The current political crisis, conflicts and riots in many Middle Eastern and African countries have led to massive migration waves towards Europe. European countries, receiving these migratory waves as first port of entry (POE) over the past few years, were confronted with several challenges as a result of the sheer volume of newly arriving refugees. This humanitarian refugee crisis represents the biggest displacement crisis of a generation. Although the refugee crisis created significant challenges for all national healthcare systems across Europe, limited attention has been given to the role of primary health care (PHC) to facilitate an integrated delivery of care by enhancing care provision to refugees upon arrival, on transit or even for longer periods. Evidence-based interventions, encompassing elements of patient-centredness, shared decision-making and compassionate care, could contribute to the assessment of refugee healthcare needs and to the development and the implementation of training programmes for rapid capacity-building for the needs of these vulnerable groups and in the context of integrated PHC care. This article reports on methods used for enhancing PHC for refugees through rapid capacity-building actions in the context of a structured European project under the auspices of the European Commission and funded under the 3rd Health Programme by the Consumers, Health, Agriculture and Food Executive Agency (CHAFEA). The methods include the assessment of the health needs of all the people reaching Europe during the study period, and the identification, development, and testing of educational tools. The developed tools were evaluated following implementation in selected European primary care settings. The current political crisis, conflicts and riots in many Middle Eastern and Sub-Saharan countries have led to an increase of massive migration waves towards Europe. In 2015, 1,255,600 first-time asylum seekers applied for international protection in the Member-States (M-Ss) of the European Union (EU), a number more than double compared to the previous year [a deep awareness of the suffering of another, coupled with the wish to relieve it” [The EUR-HUMAN Project focuses on strengthening PHC given this represents the first contact with the POE countries healthcare system for refugees and migrants, and also other countries accepting them at later stages, with the aim to provide affordable, comprehensive, person-centred, culturally appropriate and integrated care for all ages and ailments. However, the focus of the project, was exclusively on refugees, given Europe experiences the peak in the massive waves of refugee influx during the implementation period of the project, although, migrants were also representing target populations for the reported actions. Although reports on refugee needs were available prior to the period of the crisis, no systematic review and analysis of the refugee needs, wishes and preferences at their arrival had been attempted. Many efforts have been undertaken to address refugee health needs, both physical and mental including extensive efforts of non-governmental organisations (NGOs) in Greece and Italy. Nevertheless, such efforts have been disjointed and fragmented, as well as isolated from existing PHC structures and paths of care; an essential parameter for in the facilitating the smooth entry of refugees into the healthcare system and a foreign, on multiple levels, new society. A systematic approach based on evidence for the role of PHC and for the rapid capacity building of this pillar of care has not been previously discussed and studied. Compassionate care is also currently receiving a lot of attention in the context of PHC , with coieve it” .Although the notions of integration, patient-centredness, comprehensiveness and compassion have received high attention, with reports in the body of scientific literature emerging from all European countries, they still largely remain rhetoric, with a urgent need of evidence-based interventions encompassing these elements. Lack of integrated PHC in many European settings, including Greece, poses additional obstacles when it comes to implementing coordinated action and capacity-building .Most importantly, efforts assessing prior capacity-building actions and systematic efforts leading to evidence-based interventions are scarce in Europe. The European Commission has repeatedly highlighted the need for implemented evidence-based interventions with sound evaluation to inform and facilitate decision-making in terms of healthcare policymaking. This correspondence article reports on methods used within the EUR-HUMAN project for enhancing PHC for refugees through rapid capacity-building actions, including i) the assessment the health needs of all people that have reached Europe in the period of the project implementation independently their legal status with a more focus on refugees in Europe using a refugee-centred approach, ii) the identification, development and testing of tools and educational interventions to give appropriate healthcare to this population group within the frame of European PHC, and iii) the evaluation of tools developed and implemented in selected European primary care settings. The evaluation was aimed at gaining information on how to better facilitate uptake of successful practices across other M-Ss and beyond the duration and scope of the project.To meet all abovementioned project objectives, several resources of information had to be incorporated, including a refugee needs assessment, the systematic search of the current literature, and extensive consensus debate about the components of essential tools and interventions for care provision. A graphical representation of the Work Packages of the EUR-HUMAN project is depicted in Fig. Qualitative research techniques applied in the fieldwork in WP2 were typical for Participatory and Learning Action (PLA) . PLA tecNPT is a theoretical framework concerned with the work that individuals and organisations have to carry out in order to embed and normalise new, complex ways of working into routine practice” [This method facilitates the dialogue with national, regional and local stakeholders, as well as with the refugees themselves, in order to assess refugee needs, understand their wishes and elicit their preferences, with the aim of incorporating all these relevant elements in relevant healthcare service delivery. The initiated PLA-brokered dialogue with stakeholders has been anchored in a theoretical framework based on the four constructs of the Normalisation Process Theory (NPT)  with theractice” . It has Prior to introducing this PLA-dialogue, meetings with stakeholders and healthcare providers were held in order to capture the voice of the refugees and to facilitate the implementation of the project by making all stakeholders aware of the aims and proposed actions and communicating in a transparent manner how their cooperation was critical for timely implementation. A meeting with local stakeholders  took place in the island of Lesvos to that effect. To further facilitate the smooth operationalization of implementing PLA techniques, a two-day training of the PLA actors was arranged. These PLA sessions were held in refugee reception centres between February 2016 and March 2016. During the sessions, cultural mediators or interpreters were engaged to ensure cultural background barriers were eliminated and linguistic requirements, across a wide spectrum of languages, were met. In total, forty-three (43) such training sessions were conducted with ninety-eight (98) refugees and twenty-five (25) healthcare workers, across several countries.By using the PLA method, a qualitative, comparative case study was performed in hotspots, transit, intermediate -and longer- stay reception centres in seven EU countries  in order to enhance the data collection process. This study was a preliminary effort to assess the current conditions and feasibility of deploying an intervention in the participating countries, while at the same time determining the particular characteristics of each site, which, of course, needed to be taken into consideration. Data have been illustrated on PLA charts. In this manner, it was ensured that verbal and visual forms of data were recorded in a consistent manner across all stakeholder groups. All PLA charts have been digitalised after each data generation session in order to preserve the data. Verbal data have been recorded on post-it notes, in point form or short phrases rather than in full verbatim quotes. All sites analysed their data thematically, individually, on the basis of a universal, for all sites, coding framework provided. All sites reported on their fieldwork using a universal, for all sites, fieldwork evaluation form provided for this purpose. The results of the fieldwork helped to develop the flowchart and the training in Lesvos to address training needs for the local health professionals.http://eur-human.uoc.gr/work-package-5/). This included practical handbooks, manuals and reports, and validated tools that were identified through a systematic search in the literature. The proposed procedure consisted of triage, i.e., identification of MH conditions requiring immediate specialist attention in the circumstances of very high demand and constrained PHP resources, screening, i.e., identification of individuals who are under increased risk for developing serious MH conditions, immediate assistance based on the PFA principles, and ultimately, referral for full MH assessment and care as needed. This protocol also guided the development of the modules on MH within the online training course and the specific MHPSS face-to-face training. Furthermore, the consortium worked on the development of a protocol for the rapid assessment (RA) of mental health and psychosocial needs of refugees on the basis of an appropriate support model (MHPSS purposes) that was identified through the data collection and review activities  [Specific attention was given to the assessment of mental health (MH) needs of refugees, this being the task of a separate dedicated work package of the EUR-HUMAN project. For this purpose, a protocol for the rapid assessment of MH and psychosocial needs of refugees, including tools, guidelines and procedures and interventions for provision of Psychological First Aid (PFA) was developed. This protocol was developed on the basis of a hierarchical approach and according to expert guidelines on addressing the overall approach on MH and Psychosocial Support (MHPSS)  .PsychINFO; Sociological Abstracts; Cochrane; Pilots; PubMed; Embase). In total, 5492 articles were identified. After discussion, consensus was reached on selecting 264 articles for full text screening. All articles were primarily qualitative, descriptive or reporting on research employing mixed methods.In addition to this fieldwork, different literature databases were systematically accessed, searched and used for data retrieval during the project to identify relevant literature on suitable interventions, tools and known implementation factors to optimise healthcare provision for refugees in different European settings, focusing on a broad diversity of implementation factors. Search-strings comprising combinations of refugee-related terms and implementation terminology were formulated in English. The search strings were run through six (6) databases  participating professionals and experts, which were involved in managing the refugee crisis at the different work locations of the partners in the EUR-HUMAN consortium across countries, i.e., in Austria, Croatia, Greece, Hungary, Italy, and the Netherlands. To the same, effect, ten expert interviews  and Médecins du Monde (MdM) were conducted, to collect information on the context, meaningful structures, process characteristics and challenges of healthcare optimisation for refugees.. Apart from the experts that were proposed by the consortium partners, a refugee representative also participated.Based