Arabidopsis, trichoblast and atrichoblast cell fate results from intra- and intercellular position-dependent signaling and from complex feedback loops that ultimately regulate GL2 expressing and non-expressing cells. When epidermal cells reach the end of the root expansion zone, root hair promoting transcription factors dictate the establishment of polarity within epidermal cells followed by the selection of the root hair initiation site at the more basal part of the trichoblast. Molecular players in the abovementioned processes as well as the role of phytohormones are discussed, and open areas for future experiments are identified.Plant roots fulfill important functions as they serve in water and nutrient uptake, provide anchorage of the plant body in the soil and in some species form the site of symbiotic interactions with soil-living biota. Root hairs, tubular-shaped outgrowths of specific epidermal cells, significantly increase the root’s surface area and aid in these processes. In this review we focus on the molecular mechanisms that determine the hair and non-hair cell fate of epidermal cells and that define the site on the epidermal cell where the root hair will be initiated (=planar polarity determination). In the model plant  Figure 1). In the meristematic zone cells undergo mitotic divisions. As such, this zone mainly determines the root’s cell number. Upon leaving the meristem, cells modulate their physiological state and architecture to prepare for rapid elongation: they progressively develop a central vacuole, polarize their cytoskeleton and remodel their cell walls. Parallel deposition of cellulose microfibrils, transverse to the future long axis of the cell creates anisotropic cell wall mechanics that accommodate longitudinal cell expansion  how epidermal cells know whether or not to initiate a root hair, (2) how the position of the root hair bulge is defined, and (3) which mechanisms are required to form the actual bulge. We will further identify future challenges to even better understand the mechanism and control of root hair development.All cells that arise from the meristem go through the elongation zone before entering the differentiation zone. How do epidermal cells that all pass through the same developmental zones, perform similar functions, and initially look morphologically similar  gain a different cell fate?Arabidopsis the root epidermis is arranged in clearly distinguishable cell files arising from 16 initial cells. Upon maturation the epidermis consists of two distinct cell types: root hair cells (trichoblasts) and non-hair cells (atrichoblasts) .In oblasts) . The ideThe patterning of both epidermal cell types is first established during the early stages of embryogenesis, which is far before the first signs of epidermal differentiation. The expression of GLABRA2, a transcription factor required for specification of the atrichoblast cell fate, can already be detected in the heart stage embryo . In addiFigure 2B). Many studies suggest that the root hair cell fate depends on the absence of an inhibitory pathway that prevents differentiation into a hair cell  and CAPRICE (CPC) that act in opposition to each other . It is pAdditionally, in atrichoblast cells, the WER/GL3/EGL3/TTG1 complex positively regulates CPC and two other root hair fate-promoting transcription factors, TRIPTYCHON (TRY) and ENHANCER OF TRY AND CPC1 (ETC1) . The latThe current understanding is that, upon activation by the WER/GL3/EGL3/TTG1 complex, CPC, TRY, and ETC1 migrate to the adjacent root hair cells and competitively inhibit binding of WER to GL3/EGL3 , which rThis description demonstrates that the network of root epidermal cell patterning is highly complex and employs, besides regulatory mechanisms such as lateral inhibition, several feedback loops and interactions . RHD6 directly targets RSL4, which is now thought to form the first auxin-responsive component in the root hair signaling pathway. RSL4 protein starts accumulating 2 h before root hair initiation and reaches a maximum during the early stages of root hair development, after which it is slowly degraded by the 26S proteasome during tip growth , influence root epidermal cell fate determination. Pharmacological studies have shown that treatment with aminoethoxyvinylglycine (AVG), an ethylene biosynthesis inhibitor, or Agormation . Alternaormation . In addiormation  by bindiormation , causes ormation . In contormation  even thoormation . On the pathways . RHD6 itp growth . Not onlhairless . Based oaxr2 and axr3) do not form root hairs  treated plants had reduced WER and GL2 transcription, showed aberrant patterning of WER and GL2 expression in the epidermis, and that they contained an increase of non-hair cells in the H-position. The ectopic expression of WER and GL2 in bri1 roots was shown to be due to decreased expression of CPC  showed that BIN2 also directly binds to WER, EGL3, and TTG1 and phosphorylates the latter two. TTG1 phosphorylation inhibits WER/GL3/EGL3/TTG1 complex activity and subsequently GL2 transcription, whereas phosphorylation of EGL3 (which is transcribed in H-cells only) was shown to be responsible for both intra- and intercellular (H- to N-cells) EGL3 transport together with GL3 ?The decision to initiate a root hair is made by the abovementioned mechanism(s) and promotes local cell wall outgrowths. But, how does a cell wall that has just adapted the architecture to prevent expansion at the end of the elongation zone, accommodate polar expansion  is tightly linked to the early events of root hair formation and occurs before the cell wall bulges out  mutant that contains abnormally expanding trichoblast cells  that control cycling between GTP-bound (on) and GDP-bound (off) states of ROPs and the early localization of ROP at the plasmamembrane of the future initiation site  and regulation of TGN components VHA-a1 and SYP61 . The hemispherical apex is filled with densely packed vesicles while small organelles such as Golgi stacks, mitochondria, endoplasmic reticulum and plastids are present in a sub-apical region. The more basal part of a hair contains a large vacuole that occupies most of the space. The nucleus enters the hair and follows the growing tip at a constant distance. When a root hair becomes mature and growth ceases, this highly polar organization of the cytoplasm disappears (When the bulge is fully formed, the transition from initiation to tip growth begins with the accumulation of secretory vesicles at the apical part of the bulge. A tip growing root hair has a highly organized cytoarchitecture (sappears . SeveralArabidopsis thaliana root is being successfully exploited as a model system to elucidate complex molecular pathways leading to cell fate determination, planar cell polarity, and tip growth. Many players in these pathways have been described. However, undoubtedly several are yet to be identified. The influence and action of phytohormones such as auxin, ethylene and brassinosteroids on root architecture and development is generally recognized, yet their roles in the different developmental processes are far from fully understood. Several open questions thus still remain, and answers are needed to understand the intriguing complexity of development on the single cell level.Abundant genetic, molecular, and mutant resources have made that the DB, SS, and KV studied the literature, critically reflected on the current knowledge and wrote the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS) from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH). More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα), were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C) recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKA's role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway.Cyclic-AMP (cAMP)-dependent protein kinase (PKA) is the main effector of cAMP signaling in all tissues. Inactivating mutations of the  CS leads to central obesity and metabolic abnormalities and several other manifestations including moon face, buffalo hump, striae, and opportunistic infections , adrenocortical adenomas (ACA) and cancer (ACC). BAHs account for 10–15% of AICS and are classified in two subtypes: macronodular (nodules >1 cm) and micronodular (nodules <1 cm) Lacroix, . MacronoMicronodular hyperplasias include a pigmented form named primary pigmented nodular adrenocortical disease (PPNAD), which is typically diagnosed at a younger age. PPNAD is the most common endocrine lesion of Carney complex (CNC), occurring in more than 60% of CNC patients  to cyclic adenosine monophosphate (cAMP). cAMP activates its main intracellular mediators, EPAC (Exchange Protein Activated by cAMP), and a serine/threonine kinase called cAMP-dependent protein kinase A (PKA)  , as well as in ACAs  PRKACA encodes the most highly expressed catalytic PKA isoform in the human adrenal and the functional consequences of two mutant variants have been predicted using different modeling approaches based on mouse PKA crystal structure    is an endogenous thermostable peptide that interacts with the catalytic domain and is able to inhibit Leu206Arg variant in vitro  and the even higher presence of PKA function alterations (Bimpaki et al., PRKACA is ubiquitously expressed, its mutations may also be found in other tissues with PKA-dependent tumorigenesis. Future analysis and use of animal models will provide useful information to help answer this, and other, questions.The discovery of somatic mutations in 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.


