Retinopathy of prematurity (ROP) is a blinding morbidity of preterm infants, which represents a significant clinical problem, accounting for up to 40% of all childhood blindness. ROP displays a range of severity, though even mild disease may result in life-long visual impairment. This is complicated by the fact that our current treatments have significant ocular and potentially systemic effects. Therefore, disease prevention is desperately needed to mitigate the life-long deleterious effects of ROP for preterm infants. Although ROP demonstrates a delayed onset of retinal disease following preterm birth, representing a potential window for prevention, we have been unable to sufficiently alter the natural disease course and meaningfully prevent ROP. Prevention therapeutics requires knowledge of early ROP molecular changes and risk, occurring prior to clinical retinal disease. While we still have an incomplete understanding of these disease mechanisms, emerging data integrating contributions of maternal/placental pathobiology with ROP are poised to inform novel approaches to prevention. Herein, we review the molecular basis for current prevention strategies and the clinical outcomes of these interventions. We also discuss how insights into early ROP pathophysiology may be gained by a better understanding of maternal and placental factors playing a role in preterm birth. Retinopathy of prematurity (ROP) represents a significant clinical problem accounting for up to 40% of childhood blindness worldwide with an estimated incidence of 68% in infants born less than 1,250 g . With inROP is a disorder of retinal vascular maturation with unclear molecular etiology; early birth, low birth weight (BW), and post-natal oxygen exposure are the only universally accepted independent disease predictors –12. In hOn the basis of this understanding, intervention in the “pre-clinical” phase 1 disease would allow for facilitation of normal retinal vascularization and prevent progression to the second phase of ROP. This necessitates an understanding of early ROP risk and disease mechanisms, occurring prior to and within the first phase of ROP. While overall these are incompletely understood, there has been a significant effort to better delineate early ROP pathological mechanisms , 14–16. Herein, we review key early molecular disease pathogenesis that has informed treatment and prevention therapies, clinical findings based on these interventions, novel insights from maternal, and placental pathobiology to identify ROP disease prevention strategies.While ROP early risk and mechanisms are incompletely understood, we have greater understanding of the “final common pathway” mediating neovascular retinal disease.Studies have shown that birth and the consequent switch to lung ventilation produces a change in arterial blood oxygen tension significantly above that of the intrauterine fetus . In the This neovascular event has the potential for greatest visual harm as the vitreally-directed vessel growth can result in tractional retinal detachment and irreversible blindness. The central events and molecular changes that form the framework of ROP pathology, as summarized in Fluctuations in angiogenic growth factors occurring in the preterm retina and mediating ROP disease are thought to primarily arise from avascular retina . This unEarly studies in animal models of oxygen induced retinopathy (OIR) showed that supplemental VEGF at the onset of hyperoxia significantly decreased both initial vaso-obliteration and the subsequent neovascularization characteristic of ROP, and that inhibition of VEGF was a viable treatment for neovascular disease once established –34. ThesCryotherapy and laser photocoagulation are considered the gold standard ROP treatment , 52, 53.Taken together, our current therapeutic interventions have significant limitations, side-effects, and systemic concerns. Furthermore, they largely lack the ability to restore normal retinal architecture and function through preservation of physiologic retinal vascularization. To this end, work has been done to elucidate mechanisms informing early ROP pathogenesis, within the first phase of ROP, which would allow for intervention prior to development of neovascular disease. These interventions, outcomes and potential for further advancement are reviewed below.Gestational age, BW, and post-natal oxygen supplementation are well-established to be the three greatest risk factors independently informing ROP risk , 17. Of IGF-1 is a growth factor critical for normal vascular development, provided in part by the placenta and amniotic fluid during gestation. After term birth, neonate serum levels of IGF-1 decrease; this change is exacerbated in the setting of preterm birth . FurtherHypoxia-inducible factor 1 (HIF-1) is a transcription factor recognized as a master regulator of oxygen response under developmental, physiologic, and pathologic conditions , 76. UndCaffeine was first observed to reduce severe ROP outcomes in a randomized multicenter clinical trial examining neurodevelopment and growth in preterm infants treated for apnea of prematurity. At 18–21 months, the surviving caffeine treated infants had significantly less severe ROP than placebo infants, with no difference in mortality . Two recPropranolol, a nonselective beta-adrenoreceptor blocker (B-AR), was initially proposed as a treatment for neovascular ROP disease after it was found effective in reducing the growth of infantile capillary hemangiomas, which demonstrate VEGF-mediated pathogenesis similar to ROP . PropranPerinatal inflammation –107 and The first nutritional and antioxidant supplement to be tested in preterm neonates for improving ROP outcome was vitamin E . HoweverWork has also been done to investigate the role for non-maternal milk sources of dietary supplements to improve preterm neonate health and prevent development of ROP, though there are mixed results. For instance, while two early clinical studies testing inositol supplementation reported an associated decrease in ROP incidence , 124, a Vitamin A supplementation demonstrates greater promise for ROP prophylaxis. Systemic concentrations of the vitamin A metabolite retinol are low in preterm infants , 129, anNCT02486042).Some of the most promising and least contentious supplements currently in clinical trials are the ω−3 long chain PUFAs DHA and eicosapentaenoic acid (EPA). Both have well established benefits in protecting against inflammation and oxidative stress contributing to neurodegeneration . MoreoveAs depicted in ROP is typified by a multifactorial etiology with most of our current investigation directed at genetic and environmental risk. Certainly, numerous candidate genes have been identified, including brain derived neurotropic factor, and key factors involved in the canonical Wnt signaling pathway, such as frizzled class receptor-4 and low density lipoprotein receptor-related protein 5. However, none of the allelic variants identified thus far sufficiently impact ROP pathogenesis to inform novel screening, prevention or treatment paradigms as reviewed recently by Swan et al., . The conThere is precedent for the influence of placental pathobiology on post-natal preterm infant diseases including bronchopulmonary dysplasia, necrotizing enterocolitis, neurocognitive and neurovascular development –163. FurWhile simply understanding the influence of maternal/placental physiology and pathobiology on preterm infants may provide a valuable framework for understanding ROP risk and pathological mechanisms, doing so within a natural model of placentally-mediated ROP protection may add further insight into potential therapeutic approaches. This idea is supported by evidence showing that placental pathologies associated with preterm birth, particularly those with intra-uterine infection/inflammation such as chronic or acute chorioamnionitis are generally strongly associated with increased ROP risk , 169–171There are varied hypotheses regarding mechanisms of ROP protection in the setting of preeclampsia. Preeclampsia is a complex maternal-fetal-placental pathology typified by placental insufficiency of unclear molecular basis; however, a recent systems biology analysis of preeclampsia confirmed prior work and suggested a more comprehensive maternal etiology for preeclampsia . TherefoWhile placental evaluation is possible in human disease and demonstrates the greatest translational value, manipulation of risk variables and local tissue  biochemical and genomic analysis is prohibitive. Preeclampsia/placental insufficiency can be modeled in rodents and ROP outcomes subsequently assessed using the OIR model of ROP. The “gold standard” approach for placental insufficiency is uterine artery ligation, which directly modulates blood flow to the placenta, thereby achieving insufficiency. While, as noted previously, this model of placental insufficiency does demonstrate decreased severity of pre-retinal neovascularization in the OIR model, there are limitations to this approach including the extreme degree and abrupt onset of hemodynamic insufficiency, which likely does not fully recapitulate preeclamptic pathophysiology now believed to be a systemic condition . AlternaROP has significant clinical impact on the life-long vision of preterm infants, even in the setting of mild disease. Our treatments are only indicated in the most severe presentations and are associated with significant visual comorbidity and possible deleterious systemic effects. We currently lack the ability to meaningfully prevent ROP disease and facilitate normal retinal vascularization. While numerous strategies have been tested, to date none have demonstrated sufficient efficacy for complete ROP prevention. Emerging evidence suggests an important role for maternal and placental physiology and pathobiology in preterm infant post-natal and lifelong disease burden, including ROP risk and mechanisms. As depicted in


Breast cancer is the most common cancer in women worldwide. In south-east Asia, both the incidence and mortality rates of breast cancer are on the rise, and the latter is likely due to the limited access to large-scale community screening program in these resource-limited countries. Breast cancer awareness is an important tool which may, through increasing breast self-examination and the seeking of clinical examination, reduce breast cancer mortality. Investigating factors associated with breast cancer awareness of women is likely to help identify those at risk, and provide insights into developing effective health promotion interventions. To investigate factors associated with breast cancer awareness in Thai women. A cross-sectional sample of Thai women aged 20-64 years was collected during August to October, 2015 from two provinces of southern Thailand (Surat Thani and Songkla). A questionnaire including the Breast Cancer Awareness Scale along with demographic characteristics was administered and Proportional Odds Logistic regression was then used to investigate factors associated with breast cancer awareness. In total, 660 Thai women participated in this study. Factors most often associated with the various breast cancer awareness domains were age and rurality. While rural women had poorer knowledge of breast cancer signs and symptoms, they also had lower levels of perceived barriers and considerably better breast cancer awareness behaviors. Despite lower knowledge of breast cancer risk factors and no evidence of better knowledge of signs and symptoms, we found rural Thai women had considerably better breast cancer awareness behavior. This may be due to these women’s lower levels of perceived barriers to breast cancer screening services. Indeed this suggests, at least in Thai women, that interventions aimed at lowering perceived barriers rather than enhancing disease knowledge may be more successful in engaging women with breast cancer screening services and increasing breast self-examination.  