on the aforementioned, an Expert Consensus Panel from various European countries assembled all the selected and appraised material. The Expert Consensus Panel (ECP) aimed to engage experts in a two-day decision-making process, with the purpose to reach consensus agreement on best practice guidelines, tools and services for the early arrival and longer-term settlement of refugees in European host countries, not excluding groups of refugees that were stranded in “transit” countries for longer periods of time. In total, thirty (30) experts from fourteen (14) different countries attended the meeting. Initially, participants discussed in small thematic groups and then reconvened with the full group to present their conclusions and suggestions and discuss in an extended plenary session. Experts focused on four (4) overarching topics  and in 5 specific areas The consensus approved an operational workflow Fig.  to facilex ante and ex post questions in order to evaluate the knowledge gained. It was important to ensure that context-specific parameters were taken into consideration given the high degree of heterogeneity across the settings. Therefore, each country translated and adapted the training material according to their local situation and their needs.Based on the information gathered from the different sources including data and information from the PLA approach with refugees, the literature review and interviews with experts, the MHPSS, and insights gained from the output generated through the Expert Consensus Panel guided, the evidence-based training material was made available online. This training material covered eight different areas (modules), namely triage, mental health, communicable diseases, non-communicable diseases, vaccination, mother and child care, cultural and legal issues, and health promotion. Each module consisted of The educational intervention in the six PHC settings was performed through the deployment via an online course in an interactive platform. The core aim of this course is to support -building of the PHC providers by minimising knowledge gaps regarding different issues of PHC for refugees in the respective settings. In addition to this online course in an interactive platform, a face-to-face training carried-out across the different settings in partner countries . Austria, Hungary and Italy implemented the training material only via the interactive platform, while Croatian, Greek, and Slovenian PHC personnel were trained utilising both approaches, i.e., face-to-face training and interactive online platform. GPs, community nurses, midwives, health visitors/social workers, as well as refugees that were health providers in their country of origin, participated in is this training. Each setting was invited to determine the location and manner, multidisciplinary target group teams and the training topics given the adaptable modular structure of the course in the interactive online platform, as well as to adjust the educational intervention to the healthcare context system context, identified needs and expectations of refugees, and the local the PHC capacity.http://eur-human.uoc.gr/online-courses/) became gradually available from the end of October 2016 onwards and across six (6) implementation settings. People that actually provided health care services to refugees and serve the national healthcare systems were eligible to participate in the course. Course participants were mainly PHC practitioners serving the national health systems or NGOs who deal with refugees. The course is still online, readily accessible via cross-linking in the project website (http://eur-human.uoc.gr/online-courses/). Nearly four hundred (400) primary healthcare workers registered in the course, with more than one third of them having successfully completed the course prior to January 3, 2017 . Most users needed between eight (8) and sixteen (16) hours to complete the full training.This educational intervention was conducted for approximately 1 month. The online course , nurse, midwife and cultural mediator, a team that was formed to provide healthcare services to them according to their needs. In total, thirty (30) refugees (three (3) men, fifteen (15) women and twelve (12) children) participated in this smaller pilot. All patients received feedback on their health status and recommendations and advice regarding the necessity of the proposed treatment (s) (if any), with further referral to secondary care or specialist care, as needed. The pilot study was evaluated by qualitative research methods .The six intervention countries evaluated the selected educational interventions in order to provide answers to questions on their feasibility and acceptability. For this purpose, the NPT method and the NoMaD questionnaire have been utilised , 13. TheFurthermore, recommendations to policymakers were formed on the basis of findings of the EUR-HUMAN project. A meeting with all Consortium partners of the EUR-HUMAN project was held in Crete to conclude the final evaluation and to discuss future actions on establishing a Network for the Care of Refugees in a compassionate manner, and with emphasis on capacity-building actions and MHPSS care.Approval from bioethical committees from all implementing settings  has been sought and received according to the existing legislative framework in each participating country . In Lesvos, where selected refugees have participated in the pilot intervention study, written information and the respective informed consent form had been provided. Every participant filled in the informed consent form. The informed consent forms were translated into English, as well as Arabic and Farsi, the languages of the countries the majority of refugees were coming from at the time of the project implementation. In the cases where refugees were from a different country and not speaking English or the other two main languages, a translator/cultural mediator informed them. The sessions were audiotaped and transcribed (after requesting and being granted permission). PHC professionals agreed to participate in the training procedure, as well as in its evaluation. Study participation was voluntary for refugees and healthcare professionals alike.This article describes the implementation of an innovative design to develop tools and educational interventions with the aim of strengthening PHC, indeed it moved to the direction of ‘co-creation’ working with the refugees and other stakeholders. It reports on methodology on how it assembled knowledge from several resources by using evidence-based approaches and through eliciting information from the refugees themselves regarding their needs, wishes, and preferences, but, also, by brokering dialogue with all relevant stakeholders, systematically searching the body of literature and building consensus through bringing together experts from across the EU and in an interdisciplinary fashion. It also focuses on how all these efforts have been translated into training modules and what methodology was used in evaluating its feasibility and acceptability.Despite several contextual barriers and diverse challenges the EUR-HUMAN project implementation encountered in each of the European settings, it implemented the development and deployment of a context-relevant educational intervention, with a focus on an easy-to-use course in an online interactive platform. This modular course, which was also easy to adapt to local context, was utilised as an instrument of engaging and enabling healthcare providers involved in refugee care, to make knowledge, tools and guidelines available for PHC providers, but, also, other stakeholders. The online platform will continue running beyond the EUR-HUMAN project cycle completion in an effort to contribute overcoming barriers in the provision of high quality, person-centred and integrated healthcare for refugees.There is currently much discussion on both national and European levels about how the produced material, the output generated by this project, could be utilised on the benefit of care services for refugees and migrants. In Greece, the online course has been given to the authorities of the Second Health Region (the most affected by this crisis in Greece) to disseminate among healthcare personnel, while the UoC team is currently training PHC professionals in the island of Crete; these PHC professionals will be providing services to this vulnerable population. One of the parts of which effectiveness can be ameliorated was the registration procedure, as it was mentioned this was difficult and unnecessarily formal. Another aspect that was considered important for future work, was that of a comprehensive economic evaluation with relevance, on a system level and societal perspective, and extending well beyond economic analysis to policymaking; such an evaluation ought to include the cost of inactivity, degradation of health, longer-term outcomes and many other factors extending well beyond the health services in scope and with an appropriate time horizon for these groups and across geographies.It is true that this European project met several challenges during its implementation, since many changes in the national policies of the countries where an implementation had been designed and given the impact of the agreement between EU and Turkey in March 2016 on the flow and movement of refugees. It was a capacity-building project that was running in a short period . It is also clear that although the focus of this project was on refugees, other migrants will benefit, as the approach, tools and training is relevant for all these groups and is not limited to refugees. However, a pilot intervention to test how the developed tools and material work in the real world was deployed. Although, this pilot involved a small number of refugees, its findings could be used when future actions and interventions will be debated. Certainly, more specific refugee- or migrant-relevant projects need to be designed. In addition, although the initial EUR-HUMAN project plan to utilise a very robust evidence-based and validated approach, the methods used in this capacity-building project are not considered as the most rigorous ones to create evidence (at least not type A or even B).The EUR-HUMAN project is anticipated to influence the working conditions and satisfaction of healthcare workers, as well as the interaction and collaboration of the three target groups . These can also contribute significantly to the development and enhancement of capacity building for PHC providers and to promote compassionate first contact, and, ultimately, comprehensive and integrated health care for refugees and migrants.