Melatonin is an indoleamine produced by the pineal gland and secreted in a circadian manner. In the past few decades, research over this topic has been enhanced. Melatonin has many important roles in the human physiology: regulator of the circadian rhythms, sleep inducer, antioxidant, anticarcinogenic. This paper reviews the involvement of melatonin in embryo fetal development. The pineal gland develops completely postpartum, so both the embryo and the fetus are dependent on the maternal melatonin provided transplacentally. Melatonin appears to be involved in the normal outcome of pregnancy beginning with the oocyte quality and finishing with the parturition. Its pregnancy night-time concentrations increase after 24 weeks of gestation, with significantly high levels after 32 weeks. Melatonin receptors are widespread in the embryo and fetus since early stages. There is solid evidence that melatonin is neuroprotective and has a positive effect on the outcome of the compromised pregnancies. In addition, chronodisruption leads to a reproductive dysfunction. Thus, the influence of melatonin on the developing human fetus may not be limited to the entertaining of circadian rhythmicity, but further studies are needed. This system has an endogenous rhythmicity of approximately 24 hours. Peripheral systems are synchronized by SCN. Melatonin represents the key linkage-molecule between the SCN and the peripheral biological clocks and it is produced and secreted in a circadian fashion, its secretion being stimulated by darkness and inhibited by light [3]. The purpose of the current paper is to review the roles of melatonin in embryonic and fetal development up to date. The role of melatonin in pregnancy and embryo fetal development has hardly been discussed, but there is clear evidence of a strong connection between fetal normal development and melatonin.In humans, the hormone secretion increases soon after the onset of darkness, peaks in the middle of the night, between 2 and 4 a.m., and progressively falls during the second half of the night [4] and has been involved in placental function in animals and human [5]. Second, in humans, SCN expresses melatonin receptors, both in adult and fetus. Maternal melatonin enters the fetal circulation transplacentally providing photoperiodic information to the fetus and by that influencing the internal rhythms of the offspring.First, the embryo and fetus are dependent on maternal melatonin, as the pineal gland becomes mature after birth. Melatonin crosses all physiological barriers without being modified, including the placental one [6].Melatonin concentrations increase in maternal blood during pregnancy, reaching a maximum at term. The presence of melatonin has also been demonstrated in amniotic fluid [7]. Melatonin treatment during human normal or abnormal pregnancy has been studied for a large range of conditions and at different times during the gestational period. Considering the ethical issues, it is more difficult to study a normally occurring pregnancy, than an in vitro fertilization (IVF) one. Melatonin administration started prior to IVF-cycles, continued during pregnancy and was associated with improved pregnancy outcomes [8]. Melatonin receptors are widespread in the human fetus from early fetal development. In addition, it appears that the fetuses’ sleep patterns develop in the late pregnancy, melatonin being the regulating factor. A normal sleep pattern is involved in the neurodevelopment and there is solid evidence that melatonin is involved in fetal neuroprotection [9]. Thus, the influence of melatonin on the developing human fetus may not be limited to entertaining the circadian rhythmicity.Chronodisruption leads to reproductive dysfunction and appears to be a key contributor to offspring diseases that develop in adult life . Melatonin decreases in conditions associated with serious outcome for the fetus and seems to be involved in preeclampsia and intrauterine growth restriction .Serum melatonin concentrations exhibit changes both in physiological and pathological pregnancies compared to non-pregnant controls. Moreover, melatonin titres are not constant during the 40 weeks of pregnancy, but they show specific dynamics  and ML2 (low affinity) receptors. There are also at least 3 subtypes of ML1 receptors: Mel1a, Mel1b, and Mel1c. To date, Mel1c receptors appear to be lacking in mammals. Mel1a and Mel1b receptors are now named MT1 and MT2 receptors, respectively. These receptors are classic G-protein-linked receptors that inhibit adenylate cyclase [13]. Another study localized specific, guanosine triphosphate (GTP) sensitive, binding sites of 2-[(125)I]iodomelatonin in the leptomeninges, cerebellum, thalamus, hypothalamus, and brainstem. In the hypothalamus, a specific binding was present in the SCN, as well as the arcuate, ventromedial and mammillary nuclei. In the brainstem, a specific binding was present in the cranial nerve nuclei including the oculomotor nuclei, the trochlear nuclei, the motor and sensory trigeminal nuclei, the facial nuclei, and the cochlear nuclei [14].Melatonin receptors have been identified in embryo and fetus, both in the nervous system and peripheral organs, especially in the endocrine system. The presence of 2-[125I]iodomelatonin binding sites in the developing of the human fetal kidney has been detected, correlated with the expression of the mRNA for the G protein-coupled melatonin receptors, Mel1a and Mel1b. Specific 2-[125I]iodomelatonin binding sites were located at the outer periphery of the developing kidney cortex in the nephrogenic region, consisting of differentiating and developing nephrons MEL binding until birth. From GD 16 until birth, the binding was no longer detectable over the thyroid gland, but persisted over the nasal epithelium [15].In Siberian hamsters, melatonin-binding sites were first apparent in the gestational day (GD) 10 over the primitive oral pharynx. From GD 12 to 14, the 16].The presence of specific melatonin binding sites was demonstrated in the middle fetal sheep cerebral artery, brown adipose tissue and adrenal gland. Melatonin concentrations in the nanomolar range or lower have direct inhibitory effects in the middle fetal sheep cerebral arteries and the fetal sheep brown adipose tissue response to noradrenalin, and also in the fetal sheep adrenal gland response to ACTH [17].Melatonin receptors were also identified in the pituitary gland and the median eminence. The pituitary concentration of [125I]melatonin binding sites was highest in the 20-day-old fetuses and then it gradually decreased in the course of postnatal development, until it reached 10% of that value in 29-day-old males. In contrast, the concentration of melatonin receptors in median eminence did not change markedly in the course of development . Melatonin has a beneficial role in the in vitro development of rodent embryos found in the 2-cell stage [19] and helped the maturation of ovine blastocysts [20].The ability of melatonin to promote embryo development in different species has been reported. When mouse embryos were cultured in a medium containing melatonin, increased blastocyst development rates were observed [21].Suppression of maternal plasma melatonin circadian rhythm by continuous light exposure during the second half of gestation showed several effects on fetal development. First, it induced intrauterine growth retardation. Second, in the fetal adrenal in vivo it markedly affected the mRNA expression level of the clock genes and clock-controlled genes, as well as it lowered the content and modified the rhythm of corticosterone. Third, an altered in vitro fetal adrenal response to ACTH of both, corticosterone production and relative expression of clock genes and steroidogenic genes was observed. All these changes were reversed when the mother received a daily dose of melatonin during the subjective night [22]. In another study on sheep, they found that melatonin had direct inhibitory effects on the noradrenalin-stimulated fetal cerebral artery contraction, the release of glycerol by brown adipose tissue, and on ACTH-induced secretion of cortisol by the fetal adrenal gland. Low levels or a lack of a circadian rhythm of the fetal corticosterone may be the cause of the intrauterine growth retardation that has been previously reported.Torres-Farfan et al. reported that the maternal melatonin decreased cortisol production in the fetal adrenal gland of the capuchin monkey [23]. Melatonin is important in normal placental development and function, a function supported by the placenta melatonin receptors expression during early pregnancy [24].The lack of maternal melatonin  during the early stages of gestation was found to disrupt drinking behavior of rat pups, an effect reversed by the administration of exogenous melatonin to the dam [Chronodisruption leads to reproductive dysfunction25]. These symptoms are not subjective, as they correlate with changes in the hormonal profile of ovarian and pituitary origin, such as a high follicular stimulating hormone (FSH) and prolonged follicular stage of the ovarian cycle [26].Women working an evening shift or a night shift, or who are transmeridian travelling, report an increased or decreased menstrual cycle length, changes in the duration and amount of menstrual bleeding and dysmenorrhea [27]. Shift work and jet lag effects on health may be explained by the secondary reduction of the total amount of sleep. FSH concentration in women who sleep less than 8 h/night lowers with 1/5 compared to women with longer sleep duration [28]. Total or partial sleep deprivation increases LH amplitude, estradiol and FSH concentrations in normal cycling women and increased estrogen is associated with a high risk of breast cancer [29].The working environment also affects the pregnancy outcomes. The shift work while pregnant is also associated with a high risk of prematurity and/ or low for gestational age babies, spontaneous abortion and subfecundity .One important cause of female infertility is poor oocyte quality. Reactive oxygen species (ROS) are normally produced within the ovarian follicle, during ovulation, and an increased production may be a cause of impaired oocyte maturation. Considering its well established role in scavenging free radicals, melatonin treatment during human pregnancy may help lower the high oxidative stress and may be a possible treatment in some forms of infertility. Melatonin has been studied in assisted reproductive technology, aiming to improve the oocyte quality and pregnancy rates following in vitro fertilization (IVF). Melatonin administration, started before IVF-cycles and continued during pregnancy, was associated with improved pregnancy outcomes. Fertilization success and pregnancy rate were improved by melatonin treatment. Fertilization rate was 50% higher in melatonin treatment cycle compared to the previous melatonin-free cycle (20.2%) [32]. No maternal and/ or embryo-fetal toxicity effects, due to melatonin treatment, have ever been reported. A median lethal dose in mice could not even be established because an increased mortality rate was not observed, even following the administration of extremely high doses of up to 800 mg/ kg melatonin [33].In 2012, Unfer et al. have reported in a personal communication that all the babies born from melatonin-treated mothers were healthy and with no congenital abnormalities. More than that, maternal melatonin treatment significantly improves placental antioxidant enzyme gene expression [Melatonin and the ovarian function34]. Studies have shown that high levels of melatonin are found in human preovulatory follicular fluid in concentrations that are much higher than those in serum [35].The role of melatonin in the production of female gametes focused on its direct actions in the ovary. Melatonin specifically concentrates in the ovary when injected systemically . Neuroprotection efficacy of melatonin in the fetal and neonatal brain was reported in many animal studies. Melatonin was given 10 minutes after the hypoxic acidemia episode in the fetal sheep and decreased the incidence of cell death and the number of activated microglial cells present in the brain [37]. The administration of melatonin at the time of induced hypoxia in the fetal sheep brain also reduced the inflammation and cell death.Melatonin