However, a significant oversight in the use of SERMs in these diseases is the differential effect of SERMs, including tamoxifen and raloxifene, on the transcriptional activity of the ERα  as well inically  and no sER and the UPRmt in C. elegans (Resveratrol, a compound derived from red wine was shown to reduce accumulation of b-amyloid protein, an hallmark of Alzheimer’s disease. In a recent study the Wenzel group investigated the mechanism by which resveratrol mediate this effect. They found that resveratrol activates both the UPR elegans . Further elegans . However elegans .mt and the ISR has been fascinating and the complexity of these pathways and the respective roles of distinct axes remain to be clearly defined. In this review, we have attempted to argue that mitochondrial stress leads to the activation of a combination of axes of these pathways that ultimately leads to a comprehensive control of cytosolic proteostasis at all levels; translation, folding and degradation by the proteasome as well as autophagy (The history of the discovery of the UPRutophagy .mt and the direct upregulation of the proteasome, respectively. The picture that emerges is that like the UPRmt, the ISR may actually consists of several axes.Of particular interest is the observation that proteins localized to the membrane of the endoplasmic reticulum notably PERK and NRF1/NFE2L1 contribute to the maintenance of mitochondrial function by regulating the ISR and activation of the UPRThe fact that drugs currently used clinically begin to emerge as potential therapeutics against proteopaties by exerting moderate mitochondrial stress and activating cytosolic proteostasis represent an exciting avenue for future research. However, the success of these drugs is likely to be tissue specific. Notably, while the expression of the mitochondria import machinery is ubiquitous , it was DG and EJ made the figure. All authors have participated in the writing of this review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Principles of 3Rs are the backbone of today’s animal research. Applying 3Rs in practice requires proper education and training. Although this could be achieved via long-term courses ranging from several weeks to years, in some circumstances a short-term course may be the only viable option. In this paper we define scientific and ethical objectives for running short-term 3Rs workshops. To meet these objectives, we propose a 2-day workshop curriculum and an ethical framework. The curriculum comprises theoretical and practical sessions and covers Replacement, Reduction, and Refinement strategies. The ethical framework defines the responsibilities of lecturers and attendees, and proposes the animals and facilities requirements to run the proposed workshop curriculum. The attendees will be assessed at the end of the workshop and they receive certificates for working under supervision until they are deemed competent at their intended functions. The proposed curriculum and framework are not subscriptive, instead they share the experience gained through running more than 60 laboratory animal 3Rs workshops.The online version contains supplementary material available at 10.1186/s42826-022-00120-9. The proper care and use of laboratory animals are best described by 3Rs principles that emphasize Replacing, Reducing, and Refining the use of animals for scientific purposes . AdherinHowever, research shows that there are unmet educational and training needs among animal research workers . In factIn this regard, we developed a framework for holding intensive two-day workshops. This framework is intended to assist attendees in acquisition of required knowledge and skills for properly and ethically performing supervised laboratory animal experiments. Additional training and supervised practice will be necessary to gain proficiency in the techniques for unsupervised work with animals. This workshop framework is not meant to be a prescriptive document. Instead, it is aimed to reflect the knowledge and experience gained through performance of a large number of previous workshops, and to incorporate this knowledge and experience into the planning and execution of future courses.describe how 3Rs principles affect the scientific quality of animal researches,describe the alternative methods that could be used instead of animal testing in projects,interpret the basic principles of animal research methodology,summarize the biological characteristics of common species and strains of laboratory animals,describe proper standards of care and husbandry of animals in research settings,identify the principles of safety and security in working with laboratory animals and naming the commonest zoonotic diseases,interpret the principles of sampling or materials administration,interpret the methods of recognizing pain and distress in laboratory animals,differentiate anesthesia and analgesia and describe proper methods of anesthesia/analgesia in common laboratory animal surgeries,summarize humane endpoints, and define proper methods of euthanasia and management of unexpected animals’ deaths,demonstrate proper handling methods of mice and rats,perform proper sexing of animals,perform dose calculation, and demonstrate proper methods of administrating substances to animals, andapply proper techniques for blood sampling.For developing the workshop plan,we used the experience gained through holding more than 60 laboratory animal workshops in the country. We also adhered to the principles outlined in our Guideline for the Care and Use of Laboratory Animals . We firsThen we developed an intensive 2-day workshop curriculum to achieve the scientific objectives via theoretical and practical sessions. This workshop curriculum is presented in Supplement 1 to this paper  We defined four major components of a standard laboratory animal workshop as: animals; attendees; human resources (i.e. lecturer and supervisors); and facilities . 