Allthese studies prove the potential of stem cells for transplantation therapy inHD, demonstrating cell grafting and the ability to differentiate into matureneurons, resulting in behavioral improvements. We claim that there are stillmany problems to overcome before these technologies become available for HDpatient treatment, such as:Huntington's disease (HD) is a fatal genetic disorder, which causes theprogressive breakdown of neurons in the human brain. HD deteriorates humanphysical and mental abilities over time and has no cure. Stem cell-basedtechnologies are promising novel treatments, and in HD, they aim to replace lostneurons and/or to prevent neural cell death. Herein we discuss the use of humanfetal tissue (hFT), neural stem cells (NSCs) of hFT origin or embryonic stemcells (ESCs) and induced pluripotent stem cells (IPSCs), in clinical andpre-clinical studies. The a) safety regarding the use of NSCs and pluripotent stem cells, whichare potentially teratogenic;b) safety regarding the transplantation procedure itself, whichrepresents a risk and needs to be better studied; and finallyc) technical and ethical issues regarding cells of fetal andembryonic origin. This disease induces destruction of neurons in the subcorticalparts of the brain hemispheres (mainly – striped body): mostly the caudate andlenticular nuclei. HD leads to the expansion of the anterior horns of the lateralventricles - a sign used to identify HD during neuroimaging.4 However, it is not clear how mHTT accumulates in the neuronalprocesses and induces an early pathological event in the brains of HD carriers.Neuronal death in HD occurs as a result of abnormal synthesis of the Huntingtinprotein (HTT), which is particularly prone to misfold and accumulates, leading toaxonal and synaptic dysfunction. This accumulation of the misfolded mutantHuntingtin protein (mHTT) may affect intracellular transport of the protein orimpair protein degradation by the proteasome, leading to autophagy.3 HD is characterized by a combinationof motor and neuropsychiatric disorders that can occur simultaneously or insuccession. Movement disorders include involuntary grimace, excessive gestures andothers. Neuropsychiatric disorders, which usually appear after the motor disorders,may affect both cognitive and intellectual functions, as well as emotional status.Cognitive dysfunction is most often expressed in problems of visual-spatialorientation.5 Memory can also suffer, especiallyin the ability to retain the information necessary for workflow. Patients may not beable to plan and organize their activities. With time, symptom severity increases,reaching that of dementia. Emotional and behavioral disorders primarily presentthemselves by the emergence of unmotivated aggression and irritability .4 Approximately one third of patientssuffer from depression. Remarkably, the cause of death among HD carriers is not thedisease itself, but accompanying illnesses, such as pneumonia and heart failure;approximately 30% of patients die from suicide. Juvenile HD is a less common,early-onset form of HD that starts in childhood or adolescence. Juvenile HD evolvesvery rapidly and presents severe symptoms that quickly lead to disability.7There are two types of disease: adult-onset HD or juvenile HD, defined by themanifestations of the first signs of the disease. In adult-onset HD, the firstsymptoms usually appear after age 30, but possibly may start earlier.8Currently, the diagnosis of HD is based on clinical (see above), genotypic andimaging findings. Since the discovery of the HD gene in 1993, a genetic test wasdeveloped that analyzes the HD mutation by counting the number of CAG repeats in theHD gene. This analysis enables direct confirmation of the diagnosis of HD inpatients that exhibit HD-like symptoms. Using a blood sample, it is possible todemonstrate that an individual who does not have HD usually has 28 or fewer CAGrepeats. HD carriers have 40 or more repeats, while a small proportion ofindividuals show a number of repeats that fall within a borderline region .814Magnetic Resonance Imaging (MRI) is a neuroimaging modality that provides thegreatest spatial and contrast resolution for assessing the type of lesion associatedwith HD and allows the most precise diagnosis. Magnetic resonance spectroscopy (MRS)can also be used to show impaired brain energy metabolism associated with, forexample, increased regional brain lactate that plays an important role in thepathogenesis of HD. In addition, computerized tomography (CT) scan, which determinesthe damaged areas of the brain, is also useful in HD diagnosis.In conclusion, neuroimaging, particularly MRI, remains a keystone in diagnosing andassessing the severity of HD. Genetic testing is extremely important to confirm thediagnosis, especially if the family history is not forthcoming and, moreover,genetic counseling of HD patients may provide guidance on the implications of thedisease.No cure is currently available. All HD treatment focuses on symptom relief, thusachieving only temporary improvement of the patient's neurological status. Althoughsuch treatment improves patient quality of life, the progress of the disease isunchanged and the neuronal loss persists.Neuronal cells and different types of stem cells are a promising raw material for thedevelopment of new therapeutic strategies in HD: they may prevent neuronal loss andconsequently delay the disease progress.17 Different celltypes can be utilized in these therapies, including fetal cells and tissues,progenitor cells or primary stem cells isolated from different tissues of an adultorganism.21 This review covers the current state of cell-basedtherapies designed for HD. Some of these therapies are still at an initial stage ofdevelopment and must be tested in animal models  before theycan be approved by regulatory agencies for use in clinical trials (involvinghumans). On the other hand, only a few studies have already reached clinical trialsas will be discussed later.The main goal of cell-based technologies is to repair the mechanisms underlyingdisease initiation and progression, achieved by replacement of dead or defectivecells and through the trophic effect, which some cell types may confer after theirtransplantation into the injured site.18 Clinical studies involving hFT aimto rebuild brain structures and neural circuitries by transplantation of hFT intothe damaged central nervous system (CNS) in HD patients. The hFT is derived fromelective surgical terminations of pregnancy in fetuses at between 6 and 12 weeks ofgestation. In the case of the hFT used for clinical transplantation, the tissueconsists of the whole ganglionic eminence, corresponding to the striatal primordiumthat ultimately develops into the caudate and putamen.26 HDpatients may receive unilateral or bilateral hFT cell transplantation from severaldonated embryos (between five and nine).Human fetal tissue (hFT) has been used in basic research for decades.26 However, onestudy reported that three out of seven HD patients that underwent bilateralstereotactic transplantation developed subdural haemorrhages and two others requiredsurgical drainage.24 No patientshad adverse effects to the associated cyclosporin immunosuppression, nor did anypatients exhibit deterioration following the procedure.26 The immunosuppressive protocol adopted in thesestudies efficiently prevents immune rejection of the graft. Histological evidence ofimmune rejection, including the appearance of microglia and macrophages in the hFTtransplantation sites, has never been observed.26Generally, the surgery and procedure of hFT transplantation into the brain of HDpatients is considered to be safe.26All these studies show the ability of hFT cells to delay disease progression andprovide stabilization or improvement in several neurological indices of cognitivefunctioning, although these changes were not uniform across HD patients.MRI analyses showed hFT graft survival and even growth without damage to thesurrounding tissue.27Although the majority of the studies do not show tumor formation after hFTtransplantation,26recently, one study emphasized the instability and risk of hFT grafts: the patientenrolled in a NIH-funded study did not show any behavioral improvement after hFTtransplantation into the brain, yet presented a growth of tissue mass, termed "graftovergrowth".28 Such graftovergrowth may be explained by the presence of immature neuroepithelial cellsactively dividing cells that express NSC markers such as Sox2, which areoccasionally preserved in the hFT-derived cell suspension used fortransplantation.28 Boththese studies28 compromised the safety of hFT transplantation andindicate the need for further in-depth studies using chemical and transgenicpreclinical models and a large number of animals. Additionally, it has been shownthat not only hFT transplantation, but also fetal stem cell transplantation, maysporadically lead to brain tumor formation at the site of transplantation.29 Although these cells are subjectto restrictions regarding safety, they are able to recover medium spiny neurons(MSN).Another key concern that needs to be considered is the safety of hFT transplantationregarding tumor formation.28 These results are important given that HD is considered adisease of the striatum, characterized by vulnerability to degeneration and death ofMSNs.2 Also, hFT graft-derived astrocytes have also beenobserved after transplantation.20Astrocytes are a sub-type of glial cells in the CNS that are protective neuronsagainst excitotoxicity by removing excessive glutamate from the extracellular space.Astrocyte recovery after hFT transplantation is a highly beneficial effect of theprocedure, since in the HD brain, where mHTT accumulates in glial nuclei, there is adecrease in the expression of glutamate transporters in neurons and astroglialcells.30A postmortem analysis of a brain carried out six months after a patient underwenttransplantation demonstrated that hFT-derived cells were able to differentiate intoMSNs which expressed dopamine-receptor related phosphoprotein (32 kDa) (DARPP-32),the neuronal nuclear antigen (NeuN), calretinin that can protect the MSNs againstneurodegeneration in HD, and somatostatin, which is increased in the basal gangliain HD.4 As we mentionedabove, mHTT aggregates clump together and damage neurons. Recent research suggeststhat these mHTT proteins may also be transmitted from neuron to neuron.Interestingly, this hypothesis