has a neuroprotective effect both in adult and fetal brain. In adults, the most important poof is its positive effect in the treatment of neurodegenerative diseases like Alzheimer or Parkinson disease [38].In a recent study, melatonin treatment before and during transient severe fetal asphyxia lowered oxidative stress and stopped the formation of hydroxyl radicals within the fetal brain, reduced lipid peroxidation and cell death and stabilized the blood–brain barrier [39]. The neuroprotective potential of melatonin is sustained by the fact that melatonin inhibits middle cerebral artery constriction produced by norepinephrine [40] and induces umbilical vasodilation in ovine models [41].Melatonin maternal administration of melatonin reduced the fetal hypoxia in an animal model of fetal growth restriction, improved neurodevelopment and decreased brain injury and oxidative stress in newborn lambs [42].In a small clinical trial, melatonin has been orally administrated to newborn babies with birth asphyxia, and was shown to significantly reduce oxidative stress by decreasing plasma levels of malondialdehyde and nitrate/ nitrite. This study holds very strong evidence of melatonin efficiency as 3 of 10 asphyxiated babies died in the non-melatonin treated group, while no deaths were recorded in the melatonin treated group [Fetal programming concept and melatonin43].Fetal programming is an emerging concept that links environmental conditions during embryonic and fetal development with risk of diseases later in life. Compromised pregnancies  may affect fetal development. The pathophysiological basis of this outcome may be the induction of high oxidative stress in these pregnancies, which secondary alters fetal development. Melatonin night levels in pregnant women with preeclampsia are significantly lower compared with normal pregnancies (48.4 ± 24.7 vs. 85.4 ± 26.9 pg/mL). Circadian blood pressure and melatonin secretion rhythm followed a parallel course in preeclampsia, both during pregnancy and, at least 2 months after delivery [44].Another theory is the existence of epigenetic inheritance system, through DNA superstructure changes occurring as a response to pregnancy environmental conditions. It is believed that melatonin is the key regulator of this kind of transmission and that it acts through nuclear melatonin receptors, producing DNA bending in the oocyte [Melatonin is an important regulator of the complex embryo-fetal developmental processes. First, it induces the circadian rhythmicity in the offspring. Second, it appears to have a direct developmental effect on nervous and endocrine system. It also protects the highly metabolic ROS producing organs in the development from the oxidative stress damage.Melatonin is currently an over-the-counter drug, with a high availability and apparently without acute adverse effects. Even though there are a few clinical studies on pregnant women that show melatonin as being risk-free, we consider that regarding the extensive and yet not known effects on development, it should not be used by pregnant women before further studies.It has been shown that melatonin has neuroprotective effects in animal models. Considering the newly investigated key-role of melatonin as an epigenetic transducer, further studies are required to confirm that there are no late onset adverse reactions in adult life.Sources of fundingFounding through POSDRU/107/1.5/S/82839.DisclosuresNone


Pancreatic cancer carries a poor prognosis as most patients present with advanced disease and preferred chemotherapy regimens offer only modest effects on survival. Risk factors include smoking, obesity, heavy alcohol, and chronic pancreatitis. Pancreatic cancer has a complex relationship with diabetes, as diabetes can be both a risk factor for pancreatic cancer and a result of pancreatic cancer. Insulin, insulin-like growth factor-1 (IGF-1), and certain hormones play an important role in promoting neoplasia in diabetics. Metformin appears to reduce risk for pancreatic cancer and improve survival in diabetics with pancreatic cancer primarily by decreasing insulin/IGF signaling, disrupting mitochondrial respiration, and inhibiting the mammalian target of rapamycin (mTOR) pathway. Other potential anti-tumorigenic effects of metformin include the ability to downregulate specificity protein transcription factors and associated genes, alter microRNAs, decrease cancer stem cell proliferation, and reduce DNA damage and inflammation. Here, we review the most recent knowledge on risk factors and treatment of pancreatic cancer and the relationship between diabetes, pancreatic cancer, and metformin as a potential therapy. Pancreatic cancer is the twelfth most common cancer in the US but represents the fourth leading cause of cancer death in both men and women  increases risk for pancreatic cancer by 22%, independent of tobacco use, whereas moderate alcohol consumption did not carry an increased risk  or glucose intolerance may be present in up to 75% of patients with pancreatic cancer, a figure much higher than in other cancer types in whom the prevalence is no more than 30%  is also dependent on glutamine metabolism reprogrammed by oncogenic Kras via a unique pathway involving aspartate transaminase (GOT1) that leads to a maintenance of essential cellular redox states in the mitochondria  like the KrasG12D and embryonic stem cell (ESC)-based mouse models .Rapamycin and active-site mTOR inhibitors have been shown to increase Akt phosphorylation and ERK activation in pancreatic cancer cells in vitro studies showed that metformin alters profiles of microRNAs that regulate apoptosis, inhibit proliferation and invasion, and are linked to reduced expression of the oncogene HMGA1  transcription factor family and target genes involved in tumorigenesis including Bcl-2, survivin, cyclin D1, vascular endothelial growth factor, and fatty acid synthase  involving metformin in the treatment of pancreatic cancer. The majority involve metformin in combination therapies, given that metformin is unlikely to produce a desired efficacy to serve as monotherapy in pancreatic cancer. However, its attractiveness as part of combinatorial therapy lies, in part, in its inexpensiveness (as low as $4 per month) and well-tolerated toxicity profile . As discussed above, metformin offers synergistic activity across several but converging signaling pathways important to tumorigenesis. Addition of metformin may also reduce effective doses of other chemotherapeutic agents needed to treat a variety of cancers  anticancer mechanisms of metformin, particularly those involving, but not limited to, mTOR signaling (upstream and downstream) and mitochondrial energy metabolism, (2) pharmacokinetic and pharmacodynamic properties of metformin, and (3) relationships between risk factors such as DM and development and progression of pancreatic cancer to identify further molecular targets and advance potential therapies. For now, the future remains bright for metformin as the scientific community eagerly awaits the results of its continued development as a treatment for pancreatic cancer.The Guest Associate Editor Guido Eibl declares that, despite being affiliated to the same institution as author Stephen J. Pandol and having collaborated with him, the review process was handled objectively and no conflict of interest exists. 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.


Retinal degeneration arises from the loss of photoreceptors or retinal pigment epithelium (RPE). It is one of the leading causes of irreversible blindness worldwide with limited effective treatment options. Generation of induced pluripotent stem cell (IPSC)-derived retinal cells and tissues from individuals with retinal degeneration is a rapidly evolving technology that holds a great potential for its use in disease modelling. IPSCs provide an ideal platform to investigate normal and pathological retinogenesis, but also deliver a valuable source of retinal cell types for drug screening and cell therapy. In this review, we will provide some examples of the ways in which IPSCs have been used to model diseases of the outer retina including retinitis pigmentosa (RP), Usher syndrome (USH), Leber congenital amaurosis (LCA), gyrate atrophy (GA), juvenile neuronal ceroid lipofuscinosis (NCL), Best vitelliform macular dystrophy (BVMD) and age related macular degeneration (AMD). Retinal degeneration is the end point of many differing disease processes. In 2014, inherited retinopathies overtook diabetic related causes for blind registration in the working population in the UK Over the past decade, stem or progenitor cell transplantation as a means of replacing tissue has evolved rapidly, with the development of protocols to drive embryonic stem cells (ESCs) towards the fate of both photoreceptors or the RPE in vitro. In a phenomenal series of experiments, Takahashi and Yamanaka identified the embryonic transcription factors that are required to turn an adult somatic cell into a pluripotent stem cell. The group was able to reprogram mouse In 2006, a major advance in stem cell technology produced a new means with which to investigate inherited diseases, such as retinopathies, in diseased patient cells 2IPSCs can be generated from a wide array of sources including fibroblasts, keratinocytes, and T cells. The cell source can contribute to the epigenetic memory and serves as a challenge for IPSC research. Numerous studies have highlighted the varied growth and differentiation characteristics of IPSC lines. This is thought to be caused by a combination of genetic and epigenetic variation leading to subtle but significant differences in endogenous signalling. e.g. Pax6, Chx10). Similarly, markers that are specific to a level of cell maturity are important. For example Recoverin is expressed early in human retinal development de facto inclusion criteria for IPSC derived photoreceptors would include a range of cell types from developmentally immature progenitors to photoreceptors. If these potential pitfalls are not properly accounted for, incorrect conclusions about disease aetiology may be drawn.Patient IPSC-derived cells/tissues can then be used to span a range of applications from elucidating the mechanism of disease causing mutations to drug and gene therapy testing . The expThe pure population of differentiated cells often has a limited proliferative capacity necessitating continued derivation from the original pluripotent IPSC bank Thus far IPSCs have been used to generate several cell types that are implicated in retinal degenerative diseases, including RPE e.g. basic fibroblast growth factor). While this technique has repeatedly proven to be a reliable and cost effective method for generating RPE, the production of neural retinal cell types requires a more directed procedure. Such methods commonly involve the agonism or antagonism of developmentally critical signalling pathways with small molecules or recombinant growth factors. Photoreceptor generating protocols are notoriously laborious, time consuming and highly dependent on the cell line used and epigenetic status, which can vary over time in culture Protocols for the generation of retinal cells from IPSCs employ either spontaneous or directed methods Frequently the cell type of interest emerges alongside a myriad of contaminating cell types — being able to identify and isolate the cells of interest is critical to the success of these studies. The highly pigmented RPE can be easily identified visually and separated manually or by fluorescence activated cell sorting equipment (FACS). The isolated RPE cells then have a degree of proliferative potential over a limited number of passages, making it possible to generate sufficient material for experiments. In contrast, non-pigmented neural retinal cells require more innovative methods for their visual identification; for example ESC lines have been developed with GFP tagged expression of early eye field marker proteins to allow easy identification and purification Additionally once the desired cell type has been generated, the disease model may be hampered by the relative immaturity of the cells generated. For example studies in human ESC derived RPE have found its transcriptome to more closely match human foetal RPE than adult cells In vivo these cells would be exposed