Over recent decades, epidemiological studies have been reporting worrisome trends in the incidence of human infertility rates. Extensive detection of industrial chemicals in human serum, seminal plasma and follicular fluid has led the scientific community to hypothesise that these compounds may disrupt hormonal homoeostasis, leading to a vast array of physiological impairments. Numerous synthetic and natural substances have endocrine-disruptive effects, acting through several mechanisms. The main route of exposure to these chemicals is the ingestion of contaminated food and water. They may disturb intrauterine development, resulting in irreversible effects and may also induce transgenerational effects. This review aims to summarise the major scientific developments on the topic of human infertility associated with exposure to endocrine disruptors (EDs), integrating epidemiological and experimental evidence. Current data suggest that environmental levels of EDs may affect the development and functioning of the reproductive system in both sexes, particularly in foetuses, causing developmental and reproductive disorders, including infertility. EDs may be blamed for the rising incidence of human reproductive disorders. This constitutes a serious public health issue that should not be overlooked. The exposure of pregnant women and infants to EDs is of great concern. Therefore, precautionary avoidance of exposure to EDs is a prudent attitude in order to protect humans and wildlife from permanent harmful effects on fertility. Infertility, which is defined as the inability to conceive after 1 year of unprotected intercourse, has a global prevalence of 9% Since the mid-20th century, numerous studies have reported an increasing incidence of human reproductive diseases and a consequent decline in reproductive function worldwide p-dioxin (TCDD) One of the most significant landmarks in endocrinology over the past century was the recognition that some of these chemicals are able to disrupt the closed feedback loops of the hormonal and homeostatic systems, thus being named endocrine disruptors (EDs) Consistent detection of ED residues in human serum, seminal plasma and follicular fluid has raised concern that environmental exposure to ED is affecting human fertility The main purpose of this paper is to review and summarise the major scientific developments on the topic of human infertility associated with ED exposure, integrating evidence from epidemiological and experimental studies. Examples of well-known and hypothetical ED are selected to highlight the potential effects of ED on human fertility, identifying future research directions.The PubMed database was used to search for articles published up to 31st May 2013, using the following MeSH keywords: endocrine disruptors, fertility and infertility. Only studies using the English language were considered. Altogether, 368 papers were retrieved. The abstract of every article was read. The leading review criterion was human epidemiological studies in which a link between ED exposure and infertility was evaluated. Moreover, as the interpretation of the scarce epidemiological data may be biased by many confounding factors, supporting experimental research in animal models was also considered. Although there has been an effort to list and rank all possible ED Given the complexity of the endocrine system, the mechanisms of action of ED are difficult to unravel. So far, most EDs are known to act as imperfect ligands (either agonists or antagonists) to nuclear and membrane receptors , thus interfering with hormone-regulated cell signalling pathways and gene expression Most EDs are supposed to act through several mechanisms, which may have synergistic or antagonistic outcomes Apart from ER and AR, the aryl hydrocarbon receptor (AhR) is the protein most studied regarding its interaction with ED. This orphan receptor acts as a transcription factor for detoxifying enzymes Some EDs are also capable of modifying hormone bioavailability by interfering with its secretion and transport or disrupting the enzymatic pathways involved in hormone synthesis and metabolism in vivoThe principle of endocrine disruption has always been controversial: it has been difficult to determine the lowest observed adverse effect level (LOAEL) and whether it is likely to be found Perhaps expectably, there is a sharp division between those who report detrimental effects of ED at environmental levels (micro- to picomolar range) – mostly academic experts – and those who appear unable to do so at any concentrations – industry corporations Hormone-mimetic ED, similarly to endogenous hormones, may have non-monotonic tissue-specific effects due to: receptor selectivity, down-regulation/desensitisation, competition and negative feedback loops Reliable evidence of both non-monotonic curves and low-dose detrimental effects has been gathered for BPA, many pesticides Populations are exposed to ED in air, water, food and in a variety of industrial products, including personal care goods. The mixture of ED that leaches into the soil and waterbodies  accumulates in the environment and in animals higher up on the food chain Although there is chronic exposure to ED through inhalation and skin contact Recent studies have concluded that plastic packaging is an important source of ED in the average human diet The average diet also contains natural ED such as phytoestrogens, which are compounds possessing strong oestrogen-like activity Human susceptibility to disruption during development has been proven The prenatal period has become a significant research topic regarding ED exposure because the placenta causes accumulation of ED in the foetus Epigenetic modifications may have an important role in the observed ED effects in gametogenesis and foetal development (see below). The epigenome refers to changes made in gene expression by altering DNA structure through DNA methylation and microRNA, among other mechanisms, without changing the actual genomic sequence EDs have been shown to disrupt the development of the human reproductive system, impairing fertility not only in directly exposed offspring but also in subsequent generations. A vast array of reproductive abnormalities has been reported in the offspring of women treated with DES during the mid-20th century, for miscarriage prevention in utero to environmental levels of PCB presented morphological reproductive abnormalities and impaired gamete quality Other ED have multigenerational effects: the offspring of TCDD-exposed mice show fertility disorders up to the third generation Male germ cells are considered as the most vulnerable cells, as they have distinctive methylation patterns and epigenetic markers ED exposure in pregnant females can directly cause detrimental effects in the next two generations through the foetus and its germline, which is already formed. Only adverse effects in the third generation and beyond are considered truly transgenerational, as they are transmitted solely through the germline As current assisted reproduction techniques do not necessarily address the underlying infertility problem, their escalating use may accidentally convey serious genetic and epigenetic anomalies Millions of children are conceived by women while on contraceptive pills containing EE. Albeit most do not show conspicuous congenital abnormalities, long-term reproductive consequences may ensue in adulthood ED may act synergistically to produce adverse effects at doses far below individual LOAEL, if there is enough overall exposure Additionally, it is hypothesised that phytoestrogens, among other EDs, may be capable of altering cell responsiveness to endogenous hormones and other ED, thereby inducing wider negative effects when there is concomitant exposure Over the last decades, epidemiological studies have reported an ominous growth in the incidence of male infertility, accompanied by decreasing sperm quality, thus reflecting impaired spermatogenesis Studies comparing male reproductive disorders in the Nordic–Baltic countries have reported an East–West gradient showing higher reproductive tract abnormalities and infertility rates in Denmark compared with Finland Actually, several epidemiological studies have found an association between inferior semen quality parameters and increased urinary and serum levels of phthalates There is an epidemiological correspondence between lower semen quality and higher incidences of cryptorchidism, hypospadias and testicular cancer Epidemiological data suggest that human developmental exposure to environmental levels of ED  is indeed connected to an increased risk of TDS features such as hypospadias and cryptorchidism Assuming the same circumstances of exposure, deleterious effects of ED may be more severe in individuals with genetic susceptibility. There are AR and ER-α genetic polymorphisms that cause mild functional impairments Hypospadias, a condition in which the urethral meatus is on the ventral side of the penis, affects about 0.4% of males at birth and has been reported to have increased significantly over recent decades Cryptorchidism is defined as the failure of one or both testicles to descend into the scrotal sac and is the most common congenital abnormality in male children, affecting 2–4% of full-term males Testicular migration is a complex process involving a transabdominal stage and a transinguinal one. Developmental exposure to ED may act on Leydig cells thus disrupting both stages by i) reducing insulin-like factor 3 expression The differentiation of the male reproductive system is entirely dependent on foetal testicular androgen production A recent study including a thousand new borns has found a linear correlation between maternal exposure to ED (e.g. pesticides and phytoestrogens) and lower testosterone levels, smaller penile length and higher incidences of reproductive anomalies including hypospadias In animal models, pregnant mice orally exposed to phthalates at doses as low as 1 μg/kg BW per day consistently gave birth to male offspring presenting a syndrome of reproductive anomalies including cryptorchidism, testicular injury, reproductive tract malformations and shorter anogenital distance (AGD) Similarly to rodents, human male infants exhibit twice as long an AGD than females in utero to 20–100 mg/kg BW per day of VCZ showed hypospadias and minute sperm counts Other anti-androgenic ED can induce TDS in animals: rats exposed to 150 mg/kg BW per day of acetaminophen during foetal development had AGD reductions comparable to those induced by phthalates Though average human ED exposure levels may be lower than those customarily used in animal studies, certain population clusters may be exposed to higher levels. Actually, occupational pesticide exposure has been connected to male infertility Data concerning ED effects on the