of pathogenic protein spread during neurodegenerationhas been critically evaluated recently by Walsh,31 who suggests experimental approaches to rigorously test itsfundaments. However, the formation of mHTT aggregates does not seem to be aprerequisite for HD, as shown by a clinical study carried out with transplanted hFTin HD patients, whereby a postmortem histological analysis performed 18 months afterthe treatment did not show the presence of neuronal protein aggregates of mHTTwithin the hFT graft.32 In thiscontext, all experimental data that support or detract from this hypothesis are ofgreat importance.Misfolded mHTT protein aggregates, which accumulate as cytoplasmic aggregates andnuclear inclusions, are a hallmark feature of HD.33 Chemical HD animal models are commonly induced by treatmentwith quinolinic acid (QA)35 or 3 nitropropionic acid (3-NP)39 Both ofthese chemical agents induce rapid development of progressive HD, early diseaseonset, abnormal behavior, and neuropathological features. These models are usefulfor rapid evaluation of the therapeutic hypothesis (acute models).15 In contrast, transgenic models areproviding more precise systems for disease replication that allow evaluation of thetherapy of interest in slow progressive degeneration.40To date, of the possible transplant options that can benefit HD patients, only hFThas been used in clinical HD studies. Thus, for the purposes of the present review,we have provided a short explanation about animal HD models, which are most commonlyused in preclinical studies for other treatment approaches. Preferably, the HDanimal model should provide similar genotypes and phenotypes as human HD.HTT gene containing the coding region for the polyglutaminerepeat into the mouse genome, under control of several promoters. The most commonlyused mouse HD models are R6/1, R6/2 and N171-Q82, which express truncated N-terminalfragments.41 Moreover, incontrast to the chemical models, the transgenic HD models are powerful researchtools because they recapitulate specific HD features such as the accumulation ofintracellular aggregates of mHTT in the cytoplasm or nucleus of neurons, known asinclusion bodies (IBs) (Transgenic models result from the random insertion of a portion of the humanes (IBs) .40 Morerecently, a transgenic nonhuman primate model in rhesus monkeys (Macacamulatta) of HD was developed.42 Also, transgenic HD models were established in large farmanimals such as sheep (Ovis aries)43 and Tibetan miniature pigs.44 However, transgenic HD models inlarge animals are high cost and technically difficult to use in routine cell-basedtherapy testing.Rodent HD models such as rats and mice are widely used to investigate cell-basedtherapy technologies.46 NSCs are self-renewing cells ableto generate neurons, glial cells and astrocytes. NSCs can be derived from embryonic,fetal or adult tissues.48 Embryonic-fetal neural precursorcells (NPCs) are composed of a mixed population of multipotent NSCs and transitamplifying/intermediate progenitor cells (NPGCs)  .4950 NSCs can be isolated at different stages ofdevelopment and cultivated in vitro under appropriate cultureconditions as neurospheres, which are free-floating clusters of round multicellularspheroids, or, alternatively, as adherent NSCs . NSCs exhibit robust engraftment at the site of injury.However, the differentiation of these cells into neural cells is stillcontroversial.53Discrete populations of NSCs can be found in specific areas of the postnatal humanbrain and seem to play an important role in postnatal growth as well as in recoveryof neural tissue from injury, anoxia, or disease.ent NSCs . Neurospent NSCs , which a15The evaluation of teratogenicity of embryo-fetal derived NSCs is still lacking,although it is essential for the further translation of these studies into clinicaltrials. Due to the commitment of NSCs to neural fate, they may be considered anideal cell type for the treatment of neurological diseases. However, NSCs aredifficult to obtain in therapeutically significant quantities and pose seriousethical and religious limitations given that they are obtained from abortedfetuses.54 We believe that these ethicalissues, associated with the limited number of experiments carried out so far, onlyminor success in these experimental therapies, as well as the potentialteratogenicity of fetal and embryonic cells, are reasons why the use ofethically-accepted alternatives is starting to dominate the field, especially inBrazil.There is no doubt that hFT and NSCs are important in many research areas, especiallyin studies of human neurodegenerative diseases. HFT and NSCs help scientistsinvestigate many aspects of basic science that cannot be studied in any other way.However, isolation of hFT and NSCs from hFT raises major ethical, political andreligious controversies.56Embryonic stem cells (ESCs) are pluripotent cells isolated from the inner cell massof early embryos. These cells can generate a whole organism when re- introduced backinto the embryonic environment (e.g. blastocysts) . ESCs ar57Due to this feature of ESCs, they cannot be used unspecialized in preclinical andclinical studies. To analyze the therapeutic potential of human ESCs in HD animalmodels, they have been reversed to NPCs that have been transplanted directly intothe striatum of the animal models. Different types of NPCs may be obtained, whichdepends on the protocol of human ESC differentiation.60Rosette-forming ESC–derived NPCs ,which is a principal target of HD therapies.59As mentioned before, tumor formation is a major safety concern for those who hope touse the cells therapeutically.ved NPCs  areunab60 The main problem in using ESC-derived NPCs in the clinic isthe need to control their proliferation in order to avoid neural cell graftovergrowth.60However, they have the capacity to differentiate into other neuron types and intoastrocytes. Preclinical short-term studies show effective recovery of motor deficitafter hESC-derived NSC transplantation in the QA rat model.invivo.62 However, the pluripotentialability of the iPSCs may be influenced by the adult donor cell source.64 The therapeutic potential of hiPSCs, as well as that ofhiPSC-derived NPCs, has also been investigated using HD animal models.67 These cells have been transplanted into bothchemical65 and transgenicHD rodent models via the ipsilateral ventricular route.67 These cells graft into recipient brains anddifferentiate into GABAergic MSNs68 and astrocytes.68 After transplantation, a modest reduction instriatal neuronal atrophy, a hallmark of HD disease that appears before the onset ofmotor symptoms, can be observed.69Laboratory-grown induced pluripotent stem cells (iPSCs) are a type of pluripotentcell that can be generated directly from adult cells . Current68 However,long-term motor functional recovery from HD followingESC- and iPSC-derived NSCtransplantation still needs to be further evaluated.After transplantation of NSCs derived from iPSCs, short- and medium-term functionalmotor improvements have been documented in comparison with sham groupanimals.73 However, a growing number of studies has demonstrated theircapacity to also differentiate into neural cells.74 Furthermore, the number of clinical trials usingMSCs is rising every year and, at least, the clinical safety of these cells has beenconfirmed.75MSCs can be found in virtually all postnatal tissues, including the embryonic annexes, bone marrow and adipose tissue. Isolation of thesecells is associated with fewer ethical concerns when compared with ESCs. MSCs have afibroblast-like morphology , express76 It has beenalso suggested that these cells can be recruited to nascent microvascular wallsduring development and postnatal growth.77 Accordingly, MSCs in their anatomical sites – stem cellniches – have their growth arrested until they are triggered to restartproliferation and even differentiation in response to physiological cues, such astissue turnover or repair, or experimentally, when isolated and cultivatedin vitro. In bone marrow, for example, subendothelialosteoprogenitors become stem cells acting as pericytes in different postnataltissues. Therefore, as an alternative to differentiation, tissue-specific MSCs mayfunction to support the regeneration of other local cell types. Such support iscarried out by MSCs after transplantation into the unhealthy organism via thesecretion of a variety of bioactive molecules such as cytokines, which have 'trophicactivities' that can promote a regenerative microenvironment.17 MSCs also benefit tissue reconstruction by stimulatingangiogenesis, the production of immunomodulatory mediators and even by deliveringother molecules to the injured site. MSCs also act by reducing chronic inflammation,inhibiting apoptosis and decreasing scar formation. They are able to stimulatemitosis of tissue-intrinsic stem cells and reduce the destructive effects ofoxidative stress.78 MSCs express very low levels of MHC class I proteins andlack MHC class II proteins, and can therefore be transplanted into other organismsof the same or different species without rejection.79The biological function of MSCs is to replace tissues in response to normal cellularturnover or trauma.83 Although the actualdifferentiation of MSCs into neurons is controversial, it is not required to justifythe beneficial activity of these cells in HD. Furthermore, MSC transplantation hasnever been shown to lead to teratoma or graft overgrowth formation, indicating thesafety of these cells, at least in animal models.MSCs isolated from bone, adipose tissue and umbilical cord have been used indifferent chemical (QA and 3-NP) and genetic animal models of HD, which were transplanted using the intracerebral route (directly into thestriatum). These studies demonstrate that MSC transplantation leads to behavioraland memory improvements, reduced brain damage, improvement of striatal degeneration,and enhanced expression of several striatal growth factors, which are attributed tothe neuroprotective effect of MSCs. MSC transplantation shows robust cellengraftment at the site of lesion, as well as the ability of these cells to migrateto adjacent areas.85 Undernon-pathologic conditions, BDNF is synthesized in the cortex, the substantia nigrapars compacta, the amygdala, and in the thalamus. All these regions supply thestriatum with BDNF.87 In HD, the deficit of BDNF in thestriatum may be due to reduced BDNF gene transcription in the cerebral cortex