to exogenous stresses, from a neighbouring cell type, the immune or vascular system. Exposure to these types of stresses may be required to bring out the disease phenotype in cultured cells, which may limit the utility of the IPSC ‘disease in a dish’ paradigm.Finally, once the desired cell type has been generated in culture, the use of this cell as a disease model may be affected by its isolation as a single cell type. 33.1Retinitis Pigmentosa is the most common form of inherited progressive retinal dystrophy that is both clinically and genetically heterogeneous. It is characterized by the progressive loss of rod photoreceptors then RPE cells. Symptoms include night blindness and progressive visual field loss, often leading to complete blindness. No definitive treatment has been established yet. RP can be inherited in autosomal dominant, autosomal recessive, X-linked, mitochondrial and genetically more complex modes in vitro model for disease discovery. In effect, they enable a better appreciation of the molecular and histological basis of diseases, including, RP, but also deliver a means of identifying possible therapeutic strategies RP1, RP9, PRPH2 or RHO gene, and generated patient-specific IPSCs that were then differentiated into photoreceptor-like cells, which expressed characteristic immunocytochemical markers and electrophysiological properties. They found that, as occurs in disease, the number of the patient-derived photoreceptor-like cells decreased more rapidly in vitro than those derived from control lines. Additionally, cells derived from patients with RP9 or RHO mutations expressed markers for oxidative or endoplasmic reticulum (ER) stress.IPSC-derived retinal cells are an ideal RP9 mutation. In effect, using IPSC-derived cells in drug screens may ultimately help to narrow the disease targets for experimental drugs, identify drugs for repurposing and facilitate clinical trial design. Using IPSC-derived cells as a novel platform for in vitro Phase 0 clinical trials would also provide important information about the efficacy and efficiency of potential therapies long before expensive clinical trials in patients. Drawbacks of this study include the use of limited photoreceptor markers and the small number of mutations analysed. In effect, the four mutations are not representative of this heterogeneous disease entity. In addition, the study does not take into account other factors that may have induced accelerated photoreceptor cell loss.The study also looked at stem cell therapy as a useful tool for screening of drug responses in RP. The authors identified α-tocopherol as a potential therapeutic that could preserve RHO-positive cells in IPSC-derived retinal cultures carrying the RHO gene (E181K). This IPSC line was subsequently used to derive rod photoreceptor like cells with the same mutation. These cells were used to demonstrate that the mutation was indeed a pathogenic mutation and were used to explore the underlying molecular mechanisms and potential therapeutic approaches. With the use of an adenoviral vector gene transfer, the mutation was amended in the patients' IPSCs and the cells were then differentiated to photoreceptor-like cells. The study found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was associated with a higher expression of ER stress and apoptotic markers. Furthermore, it was shown that numerous reagents  promoted the survival of the patients' IPSC-derived photoreceptor-like cells, with a concurrent decrease in ER stress and apoptosis markers. This study is valuable as it draws attention to the use of IPSCs for review of disease pathophysiology, as well as for development of drug and gene therapeutics.Similarly, Yoshida et al. via exome sequencing. In this study, the authors demonstrated that the insertion of the Alu sequence into exon 9 of the patient's male germ cell-associated kinase (MAK) gene blocked the generation of a splice variant of MAK, which is normally expressed only in retinal precursors. The abnormal splicing of MAK impeded normal photoreceptor development, leading to irreversible cell loss. In this study, IPSCs offered an effective means of recognizing the disease mechanism by enabling the ex vivo study of patient-specific retinal cells. Even though mutations in MAK only represent 1% of RP causes in the general population, they are quite common among individuals of Ashkenazi Jewish descent (reported in one third of cases) e.g. ABCA4 and USH2A) cannot be discounted.Furthermore, Tucker and colleagues RP2 gene. RP2 protein was undetectable in the patient cells, implying that the mechanism underlying disease progression is caused by complete lack of RP2 protein. Using translational read-through inducing drugs, Schwarz et al. Schwarz et al. Li and colleagues 3.2USH2A gene that is expressed in the photoreceptor. Zahabi et al. USH2A. Sanger sequencing of the USH2A gene introns revealed a second mutation in intron 40. In order to further interrogate the pathophysiology, they reprogrammed the keratinocytes of the patient into IPSCs and then used direct-differentiation protocols to produce bilayered optic vesicle-like structures, comprising RPE and primitive photoreceptor like cells. cDNA analysis confirmed that the mutation in intron 40 caused expression of this intronic region, a frameshift and premature stop codon. Scrutiny of the protein expression pointed towards misfolding and potential endoplasmic reticulum stress.Usher syndrome is an autosomal recessive hereditary disorder characterized by RP and congenital sensorineural hearing loss, with a varying age of onset and extent of vestibular dysfunction. A number of causative genes have been identified including the 3.3CEP290, a gene which normally produces a cilium-associated protein that is involved in photoreceptor outer segment (POS) trafficking and ciliogenesis Leber congenital amaurosis is a rare degenerative inherited eye disease that can lead to severe visual impairment before the age of one CEP290-associated LCA patient fibroblast cells. They showed that lentiviral delivery of CEP290 to patient fibroblasts increased the proportion of ciliated cells and the length of cilia in two out of three patient fibroblast lines compared to untransfected controls. This study was also able to demonstrate that the range of CEP290 therapeutic dosage was limited; with higher doses of wild-type CEP290 becoming toxic to fibroblasts cells in culture. Patient-specific IPSCs were then generated and differentiated into cells that were immunoreactive for Otx2  and cone opsin — although no photoreceptor morphology primitive or otherwise was demonstrated. These cells were successfully transfected with a lentiviral vectors containing full length CEP290 and the authors demonstrated expression of the full-length transcript and presence of the protein by western blot in patient derived photoreceptor precursor cells. However, the authors did not demonstrate a ciliogenesis defect in the photoreceptor precursor cells following transfection with full length CEP290, citing difficulties in identifying cilia in differentiated structures.Understanding the pathophysiology and mutation-specific disease severity in LCA patients is key to refining treatment and improving outcomes. A study by Burnight et al. in vitro, thereby facilitating disease modelling and drug screening. As mentioned previously, Zahabi et al. tripartite motif containing 61 (TRIM61) gene, the zinc finger protein 558 (ZNF558) gene, glutathione S-transferase theta 1 (GSTT1) and neuronatin (NNAT).Several studies were able to review pathologic formation of human retinal cells 3.4Gyrate atrophy is a progressive autosomal recessive disorder that starts in childhood and induces diffuse atrophy of the choroid, RPE and sensory retina. In GA, stem cell-based disease models are useful for pharmacological screening and the evaluation of new therapeutics.Meyer et al. 3.5ceroid lipofuscinosis 3 (CLN3) Juvenile NCL, also known as Batten disease is a group of severe neurodegenerative diseases characterized by intracellular accumulation of autofluorescent wax-like lipid pigments (ceroid-lipofuscin) in neurons. There are several subtypes based on mutations of the various genes, disease onset, and severity of the neurological defects such as progressive dementia, seizures and visual failure. The latter is normally central in nature, due to degeneration of cones in the macula. Disease progression is very aggressive and patients often die during their second or third decade. It is inherited as an autosomal recessive genetic disorder and often caused by a genomic DNA deletion in the gene, CLN2 mutation) and juvenile NCL (CLN3 mutation). Patient IPSCs were differentiated into neuronal  tissue. Abnormalities in endosomal-lysosomal system were detectable in the patient IPSC, but disease-subtype specific lysosomal storage was only evident in their differentiated neuronal derivatives. This clearly shows the necessity of IPSCs and IPSC differentiation technology to effectively model these diseases.Stem cell technology is a powerful tool for analysing the pathophysiology and for enhancing drug screening. A recent study by Lojewski et al. CLN2 or CLN3 gene, confirming previous outcomes in animal models www.clinicaltrials.gov, NCT01161576). These IPSC-derived neural progenitor cells were also used to screen potential pharmacological modulators of the CLN2 encoded protein, demonstrating the use of patient-derived IPSCs as a platform for testing new therapeutic candidates.The abnormalities could be corrected in patient cells by using adenovirus vector to overexpress wild type protein encoded by the 3.6Best vitellliform macular dystrophy is an autosomal dominant hereditary maculopathy with childhood-onset accumulation of liposfuscin in RPE. Affected individuals develop progressive central acuity loss, and metamorphopsia, due to mutations in bestrophin, a chloride channel Singh et al. 3.7AMD is a complex disease that can be subdivided into a “dry” and “wet” type. It involves loss of the RPE/photoreceptor layers, thinning of the outer plexiform layer, thickening of Bruch's membrane and atrophy of the choriocapillaris. In dry AMD, there is an accumulation of lipid-like deposits (drusen) between the RPE and Bruch's membrane, occurring typically in individuals over 50. Geographic atrophy is more prominent in later stages, leading to photoreceptor cell loss. In wet AMD, the choroidal vasculature can grow in the subretinal space, termed “choroidal neovascularisation.” In view of the pathophysiology, cell replacement therapy for retinal cell loss is very appealing, with several clinical trials underway.complement factor H gene and subsequently the complement cascade. However, the mechanism by which the chromosome 10q locus leads to increased AMD risk remains unknown. Yang et al. 2O2-induced cell apoptosis and upregulated the expression of several oxidative stress-regulating genes. Although the systemic effects of such treatments must still be evaluated in suitable animal models, this study demonstrates the potential of IPSC-derived cells to screen for the activity of therapeutic candidates in the desired target cell type early in the drug development process.Generating a disease model for AMD may be useful in understanding its pathogenesis and in the development of effective therapeutic strategies. AMD has a strong genetic characteristic with over 50 different loci identified so far. These include two on chromosomes 1 and 10 leading to a high-risk haplotype. The mutation associated with chromosome 1 affects the 4versus abnormal retinal cell behaviour in vitro and to gain new mechanistic insights into disease aetiology. Patient specific IPSCs may be used in pharmaceutical screening and in the elucidation of new treatment options for retinal diseases  UK (G1000730); the California Institute of Regenerative Medicine (CIRM) (G1000730); Fight for Sight UK (1755); The Lincy Foundation (P12761); the Macular Society; and the National Institute for Health Research (NIHR) (BRC2_011) Biomedical Research Centre based at Moorfields Eye Hospital National Health Service (NHS) Foundation Trust and University College London Institute of Ophthalmology. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.This work was supported by funding from The authors declare no competing financial interests.