Despite a considerably higher breast cancer incidence in western populations such as those in Australia and New Zealand, North America and Western Europe when compared with Asian populations , early diagnosis and improvements in treatment have led to a considerable fall in breast cancer case fatality rates in these western populations. In contrast, breast cancer mortality is increasing in many developing countries and is the most frequent cause of cancer death in women . In south-east Asian countries, despite comparatively lower breast cancer incidence rates, the mortality rate remains high. For example, while the cumulative risk of breast cancer incidence in south-east Asian countries is about half that of North American countries (4.17% vs 9.32%), the cumulative risk of breast cancer mortality is higher . This higher mortality rate is likely to be due to the poorer screening and treatment practices associated with resource-limited health care systems in many of these countries. Indeed, many women diagnosed with breast cancer in resource-limited Asian countries are diagnosed at an advanced stage of the disease , and this is likely to be one of the main causes of the higher breast cancer case fatality rate. Moreover, trends of breast cancer incidence in many developing Asian countries continue to rise, potentially compounding the problem of high breast cancer mortality. In the last 10 years, countries like China and India have seen considerable increases in breast cancer incidence . As with other resource-limited countries, diagnosis of breast cancer in Thai women is typically at a comparatively advanced stage of the disease .Several studies have suggested that the increasing incidence of breast cancer in Asian women is attributable to economic development and adaptation of a more westernized lifestyle, including delayed childbirth, reduced parity and breastfeeding, weight gain, and increased consumption of animal fat . However, it is also possible the impact of the various breast cancer risk factors may differ in Asian women. For example, peak breast cancer incidence in Asian women has been found to be between 40 to 50 years of age, whereas in Western countries the highest incidence is in women aged 60 to 70 years . Earlier detection of breast cancer leads to more effective treatment, in turn, leading to higher survival rates. Publicly funded, community-based mammographic screening has been broadly accepted as one of the best methods of reducing breast cancer mortality . However, such programs are currently infeasible in many resource-limited countries. For instance, while approximately 78% of high-income countries provide community-based mammographic screening programs, only 48% of upper-middle income countries have such programs . Other methods for the early detection of breast cancer need to be considered in the resource-limited health setting. The efficacy of breast self-examination (BSE) in reducing breast cancer mortality remains controversial. For example, in some studies BSE could not be shown to reduce breast cancer mortality . However, these studies were conducted in resource-sufficient western countries, where the impact of BSE, on top of community-based screening programs, may not have a substantial effect on reducing mortality. In countries lacking comprehensive community screening programs, BSE may have a substantial effect acting as a trigger for women seeking earlier clinical examination, thereby leading to earlier detection. In resource-limited countries, it is individual women, rather than the health-care system, that is likely to be the main agent in women seeking clinical breast examination or mammography. Several studies have shown that breast cancer awareness is associated with BSE . Indeed, one previous study demonstrated that breast cancer awareness was a strong predictor of BSE with a sensitivity of 83.3% and specificity of 60.2% in Thai women . Consequently, identifying those at risk of poor awareness is an important first step in raising breast cancer awareness, increasing breast self-examination, and subsequently, women seeking clinical breast examination in a timely manner. Several studies have investigated factors associated with breast cancer awareness, however, most of these studies have used instruments that have not been rigorously validated , or instruments that were developed for western populations unlikely to be valid outside of these contexts . In this study we investigate the factors associated with breast cancer awareness in Thai women. We employ a psychometrically-validated instrument, the Breast Cancer Awareness Scale (B-CAS), which has been shown to be valid in Thai women . Study design and sampleIn this cross-sectional study, 660 Thai women aged 20-64 years were sampled from August to October, 2015. Stratified random sampling was used to select participants from rural and urban areas of the Surat Thani and Songkla provinces of southern Thailand. Stratification was based on locality-age groups as reported by the Department of Provincial Administration statistics (2013). Women were approached at primary health care units within their respective locality  and province (Surat Thani / Songkla) districts, and upon providing informed consent, recruited into the study. Those women with a personal history of breast cancer, currently pregnant or breast feeding, or not literate in the Thai language were excluded. Permission to collect the data was obtained from the head of each community, and all participants provided informed consent. The study protocol was approved by the ethics committees of Khon Kaen University (HE 582053).Variables measuredData were collected using a self-administered questionnaire which included demographic variables along with the Breast Cancer Awareness Scale (B-CAS), an instrument previously validated in this population . The B-CAS instrument contains five domains: knowledge of breast cancer risk factors, knowledge of breast cancer signs and symptoms, attitude to breast cancer prevention, barriers of breast screening, and health behavior related to breast cancer awareness. B-CAS content was validated using a panel of twelve experts with extensive experience working in the breast cancer field . Construct validation was performed using confirmatory factor analysis where the measurement model was shown to fit the data well . Criterion validity of the B-CAS subscales was evaluated in terms of the subscales’ ability to discriminate between women who do, and don’t regularly conduct breast self-examination. All five subscales demonstrated utility in identifying women who do not perform breast self-examination (Sensitivities ranging from 65.4% to 83.3%). Test-retest and Internal consistency reliability for all subscales was also shown to be strong . Statistical analysisEpidata  was used to enter the data, and the logic check mode was used to check for data errors. Demographic characteristics of participants were summarized using means and standard deviations, for continuous variables, and frequencies and percentages, for categorical data. In the absence of clinically meaningful cut-points, the overall scale and each individual B-CAS domain was split into tertiles with women in the bottom 33% scored as low, the middle tertile as medium, and the upper tertile as high. Proportional odds logistic regression was then used to investigate the factors associated with each B-CAS domain, with bivariate and multivariable models used to generate unadjusted and adjusted odds ratios, respectively. The proportionality assumption of the proportional odds logistic regression was assessed graphically from the bivariate models of each outcome-predictor combination and the results of these models  are provided in Supplementary Tables S1 through to S6. All analysis was conducted using the R statistics package  and a significance level of 0.05 was used throughout all inferential analysis.A total of 660 Thai women completed the questionnaire (Response rate: 94.3%), and their ages ranged from 20 to 64 years old . Most participants were aged between 35 to 59 years old (58.84%), had not achieved more than a high school education (74.3%), and resided in rural areas (69.85%). Demographic characteristics of the participants are presented in The adjusted odds ratios of Breast Cancer Awareness scale  and B-CAS subscales are provided in 2=6.94, df=2, p<0.05) with married women exhibiting higher odds of better breast cancer awareness, relative to single women , although those that had been previously married did not differ significantly from single women.  cancer could be shown to be associated with knowledge of breast cancer symptoms. . The oddFew factors were associated with attitude to breast cancer prevention . HoweverOlder women were less likely to perceive barriers to breast cancer screening as a problem with the odds of higher perceived barriers of breast screening decreasing 24% for every 10 years of age . However, this decrease in mortality rate has not flowed through to many developing countries suggesting both curative and preventive intervention, such as community level screening, are central to reducing breast cancer mortality. In many south-east Asian countries, no comprehensive population screening programs exist and later diagnosis of breast cancer is the result , and this is perhaps the most probable cause for the higher mortality rate observed in resource-limited settings. Instead, in resource-limited countries like Thailand, mammography tends to be limited to diagnosis  and breast self-examination remains one of the most common ways the disease is detected in the resource-limited setting. Breast cancer awareness, which aims to raise breast cancer knowledge in women, and stress the importance of breast self-examination and seeking clinical examination, is likely to lead to earlier detection, and in resource-limited settings, is likely to play an important role in decreasing breast cancer mortality. In this study, we use the validated B-CAS instrument to identify factors associated with breast cancer awareness in Thai women. We analyzed data from a community sample of Thai women and found that women living in rural areas had lower breast cancer awareness  than women living in an urban setting. This result is consistent with some studies , but Norlaili et al., (2013) in their study of Malay women found that rural women had a better level of breast cancer awareness compared to urban women. We also found married women had better breast cancer awareness relative to single women, even after adjusting for age. This aligns with the findings of Radi (2013) who also found married women to have higher breast cancer awareness. However, Parsa et al., (2008) in their study of a similar south-east Asian population  found no association between marital status and breast cancer screening behaviors. This difference may be explained by subtle differences between Thai and Malay culture. For example, in Thai culture, it falls to daughters to look after their elder family members and it may be that younger women strongly motivate their mothers to seek regular medical checkups. Ultimately, breast cancer awareness behavior is one of the most important domains considered in the present study. It is this domain that ultimately leads to higher incidence of breast self-examination and seeking clinical examination. One of the most striking results in the present study was that rural women, despite poorer breast cancer knowledge and attitude, were considerably more likely to exhibit better breast cancer health behavior  than urban women. While other studies have found that rurality is positively associated with breast cancer awareness behavior in Australian women  and Malaysian women , no other study has observed such a strong effect size; we found that Thai rural women have almost 5 times the odds of better breast cancer health behavior relative to their urban counterparts. However, it should also be noted that there is inconsistency in the literature about rurality being positively associated with better breast cancer health behavior. Kanaga et al., (2011) reported that rural women had poorer breast cancer practices compared to urban women in Malaysia. Similar to that observed in other populations (such as Iran and the Unitied Arab Emirates), we found that age was also positively associated with better breast cancer health behavior . However, Parsa’s et al., (2008) study of breast cancer awareness among Malaysian women could not demonstrate age to be associated with breast cancer health behavior. We also found that women with higher education had better breast cancer health behavior. This has been demonstrated in several other studies of various populations  and confirms that general education plays an important role of health literacy, at least for breast cancer. This study did have some limitations. Women included in our study were limited to two southern provinces of Thailand, and whether our sample is representative of all Thai women is unknown. Furthermore, we employed a cross-sectional study design and while it makes sense that the relatively non-modifiable demographic factors we considered would necessarily precede the breast cancer awareness outcomes we studied, nevertheless such study designs are limited regarding statements of causality. The present study also had a major strength. We used a well-developed breast cancer awareness instrument that has been validated in Thai women  and other south-east Asian populations . Interestingly, our analysis found the two most important factors, those significantly associated with most breast cancer awareness domains, to be rurality and age. Higher age has been demonstrated across many studies as being associated with many breast cancer awareness domains. However, our results regarding the associations of rurality and the various B-CAS domains was surprising. In particular, despite women living in rural areas having both poorer knowledge of breast cancer symptoms and poorer attitudes to breast cancer screening, these same women had a substantially higher likelihood of better breast cancer behavior. It is not clear why this might be, but it suggests that interventions with an emphasis of disease knowledge may not be the best approach to improving breast cancer health behaviors such as breast self-examination and seeking clinical examination. It was true that we observed that rural women perceived fewer barriers to breast cancer screening than their urban counterparts, and this may be due to lifestyle differences between rural and urban Thai women. Despite the methods used for early detection differing in the two settings, the role of breast cancer awareness in both resource-sufficient and resource-limited countries is very important in decreasing the breast cancer mortality rate. Specifically, breast cancer awareness is likely to increase health-seeking behavior. Consequently, we are left with the challenge of how to raise breast cancer awareness to ensure the best possible outcomes. Somewhat surprisingly, in the present study we found that while rural women had lower knowledge of breast cancer risk factor, they had substantially better breast cancer awareness behavior and a lower level of perceived barriers to breast cancer health care. This suggest interventions wholly aimed at enhancing disease knowledge are unlikely to be effective in increasing breast self-examination among Thai women. Instead emphasis should be placed on access to breast cancer health services, and the importance of breast self-examination as a trigger for seeking clinical examination.