orreduced BDNF vesicle transport (or both).89 The decrease inBDNF expression observed in HD impairs dopaminergic neuronal function, which may beassociated with HD motor disturbances. As a result, many studies have been carriedout to examine whether increasing BDNF levels may help treat HD.88Neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), are essentialcontributors of CNS neuron function. Studies demonstrate their reduced availabilityin diseased brains, thus suggesting that they play an important role in neurologicaldisorders and, in particular, in HD.90 Studies have shown that MSCs derived from adipose tissueand bone marrow are able to secrete BDNF in vitro.93 However, MSCssecrete BDNF at low levels, which are dependent on tissue source used for MSCisolation and on donor characteristics.95NPCs and MSCs, besides their differentiation ability to produce neurons, have beenextensively investigated with respect to neurotrophic factor secretion.96Therefore, recent studies have generated NSCs derived from ESCs that overexpress BDNFin order to evaluate whether they have enhanced therapeutic abilities in HD. Neuronsderived from BDNF-GFP-expressing ESCs harbor a more complex dendritic morphology anddifferentiate into the GABAergic lineage.Great advances have been made in cell-based therapies over recent decades and it isexpected that, in the future, such therapies will be provided alone or incombination with traditional medication, new small molecule drugs and biologicaldrugs to patients who suffer from neurological conditions such as HD. Notably, all these cell-based technologies may be roughly divided into two: NSC- andMSC-based, since none of the pluripotent cells (such as ESCs or IPSCs) can be usedintact/unspecialized due to the risk of teratogenicity after transplantation intothe lesion site. NSCs derived from iPSCs look set to substitute hFT, fetal- as wellas ESC-derived NSCs, thus avoiding ethical and religious considerations.Another important conclusion is that these different cell types produce very similarclinical outcomes and therefore can be used interchangeably. The major advantage ofMSCs over NSC derived from different cell types is that they do not produceteratomas or graft overgrowth. Additionally, they express neurotrophic factors,albeit at low levels, which are apparently important for HD improvement.27 probably due to the fear that these cells will notpenetrate the BBR in significant numbers. However, we believe that MSCs can besystemically administrated and have tropism for sites of injury, even the brain, asin the case of HD. However, further studies are necessary to better understand thisadministration route and prove its efficacy and safety in HD.The major problem of cell-based technologies in HD concerns the route ofadministration, which is usually intracerebral. This will be a great obstacle forthe use of cell-based technologies in HD patients, which is a genetic disease andmay require more than one cell transplantation. None of the clinical and preclinicalHD studies have yet used the systemic intravenous route,


Mycobacterium tuberculosis (Mtb), an intracellular pathogen that infects lung macrophages and resides within modified phagosomes. Previously, we demonstrated that Mtb secretes MVs during infection that may modulate infected and uninfected immune cells. Our present data demonstrates that Mtb MVs inhibit the functions of macrophages and T cells, but promote Major Histocompatibility Complex (MHC) class II antigen presentation by dendritic cells. We conclude that bacterial MVs serve dual and opposing roles in the activation of and defense against host immune responses to Mtb and other bacterial pathogens. We also propose that MV secretion is a central mechanism for interspecies communication between bacteria and host cells during infection.The production of extracellular vesicles is a universal mechanism for intercellular communication that is conserved across kingdoms. Prokaryotes secrete 50–250 nm membrane vesicles (MVs) in a manner that is regulated by environmental stress and is thought to promote survival. Since many types of host-derived stress are encountered during infection, this implies an important role for MV secretion in bacterial pathogenesis. Accordingly, MVs produced by gram-positive and gram-negative pathogens contain toxins, virulence factors, and other molecules that promote survival in the host. However, recent studies have also shown that bacterial MVs are enriched for molecules that stimulate innate and adaptive immune responses. As an example, MVs may serve multiple, important roles in regulating the host response to  Mycobacterium tuberculosis (Mtb), participate in intercellular communication between pathogenic bacteria and the host immune system.Extracellular vesicles have been recognized in recent years as a novel form of intercellular communication that is conserved across species. Perhaps the most intriguing trend in the field is the growing number of reports of extracellular vesicle secretion by prokaryotes , anthrax toxin (Bacillus anthracis), cholera toxin (Vibrio cholerae), listeriolysin O (Listeria monocytogenes), and alpha-hemolysin (Staphylococcus aureus) , and selective targeting of cargo. MV-mediated secretion also provides an attractive mechanism to explain how lipids and membrane proteins are released by bacteria or how pore-forming toxins traffic to host membranes. Thus, MVs may orchestrate the trafficking of microbial molecules to specific locations or host targets during bacterial infection. The secretion of virulence factors and toxins within MVs has been covered in depth by several recent reviews , activate immune cells, and elicit the production of pro-inflammatory cytokines  is an excellent example of how MVs may modulate the host immune response in both a positive and negative manner. Mtb, the causative agent of tuberculosis, is an intracellular pathogen that infects lung macrophages and establishes latent infection for the lifetime of the host  Mtb deletion strain displays a hypervesiculation phenotype and elicits increased TLR2-dependent production of TNFα and IL-12 during macrophage infection , macrophages, and T cells. To simplify the experimental model, we employed MVs that were produced by axenic Mtb cultures and purified. IZON qNano analysis Figure  and elec3CSK4 as a control and determined the effect on antigen presentation molecules. The expression of MHC-I, MHC-II, and CD86 were substantially increased, demonstrating that PAMPs in Mtb MVs induce DC maturation , the American Lung Association , and the Center for AIDS Research at Case Western Reserve University (NIH P30 AI036219).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.


This perspective paper proposes that endogenous apoplastic fructans in fructan accumulating plants, released after stress-mediated cellular leakage, or increased by exogenous application, can act as damage-associated molecular patterns (DAMPs), priming plant innate immunity through ancient receptors and defense pathways that most probably evolved to react on microbial fructans acting as microbe-associated molecular patterns (MAMPs). The proposed model is placed in an evolutionary perspective. How this type of DAMP signaling may contribute to cross-tolerance and multistress resistance effects in plants is discussed. Besides apoplastic ATP, NAD and fructans, apoplastic polyamines, secondary metabolites, and melatonin may be considered potential players in DAMP-mediated stress signaling. It is proposed that mixtures of DAMP priming formulations hold great promise as natural and sustainable alternatives for toxic agrochemicals. Throughout their lifecycle, plants are prone to different sorts of stresses, many of which cause cellular rupture. In case of biotic stress, the recognition of molecular patterns from microbes  or herbivores  is well-known, especially for MAMPs. However, more recently the importance of damaged-self recognition has come to light. The manuscript by In their latest manuscript, Agave sp., the agavins (Fructans are polysaccharides synthesized in the vacuole of 15% of flowering species . Fructos agavins .Avena sativa). Their presence in the apoplastic environment after a stress event may suggest a possible role as DAMPs. Recently, it was found that short inulin-type fructans  from Arctium lappa or burdock  prime plant defenses in different pathosystems.  . This prCichorium intybus), but other types of plant fructans such as agavins . In animals, the recognition of microbial fructans by PM-localized TLRs has been documented (see above), thus activating innate immunity. The possibility exists that the same holds true in plants, where shorter microbial fructooligosaccharides (mFOS) diffuse through the cell wall acting as MAMPs to activate immune responses. However, a receptor for fructans has not been described so far. Fructans are stored in the vacuole of fructan-accumulating plants. Within the damaged-self context, lysed cells releasing their fructan content may lead to partial fructan degradation in the apoplast. The derived plant fructooligosaccharides (pFOS) are expected to be more mobile, diffusing to neighboring cells where they are possibly sensed by ancient receptors , that are actually involved in fructan MAMP recognition .Both MAMPs and DAMPs are currently accepted as immune response inducers . So are Figure 1), mild abiotic stresses may positively influence disease tolerance. If only some cells are damaged, the released mixture of DAMPs  can prime the surrounding cells, hence priming their native immune system, thus increasing tolerance to a future pathogenic attack. The process in which resistance toward a specific stress is achieved through exposure to another (milder form of) particular stress is known as cross-tolerance. After exposure to a first stress stimulus, the plant is in a primed or hardened state, allowing it to respond to future stresses in a faster and stronger way , fructan accumulates in response to low temperatures  it has been shown that a β-glucan binding protein can recognize β-glucans of the oomycete Phytophthora megasperma biotic stress tolerance as well. The potential use of mixtures of DAMPs for priming requires further investigation and may provide promising alternatives for toxic agrochemicals.MV and WVdE defined the perspective. MV wrote the first draft, input was provided by ŁPT and WVdE.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.