NCT02048709Clinical Trial Registration Number: The enzyme Indoleamine 2,3-dioxygenase (IDO1) catalyzes the cleavage of L-tryptophan, resulting in the production of kynurenine. Tryptophan depletion and kynurenine metabolites enhance the number and function of Tregs (suppressive arm) and inhibit effector T cells (stimulatory arm), regulating acquired local and peripheral immune tolerance. In cancer, IDO can either be expressed directly by the tumor cells themselves, or induced indirectly in host antigen presenting cells by the tumor and its expression has been associated with a worse clinical outcome in a variety of cancers. Indoximod (1-methyl D-tryptophan), the first IDO pathway inhibitor to enter human clinical trials, is currently being tested in multiple Phase II clinical trials. We are here to report preliminary Phase I data with another promising IDO pathway inhibitor, NLG919, a potent direct enzymatic inhibitor of IDO. While Indoximod and NLG919 both target the same IDO pathway, preclinical data clearly demonstrates that these compounds have different mechanisms of action and are synergistic. In preclinical models, NLG919 showed dose-dependent activation and proliferation of effector T cells, producing dramatic regression of large established tumors. Additionally, NLG919 showed enhanced immune activation and tumor regression when combined with Indoximod in this system.In this Phase I trial six dose levels of NLG919 are proposed for evaluation: 50, 100, 200, 400, 600 and 800 mg orally q12 h for 21 of 28 days in repeating cycles. Patients with advanced solid tumors that have progressed following standard therapy are eligible unless prior Ipilimumab or other CTLA4 targeted therapy has been administered. A single patient was treated at the first dose level and patients have been enrolled on subsequent dose levels with a standard 3 + 3 escalation scheme. Pharmacokinetics are evaluated for 48 hours following the first dose with no intervening doses at the initiation of the first cycle, and also at the end of the treatment cycle, day 21.To date, no dose limiting toxicities have been observed and detailed updated study results will be presented. The primary objectives of the study are to evaluate the safety and toxicity of NLG919 in patients with advanced solid tumors and to define the maximum tolerated dose and recommended dose for Phase II studies. Secondary objectives are to measure pharmacokinetics, pharmacodynamics and evaluate any potential antitumor responses.Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Primary and metastatic liver tumors are an increasing global health problem, with hepatocellular carcinoma (HCC) now being the third leading cause of cancer-related mortality worldwide. Systemic treatment options for HCC remain limited, with Sorafenib as the only prospectively validated agent shown to increase overall survival. Surgical resection and/or transplantation, locally ablative therapies and regional or locoregional therapies have filled the gap in liver tumor treatments, providing improved survival outcomes for both primary and metastatic tumors. Minimally invasive local therapies have an increasing role in the treatment of both primary and metastatic liver tumors. For patients with low volume disease, these therapies have now been established into consensus practice guidelines. This review highlights technical aspects and outcomes of commonly utilized, minimally invasive local therapies including laparoscopic liver resection (LLR), radiofrequency ablation (RFA), microwave ablation (MWA), high-intensity focused ultrasound (HIFU), irreversible electroporation (IRE), and stereotactic body radiation therapy (SBRT). In addition, the role of combination treatment strategies utilizing these minimally invasive techniques is reviewed. This has been, in part, driven by the limited effectiveness and range of available systemic treatments, particularly for HCC. Limitations of the effectiveness of systemic therapy are thought, in part, to be due to the frequent coexistence of HCC with cirrhosis and its high molecular heterogeneity. A significant advance was made in systemic therapeutic options with the randomized prospective validation of sorafenib in providing survival benefit in patients with HCC,,-Surgical resection has been shown to result in improved survival outcomes and thus is the mainstay of curative therapy whenever amenable for both primary and metastatic liver tumorsIn addition to the increased incidence of HCC, improved screening, follow-up and imaging algorithms have led to the earlier detection and diagnosis of low volume disease primary and metastatic liver tumors amenable for minimally invasive local techniques. These factors have fueled an ever increasing need for local treatment options for both primary and metastatic liver tumors. The improved imaging algorithms have occurred in conjunction with advances in ablative techniques towards targeting of lesions. For percutaneous ablations, a wide range of guidance techniques have been developed including using ultrasound, computed tomography, magnetic resonance, fusion of imaging modalities, and needle tracking for optimal applicator positioning. Improved guidance and lesion localization has improved upon the technical feasibility of all minimally invasive local treatment options, further solidifying their use in current practice.A wide range of minimally invasive local treatment techniques such as laparoscopic liver resection (LLR) and ablation have been developed. This article will highlight their role in current treatment paradigms, their principles of action, their relative differences in implementation, and the evidence for their use.,,,The management of both primary tumors and metastases confined to the liver is best served with a multidisciplinary approach as adopted by multiple consensus guidelines, including the National Comprehensive Cancer Network (NCCN) and the European Association for the Study of the Liver (EASL)Volume of disease is an important determinant of prognosis and treatment options in patients with disease isolated to the liver. In the BCLC treatment strategy, tumor size and number directly alters disease staging, with a single tumor <2 cm considered very early stage (0), single or up to three tumors <3 cm considered early stage (A), or a large and/or multinodular tumor considered intermediate stage (B) in patients with equivalent Child-Pugh scores (A) and Performance Status (0). Potentially curative options such as surgical resection, liver transplantation (LT), or locally ablative therapies are reserved for patients with low volume disease . For large or multinodular tumors with preserved liver function, locoregional therapies  serve as the preferred treatment option.-Numerous locoregional treatment options have been explored for the treatment of large or multinodular tumors. Transarterially delivered therapies include bland embolization, chemoembolization, drug-eluting beads, and radioembolization, each with their relative advantages and drawbacks which are beyond the scope of this reviewet al. recently analyzed a series of 3,856 patients from a single Asian center and created a new prognostic classification system and treatment strategy through statistical modelling methods termed the Hong Kong Liver Cancer (HKLC) schema . More importantly, HKLC identified subsets of BCLC intermediate and advanced stage patients who demonstrated a significantly increased survival benefit if classified under the HKLC system, where they would be stratified to receive more aggressive therapies such as surgical resection or local ablation. This new classification system further validates the effectiveness of surgical resection and local ablation in improving the overall survival in patients with primary liver cancer, and expands upon the eligibility criteria for patients to receive these more “aggressive” therapies.Yau ,-Surgical resection has been shown to result in improved survival outcomes and thus is the mainstay of curative therapy whenever amenable for both primary and metastatic liver tumorset al. prospectively evaluated 389 patients eligible for either surgical resection or percutaneous RFA for solitary HCC <3 cm in diameter for quality of life measuresTraditional, open liver resection (OLR) is also affected by significant morbidity which directly impacts upon patient’s quality of life. As compared to surgical resection, local ablation is of relatively low cost, has minimal morbidity, and can be usually performed with shorter hospital staysTechniques within the treatment armamentarium are not mutually exclusive and there is extensive reported evidence on the role of ablation in conjunction with surgical techniques.-,,For example, ablative techniques are commonly used to limit disease progression in patients awaiting transplantation (“bridge to transplantation”), with multiple studies demonstrating its utility in this settingMultiple locally ablative techniques for the treatment of tumors have been developed and adopted into standard clinical practice. Each technique is based upon a different principle of action to induce cytotoxicity , fewer postoperative days of narcotic medication , quicker resumption of diet , and decreased time to ambulation . While length of stay (LOS) information for liver surgery is variable secondary to cultural and institutional biases, almost all of the studies reported significantly reduced LOS after LLR (50% shorter) compared with OLR. Shorter LOS translated to reduced costs for LLR. The majority of studies reported comparable morbidity rates, while seven showed significantly lower complication rates with LLR . There were no differences in operative time or mortality. While this review harbors the limitations inherent to any retrospective analysis of selected patients, it supports LLR as safe, feasible, and cost-effective with demonstrable benefits.Nguyen et al. reported the only matched prospective comparison in patients undergoing resection of CRC liver metastases via laparoscopic (n=60) and open (n=60) approachesvs. OLR , while there was no significant difference in overall and disease-free survival. The 1-, 3-, and 5-year overall survival was 97%, 82%, and 64%, respectively, with LLR, versus 97%, 70%, and 56% with OLR (P=0.32). Furthermore, 1-, 3-, and 5-year disease-free survival was 70%, 47%, and 35%, respectively with LLR, versus 70%, 40%, and 27% in OLR (P=0.32).Of the nearly 3,000 published reports of LLR, approximately 50% were performed for malignant lesions, 35% of which were CRC metastasesThe nearly 3,000 reported cases of LLR indicate that it is safe, feasible, and cost-effective with demonstrable short-terms benefits and no negative impact on long-term outcomes. LLR is a reasonable first-line approach in the treatment of CRC liver metastasis in select patients. LLR should only be performed in specialized centers by surgeons intimately acquainted with open and minimally invasive techniques. While a randomized prospective trial would be optimal to validate these results, no such study has been reported to date.,,,The greatest accumulated evidence exists for RFA as compared to the other locally ablative techniqueset al. recently published a Cochrane database analysis on the use of RFA for the treatment of HCCvs. hepatic resection  with HCC meeting Milan criteria and found similar disease-free survival, cumulative survival, and complication rates between the two groups,et al. compared the efficacy of MWA versus RFA in a series of 72 patients (36 RFA/36 MWA) in a randomized fashion from a cohort of patients with equivalent background demographics and mean tumor size and concluded that therapeutic effects, complication rates, and rates of residual untreated disease were equivalent between the two modalitieset al. evaluated overall survival, complete ablation, local tumor progression and distant recurrence in a series of 155 patients (mean tumor size 2.3±0.4 cm RFA cohort and 2.2±0.4 cm MWA cohort) and also found that RFA and MWA were equivalentGiven its increased efficacy of ablation and shorter time to achieve ablations, MWA has increasingly been used in the treatment of both primary and metastatic tumors of the liver , SBRT can create a rapid radiation dose fall off, allowing ablative radiation doses to be delivered to gross tumors while sparing adjacent tissue  and small risk of fiducial migrationTypically, treatment is delivered in 3-6 fractions, with minimum 1-3 days between each fraction. Depending on location, it is possible to target multiple tumors in a single fraction. The actual radiation treatment is less than 1 hour in duration. Because it is non-invasive and painless, no sedation or anesthesia is required.