We examined age differences in metacognitive monitoring of emotionally-valenced stimuli. If older adults (OAs) are more focused on emotionally meaningful goals in late life , then they should demonstrate attentional and memory biases for positive stimuli over neutral and negative stimuli and, arguably, these cognitive biases should be reflected in their metacognitive judgments of learning. Judgments of learning (JOLs) for memory of positive, negative, and neutral words were collected. Younger adults (YAs) aged 18-23 years and OAs aged 65-90 years (N = 85) studied words in each valence category and made immediate JOLs, followed by a two-alternative forced choice (2AFC) recognition memory task. Analyses of JOLs revealed evidence for a positivity effect  in metacognitive confidence for OAs and an emotional salience effect in YAs . Predictably, YAs recognized more words than OAs, but valence did not affect number of words recognized and valence did not moderate age differences in recognition memory (p = .055). Memory monitoring as measured by resolution accuracy was equivalent in YAs and OAs . Positive affect was higher and negative affect was lower in OAs relative to YAs , lending additional evidence to an orientation toward the positive in older adulthood. These results are novel in that they demonstrate an age-related positivity effect that extends beyond the domains of memory and emotion to the domain of metacognitive aging. Discussion will focus on theoretical, methodological, and applied implications.


Nanotechnology, as an interdisciplinary science, combines engineering, physics, material sciences, and chemistry with the biomedicine knowhow, trying the management of a wide range of diseases. Nanoparticle-based devices holding tumor imaging, targeting and therapy capabilities are formerly under study. Since conventional hematological therapies are sometimes defined by reduced selectivity, low therapeutic efficacy and many side effects, in this review we discuss the potential advantages of the NPs’ use in alternative/combined strategies. In the introduction the basic notion of nanomedicine and nanoparticles’ classification are described, while in the main text nanodiagnostics, nanotherapeutics and theranostics solutions coming out from the use of a wide-ranging NPs availability are listed and discussed. The word “nanotechnology” generally states the production of new tools with nanometric dimensions . Most ofNanotechnology is demonstrating its great potential in healthcare for theranostics, preventive applications and medical-designed novelties that are referred to as “nanomedicine” by the USA National Institutes of Health . More inNanoparticles (NPs) represent one of the most successful solution that nanomedicine proposes to solve huge biomedical questions. With their size ranging from 1 to 100 nm, NPs are characterized by a considerable surface area-to-volume ratio. As described in  for diagRegarding their chemical composition, NPs can be distinguished into three main groups: carbon-based, organic and inorganic materials.The first group  includesFullerenes, water insoluble sphere containing 60 carbon atoms, represent the third allotophic form of carbon with respect to diamond and graphite. Fullerene derivatives have been successfully used for different diagnostics and therapeutics applications, many groups reported the use of these carbon allotropes for medical imaging and drug delivery purposes together with photodynamic, hyperthermia and acoustic wave assisted therapies ,13.Carbon Nano Tubes (CNTs) were classified into single-walled carbon nanotube (SWCNT) and multi-walled carbon nanotube (MWCNT) since they can be constituted by rolling up respectively one or more graphitic sheets. CNTs are applied in a wide range of biomedical applications as tissue engineering scaffold, biosensors and as labelling, imaging, drug delivery and therapeutic agents ,15.Concerning organic nanoparticles, we mainly refer to polymeric nanoparticles (PNPs), liposomes, and extracellular vesicles (EVs) .PNPs can be made from natural or synthetic polymers and thanks to their biocompatibility and biodegradability represent one of the most considered organic approaches for solve some nanomedicine challenges . They caLiposomes are sphere-shaped vesicles made up of a lipid bilayer. They can be prepared starting from cholesterols, phospholipids, surfactants and proteins . LiposomRegarding the extracellular vesicles (EVs) application in the biomedical contest, one of the most applied EVs categories is undoubtedly the nanosized exosomes (70–150 nm). They are released by all cell types, fine-tuning physiological and pathophysiological intercellular statements . At presThe inorganic nanoparticles category includes Quantum Dots (QDs), Metallic (MNPs) and Metal Oxide (MONPs) Nanoparticles .QDs are semiconductor materials consisting of a core overcoated with a shell that is usually conjugated to peptides, proteins, polysaccharides and other biomolecules to prevent the leakage of the toxic-heavy metals and increase the overall NP stability in biological fluids. These kind of nanoparticles are the most used in bioimaging and biosensing strategies: gold quantum dots (GQDs), indium–phosphate (InP), cadmium–selenium (CdSe), indium–arsenate (InAs) cadmium– tellurium (CdTe) can be differently applied for real time cell tagging and cellular apoptosis recognition .MNPs comprise magnetic and precious metals: MNPs such as Palladium (PdNPs), silver (AgNPs), gold (AuNPs), and copper (CuNPs) unveiled wide applicability as theranostics agents while magnetic ones exhibiting high stability in hypoxic tumor conditions and are successfully applied as contrast imaging and bio-sensing agents .2), mesoporous silica nanoparticles (MSNs), iron oxide (Fe3O4), zirconia (ZrO2), zinc oxide (ZnO) and titania (TiO2) show high chemical stability, antioxidant and catalytic actions that make them right for medical implants, drug delivery and bioimaging applications [Biocompatible MONPs such as ceria  from complex biological fluids.In this contest, lymphoma cells present different non-glycosylated antigens on their surface. In particular, the CD20 antigen plays a critical role in the B lymphocytes activation and differentiation processes. The overexpression of this antigen on tumoral B cells made it a valid target to successfully isolate CTCs in blood and other human fluids. Many detection methods are based on active targeting moieties, such as ligands and monoclonal antibodies. In particular, anti-CD20, e.g. Rituximab, directed against CD20 antigen, associated with different types of nanoparticles, i.e. QDs or magnetic nanoparticles, aim to isolate lymphoma cells exploiting the high affinity between antigen and antibody.et al. proposed a new tool for the detection of non-Hodgkin lymphoma: Rituximab conjugated QDs bind specifically to tumor cells, allowing their detection through flow cytometry. Results display a higher sensitivity and specificity compared with immunohistochemistry, which is the current gold standard test [Shariatifar, ard test .QDs can be also conjugated with Sgc8c aptamer for an effective diagnosis of leukemia at the early stage by imaging tumor cells in vitro or in vivo .Magnetic NPs characterized by having a high biocompatibility, stability, surface-to-volume ratio, binding-capacity and specificity were also successfully conjugated with anti-CD20 antibody to isolate lymphoma cells from biological fluids with an efficiency above the 95% . At the CTC can also be detected with a customized device (biosensor), composed by a biological receptor and a physicochemical detector. They offer ease, quick and high sensitivity and specificity measurements of complex biological samples, in a very cost-effective process.et al. created a biosensor for acute lymphoblastic leukemia (ALL) early detection. By combining graphene sheets and an electrosynthesized conductive polymer, poly(cathecol), a high electrically conductive surface was realized and AuNPs were deposited on it in order to use their affinity with thiol-modified DNA to immobilize the target DNA [AuNPs have attracted attention as probes in biological detection for their biocompatibility, surface-to-volume ratio, ease of synthesis, surface functionalization and unique properties, among which the most important is their localized surface plasmon resonance (LSPR). AuNPs functionalized with thiolate oligonucleotides (Au-nanoprobes) can be used for the detection of bioanalytes, such as ions, proteins or target DNA at a lower cost comparing to traditional methods. The detection is carried out exploiting the LSPR, according to which their intense color changes together with the modification of the dielectric medium. Au-nanoprobes are efficiently applied to the direct detection of the molecular hallmark of chronic myeloid leukemia, BCR-ABL fusion transcripts, allowing the discrimination between the most frequent isoforms of this genetic abnormality, e13a2 and e14a2 . Anotherrget DNA .AuNPs-based biosensors can be used also for the detection of specific factor of the coagulation cascade: thrombin-binding aptamer-conjugated AuNPs can detect thrombin ,45, whilGraphene sheets, combined in different ways with aptamers, are also successfully used for the detection of blood cancer cells. Aptamers are artificial single-stranded oligonucleotides, having great affinity and selectivity with their targets, i.e. cells, proteins, drugs or small molecules, lower immunogenicity and toxicity, higher chemical and thermal stability and smaller dimension compared with antibodies. They can be used conjugated to nanoparticles for the detection of specific cancer cells, for example linked to carbon QDs coated with zinc oxide nanospheres  or to quA portable sensor for a rapid analysis and diagnosis of acute myeloid leukemia was developed by using Surface Enhanced Raman Scattering (SERS). Hollow core photonic crystal fibers are integrated with silver NPs in order to enhance the weak Raman signal of cancerous cells even at very low cells counts up to 300 cells/ml . SERS Auet al. proposed a new approach which is more rapid, sensitive and economical if compared