Chlamydiae are one such group of obligate intracellular microbes whose characterization has been hampered by this requirement. To circumvent these challenges, researchers have developed culture-independent sample preparation methods that can be applied to the sample directly or to genomic material extracted from the sample. These methods, which encompass both targeted [immunomagnetic separation-multiple displacement amplification (IMS-MDA) and sequence capture] and non-targeted approaches , have been applied to a range of clinical and environmental samples to generate whole genomes of novel chlamydial species and strains. This review aims to provide an overview of the application, advantages and limitations of these targeted and non-targeted approaches in the chlamydial context. The methods discussed also have broad application to other obligate intracellular bacteria or clinical and environmental samples.The expanding field of bacterial genomics has revolutionized our understanding of microbial diversity, biology and phylogeny. For most species, DNA extracted from culture material is used as the template for genome sequencing; however, the majority of microbes are actually uncultivable, and others, such as obligate intracellular bacteria, require laborious tissue culture to yield sufficient genomic material for sequencing.  Chlamydiae. Here, we have summarized six key culture-independent methods for chlamydial genomics. These methods can be used on human, veterinary and environmental samples to select for chlamydial DNA prior to genome sequencing and analysis. This review may be used as a guideline for the selection of appropriate culture-independent technique(s) depending largely on sample type. We suggest that targeted genome capture methods should be applied if the pathogen of interest is known and a reference genome is available. However, if the pathogen is unknown, and novel species discovery is the aim, non-targeted (meta)genome capture methods should be used. The methods discussed in this review also have broad application to other microorganisms or clinical and environmental samples.Until recently, our understanding of the diversity of the bacterial kingdom has been largely limited by a reliance on the use of technically challenging and time-consuming methods whereby bacteria are cultured on media or in host cells. However, the recent advancement of culture-independent methods combined with next-generation sequencing has revolutionized our ability to study microbial biology, diversity and evolution. In particular, the expanding application of these techniques has greatly advanced our understanding of the biology and genomic diversity of the obligate intracellular bacterial phylum Microbial community profiling and ecology analysis has proven to be a useful tool for discovering diverse, novel microbial taxa in the far reaches of our biosphere. While initial microbial diversity studies involved culture-dependent methods , the devChlamydiae.Besides a greater understanding of microbial diversity, the introduction and subsequent extensive use of microbial genomics has implications for food safety Chlamydiae are globally significant, widely distributed human and animal pathogens. Chlamydia trachomatis remains the cause of the most common bacterial sexually transmitted infection worldwide, as well as the leading cause of preventable blindness . Th. Thet alC. trachomatis has also been the subject of another culture-independent approach which uses biotinylated RNA probes (‘baits’) to hybridize chlamydial DNA away from a complex DNA mixture to ‘capture’ the chlamydial DNA  following just a few passages of chlamydial cell culture  and (b) C. trachomatis genome is multiplexed microdroplet PCR, which uses 500 primer pairs to generate overlapping 1–1.3 kbp amplicons spanning the selected region [C. trachomatis phylogenetic and epidemiological studies, and the method is capable of discriminating between strains in a sample [A final targeted approach that has so far only been used to amplify a 100 kb region of the d region  (Table 1d region . Like IMd region . The mica sample . ImportaThis review has summarized the key culture-independent approaches for chlamydial genome and metagenome sequencing. As outlined in Throughout this review, we have categorized these methods as targeted and non-targeted approaches, which is determined primarily by whether the target bacterium is known . The nonC. trachomatis-positive urogenital swab in lysis buffer, we could apply MDA, depletion-enrichment, sequence capture or multiplexed microdroplet PCR (Chlamydiales-positive tissue sample but no species-level identification or it represents a putative novel species, we would either choose MDA or depletion-enrichment (As an example, if we have a discarded diagnostic plet PCR . Of thesplet PCR , and it plet PCR . Howeverplet PCR . On the richment . In thisrichment .Although chlamydial genomics has come a long way in only 20 years, there are several avenues yet to be explored. Our knowledge is currently limited only by the sample types to which culture-independent genome sequencing has been applied in the chlamydial field to date , and theC. trachomatis, C. pecorum, C. psittaci and C. pneumoniae. Reference genomes now exist for all Chlamydia species, so targeted approaches to enable high-throughput sample processing and sequencing of other species is plausible. As many CRB genomes are also now sequenced and complemented by proteomic and transcriptomic studies, these approaches could also be extended into CRBs [in vitro culture was undertaken prior to sequencing. A limitation to this method is the bias against detecting novel genes or extrachromosomal elements, as was the case for C. pecorum, for which the chlamydial plasmid sequence was only resolved due to the sequence similarity between chromosomal and plasmid genes captured using the RNA baits [To date, the targeted approaches described here have only been applied to nto CRBs . ImportaNA baits .Chlamydia field but holds great promise as it is highly specific and sensitive (Non-targeted culture-independent genomic approaches will be fundamental in identifying novel species in novel hosts or environments and hence in further characterizing chlamydial diversity. Coverage uniformity appears to be a common limitation for MDA . MDA couensitive : it is yWe did not delve into the downstream bioinformatics methods required to assemble and analyse genomes generated from these culture-independent methods, as we feel this would warrant a separate review. However, it is worth noting that different approaches may require different bioinformatics methods, of which there are a plethora.in vitro culture, but chlamydial culture-independent genome sequencing will contribute more, diverse genomic data to our knowledge of this unique, expanding phylum.Sequencing and characterization of chlamydial genomes has revolutionized our knowledge of chlamydial biology, diversity and evolution. The advent of chlamydial genomics was stunted by the requirement for All data referenced in this review have been properly cited and is detailed in the reference list.


Exosomes are endosome derived extracellular vesicles of 30–120 nm size ranges. Exosomes have been identified as mediators of cell-to-cell communication by transferring bioactive molecules such as nucleic acids, proteins and lipids into recipient cells. While exosomes are secreted by multiple cell types, cancer derived exosomes not only influence the invasive potentials of proximally located cells, but also affect distantly located tissues. Based on their ability to alter tumor microenvironment by regulating immunity, angiogenesis and metastasis, there has been growing interest in defining the clinical relevance of exosomes in cancers. In particular, exosomes are valuable sources for biomarkers due to selective cargo loading and resemblance to their parental cells. In this review, we summarize the recent findings to utilize exosomes as cancer biomarkers for early detection, diagnosis and therapy selection. Exosomes are small sized 30–120 nm) extracellular vesicles (EVs) with a multi-vesicular endosomal origin 20 nm ext,5. ExosoDue to their presence and stability in most bodily fluids and resemblance of their contents to parental cells, exosomes have a great potential to serve as a liquid biopsy tool for various diseases ,13. In pg centrifugation. Next, over 100,000× g ultracentrifugation is used to pellet crude exosomes and sMVs mixtures [The major hurdle in the clinical utilization of exosomes has been the lack of consistent and dependable methods to isolate a pure exosome population . Therefomixtures . The dowmixtures ,23. Densmixtures .As an alternative strategy to ultracentrifugation, nanomembrane based ultrafiltration and microfiltration were used to successfully isolate urinary exosomes ,25. ReceAffinity capture (AC) approaches rely on exosome surface antigen targeting monoclonal antibody (mAb) that is covalently fused to either magnetic or agarose beads. The multiple reports demonstrated the success of mAbs as baits to isolate exosomes ,30,31. IAnother challenge in exosome biology is how to accurately measure the quantity and purity of exosomes. Exosome purity is assessed by measuring exosome-specific marker antigen or protein  as a ratio of exosome concentration  using ELISA assay . Optical6 to 109 particles per mL. NTA method becomes a gold standard to measure the concentration of exosomes and sMVs. The product called Zetasizer Nano ZS  involves DLS technology that determines diameter of EVs including exosomes by measuring the fluctuations from scattering of coherent laser light through suspension of EVs [Malvern has developed NTA-based instruments called Nanosight  that allow exosomes to be counted and sized by combining light microscopy and the software that tracks Brownian motion of exosomes. The light scattering mode of Nanosight is used to measure size and its fluorescence mode is used to profile labeled markers in exosomes ,37. Nanon of EVs . Nano ZSn of EVs . HoweverRegarding to non-optical methods, TEM overcomes the limitation of the conventional light microscope to resolve exosomes as diameter of exosomes is less than optical wavelength. TEM can resolve individual exosomes due to short wavelength of electrons . TEM canExosomes contain a variety of proteins that reflect their origin and alteration of the parental cells. Based on endosome-based biogenesis pathway, exosome specific protein markers include endosome associated proteins , proteins involved in exosome biogenesis , tetraspanins , heat shock proteins  and epithelial cell adhesion molecules (EpCam) ,47,48,49A number of exosomal breast cancer markers have been identified in recent years. Rupp et al. characterized CD24 and EpCAM as tumor-derived exosome markers by using immune-affinity isolation techniques involving anti-CD24 and anti-EpCAM magnetic beads . In exosIn addition to breast cancer, exosomal proteins provide the useful source for biomarkers for a number of other cancers. Khan et al. found that Survivin, a member of inhibitor of apoptosis (IAP), was detectable in plasma-derived exosomes from both normal and prostate cancer patients, but the relative amount of exosomal Survivin is significantly higher in plasma of prostate cancer patients . Plasma-A number of recent studies reported the success of developing the rapid and high-throughput platform for clinical utilization of exosome-based diagnostics without a purification of exosomes. Kanwar et al.  developeInitial analyses of nucleic acids from isolated exosomes identified microRNAs (miRs) and mRNAs as the major components of exosomal cargo ,63. SubsSince the discovery of exosomal miRNAs by Valadi et al. in 2007 , pioneerDuring the last three years, more exosomal miRNAs were identified by using a combination of ExoQuick (System Bioscience) precipitation, ultracentrifugation and commercialized Exo-miR kit  and RNA seq-based miRNA profiling methods in a number of cancer models including breast, colon, prostate, pancreatic cancers and glioblastoma ,78,79,80Exosomal DNAs merit consideration as a diagnostic tool as well, due to their ability to carry information regarding cancer specific mutations ,81. KahlCancer-derived exosomes contribute to cancer progression through enhancing intercellular transfer of cargo that contains proteins, lipids and nucleic acids within the tumor microenvironment. The cargo of exosomes reflects the altered state of original cancers, which qualifies exosomes as minimally invasive biomarkers for early detection, diagnosis and prognosis. Compelling recent literature supports that exosome-based diagnostics provide higher sensitivity and specificity over conventional biopsy or liquid biopsy biomarkers due to their stability in biofluids. In addition, exosomal markers are readily available from most biofluids and recent advances in technology in exosome isolation make exosome-based diagnostics cost and labor effective.The major obstacle to overcome in the field of exosome diagnostics is to develop optimal methods to isolate pure exosome population. Microvesicle themselves can be a useful diagnostic tool to detect cancers, but the comparison studies will only be possible with isolation techniques that separate these two major EVs into pure exosomal vs. microvesicle populations. Another challenge is to understand the mechanisms that regulate the heterogeneity of cancer exosomes, which will affect the contents of cancer-derived exosomal cargo, and therefore influence the reproducibility of diagnostic outcomes. Despite these concerns, future efforts that combine next generation sequencing of exosomal RNAs and DNAs, proteomic analysis of exosomal surface proteins, and immune-affinity capturing techniques will reach to the next level of exosome utilization for cancer diagnosis.