female reproductive system and fertility are scant. Still, a correlation between developmental ED exposure and long-term effects is suggested The array of female reproductive disorders where ED have been implicated includes endometriosis, disorders of the uterus and disorders of the ovary, such as premature ovarian failure (POF) and polycystic ovary syndrome (PCOS) in utero may have an 80% higher risk of endometriosis than unexposed women Endometriosis affects up to 10% of women of childbearing age, causing infertility in about half those women Experimental studies support this hypothesis, as intrauterine exposure of mice to BPA There are growing concerns about the reproductive outcomes of ovarian exposure to ED during foetal development and after birth Granulosa and theca cells, which are crucial for ovarian steroidogenesis and oocyte development, are also a target for ED PCOS, consisting of hyperandrogenemia and chronic anovulation, affects 5–8% of women of childbearing age often leading to infertility Proper differentiation of the female reproductive system is regulated by oestrogens, but it proceeds even in their absence – it is the default developmental pathway HOX genes in the Müllerian duct HOXA10 by ED that either up-regulate (e.g. BPA) or down-regulate (e.g. DES and MXC) its expression has been shown to lead to uterine abnormalities and infertility WNT7 or MSX2 genes The development of the female reproductive system is regulated by the differential expression of in utero and during the first year of life ED may modify steroidogenesis both locally and through the HPG axis Kisspeptin is broadly recognised as a fundamental activator of the HPG axis, at the onset of puberty Some PCBs have been shown to alter gonadotropin-releasing hormone (GnRH) synthesis The biological ED effects through GnRH and kisspeptin neurons and the relative importance of disruption in each those cell clusters on the onset of puberty and fertility throughout life through remain unclear.Disruption of the HPG axis leading to gonadal insufficiency by reducing steroidogenesis, following exposure to DES Sex steroids have prominent roles in the differentiation of several sexually dimorphic neural circuits This paper has reviewed the existing evidence regarding ED and the rising rates of human infertility. Although the number of ED mentioned is not comprehensive, an adequate amount of data has accumulated demonstrating that EDs may have deleterious effects on human reproduction via numerous mechanisms. ED may be blamed for the rising incidence of human reproductive disorders, and may also explain some idiopathic infertility cases, both in men and women.Endocrine disruption is a serious public health problem that must not be ignored. Authorities should endorse preventive measures regarding exposure to EDs, such as limiting their production in industry worldwide, as the removal of these substances from the environment is neither simple nor cheap.Meanwhile, the general population might reduce ED exposure by following some simple yet important advice such as i) choose glass over plastics, ii) avoid using plastic containers repeatedly or plastic wrapping to microwave food, iii) reduce consumption of fatty animal products, iv) prefer pesticide-free vegetables and fruits and v) avoid excessive utilisation of cosmetics and other personal care items, particularly during pregnancy. As ED exposure at any dose may impair human development and reproduction, precautionary avoidance of exposure to well-known and putative ED is a prudent attitude.Further research is needed to improve current knowledge about known ED, and to identify potential endocrine disruptive activity by other chemicals, especially those replacing current ED before they are widely distributed. Dose–effect curves should be thoroughly studied, even at minute concentrations, as all EDs are likely to show non-monotonic responses and low-dose effects, resembling those elicited by endogenous hormones. Also, the impact of exposure to low doses of complex mixtures of ED and the prospective transgenerational effects should be evaluated, specifically concerning genetic polymorphisms, especially during gametogenesis and foetal development. It would be important to examine adult fertility and hormonal parameters of infants inadvertently exposed to contraceptive hormones during pregnancy and of infants fed cow milk/soy-based formula using baby bottles made of different substances, as opposed to breastfed infants. Clinical and laboratorial research on ED is essential, in order to protect wildlife and humans, particularly developing foetuses and children, from permanent effects on fertility.


Advanced head and neck cancers are difficult to manage despite the large treatment arsenal currently available. The multidisciplinary effort to increase disease-free survival and diminish normal tissue toxicity was rewarded with better locoregional control and sometimes fewer side effects. Nevertheless, locoregional recurrence is still one of the main reasons for treatment failure. Today, the standard of care in head and neck cancer management is represented by altered fractionation radiotherapy combined with platinum-based chemotherapy. Targeted therapies as well as chronotherapy were trialled with more or less success. The aim of the current work is to review the available techniques, which could contribute towards a higher therapeutic ratio in the treatment of advanced head and neck cancer patients.  The major goal of cancer treatment is to improve the clinical outcome by increasing the therapeutic ratio (TR). Most commonly, the therapeutic ratio is quantitatively defined as the ratio between tumour control probability (TCP) and normal tissue complication probability (NTCP). In order to maximise the therapeutic ratio, tumour control needs to increase while normal tissue complications need to decrease. As with any other malignancy, the objective in the treatment of advanced head and neck cancer is to improve TR through both components: TCP and NTCP.After decades of treatment optimisation via novel irradiation techniques, new cytotoxins and several adjuvant agents to improve tumour response to therapy, advanced unresectable head, and neck cancers are still a clinical challenge. Although the locoregional control showed improvement along the implementation of new treatment techniques, the death rate does not seem to decline for this malignancy .Several randomised clinical trials showed a significant improvement in locoregional tumour control and disease-free survival when radiation was combined with cisplatin, as compared to radiation as a single agent , 3, reasin vivo action of cisplatin as subsequent lethal mitosis may be the most significant mechanism of cell death induced by this drug [Cisplatin is a platinum compound with complex properties when it comes to radiation-drug interaction. Through inhibition of DNA repair and cell cycle arrest cisplatin demonstrates radiosensitizing properties . Furtherhis drug . One of his drug . Yoshikahis drug . Despitehis drug . At the his drug .Irrespective of the exact mechanism that leads to radiosensitization cisplatin is a powerful drug, and since its clinical implementation it remains a fundamental cytotoxic agent for the management of head and neck cancer.Tumour hypoxia and accelerated proliferation of tumour cells during therapy (both radiotherapy and chemotherapy) remain some of the biggest challenges concerning the treatment of advanced head and neck cancer. The unpredictability of acute hypoxia in tumours often leads to treatment failure, and so does the rapid proliferation of tumour cells after the initiation of therapy. Cellular recruitment from the quiescent phase, accelerated accelerated stem cell division, abortive division, and loss of asynchronous stem cell division are thought to be the main mechanisms responsible for accelerated regrowth in squamous cell carcinomas of the head and neck –13. PerhAlthough altered fractionation increased locoregional control, there were trials that showed no treatment gain because of normal tissue complications. For instance, in the EORTC 22851 randomised trial , where hWhile being one of the most potent chemotherapeutic agents, cisplatin is highly toxic to various organs. Nephrotoxicity and ototoxicity are some of the most commonly reported side effects during cisplatin-based chemotherapy. Other side effects, such as hematologic and central nervous system-related toxicities, are often dose-limiting factors or reason for treatment interruption. The rate of treatment completion is frequently reported to be below 100% due to adverse events. New radiotherapy delivery techniques might be a possible solution in reducing side effects due to better tumour conformity, as shown by studies comparing helical tomotherapy to conventional IMRT . HoweverThe risk of developing a second primary cancer after head and neck cancer treatment was shown to be strongly linked to the original risk factors, which initiated the first primary cancer, that is, smoking and alcohol consumption as well as the oncogenic human papillomavirus . Thus thHowever, there is evidence that chemotherapy, particularly cisplatin, can induce carcinogenesis in patients treated with this agent for their primary malignancy. While proven to be toxic to normal tissue since the beginning of its clinical use, cisplatin was not shown to be carcinogenic until later. A large clinical study undertaken by Travis et al. showed an increased risk of leukaemia in patients previously treated with cisplatin , 21.  