Cancer and neurodegeneration are different classes of diseases that share the involvement of mitochondria in their pathogenesis. Whereas the high glycolytic rate  and the suppression of apoptosis are key elements for the establishment and maintenance of cancer cells, mitochondrial dysfunction and increased cell death mark neurodegeneration. As a main actor in the regulation of cell metabolism and apoptosis, VDAC may represent the common point between these two broad families of pathologies. Located in the outer mitochondrial membrane, VDAC forms channels that control the flux of ions and metabolites across the mitochondrion thus mediating the organelle's cross-talk with the rest of the cell. Furthermore, the interaction with both pro-apoptotic and anti-apoptotic factors makes VDAC a gatekeeper for mitochondria-mediated cell death and survival signaling pathways. Unfortunately, the lack of an evident druggability of this protein, since it has no defined binding or active sites, makes the quest for VDAC interacting molecules a difficult tale. Pharmacologically active molecules of different classes have been proposed to hit cancer and neurodegeneration. In this work, we provide an exhaustive and detailed survey of all the molecules, peptides, and microRNAs that exploit VDAC in the treatment of the two examined classes of pathologies. The mechanism of action and the potential or effectiveness of each compound are discussed. Metabolites and ions are, thus, continuously exchanged with the cytosol through the Mitochondrial Outer Membrane (MOM), which owes its selective permeability mainly to the presence of mitochondrial porins, known as Voltage-Dependent Anion Channel (VDAC) , participate in fatty acid transport across the MOM and in cholesterol distribution in mitochondrial membranes , with conductance value of about 3.5–4.0 nS in 1 M KCl or NaCl , AIF and Smac/Diablo. In physiological conditions, apoptogenic factors are normally located in the intermembrane space of mitochondria (IMS). However, under apoptotic stimuli, they are released into the cytosol, leading to cyt c interaction with APAF-1 and the formation of apoptosome, which in turn activates the caspases cascade , led to loss of the mitochondrial membrane potential, mitochondrial swelling, and the rupture of the MOM . The two main HK isoforms, namely HK1 and HK2, are both involved in the first rate-limiting step of glycolysis. Both isoforms bind to VDAC1, obtaining a direct access to mitochondrial ATP, despite HK2 shows a higher affinity for mitochondrial binding Wilson, . The bioVDAC proteins play a crucial role in controlling mitochondrial metabolism and apoptosis. In this perspective, VDACs become interesting from a pharmacological point of view and many molecules targeted to VDAC proteins have been developed so far. Indeed, both alterations of apoptosis and of mitochondrial bioenergetics represent basal molecular mechanisms whose modulation is present in many pathologies.A cancer hallmark is the apoptosis inhibition. A combination of factors leads to raising cell resistance to death stimuli. In many tumors, several anti-apoptotic proteins are overexpressed  and Parkinson's disease (PD), while the most known neuromuscular disorder is Amyotrophic Lateral Sclerosis (ALS), which specifically affects spinal cord. Pathologies such as AD, PD, and ALS are characterized by different etiologies and symptoms. Nevertheless, at the molecular level, they are characterized by accumulation within the cells of misfolded proteins and/or peptide which can directly interact with VDAC1 , a 1:2:3 copolymer of methacrylate, maleate, and styrene, is surely one of the first listed compound able to lower VDAC's gating voltage and induce irreversible channel closure in vitro  has been reported to inhibit hexokinase binding by covalently labeling VDAC , a phytocannabinoid derived from Cannabis species and devoid of psychoactive activity. Reconstitution experiments in artificial membranes demonstrated indeed the ability of CBD to directly bind VDAC, markedly decreasing its channel conductance. This interaction, further confirmed by microscale thermophoresis analysis , a water-soluble hexavalent polycation whose effects on apoptosis are debated , a class of highly reactive molecules that induce caspase-dependent apoptosis via ROS production. VDAC was proposed as the pharmacological target of FNQs, since their anti-cancer activity was increased upon VDAC1 overexpression and decreased upon VDAC1 silencing by siRNA  is a component of the golden spice turmeric (Curcuma longa) with anti-inflammatory and antitumor activity . As for erastin, it inhibits VDAC2 and VDAC3 in cells harboring mutations in the oncogenes HRAS, KRAS, and BRAF.Compounds that are already part of clinical trials conclude this list. Among them, Clotrimazole, an antimycotic drug used in the treatment of fungal infections, is one of the best known molecule able to inhibit glycolysis by inducing the detachment of mitochondrial-bound hexokinase and therefore triggering apoptosis in various mouse models of cancer  and methyl jasmonate as drugs involved in tumor suppression through detachment of hexokinase from VDAC  that, inhibiting the activity of HK2, indirectly prevents its binding to VDAC. According to Ben Sahra et al., treatment with 2DG would promote cancer cell apoptosis when used in combination with the anti-diabetic drug metformin amino) phenylarsenoxide (GSAO) was shown to inhibit ANT and to selectively kill proliferating angiogenic endothelial cells, while being non-toxic to growth-arrested endothelial cells  superfamily, influences the expression levels of both HK2 and VDAC as well. Liu et al. proposed impaired VDAC and HK2 expression levels as responsible for HK2 dissociation from VDAC  which successfully counteracted cisplatin- or selenite-induced apoptosis in SH-SY5Y cells (a cell line commonly used as model of neurodegenerative diseases), again by diminishing the oligomerization of VDAC1   from Drosophila, in order to increase their intracellular delivery. Interestingly, modified VDAC1-based peptides were proved to reduce the anti-apoptotic effects of Bcl-2 or Bcl-xL  targets mitochondria and induces apoptosis in human cancer cells, with a mechanism in which the involvement of VDAC was not completely demonstrated  fused with a cyclic RGD motif, a member of this class. According to Borgne-Sanchez et al.  definitely represent an emerging tool in the treatment of cancer and neurodegenerative diseases. These molecules are short single-stranded RNAs of 21–24 nucleotides complementary to the 3′-end or, more rarely, to the 5′-end of mRNAs transcribed from target genes. Physiologically, microRNAs regulate gene expression at both transcriptional and post-translational level. Hence, because of their indispensable role in the control of numerous biological processes including cell cycle, cell growth, and apoptosis (Siomi and Siomi, in vitro or in cellulo and probably never will be applied in humans because of high toxicity or delivery difficulties. With regard to neurodegenerative diseases, the molecules that proved to suppress apoptosis and thus promote cell survival are still very few. This happens most likely because the molecular mechanisms underlying these disorders are still less characterized than those that define cancer. Although much remains to be to cover and uncover on the physiological role of VDAC in mitochondrial function and dysfunction, the overall data emphasize that targeted drugs and genetic approaches acting on VDAC represent encouraging strategies to treat a wide range of human diseases.This work focused on the significant contribution of VDAC in cancer and neurodegeneration, two types of diseases apparently different from each other. The detailed list reported above shows that all the pharmacological molecules, peptides and mRNAs described in the literature as targeted to VDAC are potentially effective in the therapeutic treatment of these pathologies. It is noticeable that numerous compounds able to induce apoptosis in cancer cells by modulating VDAC are already part of promising clinical trials. Many others, however, have been tested only AM and SR: collected the information and reference list for the manuscript. They also draw the figures. VDP: wrote the main part of the text and edited it.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.


While, over the past two decades, databases of the respective genes have been steadily growing and expanding to include novel, divergent sequences, belonging to a variety of taxa, somehow a view still prevails of the limited distribution of these genes, along with an evolutionary scenario in which genes for the methanogenesis pathway were horizontally transferred from Euryarchaea into Proteobacteria  the low probability of the respective genes being lost in most bacterial lineages  74 possess the genes in question . However, the Burkholderia database is heavily skewed toward the pathogenic species Burkholderia mallei and Burkholderia pseudomallei of a specialized life style typically resulting in genome reduction through gene loss  that is maintained in Archaea, Synergistetes, Firmicutes, and Candidate phylum NC10, but is not present in either Planctomycetes or Proteobacteria . The recent models of the evolution of metabolic pathways in living organisms also support spotty distribution of genes for ancient pathways  of life on Earth. Indeed, the recent reconstruction of the physiology and habitat of LUCA suggests that it possessed at least some of the genes for H4MPT/MF-C1 transfers and points to the importance of the C1 metabolites in early life (Weiss et al., 4MPT/MF-dependent reactions, the biochemistry of methylotrophy does not, and thus it is not entirely clear why so many methylotrophs maintain the pathway (Chistoserdova, 4MPT/MF-dependent reactions in non-methanogenic or non-methylotrophic species still remains enigmatic.In conclusion, the current evidence supports neither emergence of the H Koonin, , suggestThe author carried out literature and BLAST searches, made phylogenetic inferences, conceived, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics.Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism  Bacteria, like all living organisms must rapidly sense and adapt to drastic changes in their environment Roux, . These eATP-hydrolyzing proteins with all kind of activities in many cellular pathways, including replication, DNA and protein transport, transcriptional regulation, ribosome biogenesis, membrane fusion, and protein disaggregation or degradation. The AAA+ family proteins often form hexamers, and can convert the energy of ATP hydrolysis into mechanical force in order to remodel or unfold proteins or nucleoprotein complexes, to move DNA or proteins, or to facilitate membrane fusion  and Hsp60 (GroE) system, as well as other conserved chaperone systems such as ribosome-associated chaperones (Trigger factor), Hsp90 (HtpG), small heat shock proteins and redox chaperones . The tmRNA is a specialized small RNA, which aided by SmpB first acts as a tRNA and subsequently like an mRNA. This not only liberates the ribosome, but also results in the addition of a short sequence, termed an SsrA tag to the C-terminus of the unfinished protein  AAA+ protease complex  of ClpX. Under normal conditions, ClpXP and the adaptor protein YjbH suppress Spx activity by mediating its degradation  is a global repressor of protein quality control genes in B. subtilis and all Gram positive bacteria  did not complement a clpC deletion strain for survival during heat stress  regulon. ComK induces the transcription of competence genes, which encode the proteins necessary to form the DNA receptors that recognize and transport extracellular DNA into the cell. Concurrently, DNA repair and recombination systems are upregulated, whereas general transcription, translation, cell division and growth are impaired , was shown to exhibit an inhibitory effect on growth of several Gram-positive organisms, including Mycobacterium tuberculosis chaperone ClpC1, were recently discovered. While the exact mode of action is still to be discovered, it was suggested that binding of the N-terminal domain of ClpC1 by ecumicin or lassomycin leads to inhibition of degradation of natural substrates, which would eventually lead to accumulation of proteins and toxicity , the multiplication of the pathogen within macrophages, and the transcription of an actin-polymerizing protein (ActA) that is required for cell-to-cell spread (Gaillot et al., B. subtilis as an adaptor protein for specific substrate recognition by ClpCP (Turgay et al., B. subtilis is a useful model organism for the study of the role of AAA+ protease complexes.Various questions regarding the mechanism behind antibacterial activity of these newly identified compounds targeting the NTD of AAA+ proteins remain unanswered (Culp and Wright, B. subtilis are involved in many cellular processes, ranging from protein homeostasis and protein quality control to stress response pathways and the control of cellular developmental processes. Adaptor proteins play an important role in substrate recognition during both general and regulatory proteolysis (Jenal and Hengge-Aronis, B. subtilis (Fuhrmann et al., The various AAA+ protease complexes of the Gram-positive model organism 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.