Moreover, selective adenosine receptor antagonists are being assessed for their antidepressant effects in animal studies. This review focuses on how caffeine and selective adenosine antagonists can improve different aspects of depression in humans, as well as in animal models. The effects on motivational symptoms of depression such as anergia, fatigue, and psychomotor slowing receive particular attention. Thus, the ability of adenosine receptor antagonists to reverse the anergia induced by dopamine antagonism or depletion is of special interest. In conclusion, although further studies are needed, it appears that caffeine and selective adenosine receptor antagonists could be therapeutic agents for the treatment of motivational dysfunction in depression.Major depressive disorder is one of the most common and debilitating psychiatric disorders. Some of the motivational symptoms of depression, such anergia (lack of self-reported energy) and fatigue are relatively resistant to traditional treatments such as serotonin uptake inhibitors. Thus, new pharmacological targets are being investigated. Epidemiological data suggest that caffeine consumption can have an impact on aspects of depressive symptomatology. Caffeine is a non-selective adenosine antagonist for A Major depression disorder (MDD) is one of the most debilitating disorders in the world, and the most commonly diagnosed according to the World Health Organization. The Diagnostic and Statistical Manual in its last edition (DMS-5) defines this disorder as a set of symptoms including: depressed mood, decreased interest or pleasure in almost all activities nearly every day, appetite changes (changes in body weight), sleep disturbances, feelings of worthlessness or guilt, diminished ability to concentrate or indecisiveness, psychomotor agitation or retardation and fatigue or loss of energy .Although depression is typically defined as an affective disorder, it also appears that some symptoms such as psychomotor retardation, fatigue, and loss of energy are related to deficits in motivation, specifically in activational aspects of motivation. Motivated behavior is directed toward or away from particular stimuli, but it also is characterized by a high degree of activity, effort, vigor, and persistence , 2012. PThe severity of effort-related motivational symptoms in depression is related to problems with social function, employment absence, and treatment outcomes . PatientMost of the present treatment strategies for MDD focus on drugs that block the inactivation  of the monoamine neurotransmitters 5-HT and norepinephrine (NE). The classical antidepressants include monoamine oxidase inhibitors (MAOIs), which affect one of the major catabolic enzymes for monoamines , and druInterestingly, some clinical studies suggest that drugs that inhibit dopamine (DA) transport, such as the catecholamine uptake inhibitor bupropion, are relatively more effective than 5-HT uptake inhibitors for treating effort-related motivational symptoms . Further1 and A2A G-protein-coupled receptors being the most abundant  that plays an important role in the regulation of synaptic transmission and neuronal excitability . Severalabundant . A2A rectubercle , but alstubercle . Adenosipocampus . All thepocampus  that see2A receptors are highly expressed postsynaptically in DA rich areas such as neostriatum and accumbens (Acb) , which are co-localized on enkephalin-containing medium spiny neurons and converge onto the same signal transduction pathways in an antagonistic way  . In factstic way . Similar neurons , 2008.2A receptor antagonists are being tested in clinical trials for pathologies involving DAergic dysfunctions such as Parkinson disease, and positive results indicate that they can be used as adjuvant therapies  was associated with a reduced risk of depression . Also, iMultiple reports have lent support to the idea that depressed people could use caffeine as self-medication. It has been reported that psychiatric patients show a relatively high degree of caffeine consumption compared to the normal population . This apA wide range of studies demonstrate that caffeine can increase alertness and subjective energy, and also reduce fatigue , thus acPreclinical studies have been trying to elucidate the effect of caffeine and selective adenosine antagonists on classical animal models of depression . Two of 2A receptor antagonists have also been effective in these tests. Thus, SCH58261 and istradefylline (KW6002) reduced total immobility time in both the TST and the FST in mice (2A receptor knockout (A2AKO) mice showed reductions in immobility time compared to wild type (WT) animals in both tests , impaired escape latency in rats  that are being used in clinical practice, and have been demonstrated to reduce immobility in classical animal tests of depression. Thus, caffeine at low doses that do not have an effect on their own can potentiate the effects of desipramine, imipramine, duloxetine, fluoxetine and paroxetine, in animals tested on the FST . In addi2A receptor antagonists increased lever pressing in the low effort-demanding task (FI-240 s) but did not suppress the high effort task (FR20) in the dose range tested. In fact, there was a tendency for istradefylline to increase FR20 responding at a moderate dose. A1 antagonists failed to increase lever-pressing rate, but decreased FR20 responding at higher doses. These results suggest that the work potentiating effects of methylxanthines are mediated by their actions on adenosine A2A receptors, while their A1 receptor antagonistic action could be mediating the suppressant effects.Tasks measuring behavioral activation and effort-based functions have been suggested as potential animal models for the motivational symptoms of depression . Thus inProgressive ratio (PR) schedules, which require gradually increasing work output, have been also employed to explore the effect of caffeine on motivation to work for sucrose or food in rats and monkeys . Acutely2A receptor antagonists seem to be beneficial independently of the baseline performance, as demonstrated also in goals directed tasks  are highly adaptative because they enable organisms to overcome obstacles or work-related response costs that separate them from significant stimuli . An impoExtensive animal data have demonstrated that Acb DA is a key mediator of effort-based decision-making processes  tetrabenazine (TBZ), reduces selection of high effort alternatives, but animals compensate by increasing the amount of free less preferred reinforcer consumed , 2017. T2A receptor agonists into the Acb produced effects on instrumental behavior and effort-related choice that resembled those produced by Acb DA receptor antagonism or depletion  that are associated with differences in clinical symptoms . Thus, rAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.JS has received grants from Merck-Serrono, Pfizer, Roche, Shire, and Prexa. MC has received a grant from Servier. The other 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.


Additive biomanufacturing, a manufacturing method in which objects are designed and fabricated in a layer-by-layer manner, has allowed a paradigm shift in the current manufacturing of medical devices and implants. This shift from design-to-manufacture to manufacture-to-design, seen from a translational research point of view, provides the biomedical engineering and periodontology communities a technology with the potential to achieve tissue regeneration instead of repair. In this review, the focus is put on additively biomanufactured scaffolds for periodontal applications. Besides a general overview of the concept of additive biomanufacturing within this field, different developed scaffold designs are described. To conclude, future directions regarding advanced biomaterials and additive biomanufacturing technologies for applications in regenerative periodontology are highlighted.Periodontitis is defined as a chronic inflammatory condition, characterized by destruction of the periodontium, composed of hard  and soft tissues  surrounding and supporting the teeth. In severe cases, reduced periodontal support can lead to tooth loss, which requires tissue augmentation or procedures that initiate a repair, yet ideally a regenerative response. However, mimicking the three-dimensional complexity and functional integration of the different tissue components  Periodontitis is a highly prevalent disease caused by a bacterial biofilm.Over the last four decades, various attempts have been made to achieve regeneration instead of repair of the periodontal apparatus. These include root surface conditioning, graft materials, barrier membranes, gene therapy, and growth factors.Recent developments in the science, technology, engineering, and mathematics (STEM) arena allow for alternatives to address several challenges in the treatment of periodontal disease. An example of such an advancement is tissue engineering, which aims to regenerate tissues by combining cells with scaffolds and bioactive factors.,Over the years, the field of tissue engineering has made tremendous progress. Third generation biomaterials and advanced processing technologies have enabled a shift in the current manufacturing concept, resulting in scaffolds with highly tailored properties for challenging applications, including functional and load-bearing ones. This paradigm shift from design-to-manufacture to manufacture-to-design includes the integration of medical imaging with additive biomanufacturing techniques; enabling manufacturing of highly personalized medical devices and implants. However, there is a strong, unmet demand for hybrid and multiphasic materials that functionally combine spatial, mechanical, and biological benefits. In the context of periodontal regeneration, additive biomanufacturing, which referrers to the translation of additive manufacturing technologies into the field of tissue engineering and regenerative medicine (TE&RM), has significant advantages.In this review, the role of additive biomanufacturing within the field of periodontal regeneration is analyzed and discussed. After a brief description of the concept of periodontal therapy, relevant additive biomanufacturing technologies for applications within the field of periodontal regeneration are highlighted. Special emphasis is put on the design of currently available scaffolds. To bring the review to an end, future directions in the context of advanced biomaterials and additive biomanufacturing technologies for regenerative periodontology are described.et al., over the past 30 years major advances have been made within the field of periodontal therapy.,,As described by Bartold N-isoproplyacrylamide) (PIPAAm)] enabling non-enzymatic harvesting of cells and deposited extracellular matrix (ECM).in vitro by stimulating the deposition of ECM. Subsequently, the cell sheet is transplanted into the periodontal defect, enabling enhanced control over cell delivery  scaffolds to promote periodontal regeneration.In vivo evaluations revealed a significant promotion of periodontal attachment formation when using the periodontal ligament cell sheets in combination with the carrier membrane. In addition, although to a limited extent, cell sheets prepared from alveolar bone-derived cells showed to promote this attachment. Another approach involving a PCL membrane to support the cell sheet was presented by Farag et al.Cell sheet technology has already shown promising results in terms of periodontal regeneration potential in (pre)clinical trials.The design of scaffolds which mimic the complex periodontal shape and organization represents a significant challenge in regenerative periodontology. Although additive biomanufacturing may help to surmount this hurdle, adoption and long-term success of these strategies greatly rely on the biomaterials being used. Regarding periodontal regeneration, the most commonly used materials for restoring and/or replacing lost oral tissues are ceramics and polymers Table .48 CeramA wide range of different additive (bio)manufacturing technologies is described in the literature. These can be categorized into laser-assisted printing, inkjet printing, and extrusion-based printing Fig. a. CommonAnother scaffold fabrication method explored for periodontal applications is electrospinning, a broadly used micro- to nanofiber fabrication technique.,et al., multiphasic scaffolds for periodontal regeneration incorporate several beneficial