-,,,,,,,,,,,The utility of SBRT as a treatment for unresectable liver tumors was first reported in 1995 by Blomgren and colleagueset al.et al. recently explored the use of single fraction SBRT for primary and metastatic liver tumors, demonstrating comparable efficacy with low toxicityThe single institution phase I/II trial by Mendez Romero The impact of SBRT on overall survival is yet to be determined. The hetereogeneity of baseline liver function, variation in tumor size and comorbidities in the patient population of reported series precludes an accurate assessment of overall survival and local control outcomes across varying treatment modalities. This will need to be addressed in the future through randomized clinical trials. Preliminary studies suggest that liver SBRT is a non-invasive and effective treatment modality for liver tumors in non-surgical candidates who are ineligible, or have failed other treatment modalities.-et al.et al. reported outcomes of 35 patients with HCC, ranging in size from 5-12 cm, treated with HDR brachytherapy. All patients were Childs Pugh A or B. At 12 months, local control was 93% and no major toxicity was reportedRadiation has a dose-dependent effect on local control, but deliverable dose is limited by surrounding critical structures. Another technique of radiation delivery that has shown promising outcomes in single-institution series is HDR CT-guided interstitial brachytherapyHIFU incorporates multiple ultrasound beams produced by piezoelectric or piezoceramic transducers directed into a three-dimensional focal point typically 1 to 5 mm in diameter and 10 to 50 mm in length-et al. reported on the safety and efficacy of HIFU on the treatment of HCC  for a series of 49 patients who were not surgical candidates and concluded that HIFU was an effective modality in this setting, with 1- and 3-year overall survival rates reported as 87.7% and 62.4%, respectivelyet al. reported on the safety and efficacy of large HCCs  treated with HIFU and found favorable overall survival rates of 86.1%, 61.5%, and 35.3% at 6, 12, and 18 months, respectivelyet al. reported on the outcomes of HIFU performed for the treatment of HCC as a bridge to transplantation in 10 patients as compared to 29 patients who received transarterial chemoembolization and found excellent efficacy with HIFU  with none of the patients on the liver transplant list (n=5) dropping outet al. reported on the largest series of patients who have undergone HIFU ; however histologies and tumor locations were variableSeveral series have evaluated the safety and efficacy of HIFU for the treatment of HCC,IRE is an ostensibly non-thermal technique in which the direct placement of electrodes is used to create a pulsed direct current inducing cytotoxicity in tumor cells by altering transmembrane potentials which irreversibly disrupt cell membrane integrity,-et al., 13/18 (72%) of lesions were completely ablated [13/14 (93%) of lesions <3 cm] with a local recurrence-free period of 18±4 monthset al. reported on 44 patients with both primary (n=14) and metastatic  liver tumors treated with IRE; with local recurrence free survival of 97.4%, 94.6%, and 59.7% at 3, 6, and 12 months, respectivelyP=0.178). Kingham et al. reported on the use of IRE in a series of 28 patients with 65 tumors (median 1 cm) and found a local recurrence rate of 5.7% at median follow-up of 6 monthsIRE has a theoretical safety advantage over other locally ablative techniques in the treatment of tumors close to structures susceptible to thermal injury, such as major bile ducts in the liver. In addition, IRE may be more effective in cytotoxicity for tumors next to major vessels, due to reduction in the heat-sink effect. Several small series have reported on the use of IRE for the treatment of liver tumors in locations thought to be unfavorable for other ablative therapies,PEI involves the direct instillation of ethanol into tumors ultimately resulting in coagulation necrosis. As such, the technique is relatively simple and inexpensive. In practice, PEI is limited by poor and uneven distribution of the ethanol within the tumor and diffusion into the adjacent normal tissues. Ultrasound guidance allows for real-time monitoring of the ethanol dispersion and is generally used to help compensate for these limitations. Even so, multiple intra-procedural needle repositions are sometimes needed, given the uneven distribution of the ethanolet al. investigated the role of cryoablation in comparison to RFA in the treatment of unresectable HCCCryoablation involves the direct application of a cryoprobe into a tumor. The thermal contact with the tumor results in ice crystal formation and osmotic shock. One distinct advantage of cryoablation as compared to other ablative techniques is that the zone of ablation is readily visible  using non-contrast CT, ultrasound, or MRI monitoring, allowing for precise targeting of a zone of ablation to the tumoret al. reported on the use of PLA in 899 patients with 2,520 liver tumors and demonstrated a major complication rate of 2%; the majority being either pleural effusion  requiring thoracentesis or hepatic abscess requiring drainage et al. retrospectively analyzed the use of PLA for the treatment of HCC in a cohort of 432 cirrhotic patients P=0.001; RR =0.517) as an independent predictor of survival. Ferrari et al. evaluated the use of PLA as compared to RFA in a prospective randomized study of 81 cirrhotic patients with HCCvs. 78%). In addition, it was suggestive that the RFA cohort had improved overall survival as compared to PLA at 1, 3 and 5 years , however the difference did not reach statistical significance (P=0.3299). Though the evidence is still limited, there is suggestion that PLA is limited in achieving complete tumor ablation as compared to other locally ablative therapies.PLA involves the direct deposition of laser light via fiberoptic applicators to induce tissue hyperthermia in tumors. Proponents of PLA advocate that the thin flexible fiberoptic delivery fibers allow for safer and technically easier approaches to tumors as compared to other ablative techniquesThere are multiple theoretical advantages in combining locally ablative therapies with each other or to other treatment options such as transarterial embolotherapy. Several of the described modalities have different principles of action in creating cytotoxicity and combining them may potentiate their effectiveness, improving tumor control. In combination, dose and energy profiles for each locally ablative modality may be potentially reduced without comprising cytotoxicity to tumor cells; increasing the safety margin to adjacent normal tissues. Combination approaches also allow for limitations of each modality on its own to be overcome, thus expanding the number of patients eligible for therapeutic options.-et al.P=0.002; HR, 0.575; 95% CI, 0.374-0.897; P=0.009, respectively).The greatest cumulated evidence exists for the use of RFA in combination with transarterial embolotherapy. In theory, the decreased blood flow induced by transarterial embolization reduces heat loss, improving the RFA margins. In addition TACE enhances nearby control of satellite lesions,,et al., the combination of therapies were found to have increased tumor necrosis, decreased tumor growth and improved overall animal survival in a rat tumor modelThe use of localized radiation in combination with thermal ablation is of particular interest for future investigation. Hyperthermia has been proven to potentiate the cytotoxic effect of radiation and has been clinically adoptedThe combination of thermal ablation in tandem with SBRT is complimentary from a technical standpoint and is readily adaptable into clinical practice. Thermal ablative techniques offer the added clinical benefit of tissue diagnosis that can be obtained immediately prior to the procedure that is usually necessary for radiation oncologists to appropriately initiate treatments. In addition, fiducial seed placement sometimes necessary for SBRT localization can be performed at the same time. Conversely, SBRT can provide improved tumor control in areas where thermal ablation is known to have high failure rates, such as next to major vessels in the setting of RFA. In addition, doses for radiation and ablation zones for thermal ablation can be reduced to preserve normal tissue parenchyma without compromising efficacy in tumor control. The combination of these therapies serves as an exciting avenue of research with clinical studies necessary to validate proper dosing and timing regimens.Minimally invasive therapies for the treatment of both primary and metastatic liver tumors continue to highlight the fact that we are privileged to practice medicine during a time of such dynamic change and innovation. As such, both laparoscopic resection and locally ablative therapies have solidified a role in the treatment paradigms of both primary and metastatic liver tumors, including the most recently validated prognostic treatment strategy, the HKLC schema.Based upon its principle of action and technical aspects of implementation, each local therapy has its benefits and drawbacks in clinical practice. The greatest cumulative evidence exists for surgical resection and RFA. As both cost and quality of life become further incorporated into treatment paradigms, minimally invasive local therapeutic options will continue their trends towards increased utilization.As summarized in the principles of action section, locally ablative therapies harness the newest technological developments into various devices that are able to induce localized cytotoxicity to tumor cells while minimizing damage to nearby native tissues. MWA, HIFU, and SBRT are all relatively nascent technologies, though preliminary data regarding their use is promising, with overall survivals similar to RFA. These technologies expand upon the patients eligible for local ablation by offering different technical advantages as compared to RFA.Tandem approaches with combination of local ablation such as RFA in addition to locoregional therapies such as TACE have already been explored with promising results demonstrating improved overall survival as compared to RFA alone. In addition, new technologies such as IRE are currently being clinically investigated to further expand the patients eligible to safely undergo local ablation.