Uterine contractility out of the gestational phase, during the menstrual cycle and the habitual functional variations of the organ, this is one of the responsible mechanisms for reproduction and fertility, due to its direct action in the mechanisms conducting the spermatozoa to the ovule and in the decidual implantation. Pathologies such as uterine leiomyoma, endometriosis, adenomyosis, polycystic ovarian syndrome, as well as the use of intrauterine devices and oral contraceptives, may alter a functionality of uterine contractility. Thus, magnetic resonance imaging with ultrafast sequences provides a dynamic evaluation (cine-MRI) and thus the correlation of uterine contractility quality in patients with current infertility or pathologies. Uterine function is intuitively associated with the gestational period. However, the non-pregnant uterus has other vital functions, such as elimination of endometrial cells during the menstrual period and promotion of sperm cell transport to implantation sites, both related to myometrial peristalsis  and 1B. For educational purposes, these may be divided as follows: Type A  – prevailing in the follicular and luteal phases, with increased intensity and frequency in the preovulatory phase  type B  – decreasing frequency and intensity, disappearing at mid-cycle  and type C – incomplete peristaltic contractions arising from the isthmus towards the lower central portion of the uterine body. Type A and C contractions occur in the luteal phase, with decreasing frequency towards the end of the cycle. As a result, approximately one complete cervicofundal contraction occurs per second in the mid-luteal phase, regressing to one-fourth at the end of this phase and revealing a quiescent period of the uterine fundus. Different types of contractions support the hypothesis that uterine peristalsis is associated with the initial reproductive stages, i.e., sperm cell transport in the preovulatory phase and preservation of incipient pregnancy.Three types of uterine contractions  may be distinguished in women, which vary in intensity and frequency according to the functional and circadian rhythm.,2 Fory phase  and 3B;Uterine contractility is not constant over the course of the reproductive phase. Rather, it varies according to the menstrual cycle and is negatively affected by pathological conditions, such as uterine leiomyoma, endometriosis, adenomyosis and polycystic ovary syndrome, or even by intrauterine (IUD) and oral (OCP) contraceptives.-9In a study evaluating 12 women free from pelvic conditions during the preovulatory phase, Kido et al., failed to detect significant variations in uterine peristalsis during daytime, probably due to the lack of variation in serum ovarian hormone levels .1The menstrual period is responsible for one of the most common uterine conditions affecting young women: primary dysmenorrhea. Increased myometrial prostaglandin production during this period leads to increased uterine contraction frequency and amplitude, reducing uterine blood flow and ultimately causing anoxic pain. The use of functional resonance imaging to investigate dysmenorrhea was proposed by Kataoka et al.3Endometriosis, a major cause of female infertility, is characterized by the presence of stroma and endometrial glands on the outer surface of the myometrium, within the myometrium (adenomyosis), or even in the ovaries and peritoneum. In a cine-MRI study evaluating women suffering from endometriosis, Kido et al., observed significant decrease in uterine peristalsis occurrence and frequency during the periovulatory phase.4Leiomyomas are the most common form of gynecological cancer. For this reason, Orisaka et al., investigated the potential impacts of this tumor on uterine peristalsis and concluded that affected women had abnormal peristaltic patterns during the menstrual and luteal phases, leading to prolonged menses and hypermenorrhea, inefficient embryo implantation and increased risk of abortion.5 Kido et al., used cine-MRI to investigate IUD users during the periovulatory phase and detected suppression of cervico-fundal peristalsis, reversal to the fundocervical direction and thickening of the junctional zone and endometrium, findings that suggest underlying effects of IUDs on conception.Intrauterine device has been around for more than two centuries and is an effective contraceptive method; however, its exact mode of action has not been fully understood and is thought to reflect negative impacts on the implantation of the fertilized egg, along with potential spermicidal effects.6 KidoOral contraceptives are also effective and have been in use for more than 30 years. These act primarily by inhibiting ovulation via suppression of basal FSH and LH production in response to gonadotrophin releasing hormone (GnRH), with resulting suppression of estrogen and progesterone peak levels. Other potentially relevant modes of action remain to be confirmed, such as changes in tubal and uterine motility, endometrial maturation and cervical mucus production. In an effort to clarify these issues, Kido et al., evaluated fertile OCP users of reproductive age using cine-MRI and demonstrated dramatic decreases in uterine contractility intensity and frequency (down to 8.6%) in this group as compared to the Control Group (100%). Mid-cycle LH, FSH and estrogen peak suppression suggests major contraction stimulating effects of follicular estrogen. Hence, peristalsis suppression may be considered an adverse effect of OCPs.7Polycystic ovary syndrome is the most common endocrine metabolic disorder affecting women of reproductive age, and causes infertility due to oligoanovulation. Uterine morphology and function in affected women are poorly understood. According to Leonhardt et al., lack of peristalsis is more prevalent in these patients compared to controls and is thought to be associated with endometrial thickening, with no changes in myometrial morphology.8Up to the 1990s, functional assessment of the uterus in women suffering dysmenorrhea involved the use of electrodes or intrauterine catheters. These devices were capable of measuring muscle tone intensity and the amplitude of uterine contractions, but provided limited morphological detail of the uterus.,10 Radiology was going through a major global revolution at that time, with the advent and rapid growth of magnetic resonance imaging, particularly for pelvic assessment. Then, in 2004 researchers from the University of Kyoto observed that cine-MRI was able to provide more accurate images of peristaltic movements associated with the endometrium, junctional zone and myometrium. The value of cine-MRI in the assessment of pelvic floor weakness associated with cystocele, enterocele, rectocele and uterine or vaginal prolapses should also be emphasized.In 1996 and 2000, Kunz et al., described the use of transvaginal videosonography (TVVS) to assess uterine peristalsis and categorized sound waves according to their respective directions, as follows: A, cervicofundal; B, fundo-cervical; and C, isthmic.2 RadiUsing visual concepts applied in Television and Cinema, cine-MRI consists of the acquisition of multiple images in a short period of time (seconds) for future, continuous visualization at lower rates, consistent with the visual ability of the human eye (approximately 12 frames/second).10Cine-MRI has two major applications based on T2 sequences: single shot fast spin echo (SSFSE) and steady-state free precession (SSFP). Despite superior resolution, SSFP generates a lot of noise and is more sensitive to susceptibility and distortion artifacts. For these reasons, along with superior contrast and spatial resolution, SSFSE  is the s,10Therefore, when used to assess uterine contractility in infertile women suffering from associated pelvic conditions, cine-MRI may contribute additional pathophysiological information, with no significant time and financial constraints.


Epidemiology is a discipline intended to systematically investigate, and ideally quantify, disease dynamics in populations Perez, . EpidemiRecent years have seen a dramatic increase in the application of science, technology, engineering, and mathematical (STEM) tools and approaches intended to enhance such analytical epidemiology process, with the ultimate goal of supporting disease prevention, control, and eradication. The research topic here provides an update of the current state-of-the-art scientific knowledge of the application of STEM tools to the microbiology of infectious diseases, at the various components of an integral epidemiological approach, divided into the four large components of (a) experimental studies, (b) novel diagnostic techniques, (c) epidemiological characterization, and (d) population modeling and intervention.Mycobacterium tuberculosis) , its ability to form environmentally resistant structures such as biofilms and/or resist common therapeutic approaches (Staphylococcus aureus and Acinetobacter baumannii)  or the lack of effective treatments to prevent or treat infection (Trypanosoma brucei gambiense) . An in-vitro dormancy model was used to study genes that were overexpressed under hypoxia by M. tuberculosis strains recovered from disease outbreaks (as opposed to M. tuberculosis laboratory strains) . Nevertheless, complementation of evidences found in-vitro with results from in-vivo models is important because different results may be obtained in those alternative settings. As an example, murine models were used to assess antibacterial activities of a panel of 14 antimicrobial agents against multi-drug resistant A. baumannii . Using an alternative approach the efficacy of a fourth generation cephalosporin (cefquinome) against planktonic and biofilm S. aureus cells was evaluated in another study . A mouse model was also of critical importance to mimic human latent tuberculosis infection and assess the efficacy of a fungus (Ganoderma lucidum) against M. tuberculosis H37Rv . Finally, protein and gene expression found in a pathogen (S. aureus) and a vector  were analyzed in two studies that both considered different conditions [planktonic growth vs. biofilm development for S. aureus  and infected vs. non-infected specimens for the G. palpalis ].The increased knowledge gained in the last decades on the importance of host-pathogen and environment-pathogen interactions has provided a better understanding on the complexities associated with the design of effective therapeutic and preventive strategies against infectious agents, but also raised new questions on how to evaluate those potential new approaches in a capturing their complexity. Six papers looking into the use of STEM tools for the design of experimental models for replicating this environment and host-pathogen interactions and ultimately the assessment of alternative strategies to prevent pathogen replication were selected here. Those papers were focused on emerging and/or neglected pathogens whose control is impaired due, at least in part, to its fastidious nature . The combined use of magnetic nanoparticles (MNPs) and giant magnetoresistance (GMR) biosensors has recently gained attention because of their potential for simultaneous, rapid and affordable detection of multiple pathogens. Monoclonal antibodies in combination with MNPs and GMR biosensors were used to detect influenza virus in swine, with a potential application to nasal samples, which are routinely collected by the swine industry worldwide . Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is becoming increasingly popular. In the research topic here, applications of MALDI-TOF has been demonstrated in three case-studies, including antimicrobial susceptibility testing in positive body fluid cultures collected from a hospital in Shanghai, China , characterization of hypervirulent strains of Klebsiella pneumoniae , and identification of Streptococcus suis isolates obtained from multiple host species . Finally, rapid detection of emergent diseases and conditions of global importance require of novel, affordable tools that could potentially be applied at large scale in developing settings to early detect disease cases. Here, it was presented (1) a novel method using mycobacteriophage 2GFP10, which was evaluated for detecting drug resistance in clinical isolates of Mycobacterium tuberculosis , (2) nested PCR for the detection of Mycobacterium tuberculosis DNA in patients with extra pulmonary tuberculosis , (3) a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect Zaire ebolavirus in Sierra Leone using the nucleoprotein gene as a target sequence , and a novel typing method for Streptococcus pneumoniae using selected surface proteins .Eight papers were selected demonstrating the use of novel diagnostic techniques or the evaluation of established approached for the detection of global emergencies  that exemplify these challenges and include the application of molecular biology methods were selected . Use of molecular characterization techniques allow identification of the pathogen beyond the species level, thus offering a much higher degree of refinement that can help to understand better the epidemiology of a disease . Detection of virulence genes, coupled with other molecular  and conventional  was also used for a detailed characterization of a collection of V. cholerae strains recovered in a 5-year period in India, revealing a considerable diversity and thus a dynamic evolution of the pathogen that must be taken into account when evaluating individual isolates . Finally, a well-established molecular characterization technique, multi-locus sequence typing (MLST), was used to assess the genetic diversity of V. parahaemolyticum at the national (China)  and, for pandemic strains (those harboring a given set of virulence markers), global levels  by analyzing two collections of clinical and environmental isolates. While the first study demonstrated a high genetic diversity among strains recovered from the same country, comparable to what had been described at a global scale , the second evidenced the persistence of certain pandemic strains in different countries and their occasional detection in environmental samples .Once the tools for detection and characterization of the pathogen of interest have been developed the challenge shifts to the interpretation of the results obtained in different populations. Four papers focusing on three bacterial pathogens . Bayesian techniques have the potential to support the characterization of disease risk in a region because of their flexibility to adapt the coding to different data structure and variables distribution. A Bayesian multivariable logistic regression mixed model was used to assess the relation between hypothesized risk factors and African swine fever virus (ASFV) distribution in Sardinia  after the beginning of the eradication program in 1993 . Additionally, one of the challenges epidemiologists often face is the absence of control or population data. Ecological niche modeling is a tool that allows quantification of spatial risk using presence-only (case) data. An overview of the background, history, and conceptual framework of ecological niche modeling applied to epidemiology and public health was presented (Escobar and Craft). Finally, the use of Bayesian phylodinamic models was demonstrated using a large volume of porcine reproductive and respiratory syndrome virus (PRRSV) collected in the United States .Population modeling for the characterization of spatial risk is prerequisite for the design and implementation of prevention and control interventions in a region. Three techniques  have been demonstrated in this research topic (The acknowledgment that health of humans, animals, and the environment is inter-connected has resulted in the establishment of the One Health approach Perez, . Cross-cJA and AP co-edited the Research Topic and wrote this editorial.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.