2) We defined how each component would be exercised to achieve the scientific objectives of the workshop. (3) We explored how exercising each component may negatively affect the welfare of animals. (4) We applied the 3Rs principles for replacing the negatively affecting exercises where possible. (5) If full replacement was not possible, we defined strategies to reduce the number of those exercises and the number of affected animals. (6) We refined all the exercises involving animals so that they cause the least possible negative effects for the animals. (7) Finally, we combined the results of the stages 4–6 to develop the ethical framework. This framework is presented in Supplement 2 to this paper  assessment of the workshop holders, and (2) assessment of the workshop attendees. The workshop holders could be assessed from three viewpoints: executive aspects, scientific quality, and lecturer(s)’ performance. Some open-ended questions may also be considered to let the attendees freely express their opinions on various aspects of the workshop.The executive aspects may involve 5-point Likert questions on the timing, place, and facilities of the workshop. The scientific quality assessment may involve questions on the effect of the workshop on enhancement of the knowledge and attitude of the attendees, ability of the workshop in resolving the work-related problems of the attendees, and appropriateness of the workshop plan with the educational needs of the attendees. The lecturers’ performance may be assessed from at least three viewpoints: discipline, teaching skills, and interaction with attendees.Workshop attendees may be assessed from knowledge and attitude aspects. Ideally, the assessment should be performed pre- and post-workshop to evaluate the effectiveness of the workshop on enhancing their knowledge and attitude. At least one question should be derived from each theoretical topic and in accordance with the scientific objectives of the workshop. Attitude questions may cover a wide range of topics, such as animal feelings, sympathy with animals, the value of animals’ lives, replacing animals with non-animal alternatives, reduction of animals’ use, and refinement of procedures. These questions may be derived according to the ethical objectives of the workshop.The proposed workshop plan is developed to teach ethical and scientific aspects of the care and use of laboratory animals. The order and content of the theoretical session is based on the 3Rs principles. We first discuss the ethical aspects of using animals for scientific matters, aiming to morally sensitize the attendees. In this regard, we review ethics of animal use from various viewpoints: human conscience, religion, dialectics, societal concerns, scientific aspects, and law. By this, we aim to influence attendees having various viewpoints and attract their attention to the moral status of animals.The concept of 3Rs shaped the whole structure of the workshop. By discussing the Replacements to laboratory animals, we emphasize on the 1st R. Afterwards, the 2nd R is covered by teaching how various experimental designs and methods of sample size calculation could lead to more reliable data and less use of animals. The rest of the workshop discusses the 3rd R from various viewpoints.In the proposed workshop plan, no animal is used for ‘educational’ purposes and their use are only limited to training purposes. We recommend using surplus stock animals and animals from completed studies, that had been fully recovered from prior interventions with a severity score of mild to moderate . The sevIn comparison to the European framework for education and training in laboratory animal science , our proThe proposed workshop curriculum and ethical framework are provided as suggestions and are not subscriptive. We believe that they provide essential scientific and ethical structures for holding intensive 3Rs workshops in laboratory animal science.Additional file 1: Proposed workshop curriculum. Proposed workshop curriculum is presented in this supplement.Additional file 2: Ethical framework for holding intensive laboratory animal workshops. Proposed ethical framework is presented in this supplement.Additional file 3: Lab record sheet. A sample of the lab record sheet is presented in this supplement.Additional file 4: Practical session structure. The structure of the practical session is presented in this supplement.


Parkinson’s disease (PD) is a common neurodegenerative disorder classically characterized by symptoms of motor impairment , but also presenting with important non-motor impairments. There is evidence for the reduced activity of both the parasympathetic and sympathetic limbs of the autonomic nervous system at rest in PD. Moreover, inappropriate autonomic adjustments accompany exercise, which can lead to inadequate hemodynamic responses, the failure to match the metabolic demands of working skeletal muscle and exercise intolerance. The underlying mechanisms remain unclear, but relevant alterations in several discrete central regions  have been identified. Herein, we critically evaluate the clinically significant and complex associations between the autonomic dysfunction, fatigue and exercise capacity in PD. Parkinson’s disease (PD) is currently the second most common neurodegenerative disorder  and the Sympathetic and parasympathetic branches of the autonomic nervous system are crucial for homeostasis control  and are The parasympathetic preganglionic neurons are situated in the nucleus ambiguous (NA) and dorsal motor nucleus of the vagus nerve (DMV). The axons travel within the vagus nerve  and synapse at postganglionic neurons, such as those located at the cardiac plexus. While the NA contains vagal preganglionic neurons that inhibit the heart rate , the actin vivo studies have reported significantly smaller vagus nerve axons in patients with