features, including occlusive membrane properties, the need for appropriate fiber guiding, and optimization of cell sheet technology.An important aspect of appropriate periodontal regeneration is the establishment of new fiber attachment to the tooth root surface. This is mediated by the formation of neo-cementum onto the tooth surface, allowing for the insertion of the fibers. Several studies have shown that periodontal ligament tissue, in contrast to gingival connective tissue and alveolar bone, is capable of forming this type of attachment interphase.For predictable periodontal regeneration to occur, hierarchical tissue formation with the appropriate interfacial connection is required.et al. developed a biphasic PCL–PGA scaffold fabricated via computer-aided manufacturing (Fig. In vivo evaluations of the scaffold design in subcutaneous pockets in mice have shown bone and periodontal ligament regeneration capacity and generation of parallel and obliquely oriented fibers. To further resemble the periodontium, adjustments in the design were made.et al. examined the effect of controlled channel architecture in the scaffold design on the tissue interface.In vivo evaluations of this scaffold in periodontal fenestration defects in athymic rats have shown controlled and predictable periodontal fiber orientation, controlled tissue infiltration, and a better organization of the ligament interface compared with random scaffold architectures. With this image-based, fiber-guiding scaffolding system, the authors intend to predictably facilitate regeneration and integration of dental supporting tissues.To address these challenges, Park ing Fig. a.37 The As described above, another approach aiming at periodontal tissue regeneration involves cell sheet engineering. In addition to utilization of the cell sheets unaided, several groups have investigated the combination of additively manufactured scaffolds with cell sheet technology.et al. described a biphasic scaffold in combination with cell sheet technology to regenerate alveolar bone and periodontal ligament simultaneously (Fig. For example, Vaquette sly Fig. b.44 The et al. (Fig. et al., a fused deposition modeled bone component and a flexible electrospun periodontal component were taken into account.et al., the scaffold was assembled by using heat treatment.Further advancements of this strategy involved an osteoconductive biphasic scaffold devised by Costa al. Fig. c.9 In acet al. developed a triphasic scaffold (Fig. et al. and involved printing of a seamless scaffold with region-specific pore sizes for the purpose of facilitating integrated regeneration of various tissues.As an extension of the biphasic scaffolds, Lee old Fig. d.29 Thiset al. fabricated the first reported personalized additively biomanufactured scaffold for periodontal osseous defect regeneration in humans (Fig. et al. have demonstrated the potential of this additively manufactured scaffold for the treatment of a large periodontal osseous defects, the scaffold became exposed after 12 months, which ultimately led to failure from a clinical point of view. A more rapidly resorbing biomaterial with a less bulky design would probably have better suited this application. Additionally, the observed limited bone regeneration indicated the need for incorporating scaffold design imperatives, such as larger pores and pore interconnections.Recently, Rasperini ans Fig. e.41 In tAs demonstrated, multiphasic scaffolds facilitate compartmentalized tissue healing, which is essential for periodontal tissue regeneration. In general, multiphasic scaffolds for periodontal regeneration are characterized by the presence of bone and periodontal attachment compartments.Additive biomanufacturing offers favorable strategies for periodontal tissue engineering. Although encouraging improvements have been made thus far, novel strategies continue to develop at a rapid pace. Besides optimization of the biomaterials, scaffold design, and fabrication techniques, achievement of standardization is an important step towards successful implementation in the future.,Appropriate selection of biomaterials that can guide tissue regeneration plays a vital role in establishing predictable long-term results. For many years, research was focused on the development of biomaterials that simply fill the spaces of the absent tissue.There is abundant literature describing the increasing number of biomaterials for applications in periodontal regeneration.,One of the most sophisticated applications of additive biomanufacturing involves the fabrication of scaffolds. For the specific requirements of periodontal regeneration, multiphasic scaffolds have significant advantages as they facilitate compartmentalized tissue healing.However, for successful clinical translation it is important to develop a road map, which includes studies to receive the required FDA approval and CE-marking at an early stage in the process. In addition to these general approvals, there is an urgent need for guidelines and protocols for standardization in the field of additive biomanufacturing. Subsequently, these manufacturing and engineering standards need to be merged with biological considerations. Above all, it is important to take into account that bioprinting, seen from a translational research point of view, is primarily focused at patient-specific customization. Therefore, one of the biggest challenges will be establishing standardization, while still allowing for patient-specific adjustments.Additive biomanufacturing presents a powerful toolbox to enhance current scaffold-based periodontal tissue regeneration. Currently, additive biomanufacturing facilitates the development of multiphasic scaffolds, which mimic the properties and architectural configurations of periodontal tissues. For optimal exploitation of the potential of additive biomanufacturing, biomaterial characteristics play a pivotal role. By developing advanced biomaterials that influence the biological environment in a controllable manner, patient-specific treatment options with predictable clinical outcomes are envisioned.


Alternative splice isoforms are common and important and have been shown to impact many human diseases. A new study by Nellore et al. offers a comprehensive study of splice junctions in humans by re-analyzing over 21,500 public human RNA sequencing datasets. Genome Biology provides us with the most comprehensive view of human transcriptome splicing to date, having (re)analyzed over 21,500 RNA sequencing (RNA-seq) datasets and discovered 56,865 novel splice junctions [A newly published study by Nellore et al. in RNA splicing is a post-transcriptional RNA processing mechanism occurring in eukaryotic organisms whereby introns are removed from pre-mRNA leading to mature mRNA molecules, or transcripts, consisting of joined exons. The process of RNA splicing generates distinct transcript variants of the same gene, referred to as alternative transcript isoforms, the translation of which leads to distinct protein products. Thus, alternative splicing is a critical process that ensures protein diversity, with most of the multi-exon genes in humans generating multiple alternative transcript isoforms., cancer and normal cells) is a common mechanism. Recently, Sebestyén et al. [, PPARG, MITF, and MYH11) across seven cancer types that resulted in altered gene function; and (amongst many others) aberrant splicing mutations have been reported in muscular dystrophy [Dysregulation of alternative splicing can have major functional consequences through the expression of abnormal isoforms that contribute to disease progression. Isoform switching, where the most abundant transcript isoform has changed between two conditions  and their isoform structure (splicing patterns); to quantify transcript-level expression and the changes in expression under various experimental conditions; and to discover novel transcript isoforms or splice junctions; though care must be taken as accurate alignment and quantification is difficult due to the high similarity between some transcripts and genes [RNA-seq has now become the standard method to analyze the transcriptome, the complete set of transcripts expressed in a given cell. This approach is commonly used to identify the diverse set of transcript types , fewer than 80% of the observed junctions are annotated. In total, Nellore et al. report 56,865 novel junctions (18.6%) found in at least 1000 samples. Thus, comparison of multiple independent studies can reveal many unannotated junctions.Nellore et al. find that most of the reads that map to splice junctions map to junctions that are already known; specifically, in 10,090 of 10,311 datasets that met the authors’ filtering criteria, over 95% of junction reads overlap junctions found in the existing annotation. However, although most splice junctions with high read coverage have been documented, there remains a large number of splice junctions that occur across multiple samples that have not. For example, in 3389 samples from the same set (Nellore et al. confirm that variation in unannotated junction expression across samples strongly correlates with both junction sequencing depth and read length. High read coverage across splice junctions provides stronger evidence that it is real and expressed; and an increased read length allows for a larger proportion of reads to be mapped across splice junctions. Thus, both parameters, read depth and read length, strongly influence junction discovery power.From 2009 to 2013, splice junction discovery has increased over time with spikes of discovery mostly due to large-scale sequencing projects such as the Human Reference Epigenome Mapping Project  (with ovAccurate gene-level and transcript-level expression analyses often rely on the completeness of transcript and splice junction annotation, and research suffers if that annotation is incomplete. Unfortunately, such information is not at the same level of completion for species other than human—beyond human and mouse, other animal genomes can lack up to 20 megabases of annotation —and evenThe effort of Nellore et al. provides an unprecedented insight into the splice junction usage in humans through large-scale RNA-seq data analysis and further highlights the need for similar studies in other less well-characterized species . The dat


For millennia, people have cut and joined different plant tissues together through a process known as grafting. By creating a chimeric organism, desirable properties from two plants combine to enhance disease resistance, abiotic stress tolerance, vigour or facilitate the asexual propagation of plants. In addition, grafting has been extremely informative in science for studying and identifying the long-distance movement of molecules. Despite its increasing use in horticulture and science, how plants undertake the process of grafting remains elusive. Here, we discuss specifically the role of eight major plant hormones during the wound healing and vascular formation process, two phenomena involved in grafting. We furthermore present the roles of these hormones during graft formation and highlight knowledge gaps and future areas of interest for the field of grafting biology. The plant vascular tissue is crucial for transporting water, nutrients, photosynthetic products and signalling molecules throughout the plant. It provides mechanical support, thereby allowing an increase in plant stature and adaptation to various environments. As such, plants have efficient mechanisms to generate vasculature in newly formed organs, to increase vasculature in existing organs, and to heal the vasculature upon wounding or stress. There are two main types of vascular tissues: xylem, made up of tracheary elements, parenchyma cells and fibres; and phloem, made up of sieve elements and parenchyma cells, including companion cells , since this gene is specifically expressed in differentiating xylem and is also induced by drought stress and ABA treatment  is important for biotic and abiotic stress responses, including drought response, but also plays a role during plant development such as seed maturation, seed germination and the regulation of stomatal aperture . MP directly activates the transcription of a homeodomain-leucine zipper III gene, ATHB8, necessary for preprocambial cell specification and the coordination of procambial cell identity. Modulation of auxin levels in specific tissues and cells regulates vascular cell