to these current methods. They used macroporous ordered silica foams to enrich proteins in urine which are then analysed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to detect the presence of the Bence-Jones protein [The diagnosis of multiple myeloma involves the detection of the Bence-Jones protein in serum. A complementary exam is the detection of the protein in urine, however the current analyses, e.g. immunoelectrophoresis, immunonephelometry and heat precipitation, are time consuming, have low sensitivity, and provide inaccurate results. Long,  protein .Many papers reported how MNPs’ and MONPs’ chemical-physical properties affect cancer cells viability. More in details silver NPs –59 and set al. reported that L-asparaginase functionalized AuNPs are better internalized into leukemia T-cells than bare ones resulting more susceptible to localized hypertermia treatment [Shahriari, reatment .Iron oxide NPs can enter B-cell lymphoma and multiple myeloma cells through phagocytosis and electrostatic interaction inducing cells autophagy and death. This induction of cells autophagy can be further non-invasively tuned by an external magnetic field  or by thPhotodynamic therapy (PDT) is a non-invasive cancer treatment that is still under study but has shown great results. After the accumulation of a phosensitizer agent in the tumor, the diseased region is illuminated, usually with a laser source, and the photosensitizer transfer energy to molecular oxygen in order to generate ROS. ZnO NPs can act as photosensitizer  and theiMetal NPs are promising nanocarriers for anticancer drugs, proteins or nucleic acids, thanks to their small size, biocompatibility and capacity to protect and deliver high payload of drugs selectively to the tumor by active or passive targeting methods.AuNPs can deliver molecules with limited clearance, such siRNA , oligonuOther chemotherapeutics can be loaded on different types of MNPs to improve their circulation time and the uptake by cancer cells, reducing side effects toxicity. For example, magnetic nanoparticles of magnetite can be coated with daunorubicin  or magneIron or iron-based NPs are successfully used for the treatment of a non-cancerous blood disease like anemia. It is commonly treated by oral administration of ferrous sulfate supplements, but only a small part is absorbed in the upper intestinal tract and the remaining reaches the low tract where reacts with hydrogen peroxide and superoxide producing free radicals and unfavourable effects. The reduction of the size of iron to the nanometric scale, increases its bioavailability and gastrointestinal absorption . FurtherMNPs can be applied also to coagulation disorders treatments: iron-derived NPs, such as magnetite, can be used as haemostatic agents. Magnetite matrix NPs, entrapping thrombin, are applied for a non-invasive treatment of internal bleeding: they are injected directly in the bloodstream, guided through an external magnetic field to the site of bleeding, where fibrinogen is injected to accelerate the coagulation process and the combination with thrombin-entrapped NPs stops the bleeding . In contMSNs, entrapping the drug inside their pores efficiently deliver chemotherapeutics to cancer cells. Daunorubicin was encapsulated in mesoporous silica NPs functionalized with the B220 antibody to actively and selectively target acute leukemia cells . Once enMSNs can be employed as haemoglobin-based oxygen: exploiting the affinity between haemoglobin and MSNs, haemoglobin can be loaded inside the pores and replace the function of defective erythrocytes delivering oxygen to cells and tissues . To contAmong the many application of polymeric NPs in therapeutics, Poly(lactic-co-glycolic) acid (PLGA), as biodegradable polymer approved by FDA, is recognised as a valid drug delivery vehicle.Curcumin, a natural hydrophobic yellow pigment with anticancer and anti-inflammatory properties, has low aqueous solubility and bioavailability so its drug delivery capability is enhanced by PLGA NPs incapsulation ,106. TheBarasertib or AZD2811 is a potent and selective Aurora B kinase inhibitor that has a pivotal role in inducing the chromosomes alignment during mitosis and controlling the cytokinesis, thus its inhibition induces polyploidization and cell death. The drug is encapsulated in polymeric nanoparticles called Accurine, composed of poly-D,L-lactide and poly(ethylen glycol). This drug delivery system allows a scheduling of weekly (or longer periods) administrations, thanks to the high dose delivered to the tumor that increases the durability of the response and decreases the toxic side effects ,109.To overcome some of the limitations of standard anticancer drugs, they can be encapsulated inside biodegradable polymeric NPs which guarantees the delivery of the drug to the target site and a continuous and controlled release. Chlorambucil and hydroxychloroquine –112 or dA special reference needs to be made to nanogels that are colloidal hydrogel particles composed by three-dimensional crosslinked hydrophilic polymers networks. They are used for the delivery of hydrophilic large molecules, with the aim to protect drugs from degradation, and their surface can be functionalized to improve the blood circulation and the cellular uptake. Their drug delivery properties are based on the hydrogels’ capacity of swelling in aqueous environment instead of dissolving. Nanogels of linear thiolated poly(glycidol), modified with peptides sensitive to redox environment, loaded with the tumor suppressor miR-34a and a trans activator for transcription provide an effective tool for the treatment of multiple myeloma . Other kBiodegradable polymers such as PCL or PLGA can be loaded with heparin , while cTwo outstanding applications of PLGA nanoparticles have been studied for immune thrombocytopenic purpura and thalassemia, respectively. In the first case, a NP core of PLGA was covered by a platelet membranes coating, which presents all the typical platelet proteins able to partially neutralized the effects of anti-platelet antibodies and minimize the disease burden . ThalassThere are several chemotherapeutic or anti-inflammatory drugs encapsulated in lipids and this practice has been proven to be effective enhancing the local concentration in inflamed tissues, reducing the exposure of other organs and also protecting the drug from degradation.For instance, short interfering RNAs (siRNA) ,125 or aGlucocorticoids are employed in clinic for the treatment of a variety of inflammatory driven malignancies, including multiple myeloma, eventually in combination with other chemotherapeutic drugs. However, they produce severe side effects like systemic immunosuppression, osteoporosis, hypertension and others, they have also a rapid clearance and a request of frequent administrations.The therapeutic efficacy of dexamethasone is improved by liposomal encapsulation . Some chLiposomes can encapsulate drugs for different medical purposes: thrombin inhibitors, able to exert an anticoagulant activity in case of arterial acute thrombosis , mRNA toSolid lipid NPs (SLNPs) are nanocarriers used as an alternative to polymeric NPs since they present a lipid core, which encapsulates lipophilic drugs controlling their release by increasing their solubility, bioavailability and pharmacokinetic profile in case of natural-derived anticancer drugs, such as AP9-cd  and curcVarious NPs act as multifunctional nanotools and can be used both for the detection and the treatment of such haematological cancers. Referring to AuNPs, their diagnostic capability is carried out exploiting their high absorption and scattering of light, while the high surface-to-volume ratio, afford their use as nanocarriers for drugs. AuNPs, with or without further funzionalization , sustainTransferrin (Tf)-luminescent blue copper nanoclusters are coupled with doxorubicin for theranostics applications. When NPs are internalized in TfR overexpressed cells, it is possible to simultaneously detect the blue emission of transferrin into the cytoplasm and, the gradual release of doxorubicin in the nucleus through Förster Resonance Energy Transfer (FRET) .Lanthanide-doped nanoparticles can be employed as bioimaging tool for their photoluminescent properties  and loadAlso liposomes can be engineered for theranostics application: they can be loaded with superparamagnetic iron oxide NPs (SPIONs) and detected by magnetic resonance or positron emission tomography for tracking and treatment monitoring. SPIONs are covered with liposomes and then functionalized with Rituximab for increase their targeting ability and, coated with tween80 to increase their circulation time and their penetration across the blood brain barrier (BBB) to reach also central nervous system lymphoma .Core-shell chitosan-hyaluronic acid-NPs decorated with peptide pA20-36 were used to specifically target B-cell lymphoma and induce cells death in a caspase-dependent manner while fluorescent tracer and a paramagnetic agent allowed NPs internalization imaging .Also Calcium phosphosilicate NPs can be engineered, once loaded with indocyanine green and functionalized with CD96 and CD117 antibodies, they can be endocytosed by leukemic cells, allowing the detection of the disease and the application of the photodynamic therapy .To conclude here we have reviewed the broad panorama of nanoparticles, which represent one of the most useful alternative solution to manage blood diseases providing innovative non-invasive approaches for diagnosis and treatment.Although a large number of NPs-containing drugs have already received FDA approval  or are aHowever, there is no doubt that the rapid progress of the engineering of new materials and the implementation of new methods in the nanotechnology field will lead to the design and standardization of alternatives therapies specific to each patient and disease. Nanomedicine, using original and multi-faceted instruments as the NPs are, can offer the precise targeting and therapeutics tools that researchers and physicians need, to make the diagnosis and treatment techniques that they already have at their disposal even more effective and competitive.