FiThe most commonly used methods to optimise the therapeutic ratio are reviewed below. While some of these techniques are widely accepted among the medical community  others either are less successful in their clinical implementation or await more conclusive results .There is an extensive number of trials on head and neck cancer comparing the clinical effect of combined chemoradiotherapy versus radiotherapy alone. In order to collate and examine the results, a metaanalysis of the role of chemotherapy in head and neck cancer (MACH-NC) was published based on 93 randomised trials (conducted between 1965 and 2000) , which cThe advances in knowledge concerning head and neck radiobiology over the last few decades lead to clinical implementation of various altered fractionation schedules with and without chemotherapy. Altered fractionation radiotherapy combined with cisplatin-based chemotherapy became a common practice within the management of advanced head and neck cancer patients . IntensiThe studies presented in The addition of bevacizumab  or cetuxAlthough locoregional control is significantly improved with the conversion of conventional therapy into IMRT, long-term survival remains at low rates, mainly due to tumour recurrence.P = 0.18), disease-free survival , or locoregional failure . Also, there was no significant difference in the overall grade 3-4 acute mucositis (33% versus 40%) or grade 3-4 late toxicity (26% versus 21%) [The mixed results achieved with altered fractionation combined with chemotherapy suggest that there is need for a careful selection of patients who would benefit from such therapy. The RTOG 0129 phase III randomised trial showed that combining cisplatin with altered fractionation (accelerated concomitant boost) does not give superior clinical results to standard fractionation combined with cisplatin [2/day to 25 mg/m2/day. Dose limiting toxicities in the form of gastrointestinal disorders were experienced for the highest dose hence the dose recommended for phase II trial was 20 mg/m2/day. Daily small doses of cisplatin have demonstrated good tolerability in the past even in injectable form [One of the main challenges with injectable cisplatin is normal tissue toxicity. Therefore, the idea of oral administration of cisplatin was worth testing in order to investigate its level of tolerability. Tao et al. have designed a dose-escalation trial where oral cisplatin (CP Ethypharm) was administered in combination with radiotherapy to 18 head and neck cancer patients . Four ciin vitro also with less normal tissue toxicity emerges as a strong foundation for further in vivo experiments.A new platinum compound is on the horizon, namely, mitaplatin, which is a fusion between cisplatin and the orphan drug dichloroacetate previously developed to treat lactic acidosis , 32. It Image-guidance represents today an important tool for increasing the therapeutic gain by better targeting the tumour, especially during fractionated radiotherapy when tumour shrinkage is expected over time and by better sparing of the surrounding normal tissue. Image-guided radiotherapy can be optimally achieved by employing cone beam CT during the course of radiotherapy then adapting the treatment plan according to the new tumour parameters .Another imaging method, which assists in improving tumour control in head and neck cancer patients, is PET/CT. The functional properties supplied by PET together with the anatomical tumour delineation offered by CT provide a powerful tool in tumour classification and prediction of treatment outcome as well as selective targeting of hypoxic regions within the tumour. Beside 18F-FDG, which is still considered the standard radiotracer for PET imaging, there are hypoxia-specific radiotracers  as well as proliferation-specific radiotracers (F-FLT) successfully used in clinical settings .A clinical study conducted by Rothschild et al.  employinAn innovative radiation therapy trial is currently accruing patients for analysing the predictive value of biological markers and 89Zr-cetuximab uptake in head and neck cancer treated with cisplatin versus cetuximab and standard radiotherapy versus redistributed radiotherapy. The focus of the ARTFORCE trial is on individualised treatment, using functional imaging for the assessment of dose escalation to the FDG-PET positive region and adaptive replanning accounting for anatomical changes during treatment .The epidermal growth factor receptor (EGFR) plays a vital role in head and neck cancer development, growth, and metastatic spread and angiogenesis, owing to promotion of epidermal cell growth and regulation of cell proliferation. It was clinically proven that overexpression of EGFR leads to increased tumour proliferation and other growth-promoting behaviour. Of all head and neck squamous cell carcinomas a value as high as 90% exhibits overexpression of epidermal growth factor receptor .A clinical trial conducted by Bonner et al.  aimed toThe combined effect of cetuximab and cisplatin-based chemoradiotherapy was studied in a phase II trial for patients with advanced head and neck cancer . CetuximAnother anti-EGFR monoclonal antibody investigated as an agent for targeted therapies in head and neck cancer is panitumumab. As shown by preclinical studies, panitumumab has higher affinity for EGFR than other monoclonal antibodies developed to target EGFR . This miAngiogenesis inhibitors target those signalling molecules which stimulate the endothelial cells to migrate, divide, and form new blood capillaries. One such signalling molecule is the vascular endothelial growth factor (VEGF). Antiangiogenic drugs bind to VEGF before they could connect with the receptors of the endothelial cell to initiate the angiogenic process.An angiogenic inhibitor that recognises and binds to VEGF is bevacizumab, a monoclonal antibody which was the first agent of its kind to slow tumor growth in glioblastoma, nonsmall cell lung cancer, and metastatic colorectal cancer patients . Fury etAn interesting study undertaken by Wang et al.  investigin vivo studies via intratumoral injection. While the mechanism behind this behaviour is unclear, the results warrant further investigations.The same research group showed that bevacizumab has antitumour effect beyond antiangiogenesis, potentiating the cytotoxicity of cisplatin on squamous cell carcinomas . The cheThe rate and degree of normal tissue toxicity in head and neck cancer patients treated with radiation have justified the clinical need for a normal tissue radioprotector for reduction of side effects.Amifostine is a commonly used normal tissue radioprotector, which has been trialled in combination with both radiotherapy and chemoradiotherapy. Amifostine was also proven to be an effective cytoprotector against the side effects caused by cisplatin ; therefoThe future use of amifostine remains uncertain owing to conflicting results regarding normal tissue toxicities. While some of amifostine's properties such as selectivity  and safety were confirmed by several studies, the reduction in normal tissue toxicity was not always demonstrated .It is a known and accepted fact that biological functions in humans are organised around a circadian (day/night) clock. Circadian rhythms are controlled by the suprachiasmatic nucleus of the hypothalamus, also known as the “master pacemaker”. Consequently, several biological processes are dictated by this clock including sleep, hormone secretion, and cell proliferation. Experimental findings suggest the existence of crosstalk between clock gene molecules and those molecules, which are responsible for cellular progression through the cell cycle .Chronotherapy refers to treatment that is timed around this biological clock, which was shown to differ between normal and cancer cells , 55. ChrOne of the most common side effects during and after chemoradiotherapy of head and neck cancers are those involving the oral mucosa. The goal of chronotherapy is to take into account the biological clock of various tissues in trying to schedule treatment in the most opportune time for the tumour and the least harmful time for the normal cells. Therefore, investigating the peak times of oral mucosa cells could dictate the timing of treatment to diminish side effects. Bjarnason et al. have investigated the circadian variation of human oral epithelium through the expression of cell-cycle proteins  and veri2/M. During this time, normal cells would receive better protection, as they peak in G1.Experimental studies support the evidence whereby the DNA synthesis rhythm in normal cells is in phase opposition with that of tumour cells, observation that is valid for the mitotic phase as well , 57. Con1. Patients were randomised to morning (8–10 am) versus afternoon (4–6 pm) radiotherapy. The results showed a significant improvement in weight loss for the morning group as compared to patients receiving radiotherapy in the afternoon and reduced incidence of oral mucositis among male patients (49.4% versus 64.1%). It was suggested that a greater treatment time difference between the two arms could have lead to a greater advantage of morning radiotherapy.There are clinical studies supporting the evidence whereby treatment timing according to cellular circadian rhythm can improve treatment outcome in head and neck cancer patients. Bjarnason et al.  conducteIt was shown that circadian dosing time could also influence drug-related toxicities . ConsequThe results achieved with chronotherapeutics in leukaemia patients, ovarian cancer patients, and metastatic colorectal cancer patients are even more pronounced. For instance, a twofold increase in survival and disease free rate after 5 years was reported in a chronotherapy trial involving children with acute lymphoblastic leukaemia when chemotherapy (antimetabolites) was administered in the evening  versus morning treatment  , 61. TheTo obtain conclusive results with head and neck cancer chronotherapy, there is need for further studies involving a multidisciplinary approach and an open-minded attitude towards less orthodox treatment methods that showed promising results in the past.Advanced head and neck cancers are difficult to manage despite the large treatment arsenal currently available. The multidisciplinary effort to increase disease-free survival and diminish normal tissue toxicity is rewarded with better locoregional control and sometimes fewer side effects. Nevertheless, locoregional recurrence is still one of the main reasons for treatment failure.In order to increase therapeutic ratio, there are methods to improve tumour control as well as normal tissue sparing. Some of the techniques to achieve these goals are listed below.optimum fractionation schedules;optimum timing between radiotherapy and cisplatin administration based on cisplatin's pharmacokinetics and pharmacodynamics as well as the interaction between cisplatin and radiation;knowledge of pretreatment radiobiological tumour parameters such as oxygenation status and cellular proliferative capacity;image-guidance during radiotherapy;EGFR inhibitors such as cetuximab;angiogenic inhibitors;chronotherapy involving knowledge of tumour circadian rhythm.Techniques to increase TCP:optimum timing between radiotherapy and cisplatin administration;more conformal radiotherapy (IMRT);image-guidance during treatment;normal tissue radioprotectors such as amifostine;chronotherapy involving knowledge of normal tissue circadian rhythms, especially of the oral mucosa and bone marrow to diminish side effects of both radiotherapy and chemotherapy.Techniques to decrease NTCP:The latest treatment techniques combined with adjuvant and/or targeted therapies succeeded in increasing locoregional control in advanced head and neck cancer patients. The downside, however, is the increased rate of side effects. Furthermore, overall survival in this patient group has not seen any considerable improvement over the last decades. While there are promising results with targeted therapies involving monoclonal antibodies as well as with chronotherapy, the optimal treatment for advanced head and neck cancer patients is yet to be established.