Non-nutritive sucking habits may adversely affect the orofacial complex. This systematic literature review aimed to find scientific evidence on the effect of pacifier sucking on orofacial structures.A search on MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, and Web of Science databases was conducted to find all pertinent articles published from inception until February 2018, based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The quality of the studies was evaluated using the risk of bias judgements in non-randomized studies of interventions (ROBINS-I).Among the 2288 articles found, 17 articles met the selection criteria: seven prospective cohort studies, nine cross-sectional studies, and one randomized clinical trial. Using ROBINS-I, 12 studies were evaluated to have a serious overall risk of bias and five, a moderate one.These studies claimed a strong association between a pacifier sucking habit and the presence of an anterior open bite and posterior crossbite. Functional/orthodontic pacifiers were shown to cause significantly less open bites than the conventional ones.High level of evidence of the effect of sucking habits on orofacial structures is missing. The available studies show severe or moderate risk of bias; hence, the findings in the literature need to be very carefully evaluated.There is moderate evidence that the use of pacifier is associated with anterior open bite and posterior crossbite, thus affecting the harmonious development of orofacial structures.Functional/orthodontic pacifiers reduce the prevalence of open bite when compared to the conventional ones, but evidence is needed concerning the effects on posterior crossbite. Well-designed randomized controlled trials are needed to further analyze the effects of functional/orthodontic and conventional pacifiers on orofacial structures. The use of pacifiers is widespread among babies and children throughout the world. Pacifiers are frequently used to calm crying babies, to increase the well-being of the parents and babies, and to prevent thumb or finger sucking , 2. The Pacifiers were cited for the first time in medical literature in 1473, being described by German physician Bartholomäus Metlinger in his book “Kinderbüchlein,” retitled on later editions as “Ein Regiment der jungen Kinder” (“A Guide on Young Children”) , the funAccording to non-randomized studies, the use of conventional pacifiers may impair the development of orofacial structures, cause infections, shorten the duration of breast-feeding, and produce dental malocclusions , 8, 9.The effects of the use of pacifiers is duration and frequency dependent .However, the use of pacifiers has been described as having a protective effect against Sudden Infant Death Syndrome , 3, but Pacifiers designed to cause less side effects, the so-called orthodontic pacifiers, have been introduced into the market –15 or haThe purpose of this study was to systematically review the evidence of the scientific literature on the effects of pacifier sucking on orofacial structures, including the evidence of differences between orthodontic and conventional pacifiers.The present systematic review was conducted and reported according to the Preferred Reporting Items for Systematic reviews and Meta Analyses (PRISMA) guidelines for reporting studies to evaluate health care interventions and Cochrane studies method –19. The Two reviewers  independently searched the titles and abstracts of the following databases by using a standardized form: MEDLINE (via PubMed), EMBASE, Cochrane Central Register of Controlled Trials, and Web of Science. The keywords used to identify the relevant studies were pacifier, dummy, comforter, tooth, malocclusion, deciduous, tongue, swallowing, openbite, crossbite, myofunctional, muscle, and orofacial, and the different terms were combined using Boolean operators. Full texts of eligible studies were only obtained when both reviewers were in consensus.Lack of agreement between the reviewers was resolved by discussion with a third reviewer who acted as an arbitrator. The selected studies and relevant articles were checked for cross references. The remaining articles were evaluated by reading the full text independently.The types of studies intended to be included in the search comprised randomized controlled trials (RCTs), cohort studies, intervention studies, case-control studies, and cross-sectional studies. No language restrictions were applied in this search. The minimum number of participants in the relevant subgroups of the study population was 30. There were no limitations on the date of publication or the place where the studies were carried out.The participants of the reviewed articles included healthy babies and children who only had a pacifier sucking habit. The studies including subjects with craniofacial anomalies , systemic diseases, or older infants undergoing or having had previous orthodontic treatment were excluded from this review.The studies which analyzed pacifier sucking effects on orofacial structures were included.The effects of pacifier sucking should have measurable documentation relating to any malocclusion and/or other anomalies in orofacial structures.The two reviewers extracted data independently using a standardized form. The following factors were recorded when the information in the reviewed articles was available: study design, country where the study was performed, sample size, follow-up, age range, control group characteristics, examined dental and orofacial features with specific reference to anterior open bite, posterior crossbite, overjet, overbite, molar and canine relationship, and arch width. The level of evidence of each study was classified by means of the Oxford Centre of Evidence-Based Medicine . The revThe risk of bias was assessed according to the type of study available. Since only one RCT study was conducted on this topic, the assessment of the risk of bias was performed using the risk of bias in non-randomized studies of intervention (ROBINS-I) tool. It includes risk of bias due to confounding factors , selection of participants into the study, classification of interventions, deviations from intended intervention, missing data, measurement of outcomes, and selection of the reported results .The reviewers ranked independently each included study and resolved any disagreement by reciprocal consulting.The initial electronic search resulted in 2288 studies after duplicates were removed. After screening the titles and abstracts, 2227 were excluded. From the remaining articles, 61 full-text articles were eligible for assessment. Seventeen articles published up to February 2018 could be included in this systematic literature review , whilst simple tongue-thrust occurred in 16.3% and complex tongue thrust in 27.5%. In the non-pacifier group, 69.8% of the children presented with a normal swallowing pattern and simple tongue thrust occurred only in 8.3% and complex tongue thrust in 21.9% .Among the five studies that assessed the effect of different types of pacifiers on the orofacial structures, three were classified as having a moderate overall risk of bias , 15, 16 Wagner et al. found that the use of a thin neck pacifier decreases the occurrence of open bite and increased overjet . The preIn a cross-sectional follow-up study, Katz and Rosenblatt found that 23 out of 30 children who discontinued the non-nutritive sucking showed a self-correction of the open bite. However, self-correction of an open bite was observed also in children (6 out of 17) without non-nutritive sucking habits .This systematic review revealed a lack of high-quality evidence in publications dealing with the effects of pacifier sucking habits on orofacial structures.In the hierarchy of evidence, the randomized controlled trials are on the top, cross-sectional studies come in the middle, and retrospective studies are at the bottom . A singlThe ROBINS-I assessment tool is based on the Cochrane RoB tool for randomized trials and was used due to the fact that we found in our search only one study that was a true randomized trial. Other studies were defined as randomized according to the authors, but a thorough review revealed in reality severe risks of bias. The ROBINS-I assessment tool uses the domain-based assessment and has a comprehensive manual in which users can interpret the results in a similar way, thus reducing the risk of subjective evaluation.Our assessment is in accordance with the findings of Dogramaci and Rossi-Fedele , despiteWe focused specifically on the influence of pacifiers on orofacial structures, since the so-called functional/orthodontic pacifiers seem to be a promising tool to limit their negative influence on the occlusion , 14, 16.The duration and frequency of pacifier sucking was not examined in all the studies, although playing a role in the developing of malocclusions , 31, 32.Different studies disagreed on the correlation between pacifier use and increased overjet , 28, 32,A high level of evidence on the effects of pacifier habits on orofacial structures is non-existent.However, there is moderate evidence that the use of pacifier is associated with anterior open bite and posterior crossbite, thus affecting the harmonious development of orofacial structures.Taking into consideration the present evidence, it seems that pacifiers with thin neck induce less open bite than the conventional ones. Functional/orthodontic pacifiers investigated in the literature seem not to reduce the occurrence of posterior crossbite. New nipple shape and texture are needed to counteract the narrowing of the palate.Randomized controlled trials are strongly needed to further analyze the effect of conventional and functional/orthodontic pacifiers on orofacial structures.