Rhabdoviridae, Aspiviridae, Fimoviridae, Tospoviridae, and Phenuiviridae. They include both emergent pathogens that infect a wide range of plant species, and potential endophytes which appear not to induce any visible symptoms. As a consequence of biased sampling based on a narrow focus on crops with disease symptoms, the number of NSR plant viruses identified so far represents only a fraction of this type of viruses present in the virosphere. Detection and molecular characterization of NSR viruses has often been challenging, but the widespread implementation of HTS has facilitated not only the identification but also the characterization of the genomic sequences of at least 70 NSR plant viruses in the last 7 years. Moreover, continuing advances in HTS technologies and bioinformatic pipelines, concomitant with a significant cost reduction has led to its use as a routine method of choice, supporting the foundations of a diverse array of novel applications such as quarantine analysis of traded plant materials and genetic resources, virus detection in insect vectors, analysis of virus communities in individual plants, and assessment of virus evolution through ecogenomics, among others. The insights from these advancements are shedding new light on the extensive diversity of NSR plant viruses and their complex evolution, and provide an essential framework for improved taxonomic classification of plant NSR viruses as part of the realm Riboviria. Thus, HTS-based methods for virus discovery, our ‘new eyes,’ are unraveling in real time the richness and magnitude of the plant RNA virosphere.The use of high-throughput sequencing (HTS) for virus diagnostics, as well as the importance of this technology as a valuable tool for discovery of novel viruses has been extensively investigated. In this review, we consider the application of HTS approaches to uncover novel plant viruses with a focus on the negative-sense, single-stranded RNA virosphere. Plant viruses with negative-sense and ambisense RNA (NSR) genomes belong to several taxonomic families, including  Viruses are the most numerous biological entities on Earth, but the number of reported and formally described virus species, the known virosphere, is exiguous. This underestimated global virus landscape has led to a distorted view of virus diversity and function. Some aspects of virus biology including their small size, rapid rate of evolution, or lack of universally conserved viral genetic markers are likely the major cause for the lack of knowledge of most of the viruses present on our planet . Thus, aa priori knowledge. Thus, when combined with specific bioinformatics tools, HTS provides a powerful, efficient and economical alternative that has enabled not only the untargeted detection of both known and unknown viruses that inhabit a particular organism or environment but also a rapid characterization of their genomes (High-throughput sequencing (HTS) technology, also referred to as next generation sequencing, has revolutionized the nucleic acid characterization process since it allows the parallel sequencing of millions of nucleotides in a short period of time at a very high redundancy (depth of sequencing) without any  genomes . Thus, tHigh-throughput sequencing is also widely used to advance the molecular characterization not only of those viruses which have a poorly characterized genome, but also of preserved historic virus isolates which had been subjected to studies of their biological properties some decades ago. For instance, sterile stunt disease of maize, characterized by severe stunting and top necrosis of susceptible maize genotypes, was first reported in 1977 in Australia, but the genome of its etiological agent, maize sterile stunt virus was only characterized by HTS and recently reported . Similarin situ virus particle detection by electron microscopy or immunoassays, to name a few.In spite of the many advantages of HTS, there are some limitations and constraints that should be considered when using this platform for virus discovery. Given the high sensitivity of HTS, this technology may detect contaminant viral sequences or viruses that may not be actually replicating in the sampled plant tissue where they were found . In thisonly on their genomic sequences which constitutes a paradigm shift in their taxonomical classification  total plant RNA extracts, usually with a ribosomal depletion step (ii) virion-associated nucleic acids (VANA) extracted from purified viral particles, (iii) double-stranded RNA (dsRNA), enriched through cellulose chromatography or monoclonal antibody pull-down, and (iv) small interfering RNAs (siRNAs)  . These d(siRNAs) .Recent technological advances have led to the appreciation of exciting novel aspects of negative-sense and ambisense RNA (NSR) viruses, as recently reviewed by Solanum species resulted in the identification of viruses belonging to 20 different families, but only one was a NSR virus . Wuhan insect viruses 4–6 may be plant viruses based on their clear phylogenetic relationship with plant rhabdoviruses in the genus Cytorhabdovirus and that their genomes encode a P3 protein that most resembles plant rhabdovirus cell-to-cell movement proteins.Most plant NSR viruses are transmitted by arthropods in a persistent-circulative and propagative manner, thus they are adapted to infect both arthropod and plant cells. In fact, it has been suggested that plant NSR viruses may have originated from arthropod viruses that evolved to also infect plants . TherefoSince most NSR viruses replicate in both the plant host and the arthropod vector, the true virus host origins cannot be unambiguously discerned from the HTS data. Some of the newly discovered NSR viruses are highly divergent which makes it even more difficult to unequivocally determine if they are plant or plant-associated viruses, and it will require a significant amount of research to confirm their status, which is rarely carried out. This is exemplified by the recently discovered coguviruses and rubodviruses, where the host assignment based on their phylogenetic relationships is only preliminary. Thus, further biological studies will be required to determine if they are plant, plant-associated, or insect viruses .Negarnaviricota, which includes members characterized by (i) negative-sense or ambisense single-stranded unsegmented or segmented genomes, (ii) the presence or absence of a lipid membrane enveloping the capsid, and (iii) a diverse host range (Orthomyxoviridae), Zaire ebolavirus (Filoviridae) or Crimean-Congo hemorrhagic fever virus (Nairovirus). Most of the genera contain NSR viruses that infect vertebrates and only about 10% contain phytoviruses , Muvirales, Articulavirales, among others, have been created to accommodate diverse viruses which have been placed in the major phylogenetic gap between the two large groups of NSR viruses  belonging to five different families  include species of phytoviruses (Coguvirus and Rubodvirus) were recently created to accommodate novel species of NSR viruses related to members of the Phenuiviridae family , and Dichorhavirus and Varicosavirus (bi-segmented genomes) (The family genomes)   . The virgenomes)   .The increased application of HTS has seen a significant rise in the number of novel plant rhabdoviruses , as wellHigh-throughput sequencing was used successfully to complete the genome sequences of previously reported plant rhabdoviruses where only partial sequence fragments were available, such as Iranian citrus ringspot-associated virus , ivy veiLamprocephalus sp. and kalanchoe were analyzed by HTS  . FurtherFinally, partial fragments of RNAs1, 2 and 4 of a putative novel emaravirus infecting alfalfa in Australia, named alfalfa ringspot-associated virus were recently identified using HTS . PCR ampOphioviridae, contains only one genus, Ophiovirus, which is exclusively composed of members that have plants as their hosts  and their biological properties were known . DiffereAt the time of this review, only partial genome sequence of four other reported orthotospoviruses are available: groundnut yellow spot virus (S RNA), lisianthus necrotic ringspot virus (S RNA), pepper necrotic spot virus (S RNA) and tomato necrotic ringspot virus (S and M RNAs) ; HTS couPhenuiviridae includes 19 ICTV-recognized genera (Tenuivirus and likely two recently accepted new genera Coguvirus and Rubodvirus (2019.026M.A.v1.Phenuiviridae_4gen79sp.xlsx) contain species of plant-infecting viruses (Most phenuiviruses have enveloped particles with helical morphology, except tenuiviruses that have non-enveloped filamentous particles, and their genomes are comprised of two to four single-stranded, linear RNA segments. As of 2019, the family d genera . Only th viruses . Phyloge viruses .This genus is composed of two previously unknown viruses that have bi-segmented genomes, which have been discovered using HTS of citrus , while tMoreover, partial segments of a cogu-like virus were discovered when the virome of ornamental flowers was analyzed by HTS . HoweverThis genus is composed of species for two previously unknown viruses that have tri-segmented genomes which have been discovered using HTS in apple , while tTenuiviruses have a segmented genome of four to eight single-stranded RNA segments with negative-sense or ambisense polarity . The appThe number of NSR plant viruses identified so far represents a negligible fraction of the potential number of NSR viruses present in the virosphere. The discovery and in-depth molecular characterization of these viruses has been challenging given their extensive divergence, their outstanding diversity in terms of genomic architecture, and mostly, the negligible share of plant species studied for virus discovery. However, the widespread implementation of HTS, a cost-effective and efficient technology platform, allowed the discovery and molecular characterization of the genome sequences of at least 70 NSR plant viruses in the last few years. Thus, we predict that the increasing use of HTS, not only for plant samples but also in arthropod vectors, will allow the identification of many novel NSR phytoviruses which will be crucial to unravel the evolutionary landscapes of many NSR virus clades that are poorly characterized today. Nevertheless, as many more novel NSR viruses are being discovered, careful analysis will be essential to confirm their correct ecological context, to determine whether the new viral agents are plant-infecting viruses, fungi- or arthropod-infecting viruses whose RNA was co-purified along with the plant RNA, or just contamination-inadvertently sampled environmental viruses.Riboviria. In conclusion, HTS-based methods for virus discovery, our ‘new eyes,’ are unraveling in real time the richness and magnitude of the plant RNA virosphere.As soon as the virome characterization of multiple samples of cultivated and wild plants becomes more routine, more viruses with unique features and novel phylogenetic relationships will likely be discovered. As we assess the viral ‘dark matter,’ we will start to grasp the evolutionary history of plant viruses, eventually leading us to untangle the diversity of the NSR virosphere and gain increased knowledge about its complex evolutionary and phylogenetic relationships. A rich picture of the plant virome landscape will also provide an essential framework for improved taxonomic classification of plant NSR viruses as part of the realm All authors wrote the manuscript and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