Early temporary windows of heightened brain plasticity called critical periods developmentally sculpt neural circuits and contribute to adult behavior. Regulatory mechanisms of visual cortex development – the preeminent model of experience-dependent critical period plasticity-actively limit adult plasticity and have proved fruitful therapeutic targets to reopen plasticity and rewire faulty visual system connections later in life. Interestingly, these molecular mechanisms have been implicated in the regulation of plasticity in other functions beyond vision. Applying mechanistic understandings of critical period plasticity in the visual cortex to fear circuitry may provide a conceptual framework for developing novel therapeutic tools to mitigate aberrant fear responses in post traumatic stress disorder. In this review, we turn to the model of experience-dependent visual plasticity to provide novel insights for the mechanisms regulating plasticity in the fear system. Fear circuitry, particularly fear memory erasure, also undergoes age-related changes in experience-dependent plasticity. We consider the contributions of molecular brakes that halt visual critical period plasticity to circuitry underlying fear memory erasure. A major molecular brake in the visual cortex, perineuronal net formation, recently has been identified in the development of fear systems that are resilient to fear memory erasure. The roles of other molecular brakes, myelin-related Nogo receptor signaling and Lynx family proteins – endogenous inhibitors for nicotinic acetylcholine receptor, are explored in the context of fear memory plasticity. Such fear plasticity regulators, including epigenetic effects, provide promising targets for therapeutic interventions. As the brain develops, particular regions undergo different critical periods of plasticity when their underlying circuits gain heighted sensitivity to experience , 2. ExpeThe visual system offers valuable insight through the study of critical period mechanisms. In humans and animals, visually depriving one eye by obstructing it early in life yields loss in visual acuity (amblyopia) by stimulating an anatomical remodeling within primary visual cortex . ImportaEvidence from both animal and human studies suggest that the pathways underlying fear systems also undergo age-related changes in experience-dependent plasticity. Such age-related changes have been observed in both fear memory acquisition and extinction. Fear acquisition, measured through the ability to develop conditioned context-shock fear responses, does not emerge until 13–14 days . HoweverTo what extent are visual and fear system critical period mechanisms parallel? Both the visual and fear systems maintain a developmentally limited ability to rewire connections that are no longer functionally appropriate. In the visual system, critical period (but not adulthood) monocular deprivation triggers a functional re-adaptation via visual cortex restructuring that reduces the input from the visually deprived eye. Early development fear extinction modifies behavior by erasing fear memories that are no longer appropriate, and differs from adult extinction that is temporary and slower. Thus, to discuss the role of molecular regulators in critical periods of plasticity, we consider them within their functional contexts and in functional parallels between systems.To interrogate the extent to which the regulatory mechanisms of critical period plasticity in the visual system apply to the fear system, we conceptually consider “permanent fear erasure” analogously to “amblyopia,” both of which are only induced during critical period. Accordingly, we also consider “fear extinction procedure” in fear system and “monocular deprivation” in the visual system as the inducers of plasticity in each system. Because the juvenile form of fear extinction is also marked by accelerated changes in response compared to that of adult mice , we consRecent studies using rodent visual cortex have identified multiple structural and functional molecular “brakes” that actively limit plasticity and close the critical period in the adult brain , 21. StrPerineuronal nets are extracellular macromolecular aggregates associated with several subclasses of chondroitin sulfate proteoglycans (CSPGs) that surround neuronal cell bodies and proximal dendrites . In the In the fear system, the PNNs in the amygdala recently have been shown to play a central role in modifying the plasticity of fear memories and may contribute to protection against fear memory lability during acquisition. The number of CSPG containing PNNs increases in the murine amygdala between days 16 and 23 – the time coinciding with preadolescence and the developmentally related functional switch from fear erasure to less effective fear extinction  Figure . When adIn the visual cortex, experience-dependent plasticity is limited by myelin-related Nogo receptor signaling . The endIn a recent study, Nogo receptor knock-out mice were reported to show more pronounced fear extinction compared to wild type mice  Figure B. At theWhile structural brakes can limit plasticity by altering local connectivity, functional brakes can also halt plasticity by altering the neurotransmission between connections that have been formed to facilitate plasticity. In the visual cortex, a newly discovered class of proteins, the Lynx family, has been recently identified as a class of functional brakes . Lynx1, The Lynx family may also have developmental roles in fear system. Lynx1 and closed related Lynx2 are both expressed in the amygdala and change its expression levels across development in both rodent and human . Both Lyd-cycloserine (DCS), a partial NMDA agonist, facilitates extinction, and prevents the recovery of fear memories in rats, mice, and humans both before and after extinction training . C. C51). CAnother class of pharmaceutical agents shown to induce recovery of visual function in the adulthood is the acetylcholinesterase inhibitor (AchEI). In the visual system, AchEI injection can restore vision in adult wild type mice with amblyopia, a disorder in which the eye, though structurally normal, has impaired vision due to poor functional connectivity to the visual cortex  Figure C. By incAchEI treatment has not been directly examined in the context of fear extinction and memory liability, but nicotine dosing immediately before training has been reported to enhance extinction training . As AChEIn the visual system, experience-dependent modifications of histone acetylation are developmentally down-regulated, implicating epigenetic mechanisms in the regulation of critical period plasticity . AdminisIn the fear system, systemic, as well as direct, application of HDAC inhibitors into the hippocampus or medial prefrontal cortex (mPFC) prior to fear conditioning and prior to extinction enhances extinction learning –64 FiguD. Oral aEnvironmental enrichment  and darkA newly proposed behavioral intervention for treating PTSD, “reconsolidation update,” involves targeting traumatic memories as an individual reconsolidates it because the memory is rendered labile after it has been recalled Figure D. ExtingIn this review, we considered the potential contributions of “molecular brakes” identified in visual system development, the major model of critical period plasticity, to the development of fear system connections. Striking similarities between the molecular mechanisms underlying the development of these two brain regions, as well as therapeutic approaches to their dysfunction, indicate that new mechanisms identified in the visual critical period can provide both novel insights and a conceptual framework for exploring novel therapeutic approaches to aberrant fear responses in PTSD patients. Future studies examining the contributions of molecular brakes, such as myelin-related nogo receptor signaling and the Lynx family, as well as their epigenetic regulators in the context of the fear system development, may shed light on new targets for therapeutic intervention. The interactions between multiple brakes have yet to be connected – not only those mentioned in this review, but also well-established age-related mechanisms, such as hippocampal neurogenesis. Future research manipulating critical period mechanisms for clinical use will require further elucidation of systems in which these molecular brakes are involved. The mechanism of re-closure after the therapeutic reopening the critical period is another important area of investigation. Finally, better comprehension of the scope of these regulators to realize both their therapeutic potential as well as the undesired consequences of increased brain plasticity is needed.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.