PD  and sympathetic ganglia] and parasympathetic centers , and may disrupt the central components of autonomic reflex arc involved in autonomic regulation . Human s with PD . With re with PD . In fact with PD .Heart rate variability (HRV) derived estimates of cardiac parasympathetic activity have been observed to be reduced in patients with PD  independSympathetic preganglionic cell bodies are located in the IML of the spinal cord and the preganglionic fibers directed to the heart synapse at the stellate ganglion and upper thoracic ganglia (T1–T5). Postganglionic sympathetic fibers synapse in the heart  and in the vasculature. The sympathetic preganglionic neurons in the IML receive strong excitatory drive from neurons of the rostral ventrolateral medulla (RVLM) . SympathOrthostatic hypotension (OH) affects approximately 50% of the patients with PD and the prevalence may be higher when considering asymptomatic OH . NoteworThe microneurography technique provides a valuable tool for direct recording of muscle sympathetic nerve activity (MSNA) in humans . Shindo Despite the fact that most indices derived from HRV primarily reflect vagal function, the joint analysis of the low frequency (LF) component, expressed in normalized units, and the LF/HF ratio, has been suggested to be an acceptable marker of sympathetic modulation  althoughDuring exercise, several neural mechanisms work in concert to precisely control cardiovascular and hemodynamic responses . In neurThe first evidence for an attenuated cardiovascular response to exercise in PD was provided in the late 80’s by Sympatho-excitatory mechanisms are activated during exercise , where both mechanically and metabolically sensitive sensory fibers provide feedback via the dorsal horn of the spinal cord to brainstem cardiovascular areas in response to mechanical  and metabolic stimuli , respectively . The latParasympathetic activation produces negative inotropic, chronotropic and dromotropic effects on the heart . The actLowered vagal activity generated by the DMV might play a role in the attenuated cardiovascular responses, as well as, lowered exercise capacity presented by some patients with PD . In an aThere is currently no cure for PD, and although the primary resource for symptomatic control is pharmacologic-based, non-pharmacological low-cost approaches, such as exercise training, are being thought of as the universal prescription for PD . EpidemiOptimal autonomic nervous system function is crucial for evoking appropriate cardiovascular and hemodynamic adjustments to exercise. Emerging evidence suggests that cardiac parasympathetic activity may be also a determinant of exercise capacity. In PD, the pathogenesis affects both parasympathetic and sympathetic nerve activity, which in turn seems to mediate the abnormal cardiovascular responses observed during exercise. These findings may be important in the understanding the complexity of the neural control of circulation in patients with PD. Further studies are needed to better understand the pathophysiological underpinnings on the exercise response in PD.JS-C wrote the first draft of the manuscript. All the authors read and revised it critically for important intellectual content. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The use of chemotherapy drugs against tumours is associated with various drawbacks such as poor solubility, low stability, high toxicity, lack of selectivity and rapid clearance. Nanocarriers can improve the safety and efficiency of drugs by increasing their solubility, enhance their circulation time and improve their uptake into cancer cells. Natural materials can be incorporated in the fabrication of nanocarriers as a substitute to synthetic ingredients. Several studies developed different types of green based nanocarriers using materials obtained from plant or microbial sources such as polysaccharides and polyphenols without the need of toxic chemicals in the synthesis. The green components can have many roles for example as mechanical support, trigger pH response for drug release, or act as a targeting ligand. The inclusion of these green components will support the cost effective and feasible large-scale production of nanocarriers with minimum negative impact on the environment.The use of nanocarriers for biomedical applications has been gaining interests from researchers worldwide for the delivery of therapeutics in a controlled manner. These “smart” vehicles enhance the dissolution and the bioavailability of drugs and enable their delivery to the target site. Taking the potential toxicity into consideration, the incorporation of natural “green” materials, derived from plants or microbial sources, in the nanocarriers fabrication, improve their safety and biocompatibility. These green components can be used as a mechanical platform or as targeting ligand for the payload or can play a role in the synthesis of nanoparticles. Several studies reported the use of green based nanocarriers for the treatment of diseases such as cancer. This review article provides a critical analysis of the different types of green nanocarriers and their synthesis mechanisms, characterization, and their role in improving drug delivery of anticancer drugs to achieve precision cancer treatment. Current evidence suggests that green-based nanocarriers can constitute an effective treatment against cancer. Nanotechnology is an interdisciplinary field of science concerned with the processing of matter at atomic/molecular level . NanotecThe commonly used conventional chemotherapies include alkylating agents, antitumour antibiotics ), antimetabolites , methotrexate, gemcitabine), topoisomerase inhibitors, and mitotic inhibitors  . ChemothTherefore, the overall aim of including green components in the fabrication of NPs as nanocarriers is to decrease toxicity to the body, to have an environmentally benign industry process and to