High-grade gliomas (HGGs), including glioblastoma and diffuse intrinsic pontine glioma, are amongst the most fatal brain tumors. These tumors are associated with a dismal prognosis with a median survival of less than 15 months. Radiotherapy has been the mainstay of treatment of HGGs for decades; however, pronounced radioresistance is the major obstacle towards the successful radiotherapy treatment. Herein, tumor hypoxia is identified as a significant contributor to the radioresistance of HGGs as oxygenation is critical for the effectiveness of radiotherapy. Hypoxia plays a fundamental role in the aggressive and resistant phenotype of all solid tumors, including HGGs, by upregulating hypoxia-inducible factors (HIFs) which stimulate vital enzymes responsible for cancer survival under hypoxic stress. Since current attempts to target tumor hypoxia focus on reducing oxygen demand of tumor cells by decreasing oxygen consumption rate (OCR), an attractive strategy to achieve this is by inhibiting mitochondrial oxidative phosphorylation, as it could decrease OCR, and increase oxygenation, and could therefore improve the radiation response in HGGs. This approach would also help in eradicating the radioresistant glioma stem cells (GSCs) as these predominantly rely on mitochondrial metabolism for survival. Here, we highlight the potential for repurposing anti-parasitic drugs to abolish tumor hypoxia and induce apoptosis of GSCs. Current literature provides compelling evidence that these drugs  could be effective against cancers by mechanisms including inhibition of mitochondrial metabolism and tumor hypoxia and inducing DNA damage. Therefore, combining these drugs with radiotherapy could potentially enhance the radiosensitivity of HGGs. The reported efficacy of these agents against glioblastomas and their ability to penetrate the blood-brain barrier provides further support towards promising results and clinical translation of these agents for HGGs treatment. High-grade gliomas (HGGs) are malignant and incurable tumors arising from glial cells, present in the central nervous system (CNS). The World Health Organization (WHO) classifies the HGGs as either grade III or grade IV tumors, based on the histopathological criteria. HGGs can originate in various parts of the CNS and affect individuals of all age groups. Despite advances in treatment modalities, the heterogeneous nature of the tumor accounts for a notoriously poor prognosis and limits the efficacy of conventional therapies . ResistaGlioblastoma is the most frequently diagnosed and aggressive tumor of all HGGs. It represents 14.6% of all primary CNS tumors and 48.3% of all primary malignant CNS tumors . It is cSurgery is the primary standard of care for glioblastoma patients . HoweverDIPG is a deadly form of HGG primarily affecting children. It represents 10–20% of all childhood brain malignancies and accounts for the majority of brain cancer-related deaths in children . DIPG orAs a result of the sensitive brainstem location and invasiveness of the tumor, DIPGs are not amenable to surgical resection . CurrentSince all HGGs, including glioblastoma and DIPG, recur following treatment, there is an increasing need to develop new treatments in developing a cure for these deadly malignancies. Radiotherapy has been the mainstay of treatment for all malignant gliomas and has been the only standard treatment for DIPG for decades , 12. HowThis review aims to outline mechanisms responsible for radioresistance in HGGs, particularly in glioblastoma and DIPG, with an emphasis on the role of hypoxia and the associated metabolic pathways implicated in tumor recurrence, metastasis, and resistance to chemo- and radiotherapy. It will address how inhibiting mitochondrial metabolism is an attractive strategy to decrease tumor hypoxia and increase oxygenation and therefore radiosensitize the tumor cells. It further discusses the need for mitochondrial targeted therapies against the resistant glioma stem cells (GSCs) which are significant contributors to tumor recurrence and relapse. Finally, it poses repurposing anti-parasitic drugs in combination with radiotherapy as an approach to abolish tumor hypoxia and eradicate both GSCs and glioma differentiated cells, and therefore offer an approach to improve radiotherapy efficacy.Cancer cells have various mechanisms to escape cell death via radiotherapy. Some of these mechanisms include, the ability of cancer cells to repair the radiation-induced DNA damage; cell-cycle arrest; alterations in the expression of oncogenes and tumor suppressor genes; autophagy induction; variations in tumor microenvironment such as hypoxia, elevation in cytokine levels, and epithelial to mesenchymal transitions; generation of cancer stem cells (CSCs); and alterations in tumor metabolism . Of thesThe link between hypoxia and radiation resistance was first established in the 1950s by Gray and his colleagues, who demonstrated that hypoxia caused radiation resistance in various plant, microbes and malignant mammalian tissues . IonizinHypoxia is associated with malignant progression, therapy resistance, and poor prognosis of glioblastomas , 28. Pre18F-fluoromisonidazole (FMISO) being the current gold standard for hypoxia imaging [A wide range of imaging approaches have been developed to understand the dynamics between oxygenation and hypoxia in gliomas, with positron emission tomography (PET) using  imaging . Using F imaging . This id imaging .Similarly, studies have also provided evidence of the possible link between hypoxia and pediatric HGGs and DIPG , 38. RecDuring hypoxic conditions, cancer cells upregulate crucial metabolic enzymes that help them adapt to the demand for nutrients and changes in their redox status. Adaptation to the hypoxic environment is partly mediated by the activation and stabilization of transcription factors termed as a hypoxia-inducible factors (HIFs) via inactivation of HIF propyl hydroxylases (PHD) . HIFs coThe metabolic requirements of normal healthy cells are considerably different from rapidly proliferating cancer cells. As opposed to normal cells that primarily depend upon the mitochondrial oxidative phosphorylation (OXPHOS) for energy production, most cancer cells preferentially use the aerobic glycolysis, converting glucose into lactic acid, even with enough available oxygen. Known as the Warburg effect, this results in abnormal proliferation of cancer cells and facilitates malignant progression . The hypDuring aerobic glycolysis, the glucose converted into pyruvate is reduced to lactate within the cytoplasm, instead of undergoing OXPHOS in the mitochondria . This reThe production of large quantities of lactate as a by-product of glycolysis plays a pivotal role in regulating cancer phenotypes such as cancer cell migration, angiogenesis, metastasis, and immunosuppression . Studiesin vivo progression [An enhanced rate of aerobic glycolysis suggests that cancer cells should have a decreased rate of OXPHOS; however, current research increasingly suggests that along with glycolysis, some cancers also rely on mitochondrial biogenesis and OXPHOS for energy production and gression , 53. Thegression . Anothergression , and thagression . This me2, and succinate, activate the ETC complexes, and the transport of electrons across the ETC complexes leads to the pumping of H + ions from the mitochondrial matrix to the intermembrane space [The OXPHOS metabolic pathway involves the mitochondrial uptake of pyruvate being directed to the TCA cycle. This pathway regulates ATP production through the electron transport chain (ETC) complexes I-IV, present in the inner mitochondrial membrane . The elene space . Here, oCSCs, a subpopulation of cancer cells, contribute to tumor heterogeneity and play a vital role in tumor recurrence and metastatic dissemination . These fImportantly, subtypes of cancer cells within the heterogeneous tumor population could harbour CSC-like phenotypes due to metabolic symbiosis across neighboring cancer cells, promoting tumor development under unfavorable conditions . The conThe tumor microenvironment plays a significant role in maintaining the stemness of CSCs. CSCs reside in niches with adjacent cells which provide growth factors, cytokines, and other extracellular matrix components, essential for tumor development and conserving the metabolic phenotypes of CSCs that underlie therapeutic resistance and distant metastasis . A hypoxSince tumor hypoxia is identified as a barrier to successful radiotherapy treatment, targeting hypoxia is an attractive approach to overcome radioresistance and enhance the effectiveness of radiotherapy . In the A decrease in OCR could be achievable by inhibiting the mitochondrial OXPHOS . Mitocho+) [Reduced oxygen tension also increases the production of ROS, therefore increasing cellular redox stress. Cancer cells counter this state by HIF1-mediated modulation of the subunits of ETC complex IV, also known as the cytochrome c oxidase (COX) present in the mitochondria . Under n+) . This is+) . Low levHIFs can also interact with proto-oncogenes such as c-MYC, altering tumor metabolism and driving malignant proliferation . The intBesides hypoxia, HIF-1α is also regulated by the phosphatidylinositol – 4,5 – bisphosphate 3 – Kinase (PI3K)/protein kinase B (Akt)/mTOR pathway . The PI3Repurposing drugs for cancer treatment is a cost-effective approach as the knowledge of dosage, safety, and side effects profile is already available. An understanding of the radioresistance mechanisms of HGGs has revealed that both tumor hypoxia and GSCs are significant contributors to tumor recurrence and resistance to chemo- and radiotherapy. Since GSCs are highly dependent on mitochondrial biogenesis , using dVarious anti-mitochondrial drugs that inhibit OXPHOS either directly or indirectly have been explored as agents for anti-cancer therapy. These include the mitochondrial complex I inhibitors – biguanides (metformin and phenformin), BAY 87-2243, IACS-010759, Fenofibrate, and VLX600; complex II inhibitors – Lonidamine, VLX600, and Alpha-tocopheryl succinate; complex III inhibitors – Atovaquone; complex IV inhibitors – Arsenic trioxide and VLX600; mitochondrial protein synthesis inhibitors – Tigecycline and Doxycycline; and mitochondrial transfer inhibitors – NOX2 inhibitors and CD38 inhibitors . StudiesRepurposing anti-parasitic drugs that have previously displayed promising results against various cancer types, including glioblastoma, may also yield substantial results against DIPG. Anti-parasitic drugs, such as atovaquone, ivermectin, mefloquine, proguanil, and quinacrine could inhibit tumor growth by targeting various cancer pathways. These drugs have the potential to alleviate tumor hypoxia by decreasing OCR, induce mitochondrial dysfunction and oxidative stress by inhibiting the mitochondrial ETC, and upregulate mechanisms of DNA damage; all of these would lead to an increase in cancer cell death. An increase in apoptosis of GSCs and reoxygenation of radioresistant HGGs by these drugs could increase the radiosensitivity of HGG tumor tissue and result in improved radiation response , which is actively expressed in many solid and hematological malignancies . Upregul− channels in the nerve and muscle cells of the parasites [− ions, causing hyperpolarization of parasitic cell membranes, and leads to muscle paralysis and cell death [Ivermectin is FDA-approved for the management of onchocerciasis, intestinal strongyloidiasis, pediculosis capitis, and inflammatory lesion – rosacea . It beloarasites . This rell death . The safll death . Ivermecll death . Howeverll death , 134. Thin vitro and in vivo subcutaneous glioblastoma models, where it inhibited proliferation, induced caspase-dependent apoptosis, and potentiated angiogenic inhibition [Interestingly, ivermectin has shown promising anti-cancer efficacy in various cancers by increasing mitochondrial dysfunction, inducing oxidative stress, and energy crisis . By selehibition . These rhibition .in vivo xenografts [Furthermore, ivermectin was also found to induce oxidative stress in glioblastoma cells by inhibiting the Akt/mTOR pathway . The mTOnografts .Studies have also demonstrated that ivermectin induces cytostatic autophagy in breast cancer, and inhibits proliferation of ovarian cancer and oesophageal squamous cell carcinoma, by inhibition of PAK1 protein –100. MorPlasmodium falciparum and is an effective blood schizonticide for P. vivax [P. falciparum; however, the exact mechanism of action remains unknown. Recent investigation has found that mefloquine inhibits protein synthesis by interacting with the GTPase-associated center of the 80S ribosomal subunit in P. falciparum and therefore kills the malarial parasite [Mefloquine, a