Evaluating environmental health risks in communities requires models characterizing geographic and demographic patterns of exposure to multiple stressors. These exposure models can be constructed from multivariable regression analyses using individual-level predictors (microdata), but these microdata are not typically available with sufficient geographic resolution for community risk analyses given privacy concerns.We developed synthetic geographically-resolved microdata for a low-income community  facing multiple environmental stressors. We first applied probabilistic reweighting using simulated annealing to data from the 2006–2010 American Community Survey, combining 9,135 microdata samples from the New Bedford area with census tract-level constraints for individual and household characteristics. We then evaluated the synthetic microdata using goodness-of-fit tests and by examining spatial patterns of microdata fields not used as constraints. As a demonstration, we developed a multivariable regression model predicting smoking behavior as a function of individual-level microdata fields using New Bedford-specific data from the 2006–2010 Behavioral Risk Factor Surveillance System, linking this model with the synthetic microdata to predict demographic and geographic smoking patterns in New Bedford.Our simulation produced microdata representing all 94,944 individuals living in New Bedford in 2006–2010. Variables in the synthetic population matched the constraints well at the census tract level  and reproduced the census-derived spatial patterns of non-constraint microdata. Smoking in New Bedford was significantly associated with numerous demographic variables found in the microdata, with estimated tract-level smoking rates varying from 20%  to 37% .We used simulation methods to create geographically-resolved individual-level microdata that can be used in community-wide exposure and risk assessment studies. This approach provides insights regarding community-scale exposure and vulnerability patterns, valuable in settings where policy can be informed by characterization of multi-stressor exposures and health risks at high resolution. Evaluation of environmental health risks in communities has increasingly focused on the combined risks to health from multiple agents or stressors, defined by the United States Environmental Protection Agency (US EPA) as “cumulative risk assessment” While this framework is appealing, there are significant analytical challenges. One such challenge is developing exposure models, which must have sufficient geographic and demographic resolution to provide relevant contrasts within the community and to identify high-risk subpopulations given correlations (positive or negative) among stressors.It is impractical to gather detailed exposure measurements or exposure-related information from all members of a community, so in many cases investigators must leverage a limited number of exposure measurements and develop broadly applicable exposure models. For air pollutants of ambient origin, monitoring networks and public geospatial databases may be sufficient to evaluate exposures, but for most other stressors, additional information would be required. For example, mercury exposure is typically dominated by fish consumption, which may be influenced by income, race/ethnicity, and other factors A significant barrier in applying these models to specific communities is the lack of geographically resolved multivariable data on key predictors. For example, the US Census provides the distribution of home age and the distribution of household income at the census tract level, but not the cross-tabulated distribution of home age by household income. If attributes are correlated with one another, which is generally the case given strong socioeconomic interdependence for many attributes, the lack of this information will lead to erroneous exposure models. Census microdata provide the detailed individual-level data needed to determine these interdependencies, but only at very coarse geographic resolution, because of the need to limit potential identifiability. Most exposure regression models involve a blend of geographically resolved  and demographically resolved  predictors, but it is not possible to obtain multivariable demographic information with high-resolution geographic data from public data resources.Simulation approaches can be used to construct geographically resolved microdata, using optimization techniques to maximize the fit between individual-level microdata lacking geographic resolution and aggregated census data at high geographic resolution In this paper, we develop synthetic microdata for a low-income community living near a Superfund site , where these synthetic microdata include multivariable individual-level attributes and spatially resolved geographic assignment. We then constructed a new model of the individual-level likelihood of smoking in New Bedford as a function of the microdata characteristics. We linked this model to the synthetic microdata as a demonstration of the usefulness of developing synthetic microdata in providing insight about the geographic and demographic patterns of exposure to a key risk factor for the residents of New Bedford, which can have further implications for use in characterizing multi-stressor exposures.th-lowest median household income. Residents had elevated rates of adult diabetes, hypertension, high cholesterol, smoking, and obesity New Bedford, Massachusetts is a diverse urban city with approximately 95,000 residents in 2010 . The NewTwo general approaches have been used in the literature to generate synthetic microdata Probabilistic reweighting using simulated annealing has been shown to perform slightly better than alternative reweighting approaches, such as deterministic reweighting or conditional probability modeling Generating synthetic microdata using probabilistic reweighting requires two basic types of data – multivariable individual-level microdata lacking resolved geographic identifiers, and small area constraints to place the individuals in appropriate locations. We derived both from the ACS 5-year database (2006–2010). The ACS surveys a fraction of the US population each year, and the 5-year estimates are generally used to determine estimates for smaller geographic areas (like census tracts). For the microdata, we used ACS data from the Public Use Microdata Sample (PUMS), which provides geographic identifiers for Public Use Microdata Areas (PUMAs) comprising at least 100,000 individuals. The PUMA of interest for our analysis includes New Bedford as well as bordering or nearby communities . Based oFor small area constraints, we focused on the census tract as the unit of analysis, because more candidate constraints were available at the census tract level than at the block group level. There are 31 census tracts in New Bedford , with poFor a given set of constraints, the goodness of fit was evaluated using the relative sum of square Z-scores associated with individual constraint tables, in comparison with the chi-square critical value (p < 0.05). As described by Williamson Synthetic microdata need to be evaluated across three major dimensions to ensure that the individuals have been assigned to appropriate geographic areas, therefore making the microdata applicable to community-scale modeling of environmental health risks.Evaluation 1. First, the degree of error needs to be assessed for the microdata fields included in the constraints .Evaluation 2. Second, the degree of error needs to be assessed for microdata fields that are not included in the constraints but are potentially important for evaluating environmental health risks in the community. While greater error would be anticipated for fields not included as constraints, it is important to know whether the complete multivariable microdata reasonably reflect the spatial and demographic patterns described in the ACS. For this second evaluation, we focused on whether the geographically resolved microdata reasonably predict the percentage of multi-family homes and number of rooms per home by census tract. Both of these variables are potentially relevant for housing-related exposures and are not simply explained by the constraint variables .Evaluation 3. Third, the synthetic microdata must be able to characterize geographic and demographic distributions for key stressors of interest not available in the microdata, using regression models explaining variability as a function of the microdata. These distributions cannot be directly validated in the same manner as in the first two evaluations above, because the lack of geographically and demographically refined exposure data is the precise rationale for this modeling approach. However, some of the aggregated estimates can be compared with surveillance data or previous models. To conduct the third evaluation, we developed a multivariable regression model for smoking, given that direct smoking and resulting environmental tobacco smoke are associated with multiple health outcomes of concern to the community. We obtained 2006–2010 Behavioral Risk Factor Surveillance System (BRFSS) data from the Massachusetts Department of Public Health (MA DPH), which contains smoking status and extensive demographic data for 4,348 individuals living in New Bedford across the five study years. We constructed a multivariable logistic regression to predict smoking status as a function of demographic variables. We considered as candidate predictors variables that had previously been associated with smoking in the literature and were available from ACS-derived synthetic microdata, including age, gender, marital status, race, ancestry, educational attainment, household income, and employment status. All predictors were constructed to be identical in structure as the covariates in the synthetic microdata, and both the regression model and microdata are at the individual level, allowing for direct model application. While formal validation is not possible, we predicted smoking prevalence for demographic subpopulations described in MA DPH reports Because our simulation modeling involved multiple uncertainties, we used Monte Carlo analysis to quantify these uncertainties in the predicted geographic and demographic patterns of smoking in New Bedford. We incorporated uncertainty at two levels. First, we included uncertainty in the smoking regression model by randomly sampling coefficient values given estimated means and standard errors, using the variance-covariance matrix to account for correlations among coefficients. This yields an uncertainty distribution of smoking probabilities for each individual. Second, we randomly assigned smoking status to individuals given these probabilities, so that each individual had a binary (yes/no) assignment in each of the 5000 realizations. This allowed us to characterize uncertainties in estimated smoking rates for any geographic or demographic subpopulation of interest.Applying all 13 candidate constraints yielded synthetic microdata for New Bedford which perfectly matched all univariate constraints at the census tract level, with a good overall fit across all constraints . The lasThe overall total absolute error per household was well under 1 in all census tracts, exceeding 0.3 in only one census tract. For all seven univariate constraints as well as four multivariable constraints , the relative sum of squared Z-scores never exceeded the chi-square critical value, with zero or near-zero values. Only the two most complex multifactorial constraints with many categories  displayed enough error to exceed the chi-square critical value for some of the census tracts. Examining the fits for those tables, differences were generally modest and reflected small population numbers in adjacent cells along at least one dimension. When the number of constraints was loosened , the degree of error increased across both error metrics. We therefore concluded that application of all 13 constraints yielded the most accurate synthetic microdata.To determine whether the synthetic microdata appropriately assigned individuals to census tracts, we also evaluated the percentage of multi-family homes and average number of rooms per home by census tract, as predicted by the synthetic microdata and as reported in the ACS. As shown in To illustrate the application of the synthetic microdata, we combined it with a multivariable regression model of smoking to evaluate the resulting demographic and geographic patterns of smoking for New Bedford. In our multivariable logistic regression of smoking, all tested covariates were statistically significant . SmokingApplying this multivariable regression model to the synthetic microdata, there was a wide range of predicted probabilities for individuals, consistent with strong demographic patterning of smoking behaviors – about 8% of the population had a mean smoking probability above 50%, while 14% of the population had a mean smoking probability under 10%. The smoking rates for key demographic groups corresponded closely with reported surveillance data as derived from the BRFSS , in spitUsing simulation methods, we constructed geographically resolved synthetic microdata for a high-risk community and demonstrated its utility for predicting geographic and demographic patterns of smoking. The individual-level microdata were statistically robust, with only minor misrepresentation of complex cross-tabulated constraints at census tract resolution. Sensitivity analyses showed that removing those constraints led to poorer overall model performance, indicating the value of the cross-tabulated constraints in providing a more accurate characterization of the individual constraint variables. We were able to connect these microdata with a strong predictive model of smoking behavior as a function of multivariable demographic characteristics, relying on local data to ensure that demographic variables retain their local context. For example, general population smoking regression models The multivariable demographic information and corresponding regression models predicting exposures would allow us to rapidly identify high-risk subpopulations for specific health outcomes. As a simple example, previously-derived multivariable regression models constructed from New Bedford data In spite of the strong model performance and potential generalizability of our modeling strategy, there are some limitations in our synthetic microdata and corresponding smoking models, as well as in the general approach. While ACS microdata include a large number of covariates, there remain gaps in the individual-level information available, and the synthetic microdata are ultimately built on a population sub-sample that may contribute uncertainties in characterizing multiple exposures for smaller (community-relevant) areas. With an average of 3,063 residents per census tract in New Bedford , the 5% sample of the population available in the ACS microdata will not be able to fully capture all multivariable demographic nuances. In addition, ACS microdata will underrepresent undocumented populations that may be at particular risk.Our modeling approach is also predicated on the assumption that basic demographic characteristics are strong predictors of environmental exposures. While this is clearly the case for smoking, it may not hold more broadly. For example, for pollutants with strong spatial gradients, we may not adequately capture exposure contrasts using census tract resolution. However, there is nothing in our analytical approach that requires census tract resolution, and relying on individual-level data as the foundation for our modeling affords flexibility for future applications. It may also be challenging to communicate our models and findings to communities or policymakers, as the structure of the models suggests that exposures are dictated by demographics rather than being a function of complex behaviors and exposure pathways.Relatedly, there are a limited number of national-scale environmental exposure studies with sufficient geographic resolution, so an analysis that extended beyond smoking or other survey-derived behaviors in the BRFSS would need to rely on some amount of local sampling or surveying. We focused on New Bedford in part because a long-standing pregnancy cohort study In spite of these limitations, our modeling approach offers some significant advantages over other options. Geographically resolved synthetic microdata provide community-scale detail that would rarely be available for cumulative risk assessment models, including exposure predictors and vulnerability attributes. By building both the microdata and the smoking regression model at the individual level, we yield robust spatial estimates and avoid issues such as the modifiable areal unit problem. Subpopulations at high risk across multiple exposures can be identified and targeted for public health interventions. Our strategy would also allow for any exposure characterization study to rapidly yield community-scale conclusions, provided that the requisite demographic and geographic data were collected from study participants.Our simulation approach provides a method to create detailed and geographically resolved individual microdata for a low-income population that can be used in community-wide environmental exposure and risk assessment studies. We applied the method to a regression model of smoking behavior to illustrate the applicability of our approach. While our population characteristics and resulting models were specific to New Bedford, all our methods and input data generalize to any other city in the US, providing a powerful mechanism to compare health risk factors within and between major cities, and allowing for more targeted public health interventions. In future studies, these microdata will be connected with comparable regression models for multiple environmental exposures to provide detailed characterization of exposure patterns relevant to health outcomes of interest in New Bedford.


A vicarious experience is an empathetic state in response to the observation of others' sensations, emotions, and actions  and we outline similarities and differences between synesthesia and vicarious experiences Table  for the. By usinAt a first glance, mirrored sensory experiences and synesthesia seem to share many features, but there are also marked differences as already mentioned by Fitzgibbon et al. . Thus, iFor both conditions, the mirrored sensor experience and synesthesia, there is a clear relationship between an inducing stimulus  and a resulting concurrent experience . Specifically, this may be the observation of touch for mirrored touch experiences and a letter or number in grapheme-color synesthesia. In both cases the concurrent experiences are triggered automatically and involuntarily. Empirical evidence can be found with variants of the Stroop task Stroop, , where pexperiences, synesthetes generally exhibit high test-retest consistency for individual inducer-concurrent pairings, but not so controls who have to rely solely on memory  in mirrored sensory experiences prevents individual forms of mirrored sensory experiences from sharing with established forms of synesthesia the core criterion of idiosyncratic inducer-concurrent pairings. That is, while A may elicit a red color experience for one grapheme-color synesthete, it may elicit a blue color experience for another grapheme-color synesthete  seem to be very similar to socially contagious phenomena such as laughter Provine, , yawningEvidence for the similarity between mirrored sensory experiences and socially contagious phenomena can be found on a neural basis. Exactly as mirrored sensory experiences are socially contagious phenomena based on mirror neuron activity and do in fact elicit similar brain activity in the perceiving person as well as the in the observing person (Holle et al.,


Hmx1 gene is crucial for normal external ear development and when disrupted can mimic loss-of function Hmx1 coding mutations in mice and humans. These findings provide important support for conserved developmental roles of CNEs in divergent species, and reinforce the concept that CNEs should be examined systematically in the ongoing search for genetic causes of human developmental disorders in the era of genome-scale sequencing.Comparison of genomic sequences from diverse vertebrate species has revealed numerous highly conserved regions that do not appear to encode proteins or functional RNAs. Often these “conserved non-coding elements,” or CNEs, can direct gene expression to specific tissues in transgenic models, demonstrating they have regulatory function. CNEs are frequently found near “developmental” genes, particularly transcription factors, implying that these elements have essential regulatory roles in development. However, actual examples demonstrating CNE regulatory functions across species have been few, and recent loss-of-function studies of several CNEs in mice have shown relatively minor effects. In this Perspectives article, we discuss new findings in “fancy” rats and Highland cattle demonstrating that function of a CNE near the  Genomic regions that can be recognized by the alignment of distantly related vertebrate species, such as fish, rodents, and humans, have been termed “conserved non-coding elements” or CNEs  mutation is a recessive trait causing ventral displacement and rotation of the external ear (pinna), leading to protruding ears akin to the cartoon elephant that gives the strain its name , and one called “misplaced ears” , characterized by deformities of the pinna and also variable eye defects  between all mammalian species, and exhibits a core of conserved sequence that is retained in reptiles, fish and amphibians (Figure Remarkably, the rat t Figure . In the s Figure . The bovdmbo deletion region, likely conferred by this CNE, has been provided by studies of developmental gene expression in dumbo rat embryos (Quina et al., crop ear duplication will increase enhancer function or disrupt it.Supporting evidence for tissue-specific enhancer function within the rat crop ear phenotype in contrast is a marked pinna deficiency. These differences may reflect apposing effects of the mutations: i.e., loss of Hmx1 expression in rats and increased expression in cattle. With respect to this, it is also interesting to note that the bovine crop ear trait is partially dominant, whilst the rodent Hmx1 alleles, including the rat dmbo CNE deletion, are recessive. It will be interesting to see whether these distinct modes of transmission result from the specific effect of the crop ear duplication event on Hmx1 enhancer function, or interspecies differences in the effects Hmx1 gene dosage on pinna development. Regardless, these cross-species examples reinforce the role of CNEs in conserved developmental processes and the notion that they can be a cause of developmental disorders.The ear phenotype of dumbo rats closely resembles that seen in the mouse loss-of-function alleles, with pinnae that are slightly dysmorphic and ventrally displaced and rotated, giving the appearance of larger “floppy” ears. The bovine Hmx1 coding and CNE mutants in mice, rats, cattle and humans indicate that CNE mutant phenotypes will not necessarily be subtle or polygenic, but instead may show Mendelian inheritance with high penetrance, and may phenocopy at least some of the features associated with null mutations in the genes they regulate. Although work in this area has just begun, three general strategies can advance the identification of functional mutations in CNEs that underlie human disease.Extensive evidence for conserved CNE enhancer function probably exists in the gene pool of the diverse vertebrates for which we now have nearly complete genomic sequence. The similarity of the phenotypes seen in First, functional variants in CNEs will escape detection using the current methods of genomic analysis, particularly whole exome sequencing and transcriptome sequencing, since CNEs reside in introns and intergenic sequences. The CNEs identified in the human genome should therefore be systematically added to whole exome analysis of human genetic disorders, and examined for copy number variation, in order to appreciate the full range of sequence diversity in these regions that may cause disease phenotypes. The recognition of functional changes in CNEs would be greatly facilitated by a complete catalog of the genetic diversity in these loci, in the human population as well as across species.Second, even when sequence variants in CNEs are identified, our ability to decode changes in CNE function based on sequence variation is still quite limited, especially for single nucleotide polymorphisms. General evidence for the enhancer function of a CNE, although not its specific regulatory role, can be derived from the analysis of local chromatin states by chromatin immunoprecipitation of modified histones and DNA methylation analysis (Schones and Zhao, in vivo vertebrate model, such as transgenic mice (Pennacchio et al., Finally, the definitive functional test of a disease-associated CNE can only be performed by demonstrating gain or loss of regulatory function in an appropriate 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.