increase affordability. The green synthesis may be facilitated by plants extracts and microbes or their isolated biomolecules . The greIn this review, we will highlight different examples of green based nanocarriers used in the drug delivery of conventional anticancer drugs that contain a plant or microbial-derived components in their formulation. The review will highlight the role of the different green component used in the synthesis or in the drug delivery mechanism. In addition to the safety of using these green components, the review will also include the various benefits and the advantages of using each type of the green components.2) in their structures enables them to establish non-covalent bonds with biological cells and to form bioadhesive polymers that can improve the efficacy of different drugs ..36].Aureobasidium pullulans fungus ..67].trans-stilbene) is a natural polyphenol, which is extensively reported with anticancer activity [Resveratrol  into these nanoethosomes increased their stability. The EGCG-nanoethosomes also showed efficient skin penetration reaching the hypodermis, whereas the basic-nanoethosomes were only located in the dermis and epidermis as exhibited by confocal laser microscopy. Docetaxel was then loaded into the EGCG-nanoethosomes and its antitumor activity was evaluated against human melanoma cancer cells (A-375) implanted in mice skin. The EGCG-nanoethosomes loaded with docetaxel led to a significant reduction in the tumour size in comparison to the basic-nanoethosomes loaded with docetaxel [Liao et al. (2016) reported the use of EGCG in the preparation of nanoethosomes for transdermal delivery of docetaxel. The basic nanoethosomes were prepared using 2% soybean phosphatidylcholine, 30% ethanol, 1% Tween-80 and 0.1% sugar esters. The loading of 0.2% EGCG  implanted on mice [The effectiveness of DOX was also enhanced by loading it on liposomes made of two layers of phospholipids of lecithin and  on mice .Zhang and co-workers (2017) developed liposomes made of phospholipids coating mesoporous carbon matrix for the oral delivery of the poorly soluble, docetaxel. The authors used carboxymethyl chitosan to protect the liposomes from the gastric pH. The incorporation of phospholipids and was found to increase the sustained release effects of the liposomes. Additionally, the carboxymethyl chitosan and phospholipids layers were shown to reduce the toxicity of mesoporous carbon matrix, which can lead to increased biocompatibility .The use of cells as drug delivery systems has been studied for their bioavailability, increased targeting effects, enhanced drug pharmacokinetics, controlled released capabilities and reduction of drug immunogenicity and toxicity . Microbi2 and Na3PO4 aqueous solutions. The yeast-hydroxyapatite nanoscaffolds were loaded DOX as a drug model and further functionalized with FA for targeting purposes (DOX-nHAP@yeasts-FA). The mice infected with hepG2 subcutaneously showed significant reduction in their tumour volume by DOX-nHAP@yeasts-FA as well as free DOX. However, the authors highlighted the safety of using DOX-nHAP@yeasts-FA because of their lower toxicity on normal cells in comparison to the free DOX [A study used yeast cells for the first time to host hydroxyapatite nanoscaffolds after the incubation with CaClfree DOX .E. coli expressing heavy metal binding proteins (HMBPs). The biogenic GNPs were found to be safer than conventional synthesized GNPs. Further, the loading of DOX on the biogenic GNPs was done and demonstrated higher apoptosis rate than the free DOX mainly due to the improved intracellular uptake as reported by the authors  reported the formation of spherical GNPs from the carriers .Different green components were shown to be key players in the synthesis and in the function of different types of nanocarriers. The roles of these green components range from being a scaffold for materials such as drugs or targeting ligand, to the reduction of metal salts and the stabilization of MNPs. These green components were derived mainly from plant or microbial sources, which led to the biosynthesis of biocompatibility nanocarriers with improved safety margin that can allow their use in biological applications. Furthermore, the shape, size, and stability of green nanocarriers could be optimized by regulating the pH, reaction temperature, reactants’ concentrations, and time of incubation during the synthesis. In most of the reports, the green nanocarriers were mainly found to increase the anticancer activity of the loaded drugs. This was done by having augmented anticancer properties, by increasing the targeting effect, or by excreting controlled release. Some reports also formulated pH responsive nanocarriers to take advantage of the different pH levels between cancer and normal tissues, which can assist in increasing the target effects of their therapeutic payloads.There are few challenges that face the potential use of green based nanocarriers in the market. Similar to other conventional nanomaterials, their synthesis remains complicated especially with polymeric NPs or liposomes. In addition, there are unsubstantial concerns of possible allergens or toxicity caused by impurities that can be found on the natural isolated components such as total plant extracts or natural phospholipids for example. However, using these natural parts can improve the safety of the nanomaterials. This is because biodegradable and biocompatible green components can reduce the toxicity that can arise from the nonbiodegradable residual parts of the conventional drug delivery systems. The green components can facilitate the synthesis without the need of toxic chemicals during the preparation of NPs, which improve the safety of the final product. Importantly, unlike the synthetic materials, these green renewable components will certainly reduce the cost of the upscaled production for commercial use.