quinolinemethanol, is FDA-approved for the treatment of malarial infection caused by chloroquine-resistant P. vivax . It has parasite .Mefloquine has demonstrated anti-cancer efficacy across various cancer cell lines and has been repurposed in the therapeutic targeting of several cancer signaling pathways. Yan et al. 2013 observed that mefloquine reduced the cell viability of prostate cancer cells by altering MMP and producing excess cytotoxic ROS, which decreased the phosphorylation of Akt and triggered JNK, ERK and AMPK signaling pathways . InteresMefloquine also stimulated apoptotic cell death in breast cancer by inhibiting autophagy and decreased the proliferative and self-renewal capacity of liver CSCs by targeting the β-catenin pathway , 109. ItFurthermore, mefloquine was also found effective in inducing cell death in AML, chronic lymphocytic leukemia (CLL), and glioblastoma through disruption of lysosomes –114. LysP. vivax malaria; it was initially used alone and later administered in combination with chloroquine or atovaquone and the combination was found to be highly effective [P. falciparum malaria; both of these drugs target the parasite P. falciparum’s pre-erythrocytic and erythrocytic stages and induces causal and suppressive prophylaxis [The biguanide proguanil was first developed as an anti-malarial drug for the management of acute ffective . It is mffective . Anotherphylaxis .Proguanil enhances the activity of atovaquone in collapsing MMP and causing mitochondrial dysfunction . DespiteQuinacrine, an acridine derivative, was initially used for the prophylaxis and management of the malarial infection . It is cThe anti-cancer potential of quinacrine has been extensively explored, revealing various therapeutic targets. Due to its low toxicity profile, minimal side effects, and interference with many cancer signaling pathways, quinacrine offers compelling evidence as a promising antineoplastic agent . The cytA study by Preet et al. 2012 demonstrated that quinacrine targets breast cancer cells and induces cell death, cell cycle arrest, and decreases cell migration through inhibition of topoisomerases and increased DNA damage . It stimin vitro and in vivo animal models [Quinacrine has also been found to act synergistically with chemotherapy drugs such as carmustine, oxaliplatin, and carboplatin, or inhibitors including cisplatin, 5-fluorouracil, paclitaxel, and sorafenib, across various cancer types , 151–155l models . Due to Quinacrine was later replaced by chloroquine for the treatment of malaria, however, it continued to be used in the treatment of diseases. Chloroquine, as an anti-malarial agent, has also shown promising anti-cancer potential. It is found to disrupt autophagy by blocking the formation of autolysosomes and activating the GRP78/BiP chaperone which enhances endoplasmic reticulum (ER) stress , 158. ImGlioblastoma and DIPG are deadly and malignant forms of HGGs, contributing to the medical health burden. Despite decades of research and investigating novel agents in clinical trials, both tumors result in high mortality rates and no cure has been established. Patients undergoing radiotherapy experience high rates of recurrence due to the intrinsic radioresistance of the glioma cells. Therapies targeting radioresistance mechanisms are urgently needed to improve radiation response of HGGs as these could increase the overall survival of patients. Tumor hypoxia and alterations in the metabolic status enable cancer cells to achieve the malignant and resistant phenotype. Targeting hypoxia and increasing oxygenation around the tumor tissue is essential for the effectiveness of radiotherapy in HGGs. Inhibiting mitochondrial metabolism could help abolish tumor hypoxia by decreasing the OCR of tumor cells and could subsequently increase the oxygen availability. Therapies targeted towards mitochondria would also be beneficial against GSCs which preferentially rely on mitochondrial metabolism and are key players in tumor recurrence and resistance to therapies . TherefoThere is considerable evidence supporting the anti-neoplastic role of anti-parasitic drugs (summarized in Table in vitro cancer cell lines and patient-derived xenograft (PDX) implanted in immunocompromised mice. It is important to consider that the successful development of anticancer therapies must consider the interaction between the tumor cells and immune cells. Since the immune system poses a major challenge to the long-term success of the anti-cancer therapies, one possible avenue could be to investigate the antineoplastic role of these drugs in ‘humanized’ mouse models that closely mimic the patient tumor microenvironment [A significant limitation in the current literature is that these anti-parasitic drugs have only been investigated in ironment . TherefoIn summary, the combination of anti-parasitic drugs and radiotherapy might serve as novel approach towards the management of malignant HGGs and may improve patient survival outcomes. There is need to study the effects of these compounds on interactions between cancer cells and immune system as this could provide further insight into the translation of this approach to clinical practice.


Hemorrhagic strokes account for half of all strokes seen in children, and the etiologies of these hemorrhagic strokes differ greatly from those seen in adult patients. This review gives an overview about incidence and etiologies as well as presentation of children with intracerebral hemorrhage and with differential diagnoses in the emergency department. Most importantly it describes how neuroimaging of children with intracerebral hemorrhage should be tailored to specific situations and clinical contexts and recommends specific imaging protocols for acute and repeat imaging. In this context it is important to keep in mind the high prevalence of underlying vascular lesions and adapt the imaging protocol accordingly, meaning that vascular imaging plays a key role regardless of modality. Magnetic resonance imaging (MRI), including advanced sequences, should be favored whenever possible at the acute phase. While accounting for a minority of adult strokes, hemorrhagic strokes account for half of all strokes seen in children, and occur in approximately 1–2 per 100,000 children per year . Trauma In this review, we therefore give an overview about the clinical presentation of pediatric patients with intracerebral hemorrhage and with differential diagnoses in the emergency department. We also describe how neuroimaging of children with intracerebral hemorrhage should be tailored to specific situations and clinical contexts and recommend specific imaging protocols for acute and repeat imaging.Presenting symptoms of ICH in children can be nonspecific, particularly without preceding head trauma to raise initial suspicion. Headache is the most common feature of ICH in non-neonates, occurring in up to 80% of children ,10. WhilThe choice of neuroimaging modality should always be adjusted to clinical presentation and suspected cause. In children with acute focal neurological deficits, most pediatric stroke guidelines recommend brain magnetic resonance imaging (MRI) as first imaging modality especially in suspected ischemic stroke, where computed tomography (CT) may be less sensitive for detection of early ischemic changes ,18. Yet,After the positive diagnosis, careful evaluation of risk factors for secondary neurological deterioration is mandated. Pediatric counterparts to the adult ICH scoring system  have beeIntracranial vascular imaging (CT or MR angiography) should then systematically be acquired as part of the initial diagnostic examination. Indeed, there is a high prevalence of readily identifiable vascular lesions such as arteriovenous malformations (AVM), arteriovenous fistulas (AVF), or aneurysms in children with ICH.  If identDigital subtraction angiography (DSA) has a central role for further characterization of vascular anomalies, and to investigate ICHs with undetermined etiology, due to its higher spatial and temporal resolutions and consequent higher sensitivity for intracranial shunts detection.  The optiIn the absence of intracranial arteriovenous shunt, the second most prevalent etiology are cavernomas, more conspicuously detected using MRI. Similarly, hemorrhagic brain tumors should be searched for using contrast enhanced MRI . To summarize, the optimal acute imaging sequence for a child with ICH would be CT or MR with vascular imaging (CTA-MRA), followed with gadolinium enhanced MRI if the initial modality was CT, finally completed DSA in the absence of identifiable cause.The specificity of ICH etiological spectrum in children makes advanced MR imaging techniques particularly useful for the routine evaluation of pediatric ICH etiology.As arteriovenous shunts are the most prominent cause for hemorrhage in children, perfusion imaging such as arterial spin labelling, and dynamic vascular imaging such as time-resolved multiphase MR-angiography can give immediate insight on the underlying etiology and help tailor therapeutic management , to detect intraventricular blood.Diffusion weighted imaging, to identify areas of restricted diffusion that can point towards a hemorrhagic transformation of an acute ischemic stroke or towards a venous infarction in the context of cerebral venous thrombosis.FLAIR or T2 (in children younger than 2 years old), to identify pre-existing brain lesions, including brain tumors, and quantify edema, to evaluate ventricles volume and hydrocephalus.Pre contrast T1.Whole brain time-of-flight angiography, to identify macro-vascular lesions.Arterial spin labeling (ASL) perfusion, to identify arteriovenous shunts.Dynamic MR-angiography, to identify and characterize high flow arteriovenous shunts.Post-contrast T1, to identify enhancing tumors, delineate an arteriovenous malformation or other vascular malformations.In most vascular pathologies  except cavernous malformation (CM), DSA will be key for further lesion characterization but a CM may also be accompanied by a second vascular lesion, such as an AVM, in which case a DSA may be useful. The major differential after diagnosis of ICH in children is the presence of an AVM as it is the most common vascular lesion in this age group, with a hemorrhage risk of up to 6% per year and a mortality rate of up to 25% per hemorrhage ,25,26. TCavernous malformations (CM) is amongst most frequent causes for non-traumatic pediatric ICH. It typically appears on T2 * Gradient Echo weighted sequences as a multilobulated well-defined lesion with blooming hypointensity ,35. The CTA/MRA are often used as first screening for aneurysms ,38; howeWhen the initial imaging work-up is inconclusive, and in the absence of systemic concurrent causes, repeated brain imaging should be considered as all structural underlying etiologies may be masked by the acute mass effect caused by the hemorrhage. Vascular lesions can be masked or partially destroyed by the initial hemorrhage and the risk of rebleeding is hypothesized to be higher when the clot retracts . Some indirect arguments may point to the presence of an underlying parenchymal lesion. This most notably includes the early detection of peri-hematoma brain edema. Indeed, edema typically develops after 48–72 h, hence important edema at baseline suggest the presence of a pre-existing tumoral lesion, which should prompt early re-imaging so as not to delay etiological care.The timing of repeat imaging should be tailored to specific clinical context and should include repeat DSA. Common temporal landmarks include after mass effect contraction (within three weeks), and after complete hemorrhage resorption (within three to six months) if intermediate control remained inconclusive.Neuroimaging of children with intracerebral hemorrhage should be tailored to specific situations and clinical contexts. Yet, one must keep in mind the high prevalence of underlying vascular lesions and adapt imaging protocol accordingly. Vascular imaging plays a key role regardless of modality. MRI, including advanced sequences, should be favored whenever possible at the acute phase.