Malaria remains one of the most important infectious diseases in the world, being a significant public health problem associated with poverty and it is one of the main obstacles to the economy of an endemic country. Among the several complications, the effects of malaria seem to target the skeletal muscle system, leading to symptoms, such as muscle aches, muscle contractures, muscle fatigue, muscle pain, and muscle weakness. Malaria cause also parasitic coronary artery occlusion. This article reviews the current knowledge regarding the effect of malaria disease and the anti-malarial drugs on skeletal and cardiac muscles. Research articles and case report publications that addressed aspects that are important for understanding the involvement of malaria parasites and anti-malarial therapies affecting skeletal and cardiac muscles were analysed and their findings summarized. Sequestration of red blood cells, increased levels of serum creatine kinase and reduced muscle content of essential contractile proteins are some of the potential biomarkers of the damage levels of skeletal and cardiac muscles. These biomarkers might be useful for prevention of complications and determining the effectiveness of interventions designed to protect cardiac and skeletal muscles from malaria-induced damage. Malaria remains as the most important human infectious diseases in the World, with around 214 million cases a year, and an astounding 438,000 deaths resulting from this disease alone , in, inPlasmFew studies have been focused on cardiac effect in severe malaria –27 despiCirculation levels of cardiac proteins, such as troponin T (TnT), myoglobin and creatine kinase (CK), which are biomarkers of myocardial injury, increases with the severity of malaria, indicating myocardial impairment in complicated falciparum malaria , 25. In Cardiac troponin T is the most sensitive biomarker for detection of minimal myocardial damage . ReleaseP. falciparum glycosyl phosphatidylinositol (GPI), which act as a toxin in the malaria pathogenesis, up-regulated genes related to apoptosis and myocardial damages were found, indicating that P. falciparum GPI can also induce cardiomyocyte apoptosis [By analyzing gene expression of cardiomyocytes treated with purified poptosis . In the Plasmodium vivax infection and the appearance of cardiac complications, with diagnosis of acute coronary syndrome, tachycardia, arrhythmia and myocardial failure. However, there is the possibility of anti-malarial therapy being the cause of these cardiac complications [Some case reports have also suggested an association of ications –40.Plasmodium chabaudi, Plasmodium vinckei petteri and Plasmodium yoelii nigeriensis, following acute and chronic malaria [In a study using rodent malaria models, irreversible lesions were observed in several organs, including heart, of mice infected with  malaria .Plasmodium, (ii) the clinical condition of the patient, and (iii) the susceptibility of the parasites to the anti-malarial drugs determined by the geographic area where the infection was acquired and the previous use of the medicines.Treatment for malaria disease can only be initiated after making the correct diagnosis. Once made, appropriate anti-malarial treatment must be initiated immediately. Treatments are guided by three main factors: (i) the infecting species of Most anti-malarial drugs used are active against the blood parasite stages (the forms that causes the symptoms) and include mainly: chloroquine, mefloquine, quinine, quinidine, doxycycline, clindamycin and artemisinins. Widely-used anti-malarial drugs have a limited clinical lifespan due to increasing parasite resistance development. For instance, chloroquine, the drug of choice most used in Africa to combat malaria is no longer effective against the disease in several of the malaria endemic areas. Artemisinin-based combination therapy is currently the recommended treatment against falciparum malaria, in many parts of the world. However, there is serious concern that malaria parasites are developing resistance to this treatment. With parasite resistance continuously rising, anti-malarial drug discovery requires strategies to decrease the time of delivering a new anti-malarial drug while simultaneously increasing the efficacy of existing treatments, developing alternative treatments , as wellChloroquine is used in treatment and prophylaxis of malaria in areas where malaria is known to be sensitive to its effects. Prolonged administration of it can cause heart block and progressive myopathy . It has Cases of neuromyopathies and cardiomyopathies due to chronic chloroquine intoxication have been reported , in longQuinine, a drug of choice in areas of chloroquine resistance, can cause serious toxic effects, such as arrhythmia, angina and hypotension, circulatory defect and shock , 49. CasAnother malaria drug, artesunate, has been used as alternative to treat malaria with minimal toxicity. However, it has been shown to affect airway smooth muscles, inhibiting the proliferation of human cultured airway smooth muscle cells, causing hyperplasia and hypertrophy , which lLong-term impacts of malaria include death, disability, and significant socioeconomic burden on societies where the disease is prevalent. A better understanding of the biological processes underlying the progression of infection to disease is urgently needed to reduce the morbidity and mortality of malaria.As many diseases caused by protozoan parasites, malaria has shown to cause detrimental effect on cardiac and skeletal muscles –7, 11. MPlasmodium species, increasing cytokines levels and free radicals could also be considered as potential mechanism of damage and muscle weakness. Together, this information could be used for improved monitoring of disease progression and development of specific interventions for the protection of cardiac and skeletal muscles against the damaging effects of both the infection and the treatment.A hypothetical model is shown in Fig. Some of the findings reviewed in this article can help explain many of the symptoms in humans infected with malaria. Malaria infection induces a combination of inflammation and oxidative damage in skeletal and cardiac muscles leading to the enhanced degradation of key contractile proteins, which in turn is responsible for the compromised muscle function. Therefore, physiological key questions need to be answered: (i) What is the mechanism of direct damage to the contractile machinery? (ii) What are the genetic signaling pathways/networks regulating these modifications? (iii) Can these detrimental effects be counteracted with specific exercise training modalities, antioxidants therapy, dietary interventions or new drugs rationally designed to prevent the muscle damage?These questions and many more and the large toll exerted by malaria clearly shows the need for investigation to determine the cellular and molecular mechanisms of malaria induced muscle damage.


The world of dentistry is approaching graphene-based nanomaterials as substitutes for tissue engineering. Apart from its exceptional mechanical strength, electrical conductivity and thermal stability, graphene and its derivatives can be functionalized with several bioactive molecules. They can also be incorporated into different scaffolds used in regenerative dentistry, generating nanocomposites with improved characteristics. This review presents the state of the art of graphene-based nanomaterial applications in the dental field. We first discuss the interactions between cells and graphene, summarizing the available in vitro and in vivo studies concerning graphene biocompatibility and cytotoxicity. We then highlight the role of graphene-based nanomaterials in stem cell control, in terms of adhesion, proliferation and differentiation. Particular attention will be given to stem cells of dental origin, such as those isolated from dental pulp, periodontal ligament or dental follicle. The review then discusses the interactions between graphene-based nanomaterials with cells of the immune system; we also focus on the antibacterial activity of graphene nanomaterials. In the last section, we offer our perspectives on the various opportunities facing the use of graphene and its derivatives in associations with titanium dental implants, membranes for bone regeneration, resins, cements and adhesives as well as for tooth-whitening procedures. Tissue engineering is an interdisciplinary science which aims at developing biological substitutes to restore, maintain, or improve tissue function by using a combination of cells, scaffolds and suitable biochemical factors . ScaffolOver the years, several natural and synthetic three-dimensional 3D) scaffolds have been successfully developed and employed for various tissues, such as skin D scaffol, muscle In the last decade, many advances have been made in the dental tissue engineering field aimed at the regeneration of dental pulp , periodoIn the last few years, graphene and its derivatives have emerged as a new class of nanomaterials . GraphenSince its discovery in 2004 , many apThe present review intends to provide the reader an overview of the current state of the art of the graphene-based nanomaterials in tissue engineering in dentistry. Different aspects of the graphene-based nanomaterials will be discussed in detail. First, we highlight the role of stem cells and their interactions with graphene nanomaterials. We consider the influence of graphene-based nanomaterials both on biocompatibility, cytotoxicity and differentiation properties of stem cells. In this context, particular attention will be given to stem cells of dental origin. The interactions with immune cells and antibacterial properties of graphene, which are critical aspects to consider in the hostile environment of the oral cavity, are subsequently discussed. Finally, applications of graphene-based nanomaterials on dental implants, membranes, resins, cements and adhesives as well as on teeth-whitening are extensively presented.The first aspect to consider when developing a new nanomaterial for biomedical applications is its biocompatibility. Excellent biocompatibility is indeed essential in order to avoid any adverse effect of a material in living tissues . Apart fGraphene-based nanomaterials are rapidly spreading as scaffolds in tissue engineering applications, also in the dental field; consequently, assessments of their biocompatibility and cytotoxicity have to be done. Dosages, viability assays, type of nanomaterials, type of cells and uses need to be evaluated before developing a new reliable graphene-based nanomaterial.Over the years, biocompatibility of graphene and its derivatives has been largely discussed among authors. The comprehension of the toxicological potential of graphene-based nanomaterials, indeed, has to be carefully evaluated in order not to interrupt future in vivo studies and translational efforts . AccumulThe 3--2, 5-Diphenyltetrazolium bromide (MTT) assay, which measures the mitochondria function of the cells, is one of the preferred method for evaluating the effect of nanomaterials in cell culture . In one Shortly after, another work evaluated the toxicity and biocompatibility of GO to human lung A549 cells, a widely used cell line for toxicity studies . CompareAnother work that stresses this concept is the one by Sasidharan and colleagues . They fo2O2 decomposition and ⋅OH formation. Among various types of ROS, ⋅OH is an extremely reactive free radical and indiscriminately induces oxidative damages on various biomolecules, including DNA, lipids and proteins [The physicochemical mechanism of the oxidation-state dependent cytotoxicity of GO was proposed recently by the group of Zhang . They syproteins .Soon after, another distinct process contributing to the molecular basis of graphene cytotoxicity was unraveled by Duan and colleagues . They deWith regard to in vivo experiments, there are few studies concerning the biocompatibility of graphene-based nanomaterials since most of the investigations have only focused on the in vitro aspect of research. Nevertheless, the