Eye misalignment, called strabismus, is amongst the most common phenotypes observed, occurring in up to 5% of individuals in a studied population. While misalignment is frequently observed in rare complex syndromes, the majority of strabismus cases are non-syndromic. Over the past decade, genes and pathways associated with syndromic forms of strabismus have emerged, but the genes contributing to non-syndromic strabismus remain elusive. Genetic testing for strabismus risk may allow for earlier diagnosis and treatment, as well as decreased frequency of surgery. We review human and model organism literature describing non-syndromic strabismus, including family, twin, linkage, and gene expression studies. Recent advances in the genetics of Duane retraction syndrome are considered, as relatives of those impacted show elevated familial rates of non-syndromic strabismus. As whole genome sequencing efforts are advancing for the discovery of the elusive strabismus genes, this overview is intended to support the interpretation of the new findings. None of the authors has a conflict of interest related to any of the content of this review.Strabismus  is one of the earliest recorded genetic disorders. More than 2400 years ago, Hippocrates observed ‘Children of parents having distorted eyes squint also for the most part’. The prevalence of strabismus is 2–4% among Caucasians, 2.4% among Hispanic/Latinos, 2.5% among African-Americans, and 1% in East-Asians While the causes of non-syndromic strabismus are largely unknown, twin studies and family studies have demonstrated a substantial genetic contribution to strabismus In this review, we summarize strabismus etiology and pathogenesis, genetic studies of non-syndromic strabismus and Duane retraction syndrome (DRS), as non-syndromic strabismus occurs at elevated rates in affected families The mechanisms underlying strabismus may involve one of several systems or tissues . Past reThe age of onset distribution for strabismus is bimodal, with approximately 22% diagnosed before the age of 12 months and approximately 43% detected between 2 and 3 years of age. Non-accommodative strabismus was more common in the first group, while accommodative strabismus was more common in the second group  As indicated above, pathogenesis of infantile esotropia may result from defects spanning the visual-motor axis b 30. ResHypotheses for strabismus mechanisms have been proposed which focus on the subcortical visual pathway, brainstem vergence motoneurons, the brainstem vestibule-ocular pathway, and cranial nerves Significant strabismus risk factors include retinopathy of prematurity, low birth weight, premature birth, and smoking during pregnancy. As our focus will remain on genetic risk, the interested reader may find additional information about the other factors in the systematic review by Maconachie et al. Many early studies focused on the transmission of strabismus through families. However, findings varied in terms of heritability, inheritance mode, and the concordance of strabismic types A longitudinal study found that 18% of 34 babies born in families with a parent affected by convergent (i.e. esotropia) strabismus developed constant or intermittent esotropia by 6 months As familial clustering of strabismus can reflect either a common genetic factor or an unrecognized environmental factor, twin studies are the key to quantify the relative genetic contribution. Twin studies of strabismus have reported higher concordance rates in monozygotic twins than dizygotic twins, suggesting a predominant genetic factor Esotropia and exotropia have a strikingly different genetic risk profile. In the Podgor study, a striking odds ratio of 330 was reported for exotropia in cases of multiple birth with one affected twin, while single births had an extremely low odds ratio of 2.2, data most consistent with a strong multiple birth environmental impact on exotropia risk A key consideration arises from twin studies. Wilmer and Backus raised the potential confounding contribution of phoria to the study of strabismus genetics. Phoria is a latent misalignment of the eyes that appears when fixation on a target is broken . Wilmer and Backus observed that genetic factors were necessary for strabismus development but not for phoria development Summarizing the above information, esotropia is most closely tied to heritable factors while exotropia has a stronger environmental component. Future studies should therefore be designed in a manner that controls for the environmental component, including multiple births.Dominant, recessive, and sex-linked inheritance patterns have been proposed for non-syndromic strabismus in family studies Parikh et al. identified the first concomitant strabismus locus on chromosome 7p22.1 (STBMS1) in a linkage analysis of a large family. Among seven initially assessed multiplex families with non-syndromic strabismus, one family showed a significant logarithm of the odds (LOD) score on chromosome 7. Although the pedigree suggested an autosomal dominant inheritance pattern, the haplotype data was most consistent with an autosomal recessive model or a more complex model, such as semi-dominant inheritance The Ohtsuki group tried to identify comitant strabismus susceptibility loci through sib-pair analysis and nonparametric linkage analysis for multiple pedigrees. This initial 2003 attempt indicated multiple loci with low LOD scores A summary of reported candidate loci for comitant strabismus is presented in TableMYH13) and myosin heavy chain-1 (MYH1), and related contractile genes were downregulated; genes involved in energy balance, such as mitochondrion homeostasis or regulations of energy metabolism, were dysregulated in strabismic EOMs. The conclusions should be assessed with caution, since it was not specified which forms of strabismus were represented in the samples, although the authors suggested that the sample set may have a high portion of exotropia cases Experimental approaches to elucidate molecular mechanisms related to strabismus have been pursued. Microarray analysis showed that expression of 604 genes differ significantly between 100 strabismic EOM samples and 28 normal EOM samples. Together with PCR experiments, three major conclusions were drawn. Collagen and collagen-related genes were upregulated; specific myosins, such as EOM-specific myosin (vs cadavers), which has been recognized to cause difficulty in the interpretation of gene expression studies In another study, expression levels of seven myogenesis-related genes in EOMs from 18 concomitant strabismus patients were compared against 12 samples from a single non-strabismic individual. Six of the genes had reduced expression levels, leading Zhu et al. to suggest that altered growth of muscles may be involved. However, it was unclear whether the patients had congenital strabismus nor the nature of the deviations involved While the focus of this review is the genetics of non-syndromic forms of strabismus, there are familial syndromes in which strabismus rates are elevated in otherwise non-syndromic family members. About 70% of DRS cases do not exhibit other congenital abnormalities, and approximately 20% of cases have a family history of strabismus Three types of DRS have been described based on clinical examination. In these studies, key attributes include abduction, movement of a body part away from the midline, and adduction, movement toward the midline. Type 1 DRS is characterized by marked limitation of abduction, type 2 DRS is characterized by marked limitation of adduction, and type 3 DRS is characterized by a combination of marked limitation of both While most cases are sporadic, reports of familial DRS date back to 1896 de novo reciprocal balanced translocation t was identified in a patient with DRS. This patient had amblyopia and narrowing of palpebral fissures CPA6) gene at the previously identified DURS1 (DRS-1) locus on chromosome 8 was disrupted between the first two exons in this patient and was proposed as the causal gene CPA6 mutations were identified in a set of 18 sporadic DRS patients CPA6. CHD7 duplication was suggested to be responsible for at least part of the features in resulting from the 8q12 duplication The focus on chromosome 8q in DRS studies has progressed to the search for a causal gene in the loci, but no clear single causal gene has been established. A CHN1 gene has been more clearly demonstrated to be a causal gene for DRS2. CHN1 is located on chromosome 2 and encodes two Rac-specific guanosine triphosphatase (GTPase)-activating alpha-2-chimerin isoforms. Miyake et al. identified seven heterozygous missense mutations in seven unrelated DRS2 families co-segregating with the affected haplotypes CHN1 mutations were present in 7 of 20 (35%) examined DRS families, while no CHN1 mutations were observed in 140 sporadic DRS patients CHN1 were found in two families with type 2 DRS The While loci have been established that account for a portion of type 1 and type 2 DRS, the genetic components of type 3 DRS are more elusive. It is possible that the type 3 DRS is more heterogeneous than the other two classes. In a thin-sectioned magnetic resonance imaging (MRI) study, the abducens nerve was reliably observed in 60 eyes of 30 individuals from a control group. The abducens nerve on the affected eye was absent in 18 of 18 eyes from 16 patients with type 1 DRS, and in 2 of 2 eyes from type 2 DRS patients. The nerve was absent in only 3 of 5 eyes from five patients with type 3 DRS In addition to the ocular anomalies of the basic form of DRS, Okihiro syndrome  is associated with additional abnormalities affecting the upper limbs and, less commonly with renal anomalies and sensorineural hearing loss SALL4 zinc finger transcription factor gene were the first causal genetic alterations discovered for Okihiro syndrome patients SALL1 gene. They successfully identified mutations in SALL4 gene from five of eight Okihiro families SALL4 gene in affected individuals originally reported by Okihiro et al. in 1977, as well as 2 additional families SALL4 mutations carriers TBX5 gene. Whole mount in situ hybridization analysis of Sall4 expression during mouse embryogenesis shows prominent expression in midbrain and branchial arches and suggests that a dosage reduction of Sall4 might disrupt abducens nerve development Mutations in the Although the genetic origins of strabismus remain to be fully deciphered, several animal models of the phenotype have been studied and may serve as resources in the search for causal genes. Most of the model animals described below are albinos, with pigmentation loss ranging from partial to complete. Visual abnormalities, including strabismus, have been linked with albinism in diverse mammals such as albino primates, white tigers, and albino cats (including Siamese cats) Animals with cross-eyes have become popular images on the Internet. Joco, a cross-eyed lion at the Erfurt Zoo (Germany) is most likely to suffer from congenital strabismus. The cross-eyed opossum Heidi at the Leipzig Zoo (Germany) became a celebrity, but the condition was likely environmentally triggered. The causes of strabismus in animals vary, with only a portion deriving from genetic influence. Finding suitable animal models for the study of non-syndromic strabismus could accelerate research efforts.TYR gene encoding tyrosinase is expressed normally in cooler extremities, giving a darker color, while expression is reduced in warmer parts of the body, leading to poor pigmentation. Anatomical studies show that axons of temporal retinal ganglion cells go to the opposite side of the brain instead of staying on the same side as observed in non-albino cats TYR genes into such albino mice and rabbits corrects for axon misrouting TYR mutations contribute to strabismus in humans. While the link between strabismus and axon misrouting is unknown, genes directly involved in optic chiasm development might be considered as candidates In Siamese cats, a temperature-sensitive mutated The unusual axon wiring pattern observed in TYR defective albino animals raises concern that these animals may not be suitable models for human strabismus. Artificially induced strabismus models, such as those established by tenotomy (tendon lengthening) and by exposure to early abnormal visual experience, may be similarly ill-suited to study genetic influences on strabismus. To evaluate the relevance of artificially induced strabismic cats, the ocular dominance distributions for cats with induced strabismus and natural strabismus were compared and found to be similar. Approximately 35% of cells were monocular in either strabismus group, but a statistically significant difference was noted with normal cats, which have 81% binocular cells The causal genes predisposing to non-syndromic forms of strabismus remain to be discovered. The combination of next generation sequencing with both large-scale populations and targeted families may soon reveal critical genes and consequently confirm or expose critical molecular mechanisms. Genome-wide association studies have been reported to be underway, while exome sequencing family-specific studies of non-syndromic strabismus are likely to emerge soon