fate. For instance, high auxin levels increase the number of secondary xylem and phloem cells in petunia  6 and 8 promote the expression of the NAM, ATAF, and CUC (NAC) TF ANAC071 above the cut, while inhibiting expression of RELATED TO AP2 6L (RAP2.6L)  19 and 20, important for pith cell proliferation during tissue reunion , acts downstream of auxin and regulates xylem pole pericycle cell division and lateral root formation  or response (bri1: brassinosteroids insensitive 1) repress xylem formation  are steroid hormones involved in cell growth and plant morphogenesis (reviewed in Belkhadir and Jaillais lkb (named after the internode length locus Lk) grafts successfully , a regulator of protophloem differentiation, which is both induced by auxin and slightly repressed by BRs /WOODEN LEG (WOL)/ARABIDOPSIS HISTIDINE KINASE 4 (AHK4) alone, or in combination with AHK2 and AHK3, results in reduced cell file number in root vascular bundles, due to reduced cambial activity , a CK signalling inhibitor. Several CK signalling mutants produce extra xylem, while exogenous CK treatments reduce xylem formation  are adenine-derived phytohormones involved in various aspects of plant development including cell division, lateral root formation and meristem maintenance. Their effects often occur through interaction with the auxin signalling pathway , a wound-inducible TF, upregulates and promotes callus formation through the activation of CK signalling /TRACHEARY ELEMENT DIFFERENTIATION INHIBITOR FACTOR (TDIF) RECEPTOR (TDR) promotes vascular cell divisions, and loss of this receptor has mild effects due to a compensation mechanism dependent on ethylene , an auxin-inducible TF that interacts with the PXY/TDR signalling pathway  mutants  or blocked in ethylene response  successfully graft  is highest in the xylem of hybrid aspen  are diterpene phytohormones with an important role in plant development, particularly in regulating plant growth, as GAs promote cell expansion, cell differentiation and cell proliferation  and its derivatives are lipid-derived plant hormones primarily known for their activation during biotic and abiotic stress including wounding, but are also involved in plant development such as root development and trichome formation   are carotenoid-derived signalling molecules that regulate plant development and architecture (reviewed in Brewer et al. The role of SLs during the wound response has, to our knowledge, not been addressed. Nevertheless, the role of SLs in cambium activity together with their role in the regulation of auxin transport suggests that SLs might be involved in grafting. However, SL-deficient mutants in various plant species graft successfully (Beveridge et al. Arabidopsis where grafting is robust. Many of these hormones might have more subtle effects on enhancing or suppressing graft formation, and their effects may not be obvious until grafts are more mature. Further in-depth studies are required to fully understand the role of these phytohormones and their contribution to graft formation and to the long-term compatibility and success of the graft in both herbaceous and woody species.The development and differentiation of vascular tissues during plant growth is finely regulated by almost every known phytohormone. Nevertheless, auxin appears to be the primary regulator of vascular cell differentiation and patterning with other hormones interacting with the auxin biosynthesis, transport and/or signalling pathways to fine-tune this process Fig. . Since s


Mesenchymal stromal cells are a population of undifferentiated multipotent adult cells possessing extensive self-renewal properties and the potential to differentiate into a variety of mesenchymal lineage cells. They express broad anti-inflammatory and immunomodulatory activity on the immune system and after transplantation can interact with the surrounding microenvironment, promoting tissue healing and regeneration. For this reason, mesenchymal stromal cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Another clinical application of mesenchymal stromal cells is the targeted delivery of chemotherapeutic agents to neoplastic cells, maximizing the cytotoxic activity against cancer cells and minimizing collateral damage to non-neoplastic tissues. Mesenchymal stem cells are home to the stroma of several primary and metastatic neoplasms and hence can be used as vectors for targeted delivery of antineoplastic drugs to the tumour microenvironment, thereby reducing systemic toxicity and maximizing antitumour effects. Paclitaxel and gemcitabine are the chemotherapeutic drugs best loaded by mesenchymal stromal cells and delivered to neoplastic cells, whereas other agents, like pemetrexed, are not internalized by mesenchymal stromal cells and therefore are not suitable for advanced antineoplastic therapy. This review focuses on the state of the art of advanced antineoplastic cell therapy and its future perspectives, emphasizing in vitro and in vivo preclinical results and future clinical applications. The ultimate goal of cancer chemotherapy is to optimize patient outcomes by increasing the drug concentration in the target tissues, thereby enhancing therapeutic efficacy, while simultaneously decreasing the exposure of healthy cells and tissues to reduce toxicity. Nanomedicines shift the tissue distribution of chemotherapeutic drugs, thereby significantly reducing the dose-limiting adverse effects while maintaining or even improving their efficacy .Synthetic lipid- or polymer-based carrier systems or natural carriers like extracellular vesicles, viruses, bacteria and cells have been employed as drug carriers . MesenchMSC are undifferentiated multipotent adult cells defined as plastic-adherent, fibroblast-like cells possessing extensive self-renewal properties and the in vivo and in vitro potential to differentiate into osteogenic, chondrogenic, and adipogenic lineages when cultured in specific inducing media . MSC havInitially discovered in bone marrow, MSC can be isolated from a wide spectrum of adult and foetal tissues like umbilical cord, adipose tissue, periosteum, tendon, dental pulp, cornea, thymus, spleen, brain, liver, placenta, and synovial and amniotic fluids . BesidesThanks to these characteristics, MSC are being exploited as an experimental therapy for a wide spectrum of human diseases. Current knowledge indicates that MSC effectively impact on disease via the secretion of paracrine-acting factors to reduce local inflammation, reprogramme immune cells, and trigger host repair pathways. It was recently discovered that MSC also produce extracellular vesicles—including exosomes—carrying as cargo mRNAs, microRNAs, and proteins inducing non-autonomous therapeutic changes within the damaged host tissue  Figure .New therapeutic approaches to the cell-based delivery of chemotherapeutic drugs have been widely explored thanks to the capacity of MSC to migrate and engraft into tumours after intravenous administration . After eMany different methods of drug delivery have been described in the last decade, including immunoconjugates for targeting tumour-specific antigens, nanoparticles, and genetically modified stem cells; glycoengineering protocols to induce expression of non-natural azide groups on the surface of MSCs without altering their viability or tumour homing capacities have been reported, as well as nano-engineered MSCs were prepared by treating human MSCs with drug-loaded polymeric nanoparticles ,17,18,19However, non-modified MSC are probably the best choice for anticancer drug delivery as they readily adapt to culture conditions and home to pathological tissues when injected in vivo and possess intrinsic antineoplastic activity . This teOn the one hand, MSC hold great promise for oncology because they release active soluble factors and play an effective immunomodulatory role. They can also cross the blood brain barrier, thus representing a potential therapeutic tool for adult and paediatric brain tumours ,21. On tSeveral hypotheses have been put forward to explain MSC antineoplastic activity, including inhibition of proliferation-related signalling pathways, angiogenesis suppression, and cell cycle inhibition ,24,25.Paclitaxel and gemcitabine are the chemotherapeutic drugs best loaded by mesenchymal stromal cells and delivered to neoplastic cells. Although pemetrexed has shown promising results in vivo for malignant mesothelioma therapy, the drug is not internalized by MSC and hence cannot benefit from this method for the time being.The three lead compounds currently used in anticancer therapy, paclitaxel, gemcitabine and pemetrexed, have shown a wide range of activity focusing on different targets and still represent the first-line chemotherapy, especially against solid tumours.Taxus brevifolia  pyrimidine [Pemetrexed  is a mullutamate ,46,47,48rimidine ,50,51 orrimidine .Although paclitaxel and gemcitabine are the most common antineoplastic drugs potentially deliverable by MSC, in the last decade, several other toxic compounds have been experimentally tested.Kosaka et al. reported that MSCs expressing cytosine deaminase and concurrent 5-fluorocytosine administration could improve the survival of rats bearing 9 L gliomas . Ryu et Present results combining stem cells and nanoparticles for the induction of toxicity toward neoplastic cells demonstrate a promising proof-of-concept, but more work needs to be developed to confirm that stem cells effectively improve the efficacy of free-standing nanoparticle systems.Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, being the fourth leading cause of cancer death in the United States . PDAC reGlioblastoma—also known as glioblastoma multiforme (GBM)—is the most aggressive brain cancer, representing 15% of brain tumours . TreatmeMelanoma—also known as malignant melanoma (MM)—is a malignant tumour developing from melanocytes of the skin or other tissues. Surgery plays a curative role for local disease, whereas immunotherapy, biologic therapy, radiation, or chemotherapy may improve survival for metastatic patients, although five-year survival rates drop from 98% among patients localized disease to 17% among those in whom spread has occurred. Paclitaxel-loaded MSC (PTX-MSC) have effectively inhibited lung metastasis formation in a murine B16 melanoma model, thus representing a potential therapeutic model for MM lung metastases .Multiple myeloma is a neoplasm suppressing osteoblastogenesis by bone marrow mesenchymal stromal cells (BM-MSC). The role of MSC for multiple myeloma treatment is widely debated because of the contradictory results on MSC’s ability to inhibit or stimulate cancer growth. MSC could serve as vehicles for targeted delivery of anti-tumour agents into bone marrow. It has been experimentally demonstrated that PTX-loaded BM-MSC are active on the proliferation of a human myeloma cell line, expressing an intense suppression of myeloma cell growth. This suggests that drug-loaded MSC could represent an effective method to deliver the chemotherapeutic agent into the bone marrow .Malignant pleural mesothelioma (MPM) is a cancer related to asbestos exposure whose incidence is expected to peak in 2020–2025 in Europe and Japan. The current standard first-line treatment is a platinum-based doublet containing a third-generation antifolate like pemetrexed (PMX) or ralitrexed. There are no approved second-line treatments for MPM as its prognosis is extremely poor, making it a disease setting to test new drugs. Initial experimental observations showed that PMX is not internalized and released by MSC. However, based on previous results with PTX-primed MSC, further experimental studies demonstrated that PTX-loaded MSC showed an excellent capacity to inhibit MPM cell proliferation, hitherto representing the best in vitro combination (drug + cells) for MPM therapy ,64 Tabl.MSC antineoplastic drug loading and drug delivery represents a promising new field of chemotherapy and advanced cell therapy for oncological diseases, in particular those with a poor prognosis. Successful experimental tests on models of pancreatic adenocarcinoma, glioblastoma, melanoma, multiple myeloma and malignant pleural mesothelioma have yielded very encouraging in vitro results. Phase I clinical studies are now required for further confirmation of the feasibility and efficacy of advanced cell therapy. Adipose-derived MSC as well as nanoparticles may represent—in the future—the new frontiers of drug loading and delivery.