TGFBI gene. Furthermore, there is recent evidence that targeted cytoplasmic irradiation induces changes in mitochondrial morphology. Mitochondria are the energy center of a cell and normal mitochondria are highly dynamic organelles that move along microtubules or microfilaments and continuously fuse and divide in healthy cells. A balance between mitochondrial fusion and fission is essential to maintain normal mitochondrial function. In contrast, irradiated cells show shortened and fragmented mitochondria together with a reduction in mitochondrial functions. These included reduced cytochrome C-oxidase as well as succinate dehydrogenase activities when compared with non-irradiated controls, suggestive of reduced respiratory metabolism. Furthermore, irradiated airway epithelial cells showed an increase in mitochondrial superoxide production that was quenched by the radical scavenger, dimethyl sulfoxide. This acute mitochondrial response caused by cytoplasmic irradiation may result in the release of several stress mediators, which are necessary for mitochondria to preserve cellular homeostasis. The observation that progeny of mammalian cells that are irradiation through the cytoplasm show evidence of genomic instability highlights the functional role of mitochondrial damage in radiation carcinogenesis.Although radiation is a well-established human carcinogen, the mechanism of how radiation induces cancer is not clear. High linear energy transfer (LET) particles such as those used in radiotherapy and found in the natural radiation environment in space are potent clastogens that induce chromosomal breakages and present a potential mechanism for the loss of tumor suppressor genes. Indeed, using an immortalized human bronchial epithelial cell line, we show previously that high LET radiation, including alpha and HZE particles, induces a stepwise neoplastic transformation of these cells and a consistent downregulation of the tumor suppressor


The rate of glucose utilization in tumor cells is significantly enhanced as compared to normal cells and this biochemical characteristic is utilized in PET imaging using FDG as a major workhorse. The PET systems as well as cyclotrons producing positron emitting radiopharmaceuticals have undergone continuous technological refinements. While PET (CT) systems enable fusion images as well as precise attenuation correction, the self-shielded cyclotrons developed provide dedicated systems for in-house production of a large number of PET radiopharmaceuticals. The application of PET images in oncology includes those of pulmonary, colorectal, breast, lymphoma, head & neck, bone, ovarian and GI cancers. The PET has been recognized as promising diagnostic tool to predict biological and physiological changes at the molecular level and hence offer a potential area for future applications including Stem Cell research. Much of the enthusiasm concerning fast emerging applications of Positron Emission Tomography can be attributable to its clear-cut recognition as a unique imaging modality with proven clinical value in the management of cancer patients. Unlike other imaging modalities, Positron Emission Tomography (PET) quantitatively demonstrates scintigraphic representation of tumor physiology as well as anatomy and thus assumes unique advantage over other cross-sectional imaging methods such as Computed Tomography (CT). In order to permit tumor imaging using PET or any other tracer technique, the basic features deployed relate to differences in physiological and metabolic characteristics of tumor and normal tissues. These differences include tumor cell surface antigen phenotype versus normal tissues. In general, growth and hence use of DNA precursors such as thymidine as well as rates of protein synthesis in tumors are often increased as compared to normal tissues. Transport and incorporation of various types of amino acids as well as rates of anaerobic and aerobic glycolysis has been observed in tumor cells. In a widA positron is an antiparticle of an electron with identical mass and charge. After emission, the positron has some kinetic energy, which is lost through multiple collisions with electrons present in the neighboring tissues. The complete or almost complete loss of energy by the positron results into its combination with electron. This eventually forms a short-lived composition, i.e., positronium. The schematics are shown in Each pair of parallel and opposite detectors produces a coincidence line, which is unique in terms of location and direction. A large number of such coincidence lines form the data set and by the use of which a cross-sectional image can be reconstructed. The data pertaining to coincidence events is stored as two-dimensional matrix in which horizontal direction represents offset from the center of the field of view (CFOV), whereas vertical direction describes the projection angle. This set of data in terms of two-dimensional matrix is called ‘Sinogram’ and provides a set of projection data for reconstruction of image. Sinogram data however needs to be corrected for tissue alternations as well as detector non-uniformities. Various detector elements in a PET system are expected to exhibit variation in detection efficiency due to geometrical variation, differences in energy discrimination as well as detector gains. Such variations need to be equalized to prevent appearance of any artifacts. In addition, attenuation correction accounts for the compensation due to intra-tissue absorption of one or both annihilated photons. After necessary corrections, the Sinogram cumulatively represents all the coincidence events along a particular coincidence line. Sinogram data is then used to reconstruct the image using filtered back projection or an interactive technique.Ideally speaking, PET scanner should record only those coincidence events that originate as a result of positron annihilation along the line between the two parallel opposite detectors. Such detected events are called True Coincidences as depicted in In addition to accidental or random events, there may be a situation where both of the annihilated photons are diverted from their original direction as a result of Compton interaction and reach the detector to misposition the coincidence event as shown in In an ideal situation, only two detectors in a coincidence time window would be required to be activated. However, at higher count rates, more than one positron gets annihilated and more than two detectors may get activated. This results into ambiguity about the location of the event. Such ambiguous events can be distinguished from valid coincidence events and are usually discarded and hence result into net reduction in overall count rate.As already described, in a positron annihilation two photons are simultaneously emitted in opposite directions. If either photon is absorbed within the body, a coincidence event will not take place. Such a probability of attenuation of photon within the body exists also for all other annihilation events independent of the position. A blank scan followed by transmission scan for all possible coincidence lines using a rod or point source (that rotates around the patient) enables measurement of transmission count rates. However, transmission scan can also be performed after tracer administration.6 The traIn view of the fact that emitted photons to be detected in PET have energy of 511 KeV, the detectors to be used need to have specialized characteristics. These include stopping power, amount of light produced against each absorbed photon and time taken for the decay of light.The increased stopping power enables detector's ability to efficiently absorb the total energy of 511 keV of annihilated photons. In order to achieve this, a scintillator with high effective atomic number and linear attenuation coefficient needs be used. This would enable predominantly the photoelectric interactions within the crystal or scintillator and impart full absorption of energy.In addition, the detector should also have a capability of producing higher light output so as to have better energy resolution. The increased light output also enhances accuracy of detector element configuration. Also the decay time of the light decides how accurately coincident photons can be detected by a pair of detectors. The shorter time constant enables faster production of the signal after complete absorption. With fast scintillators, use of narrow coincidence time window would suffice and would reduce the probability of detection of unrelated or accidental coincident events. The signal is integrated for three or four times the decay constant and during this period, the detector is dead. This dead time of the system is a main factor limiting count rates, particularly at high activity levels in PET systems.Bismuth Germanate (BGO) has been predominantly used as a detector of choice for the last several years because it possesses the desired characteristics, namely, high effective atomic number as well as high density and hence high stopping power. Approximately, 95% of the annihilated photons of 511 keV undergo interaction within a 3-cm thick block of BGO detector. On the contrary, only 36% of the emitted photons undergo interaction in the 3-cm block of NaI detector. However, the light output of BGO detector is fairly poor as compared to NaI detector. In addition, BGO has longer decay time constant and therefore enhanced dead time and hence limits the count rate performance.Recently, Lutetium Oxyorthosilicate (LSO) has been identified to possess better characteristics and hence is ideally suited for use as a scintillating material (detector) in PET. It proviIn a typical PET system, there are multiple ranges of block detectors around the patient so as to record coincidence events emanating from all possible projections around the patient. BGO/LSO detectors are stated to be the best suited for a wide range of clinical studies and improved count rate performance. However, use of LSO would further improve dynamic