in vitro cell culture environment is definitely different from the in vivo complex 3D physiological condition. In addition, typical cytotoxicity assays measure the effect only during the first 12–24 h after exposure to a toxic substance although many biological reactions in vivo continue beyond 24 h.As for in vitro experiments, the in vivo effects of graphene-based nanomaterials were observed to be dependent on their concentration, physicochemical properties, exposure time, but also on administration route and on the characteristics of the animals used in the assays .Wang and colleagues observed that intravenous injection of GO at 0.1 mg and 0.25 mg showed no obvious toxicity to mice, whereas almost half animal population died after administration of 0.4 mg GO . All deaIn another study, GO or pluronic-dispersed graphene was administered by intratracheal instillation to mice . The autLater, the group of Yang tested several Polyethylene Glycol (PEG) functionalized GO materials with varied sizes and surface chemistry after oral and intraperitoneal injection to mice . The autConsidering certain inconsistencies and contradictory results in several studies, generalizations about graphene toxicity should be avoided. Indeed, it is generally accepted that the in vitro and in vivo behaviors of graphene-based nanomaterials are closely associated with their physicochemical properties, such as surface functionalization, coating, size and importantly, the administration routes. However, based on the studies presented in this section, we may summarize that the hydrophobic forms of graphene-based nanomaterials, which accumulate on the surface of cell membranes, are more toxic compared to the most hydrophilic ones. These, in fact, are more prone to infiltrate the cell membrane and to be internalized, as well as being removed from the application site. At the same time, when comparing GO with its reduced counterpart, rGO, the latter results less toxic than the hydrophilic form. Anyway, by controlling the GO reduction and maintaining the solubility, it is possible to minimize the GO toxicity and unravel the wide range of biomedical applications.As mentioned before, one of the key characteristics of the ideal scaffold is an effective and controlled guidance of the proliferation and differentiation of stem cells into specific tissue lineages, either in the absence or presence of chemical inducers and growth factors. In the past few years, graphene-based nanomaterials have been increasingly used as scaffold materials for stem cell growth and differentiation. In the specific field of dental tissue engineering, most of the studies focused on evaluating the role of graphene in driving the osteogenic differentiation of stem cells. Indeed, bone regeneration plays a fundamental role in regenerative dentistry and how incorporation of graphene-based nanomaterials can enhance osteoconductivity through stimulating both cellular osteogenic differentiation and biomineralization have been extensively investigated .In one of the first in vitro studies using stem cells, graphene produced by the Chemical Vapor Deposition (CVD) method and combined with different substrates was found to increase human Mesenchymal Stem Cells (MSCs) proliferation with no signs of cytotoxicity . MSCs arStarting from these results, another group investigated whether the graphene-induced osteogenesis of human MSCs was correlated to the chemical properties of graphene . In the Other studies have further tested the effects of GO on MSCs growth and osteogenic differentiation and the results varied. In the interesting work of Wei and colleagues, the authors compared the cellular behaviors of BM-MSCs towards pristine GO nanosheets using two in vitro biomimetic culture methods, that imitate similar situations in vivo . The seqIn recent years, also 3D graphene-based scaffolds have been actively explored and developed for the growth and differentiation of stem cells. The group of Crowder was the first to employ 3D graphene foams as substrates for human BM-MSCs culture . When seBesides being used in their pristine form, graphene nanomaterials have also been combined with different polymers, nanofibers, or nanoparticles to create novel composites for controlling the growth and differentiation of stem cells. Hydroxyapatite (HAp), for example, is a calcium phosphate ceramic commonly used for bone repair or regeneration due to its chemical similarity to that of natural apatite in bones . The recNatural bone is a composite material in which inorganic HAp nanocrystals are orderly embedded in an organic matrix made of collagen . CollageAmong others available materials, peptides containing the repeating arginine-glycine-aspartic acid (RGD) sequence, which is often found in Extracellular Matrix (ECM) proteins, have been widely applied to enhance cell adhesion to synthetic polymers or metallic surfaces ,59. In oOne last work worth mentioning is the one of Lim and colleagues . The autAll the works presented in this section demonstrate that, over the last few years, graphene has been found as one of the most promising biocompatible scaffolds for human MSCs adhesion, proliferation and differentiation, particularly towards the osteogenic lineage. These recent investigations clearly indicate a potential or active ability of graphene and its derivatives to induce the osteogenic differentiation of MSCs. Graphene alone seems to drive the spontaneous osteogenesis of stem cells; however, the presence of osteogenic inducers or growth factors in culture medium contributes in promoting this effect. Further summarizing the studies presented here, we may conclude that the association of graphene with stiff substrates, such as HAp, or with natural bone components, such as collagen, enhances the intrinsic properties of graphene in guiding the osteogenic differentiation of stem cells. Also mimicking the natural bone ECM, for example through the production of 3D graphene-based scaffolds, has proved to be a novel strategy in directing the stem cell fate towards the osteogenic lineage. Nevertheless, a deeper understanding of the ability of graphene to improve the biological properties of different scaffold materials will be essential to engineer graphene-based substrates for targeted biomedical applications.Regarding stem cells of dental origin, few data are available to date. Dental tissues are a very rich source of stem cells, which are collectively called Dental Stem Cells (DSCs) . In the Dental pulp is the soft connective tissue of the tooth and Dental Pulp Stem Cells (DPSCs) are a unique MSCs population that is present in the cell rich zone and core of the pulp. These cells were firstly discovered in 2000 by Gronthos and colleagues, thanks to their ability to differentiate into odontoblasts/osteoblasts, adipocytes and neural-like cells .Previous studies established that DPSCs and BM-MSCs are analogous cell populations, since they share expression profile for thousands of genes, among which those associated with the initiation of mineralization and bone homeostasis . Other cIn the study of Rosa and coworkers, the effect of GO-based scaffolds on DPSCs proliferation and differentiation was evaluated and compared to that of glass substrates . Cell moIn another study, the authors aimed at evaluating the potential of graphene to induce odontoblastic or osteogenic differentiation of DPSCs without the use of any chemical inducers . Dentin Therefore, authors seem to agree with the fact that graphene is a biocompatible material for DPSCs and it might be used as a valid substrate for dental tissue engineering since it is able to promote either osteogenic or odontogenic differentiation of DPSCs depending on the surface characteristics and functionalization of graphene, as also emerged in the previous section.The periodontium refers to those structures which surround and support teeth. It is composed by four parts: gingiva, alveolar bone, cementum and periodontal ligament. The function of the periodontal ligament is to literally attach the teeth to the bone and when a chronic process, called periodontitis, affects these structures, the teeth stability and health are compromised. All the periodontium is constantly maintained by Periodontal Ligament Stem Cells (PDLSCs), which have the ability to differentiate into cementoblasts, odontoblasts and fibroblasts .Thanks to their potential to differentiate into several types of specialized cells, PDLSCs can be used as substitute to BM-MSCs in order to determine the osteogenic potential and the viability onto different substrates . Even ifIn one of the few studies, the effects of GO, Silk Fibroin (SF) and GO combined with SF were investigated on PDLSCs adhesion, proliferation, viability and expression of MSCs markers . SF is wLater, the same group investigated the potential of SF and GO composites to promote human PDLSCs spontaneous differentiation into osteo/cementoblast-like cells . In the The dental follicle is the loose connective tissue surrounding the enamel organ and the dental papilla of the developing tooth germ. Its principal role is the coordination of tooth eruption through the regulation of the osteoclastogenesis and osteogenesis processes . The DenTo date, only one study evaluating the behavior of human DFSCs on graphene-based nanomaterials has been reported . In the A critical step for the application of nanotechnology in medicine is the interaction of a material with the immune system because, once administrated, it immediately enters in contact with the immune cells. Graphene and its composites are not exempt from the interaction with cells of the immune system and, as in the last few years the biological applications of graphene-based nanomaterials have grown, it results necessary to investigate the immune-related impact of these nanomaterials. Many papers about this issue underlined stimulation or suppression effects of graphene-based nanomaterials on immune system cells, depending mainly to graphene purity, oxidation level, shape dimension, functionalization and to the type of immune cells analyzed.The first extended investigation on graphene and immune cells was made by Sasidharan and coworkers . After cTo best characterize the impact of GO on immune cells, Orecchioni and colleagues studied and compared the effects of small-size (100–500 nm) and large-size (1–10 µm) GO flakes on a pool of PBMCs . The autLymphocytes, which represent about the 20–40% of the white blood cells and the 70–80% of PBMCs, are responsible for the antigen-specific and innate immune response. The most of research have been made analyzing the effects of graphene-based nanomaterials on T lymphocytes, concluding that the cytotoxicity of graphene depends on its dosage, time of exposure and type of material with which these are combined. For example, Ding and coworkers demonstrated that low doses of GO have no cytotoxic effects on T lymphocytes, while a dose over 100 µg/mL causes oxidative stress related apoptosis after 24 h of incubation . In partHalf of the studies about graphene and the immune system have been carried out on macrophages, not only because these represent the first phase of body immune reaction, but also because macrophages are the easiest long-term culturing primary immune cell population .The phagocytic activity of macrophages makes them the most exposed cells to the negative influence of graphene and its derivatives . It has Regarding the activation of macrophages, Chen and colleagues showed that pristine graphene and GO promoted the production of several cytokines, such as IL-1α, IL-6, IL-10 and Tumor Necrosis Factor alpha (TNF-α), via NF-κB pathway, that is activated via Toll-Like Receptors (TLRs) . GraphenDendritc cells (DCs), representing the 1–2% of PBMCs, play a fundamental role in the activation of antigen-specific T lymphocytes . Few datInfection is a frequent process during biomaterial implantation procedures. Since antibiotics often impact negatively on bacterial flora and pathogens are able to acquire resistance against different antibiotics, in the last few years various nanotechnologies with