Supervised approach clearly outperforms unsupervised approach. The Twosides network gained the best prediction performance regarding the area under the precision-recall curve (0.93 for both random forests and gradient boosting machine). The applied methodology can be used as a tool to help researchers to identify potential DDIs. The supervised link prediction approach proved to be promising for potential DDIs prediction and may facilitate the identification of potential DDIs in clinical research.Drug-drug interaction (DDI) is a change in the effect of a drug when patient takes another drug. Characterizing DDIs is extremely important to avoid potential adverse drug reactions. We represent DDIs as a complex network in which nodes refer to drugs and links refer to their potential interactions. Recently, the problem of link prediction has attracted much consideration in scientific community. We represent the process of link prediction as a binary classification task on networks of potential DDIs. We use link prediction techniques for predicting unknown interactions between drugs in five arbitrary chosen large-scale DDI databases, namely DrugBank, KEGG, NDF-RT, SemMedDB, and Twosides. We estimated the performance of link prediction using a series of experiments on DDI networks. We performed link prediction using unsupervised and supervised approach including classification tree,  Combined use of multiple drugs at the same time  is common in modern pharmacotherapy , particuAdverse drug reactions (ADRs) are harmful reactions that are caused by intake of medications . Many ADDDIs may be naturally represented as a network in which nodes refer to different drugs and relationships between them designate their interactions , 14. ComA plethora of statistical methods were employed and developed to predict DDIs. An extensive overview of recent approaches is presented separately in the next section. Existing methods may be categorized into three main approaches to DDI prediction: (i) a similarity-based approach, (ii) classification-based approach, and (iii) text mining approach. A similarity-based methods are based on the assumption that similar drugs may interact with the same drug. For instance, two drugs may interact if they have similar molecular profile. Classification-based techniques mimic the DDI prediction task as a binary classification problem. For example, drug-drug pairs are represented as feature vectors, while target variable is represented by presence or absence of interactions. A particular instance of classification-based methods is link prediction, which aim is to assess the probability that a relation exists between pair of nodes in a network, based on observation of topology of existing nodes and their attributes , 21. FinIn this study we examine link prediction from the viewpoint of predicting potential DDIs. The main objectives of this work are: (i) to represent the process of discovering potential DDIs as a binary classification task in which features are represented as topological and semantic measures between drugs, and (ii) to evaluate performance of unsupervised and supervised machine learning methods for predicting potential DDIs. This study is different from other related studies in the following facets: (i) we use broader set of databases for DDIs prediction including DrugBank, KEGG, NDF-RT, SemMedDB, and Twosides; (ii) besides network-based features we also include semantic-based features, for instance chemical information of a drug and assigned Medical Subject Headings (MeSH); (iii) regarding methodological considerations we assume balanced distribution of DDI pairs; (iv) in addition to unsupervised approach we also include supervised statistical learning methods; and (v) last but not least, the study relies on comprehensive statistical evaluation and on manual evaluation performed by trained pharmacist.A recent comprehensive review of DDI detection utilizing clinical resources, scientific literature, and social media is given by Vilar et al. . In prevThe similarity-based approach exploits the idea of biological profiles which are used to compare drugs and infer new molecular properties . Gottliek-nearest neighbors, logistic regression, and support vector machine) to learn interactions between pairs of drugs. Jamal et al.  AND compound_name_2[TW]’). This frequency could serve as an indicator of novelty for our discoveries. A trained pharmacist (PF) reviewed all interactions and suggests a most plausible interpretation for the cause of interaction. Below we present a subset of four interactions, which seem most interesting to us.In this subsection we study a list of top 15 predicted interactions between compounds based on DrugBank database . We offeFluindione (DB13136) is a vitamin K antagonist and is under investigation for the treatment of venous thrombosis, pulmonary embolism, permanent atrial fibrillation, and anticoagulation therapy. On the other hand, picosulfuric acid (DB09268) is a contact laxative used for constipation treatment or to prepare the large bowel before colonoscopy or surgery. As of this writing, there are no matching documents in PubMed which cite both fluindione and picosulfuric acid. However, it is well known that contact laxatives might significantly reduce absorption of orally administered drugs. Specifically, using fluindione and picosulfuric acid concomitantly, the absorption of fluindione into the systemic circulation may be reduced, lower concentrations of fluindione at its binding site may be expected and consequently, fluindione  would be less effective.Mefloquine (DB00358) is a phospholipid-interacting antimalarial drug, very effective against Plasmodium falciparum and with relatively few side effects. Orlistat (DB01083) on the other hand is a drug designed to treat obesity. Its primary function is preventing the absorption of fats from the human diet by inhibiting pancreatic lipases, enzymes that break down triglycerides in the intestine. There are no documents in the PubMed which match both mefloquine and orlistat. However, we may anticipate the pharmacokinetic interaction between both the compounds at the level of absorption. Mefloquine is a rather fat-soluble molecule and its absorption could be significantly decreased while taking orlistat concomitantly.Phenobarbital (DB01174) is a barbituric acid derivative that acts as a nonselective central nervous system depressant. It promotes binding to inhibitory gamma-aminobutyric acid subtype receptors, and modulates chloride currents through receptor channels. Atomoxetine (DB00289) on the other site is the first non-stimulant drug approved for the treatment of attention-deficit hyperactivity disorder (ADHD). In PubMed database there are only two documents referring to both compounds. Phenobarbital is a strong inducer of the liver enzymes of the cytochrome P450 system, through which many drugs are metabolized. Biotransformation of atomoxetine in human is predominantly carried out with isoenzyme CYP2D6. The isoenzyme is induced by concomitant administration of phenobarbital. Atomoxetine is thus rapidly metabolized to its inactive metabolites. Consequently, plasma concentrations of atomoxetine are decreasing rapidly with time which could result in lower atomoxetine therapeutic effect.+/K+-enhancing ATPase. Digitoxin (DB01396) is also a cardiac glycoside, phytosteroid, similar in structure, mechanism of action and effects to digoxin (Na+/K+-enhancing ATPase), although digitoxin effects are longer-lasting. There are 391 documents in PubMed which cite both ouabain and digitoxin, although manual review of the abstracts reveals no paper in which both ouabain and digitoxin are described as interacting compounds. If both compounds would be administered concomitantly without adjustment of the dosing regimen, the pharmacological effects were expected to be excessive , especially for the reason that both drugs have a very narrow therapeutic window.Ouabain (DB01092) is a cardiotonic glycoside obtained from the seeds of Strophanthus gratus and other plants and has a long history in the treatment of heart failure, angina pectoris and myocardial infarction. The drug acts as an inhibitor of the NaIn this study we performed a computational approach to potential DDIs identification using computer-based link prediction techniques. We performed link prediction on the five arbitrary chosen large-scale DDI networks from bioinformatics domain. The main conclusion of this research is that supervised link prediction demonstrated to be a plausible methodology for DDIs prediction. The predicted power is very high for all major DDI databases. In addition, results of this study clearly show that topological information is more important in predicting novel DDIs than semantic information. Our approach is especially useful for large-scale DDI networks for detection of potential associations among drugs whose biological roots are not completely explained.There is a huge interest to understand and elucidate DDIs in contemporary science. As in other life science issues, network-based pharmacology offers a convenient back-up to mechanistic and molecular modeling . Due to Despite the large number of approaches, link prediction in large-scale networks is still a very challenging problem. There are also many possibilities for further work. First and most important, we should dig into the problem of cleaning  interactions in the network. A small portion of detected interactions in the analysed networks may be also excessive. In this regard it is crucial to distinguish between potential and actual, clinically confirmed interactions among drugs. Describing the practical importance of an interaction is essential due to a myriad of potential, but clinically not significant, associations . CurrentLink prediction is a promising methodological framework for studying complex systems in different scientific disciplines, including pharmacology. We evaluate an approach to potential DDIs prediction using link prediction methodology. We study the prediction performance of unsupervised and supervised link prediction algorithms on several large-scale DDI networks. Although there exist many different approaches and algorithms, reliable prediction of links in a network is still a very challenging problem. Computational approach presented here can be used as tool to help researchers to identify potential DDIs. Overall, our results demonstrated favorable classification performance and suggest appropriateness of the presented methodology for potential DDIs identification.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 TableAbsolute and relative average feature importance for all learning models for all included networks.(PDF)Click here for additional data file.