Understanding how an adult brain reaches an appropriate size and cell composition from a pool of progenitors that proliferates and differentiates is a key question in Developmental Neurobiology. Not only the control of final size but also, the proper arrangement of cells of different embryonic origins is fundamental in this process. Each neural progenitor has to produce a precise number of sibling cells that establish clones, and all these clones will come together to form the functional adult nervous system. Lineage cell tracing is a complex and challenging process that aims to reconstruct the offspring that arise from a single progenitor cell. This tracing can be achieved through strategies based on genetically modified organisms, using either genetic tracers, transfected viral vectors or DNA constructs, and even single-cell sequencing. Combining different reporter proteins and the use of transgenic mice revolutionized clonal analysis more than a decade ago and now, the availability of novel genome editing tools and single-cell sequencing techniques has vastly improved the capacity of lineage tracing to decipher progenitor potential. This review brings together the strategies used to study cell lineages in the brain and the role they have played in our understanding of the functional clonal relationships among neural cells. In addition, future perspectives regarding the study of cell heterogeneity and the ontogeny of different cell lineages will also be addressed. One fundamental issue in Neuroscience is how the different lineages in the brain are established and what contributions sibling cells make to the nervous system and how they influence its behavior. The current belief is that there is large cell heterogeneity in the adult brain, raising the question as to how these different cell types are generated during development. However, a fundamental question is whether this heterogeneity is ontogenically determined and if so, what are the physiological implications of this? Thus, lineage tracing has developed from the need to pursue all the progeny of specific neural progenitor cells (NPCs) to determine how complete neural networks are built and the contribution of specific progenitors to these networks.Neural stem cells (NSCs) are cells that self-renew and that can produce all the lineages present in the adult brain . Thus, tAt early embryonic stages, the principal neural lineages are specified in the neural plate, a defined region of the ectoderm. In parallel, cells of other origins colonize the prospective brain, such as microglia (mesoderm) and blood vessel cells (endoderm). Initially, NSCs known as neuroepithelial cells (NECs) undergo symmetric cell divisions to amplify their pool and prior to generating bipolar radial glial cells (RGCs) . These RNew approaches in single-cell transcriptome technology have provided novel data regarding cell heterogeneity, addressing the varied gene expression in different brain populations. Specifically, a compilation of molecular markers has been described in the neurogenic niche, expressed in either active or quiescent NPCs under physiological conditions and after brain insult –19. In tTogether, defining NPC populations using specific markers has some limitations, as it is often impossible to precisely define these complex and heterogeneous cell populations solely in this manner. Accordingly, clonal analysis and lineage tracing has become crucial to further understand the biology of NSCs and their lineage progression , 28.Due to the large heterogeneity within the neural progenitor pool, clonal analysis is particularly important when studying stem cell biology. This concept arose at the end of the nineteenth century with the studies of Whitman and collaborators on leech embryos , and it C. elegans [Some of the first complete lineage tracing studies were achieved by direct microscope observation of lineage progression using either organisms with a small number of cells, or by isolating NPCs and tracking their divisions in vitro . A groun elegans  by time- elegans . The inc elegans . In addi elegans , 35, off elegans , 37 Howe elegans .E. coli LacZ gene [The discovery of the green fluorescent protein (GFP)  and of βacZ gene  revolutiacZ gene  or electacZ gene , and viracZ gene . RetroviacZ gene –46. The acZ gene . These bacZ gene .Tracking cells from their progenitors to their final destination and fate has been made possible through the development of some important genetic tools that have made permanent cell labeling feasible. In particular, the Cre-LoxP  and Flp-In addition, early lineage tracing studies used chimeric mice generated from tetraparental embryos enabled the origin of many structures in the body to be determined . MoreoveBrainbow technology [Drosophila melanogaster  [Drosophila (nBitbow) [D. melanogaster (twin-spot) [Drosophila (CLADES) [The employment of GMOs and the incremental growth of reporters led to the idea of combining different fluorescent proteins to track sibling cells Fig. . Initialchnology . Brainbochnology , 61 and Raeppli) –64 and ZRaeppli) , 66. ThenBitbow) . Other cnBitbow) , 69, andin-spot) . The priin-spot) , an impoin-spot) , 72. Newin-spot) –76. To r(CLADES) . These m(CLADES) , expandiStarTrack, which allows the progeny of single cells to be traced and analyzed thereafter. The StarTrack methodology was an attempt to develop a genetic in vivo lineage-tracing method that could track all the neural progeny of individual GFAP cells [Our laboratory has developed a stochastic clonal analysis method called AP cells . It is bAP cells . RecentlAP cells , 81. NovAP cells , helpingIn summary, multicolor image-based lineage tracing methods are among the most reliable methods to define lineage trees. By combining them with state-of-the-art approaches like cell type-specific optogenetic manipulations, single-cell transcriptomic analysis, cell ablation, live-cell imaging, patch-clamp recordings, two-photon microscopy, in vivo and brain slice preparations, they may help us to better understand how lineages are derived in the brain. In addition, they provide a more functional readout of the specific characteristics of clonally related cells derived from the same NPC, adding information regarding their spatial relationships.Next-generation sequencing (NGS) provides us with a powerful tool to sequence the whole genome and it opens a window to complete the study of whole organisms, elucidating their genomic, transcriptomic, epigenomic and proteomic profiles. NGS contributes to lineage tracing through both whole genome sequencing and whole-exome sequencing. It has revealed somatic mutations that accumulate in cells after replication that can be traced to define lineage progression . These a. These approaches permit the single-cell clonal dynamics and the transcriptomic profile of cells to be studied (Fig. Several other strategies have been introduced in recent years, including the implementation of CRISPR technology to drive genome editing and DNA targeting Fig. . The CRIied Fig. c. ImportSeveral methods have been proposed to examine lineage progression in different biological models in vivo or in vitro. However, their use to determine the relationships of lineages usually requires an arduous analysis. Some approaches for lineage tracing in the brain focus on the sparse labeling of cell clones to ensure the lineage connection among cells that are situated intimately in the brain Fig. a. The inOther clonal methods rely on the recombination of few fluorophores for lineage tracing Fig. b, such aMore recently, the use of fingerprints to accurately target a single NPC and that can be inherited by their entire progeny has revolutionized the field Fig. c. With tSingle-cell analyses has provided huge advantages when studying cell heterogeneity and cell dynamics in NSC populations. Transcriptomic approaches have recently shed new light on lineage progression and progenitor potential, and single-cell transcriptomic analysis has revealed important information about cell identity and the distinct expression patterns under different conditions that could be crucial to fine-tune lineage tracing approaches. Transcriptomic analyses have helped classify cells in terms of their specific transcriptomic expression. Several studies have shown the diversification of transcriptional profiles within neuronal , 107, asIn addition, deep genome sequencing has made it possible to trace the lineage of every single cell in a given organism Fig. d. DNA reLineage tracing techniques can also be applied in pathological situations to address the heterogeneity in terms of therapeutic responses or the different implications according to ontogeny. In this regard, clonal responses have been described in the progression of squamous carcinomas , sarcomaIn conclusion, DNA barcoding strategies and sequencing resources could help to consolidate cell lineage reconstructions and contribute to our understanding of cell dynamics, clonal expansion, and the behavior of specific cell lineages in normal conditions and disease. Moreover, this type of analysis may produce important advances in understanding the molecular events underlying lineage specification and the sculpting of progenitor potential.