antimicrobial properties have been developed and studied ,104, tryIt has been reported that some graphene-based nanomaterials possess antibacterial properties; nevertheless, the effects of graphene on bacteria structure, metabolism and viability depend on the materials’ concentration, time of exposure, physical-chemical properties, as well as on the characteristics of bacteria used in the tests ,106.A lot of studies indicated that graphene and several graphene nanocomposites have a remarkable antibacterial ability against both Gram-negative and Gram-positive bacteria ,109,110.Another way for graphene to exert antibacterial activity seems to be through the separation of microorganisms from the microenvironment. Aggregated graphene sheets in suspension can isolate bacteria from the surrounding environment hindering nutrient consumption, reducing their ability to proliferate and favoring their inactivation .Graphene antibacterial activity is also probably related to oxidative stress induction. This is mainly mediated by ROS generation, especially when GO is used, nevertheless oxidative stress can be triggered without ROS generation. Li and coworkers showed that graphene is able to act as an electron pump pulling out electrons from bacterial membranes and inducing ROS-independent oxidative stress that affects microorganisms .Obviously, these mechanisms are not specific against bacteria but can also affect other cells, although with less effectiveness. Pang and colleagues tried to clarify the ratio between biosafety and antibacterial activity of graphene and GO . They obEscherichia coli compared to smaller one, probably due to the capacity of larger GO sheets to completely cover bacteria inhibiting their proliferation and colony formation  and GO-chitosan against ectively . The resAll these studies would suggest that the antibacterial activity of graphene-based nanomaterials is variable and dependent on the conditions of the tests; in particular, results change depending on the type of material and the size of particles, on its concentration and its state (in solution or adsorbed on a support), on the type of bacteria used in the study and on the medium used for their growth.Graphene and its derivatives display many potential applications in the dental field, as can be seen from the literature. Thanks to their potentiality, particularly in driving the osteogenic differentiation of stem cells and antibacterial abilities, the application of graphene-based nanomaterials to already existing dental technologies are being studied. In addition, graphene seems to be interesting as a platform able to release therapeutic molecules to improve implants osseointegration and bone formation. In the following paragraphs, we will present several studies discussing the association of graphene with dental implants, membranes, resins, cements and adhesives, as well as strategies for teeth-whitening.Nowadays, Ti dental implants are considered as the best substitutes for missing teeth thanks to their reliability and predictability, besides to their mechanical strength and favorable biocompatibility ,130. SinTi inertness represents a disadvantage because it may induce the development of fibrous tissue which could lead to the failure of the implant . For thiAs evidenced earlier, GO-coating was found to be a successful substrate for anchoring and growing of DPSCs on its rough surface and for promoting their osteogenic differentiation through up-regulation of MSX-1, PAX-9, RUNX2, COLI, DMP-1 and DPSS in particular after 14 days of culture . To imprAnother approach to improve the osseointegration of Ti implants could be its coating with bioactive proteins. For bone regeneration, BMP are the most potent osteoinductive proteins and among these, BMP-2 has been reported to induce osteogenic differentiation of stem cells, which can enhance the osseointegration of implants by forming bone at the space between the implants and the implantation site ,134. La Staphylococcus aureus), facultative anaerobic bacteria (E. coli) and anaerobic bacteria (S. mutans) thanks to the synergic effect of GO contact-killing and minocycline release-killing [S. mutans and P. gingivalis [Implant GO-coating represents an advantage also due to its antibacterial properties. Despite the fixation of graphene on Ti implant makes it to lose a bit of antibacterial activity, there is the possibility to functionalize the coating with antibacterial substances, such as antibiotics or silver nanoparticles. As demonstrated by Quian and colleagues, minocycline hydrochloride added to GO-coating improved the antibacterial activity against aerobic or facultative anaerobic bacteria  substrates . The resTo increase efficiency of bone repair, especially in periodontal and periimplant bone defects, the use of Guided Bone Regeneration (GBR) membranes has been important for years in oral surgery. These membranes are placed on the bone defect regeneration site and their principal function is to not allow infiltration of soft tissue cells into the growing bone. Thus, these act as physical barriers that separate connective tissue and the regenerating bone providing the slow osteogenic cells migration . In the All membranes need to satisfy the five main criteria underlined by Scantlebury: biocompatibility, space-making, cell-occlusiveness, tissue integration and clinical manageability . In ordeThe same GO-coating applied on collagen membranes was tested using DPSCs to analyze osteoblastic differentiation and inflammatory response . The resS. mutans, without altering the standard adhesion properties of the dental adhesive.Resins, cements and adhesives are the most used materials for dental restoration. Nevertheless, their porosity and adhesiveness make them receptacles for bacteria in the oral cavity and in particular in proximity of dental restoration. These polymeric materials, indeed, facilitate the adhesion of bacteria and the formation of biofilms that are the main cause of dental restoration failure . An examS. mutans. The results showed that up to the 95% S. mutans cell viability reduction was obtained using ZNGs even at a very low concentration (5 μg/mL). Moreover, the biomass and exopolysaccharide production, which are necessary for the biofilm formation, were evaluated: the ZNGs showed to behave as an obstacle for the biofilm growth. The authors concluded that the use of ZNGs is a viable instrument to control caries disease as it decreases S. mutans growth.One of the main limitation of GNPs in dental application is its grey color. In order to ameliorate this aspect, Zanni and colleagues tested a hybrid material composed by Zinc Oxide Nanorods (ZnO-NRs) grown on GNPs, thus combining the antimicrobial effect of GNPs with the light color and biocidal properties of ZnO-NRs . The autx/MgO catalyst [Biris and colleagues successfully prepared graphene sheets embedded with various amounts of gold nanoparticles (Gr-Au-x) using the radiofrequency catalytic chemical vapor deposition technique over a Aucatalyst . Sarosi catalyst . The autAs a last example, Li and collaborators demonstrated that the functionalization of glass ionomer cements with fluorinated graphene is not only useful to inhibit bacterial growth but also to ameliorate mechanical properties of the cements, increasing microhardness and compressive strength and decreasing friction coefficient, all important parameters for cements .Nowadays, a lot of people would change the color of their teeth, which often is ruined by food, beverage and smoke; consequently, the level of demand for tooth-whitening is increasing. Tooth color is determined by intrinsic factors, associated with the light scattering and absorption properties of the enamel and dentine and extrinsic factors, associated with the absorption of materials  on the surface of the enamel.2O2) chemistry, because of its bleach-properties and the techniques constantly evolve to ameliorate the rate of whitening, the ease of the treatment and to decrease the side effects. The most common risks of teeth-whitening include increase dental sensitivity and gingival irritation, besides changes in tooth microstructure. These effects are directly related to the concentration of H2O2, the duration of the treatment and the non-bleach component of the products [The products used for tooth-whitening are extensively-based upon Hydrogen Peroxide  phthalocyanine, together with H purpose . Based o purpose . They thent time b. This rWhile graphene is one of the most promising materials in nanotechnology, the synthesis on large scale represents an important aspect to be evaluated. Most of the studies described so far have been carried out using small amount of uncontaminated and controlled defect-free samples , but in Another limitation on graphene biomedical application is the scarce existing information on in vivo toxicity mechanisms and more studies are needed to support safe biological applications of these materials. However, some reports showed that graphene-based nanomaterials mainly accumulate in liver, spleen and lung after intravenous administration . Few stuTherefore, the scarce synthetic control, the high variability in production and the multiple parameters that modulate activity and toxicity of graphene and graphene-based nanomaterials, together with the scarce available studies, are big limitations for large scale application of graphene and its derivatives.The research on biomedical applications of graphene-based nanomaterials has seen dramatic progress in the last few years. In this review, we focused on the recent advances in dental tissue engineering using graphene and its related nanomaterials. Graphene exhibits numerous outstanding properties. In addition to its exceptional mechanical strength, electrical conductivity and thermal stability, what makes graphene extremely interesting is the possibility to functionalize and combine it with different biomaterials and biomolecules. This confers new properties to existing materials and allows the generation of nanocomposites with enhanced characteristics.In this review, we discussed recent studies concerning the biocompatibility, cytotoxicity and antibacterial activity of graphene-based nanomaterials both in vitro and in vivo. Then, we analyzed the effect of graphene-based nanomaterials on the adhesion, proliferation and differentiation of stem cells, focusing in particular on stem cells of dental origin. We examined the potential of graphene to promote osteogenic differentiation of stem cells, which is a key point for its future application in dental field. In this context, we presented recent studies employing graphene and its related nanomaterials for surface modification of dental implants or other scaffolds used in dentistry, such as membranes, resins and adhesives. As clearly highlighted in this review, graphene-based nanomaterials have emerged as promising scaffolds for a wide range of biomedical applications and all the presented works seem to agree with the fact that existing dental materials show improved characteristics following the addition of graphene; nevertheless, their behavior is closely dependent on graphene’s physicochemical properties, such as surface functionalization, coating and size.The increased interest in graphene and its derivatives have led to concerns about the risk of exposure not only to humans but also to the environment. Therefore, evaluation of the safety and potential risks of these nanomaterials is mandatory to ensure the safe use of graphene materials in biomedical applications. However, there is still much investigation to perform in order to assess the potential long-term toxicity of graphene and its composites and their effects on different cells, tissues and organs, including those of oral cavity. Moreover, in-depth studies are necessary to understand cell-signaling, metabolic pathways and osteogenic effects triggered by graphene-based nanomaterials. Overall, we believe that the use of graphene-based nanomaterials in the dental field deserves to be deeply explored as it can lead to even more reliable dental treatments in the near future.