Drosophila, is involved in this process in the embryonic chick. The chicken homolog of Numb is expressed throughout the otocyst at early stages of development and is concentrated at the basal pole of the cells. It is asymmetrically allocated at some cell divisions, as in Drosophila, suggesting that it could act as a determinant inherited by one of the two daughter cells and favoring adoption of a hair-cell fate. To test the implication of Numb in hair cell fate decisions and the regulation of Notch signaling, we used different methods to overexpress Numb at different stages of inner ear development. We found that sustained or late Numb overexpression does not promote hair cell differentiation, and Numb does not prevent the reception of Notch signaling. Surprisingly, none of the Numb-overexpressing cells differentiated into hair cells, suggesting that high levels of Numb protein could interfere with intracellular processes essential for hair cell survival. However, when Numb was overexpressed early and more transiently during ear development, no effect on hair cell formation was seen. These results suggest that in the inner ear at least, Numb does not significantly repress Notch activity and that its asymmetric distribution in dividing precursor cells does not govern the choice between hair cell and supporting cell fates.The Notch signaling pathway controls differentiation of hair cells and supporting cells in the vertebrate inner ear. Here, we have investigated whether Numb, a known regulator of Notch activity in  Drosophila, and parallels between these insect sensillae and the mechanosensory epithelia of the vertebrate inner ear have guided our understanding of vertebrate ear development, and in particular of the role of Notch signaling in this process  cell that undergoes a stereotyped sequence of asymmetric cell divisions. After each division, the two daughter cells adopt distinct cell fates that depend on the levels of Notch activation that they experience  to generate a cluster of four neurons  have been deposited in Genbank (KP756695 and KP756696).We used RT-PCR to isolate from E6 chicken inner ear total RNA the full-length coding sequence of chicken Numb. In mammals, four Numb isoforms can be generated by alternative splicing  constructs  tables. All procedures carried out on chicken embryos were approved by University College London and the UK Home Office. Micro-electroporation of the otic cup with plasmid DNA was performed at HH stages 13–15 (2–2.5 days of incubation) as described in Freeman et al. . After eEmbryos were decapitated and their heads immersed in 4% paraformaldehyde in PBS at 4°C for 2–12 h. For whole-mount immunostaining, the membranous part of the inner ear was dissected out from the surrounding cartilage and incubated for 1 h in PBS containing 0.3% Triton X100 and 10% goat serum. All subsequent incubations and rinses were performed in PBS with 0.1% Triton X100 (PBT). Incubations with primary and secondary antibodies were carried out in PBT for 2 h at room temperature or overnight at 4°C. Antibodies and reagents used were: rabbit serum anti-Numb . Whole-mount in situ hybridization was performed as in Ariza-Mcnaughton and Krumlauf  and green (Fluo Green) channels within the nucleus of individual cells were collected using ImageJ and the Time Series Analyzer plugin  were also collected from at least five untransfected cells. Cells with Fluo Red > (Fluo Bdg + 2 × S.D Fluo Bgd) were classified as induced cells. In order to compare data across experiments, Z-scoring values for Fluo Red and Fluo Green were computed for all the cells analyzed in each confocal stack. Statistical analysis of the data and graphs were made with the OriginPro 9.1 software (OriginLab Corporation).Samples co-transfected with the pT2-Hes5::nd2EGFP and either the pTRE-Numb-FP635 or the pTRE-FP635 (control) Tol2 plasmids were fixed for 2 h at room temperature and processed for immunostaining for MyoVIIa/HCA using Alexa 647-conjugated secondary antibody as described above. Confocal stacks (12 bits) were acquired within transfected sensory regions and the mean values of fluorescence intensity for the red  in the chick embryo. The placode invaginates to first form a cup, then a hollow epithelial sphere named the otic vesicle. At these early stages, Numb protein is detected throughout the otic epithelium, in both presumptive sensory and non-sensory regions Figures . As in tAs the otic vesicle grows, parts of the epithelium become specialized to form several sensory patches: three cristae and three maculae  in the vestibule, for balance; the basilar papilla, in the cochlea, for hearing; and the lagenar macula, at the tip of the cochlea, for balance. Within each sensory patch, progenitor cells divide repeatedly and give rise to hair cells and supporting cells. Both these differentiated cell types can arise from the same type of progenitor cell . As a control, some specimens were electroporated with RCAS-GFP plasmid DNA.Following electroporation, the inner ear epithelium showed persistent long-term expression of the transgene carried by the plasmid—GFP or exogenous Numb as the case might be—as judged by anti-GFP or anti-Numb immunostaining. This was visible from 24 h until at least 10 days after electroporation (data not shown). The duration and extent of overexpression suggest that viral DNA had become integrated, giving rise to a spreading infection. Thus, we refer below to “infected” specimens even though RCAS plasmid electroporation, rather than RCAS viral particles, was used to initiate the process.n = 4 specimens analyzed) and supporting cells   had differentiated into hair cells and 78% into supporting cells , when cells in the vestibular patches are just beginning to undergo terminal mitosis and to become committed to a hair-cell fate, the majority of GFP-positive cells showed strong Numb expression Figure . Howevers Figure . Thus, tin ovo with 10 μg of Dox. The embryos were left to develop a further 48 h before their inner ear tissue was processed for immunostaining with Myo7A (expressed in hair cell cytoplasm) and HCA antibodies. The Numb-induced samples exhibited a phenotype very similar to that resulting from RCAS-Numb electroporation: none of the Numb-induced cells (identified by FP635 expression) expressed hair cell markers  treatment Figure . This co Figures . Further Figures , an obseHes5.1, a member of the Hairy and Enhancer of Split (Hes) family of genes, whose transcription is regulated by Notch activity and which thus serve as reporters of Notch activation in a number of tissues  located within (n = 5) or abutting (n = 3) sensory patches. As in the adjacent uninfected regions, some of these cells expressed Hes5.1 and others did not; there was no obvious difference between the infected and uninfected regions, neither in the proportion of cells expressing Hes5.1 nor in their levels of expression of Hes5.1  but significant at 48 h . We conclude from these results that Numb overexpression does induce a reduction in the intrinsic levels of Notch activity in the inner ear, but this effect is relatively mild and slow to develop, and Numb is therefore unlikely to significantly contribute to the regulation of hair cell fate decisions.To test further the cell-autonomous effects of Numb on Notch signaling, we next used a Tol2 vector encoding a reporter of Notch activity, pT2-Hes5::nd2EGFP , a proneural bHLH transcription factor essential for the differentiation of the mechanosensory cells in flies, is required for hair cell differentiation in the inner ear (reviewed in Jarman and Groves, Hes5.1.In the embryonic chick inner ear, we found that Numb is initially expressed by all epithelial cells, including sensory progenitors, and analysis of fixed tissue suggest that Numb can be distributed asymmetrically in some dividing cells, as previously reported for neural progenitor cells. Numb then becomes enriched in differentiated hair cells, which have escaped Notch activation in order to commit to a hair cell fate. Our results, however, argue against the idea that Numb acts as a determinant of hair-cell fate decisions, either by blocking activation of Notch, or indeed by any other mechanism. Cells overexpressing Numb at either early or late stages of development are not biased toward a hair-cell fate, and they do not show altered expression of the Notch effector in ovo can result in a much stronger inhibition of this Hes5 reporter (Freeman and Daudet, Using a genetically encoded fluorescent Hes5 reporter to quantify more precisely the effects of Numb on Notch activity, we found that Numb-induced cells do exhibit a significant reduction in the Hes5 reporter fluorescence levels 48 h after Dox treatment. Although this effect might suggest an interference with the reception of Notch signaling, several observations conflict with that interpretation. Firstly, the reduction in Hes5 reporter fluorescence in Numb-induced cells was very variable from cell to cell and did not appear to be correlated to the levels of expression of the Numb transgene in individual cells. Secondly, there was no significant reduction in the levels of Hes5 reporter fluorescence 24 h after Numb induction. In our previous work, we found that inhibition of Notch activity with DAPT led to an almost complete extinction of the same Hes5 reporter in less than 12 h (Chrysostomou et al., One surprising observation is that none of the Numb-overexpressing cells expressed hair cell markers, suggesting that Numb misexpression could interfere with normal hair cell differentiation and survival. A small fraction of the Numb-overexpressing cells also exhibited a mis-positioned nucleus in between the hair cell and supporting cell layers, suggesting a potential defect in their apico-basal polarity. It is possible that these merely reflect a non-specific toxic effect of the overexpressed protein, but Numb certainly has documented functions in other systems that might account for these abnormalities. In particular, Numb has been implicated in endocytosis (Santolini et al., Drosophila, neuroepithelial progenitor cells of the central nervous system of vertebrates can divide along different cleavage planes, and some studies suggest that the cleavage plane can be linked to the assignment of asymmetric daughter cell fates (Chenn and McConnell, Our findings add to the questions surrounding the proposed role of Numb as an asymmetrically inherited regulator of Notch activity in other vertebrate tissues, especially in the nervous system. As in This notion was encouraged by the finding that vertebrate homologs of Numb are expressed by neural progenitor cells in chick and mammals (Zhong et al., numb show abnormalities of cortical and sensory neurogenesis (Zhong et al., numb and the closely related numblike gene in the embryonic forebrain. Studies using different drivers for this double conditional knockout reported surprisingly different results, with apparently opposite effects on cell fate choices (Petersen et al., numb and numblike in the embryonic retina leads to abnormal proliferation and severe defects in epithelial organization (Kechad et al., Studies on cultured mouse cortical progenitor cells and in the mouse retina have demonstrated an involvement of Numb in asymmetric cell division, but with complex effects, including localization of Numb to the neuronal daughter cells in some circumstances (Cayouette et al., numb and numblike in their sensory ganglia have no deficit of neurogenesis, but show defects in axonal arborization (Huang et al., Perhaps, therefore, the fundamental function of Numb in the vertebrate neuroepithelium is not to control cell fate decisions through effects on Notch signaling, but rather to control epithelial architecture through effects on cell polarity, cell adhesion, and the cytoskeleton. Indeed, there is growing evidence that Numb can have other functions apart from influencing cell fate choices: for example, mice with conditional knockout mutations of 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.