capability of detection in PET systems.Even though most of the dual-head gamma cameras deploy NaI as crystal, an idea has been mooted to use the dual-head SPECT system as PET scanner, which is now a reality at many centres. Such systems can be either used as dual-head SPECT systems or as PET systems by using coincidence circuits. This is facilitated due to advanced digital electronics for prompt processing of signals from photo-multiplier tubes. In case of a dual head gamma camera being operated as PET system, pulse extrapolation local centroid positioning is used in coincidence mode. In addition, crystal thickness is required to be enhanced (about 1.5 cm) compared to 0.9 cm commonly used in conventional SPECT systems. Further inventions have revealed that use of two separate layers of scintillators, - for example, LSO (Lutetium Oxyorthosilicate) and YSO (Yttriium orthosilicate) - providGantry design of the camera requires adequate support for extremely heavy collimators needed for 511 keV photons.NaI (Tl) detectors used in SPECT systems are not suited for 511 keV photons and about 1.5% of true coincidence events are only detected for 0.95 cm thick crystal used.Spatial resolution is also substantially compromised.It would therefore not be always advisable to encourage the use of dual-head SPECT systems for coincidence imaging.In a typical PET system, the imaging planes are separated by a Lead or Tungsten Septa to disallow the detection of photons in one imaging plane being in coincidence with photons detected in other imaging planes. Such events need to be prevented and processing of such events would need detected imaging reconstruction algorithm that would enable reconstruction of data required from all angles in a three-dimensional domain. The deployment of 3D imaging reconstruction algorithm has revealed emergence of new generation PET system that would be capable of acquiring reconstruction data in 3D and overall counting efficiency would be drastically enhanced.10 The 3DThe annihilated photons may reach the detectors at different times unless the annihilation events occur at a position that is exactly located in the middle of two coincident detectors. However, this difference in arrival time is extremely small because the photons travel with a velocity that is somewhere near the velocity of light. The time difference, therefore, in a typical situation may be less than 1 nanosecond. (ns); and therefore, this is usually ignored in conventional PET systems. However, quality of image reconstruction can be further improved if necessary correction is applied for. Furthermore, the time of flight can provide the useful clue that can be used to constrain back projection - for example, a circle of few cm radius. This might help reducing noise amplification, thereby providing improved signal-to-noise ratio in the reconstructed image. The mostThe major factors that can affect the spatial resolution of PET images include size of the detector used; colinearity, or most precisely, noncolinearity of annihilating photons; and range of emitted positrons. (The Positron range is defined as a finite distance the positron is required to travel within the tissue medium before undergoing annihilation.)A typical PET scanner incorporates hundreds of discrete detectors in the form of rings around a patient. Each pair of detectors, depending on their size, constitutes a channel. This channel width is usually 0.4-0.6 cm. Any pair of annihilated photons falling within this channel width would be detected by that particular pair of detectors and hence would be delineating specific spatial location. Any reduction in size of detectors decreases the variation in a spatial positioning within the channel width. This clearly establishes that accuracy of spatial resolution is governed by the size of individual detectors.It is a common understanding that annihilated photons travel exactly in opposite directions, but this is not always true. If at the time of annihilation, both positrons and photons do not come to a complete rest, they would have some residual momentum before undergoing annihilation. In order to conserve the momentum, the annihilated photons might have some directional component rather than being emitted exactly in opposite directions, i.e,180°. This component of noncolinearity is expected to introduce some degree of error in locating the positional location of annihilated event, thereby affecting overall image resolution. In a typical case of whole body PET scanner having 100 cm diameter, noncolinearity may deteriorate image resolution by 0.2 cm.18] F may have energies peaking to 0.63 MeV and therefore may have a range of 2-3 mm within the tissue.1518] F m14C, 15O, 13N and 18F stimulates the atoms used in building blocks of biological molecules and are therefore easily incorporated. These features collectively provide a unique ability to measure absolute tracer concentration and this characteristic might be used for developing in-vivo tracer kinetic models. The spectrum of quality control parameters for effective performance monitoring include:The unique feature of coincidence detection in PET enables quantitative measurement of physiological parameters like blood flow, metabolic rate and receptor density. The count rate after attenuation correction can provide co-relative projection of absolute tracer concentration. In addition, simultaneous projection acquired in a panoramic manner provides rapid sequential images that can be used to predict dynamic variability of tracer concentration. The use of Calibration checkUniformitySpatial resolutionScatter fractionSensitivityCount rate losses and randomsScanner cross calibrationDrifts in coincidence timingDrifts in energy thresholdsMechanical movement of detector ringsRemovable Septa positioningLaser alignmentAttenuation correction accuracyData time correction accuracyScatter correction accuracyRandom coincidence correction accuracyThe scope of this article is not to describe in detail the quality control procedures related to above parameters. However, these factors are known to make considerable impact on the image resolution, count rate capability and random/scatter events, etc.A significant progress has been made in the technological arena to produce PET radiopharmaceuticals. The radionuclides that are commonly used in PET imaging along with their characteristics are shown in 76Br, 86Y, 89Zr, 64Cu and 124.Hundreds of chemical compounds incorporating positron emitting radionuclides have been developed and investigated for study of a host of biochemical and physiological processes. 2-(18F) Fluoro-2-deoxyglucose (FDG) is by far the most important and widely used PET radiopharmaceutical. (15-O) water, (15-O) carbon monoxide, (13-N) ammonia (11C)-1- methionine, etc, are the other regularly used PET radiopharmaceuticals. Several other radionuclides that are currently being investigated as a source of positrons include 15O, 11C, 18F, 13N in a dedicated manner. The major companies that are producing cyclotrons include CTI Cyclotron System, Japan Steel Works, Siemens, General Electric Medical Systems. For different radionuclides, different target materials are used to achieve the desired reaction, as depicted below in The positron emitting radionuclides are produced in the cyclotron system that are currently available commercially. The present date cyclotrons have a tailor-made design for producing four radionuclides, namely, The present day cyclotrons, for the sake of reducing cost and simplicity, are supplied as ‘Proton only Machine’ or ‘Dual particle machine.’ The most striking feature of such cyclotron is self-shielding, which allows access of technical staff to the cyclotron room even when the machine is operational. The shielding requirements of the walls are also achievable and the machine is no longer required to be housed in the basement. The configurations of cyclotron machines are therefore customized so as to meet the chemical requirements.in vivo mechanisms of various diseases. As opposed to classical diagnostic imaging, molecular imaging, in a unique manner, unfolds the molecular abnormalities responsible to form basis of many diseases.The fusion and interdisciplinary areas like cell/molecular biology, pharmacology, medical physics, image capture techniques, nuclear medicine, chemistry, biomathematics and bioinformatics together create a novel imaging paradigm called Molecular Imaging. The overall objective in molecular imaging is aimed at addressing the issues like visualization, characterization and quantification of biological processes taking place at cellular and subcellular level within the living organisms. A molecular image is thus a representative of cellular and molecular pathways including The frontline issues, that are being addressed in molecular imaging/molecular medicine include:in vivo imaging techniques depicting gene expression and protein-protein interactionsEvolving noninvasive Simultaneous monitoring of molecular eventsCell targeting and cell traffickingOptimization of gene and drug therapyMonitoring of disease progression/therapy response at molecular pathological levelThe founding principles of nuclear medicine trace forward the principles or objectives of molecular imaging. Apart from nuclear medicine, where radioactive isotopes are used to produce imaging signals. Other strategies/modalities like MRI are being adopted for molecular imaging by using molecular probes to provide image in signals other than radioactivity.The most widely used and simple probes are the ones routinely used in conventional medical imaging. These include nonspecific imaging probes like contrast agents, nuclear medicine tracer (radioisotopes) and fluorochrome reporters. Such probes are effectively utilized to image flow and perfusion, changes in blood volume and detect downstream pathological changes but are unable to characterize the changes in the earlier stages of the disease.in vivo assessment of biological and physiological processes involved in the earlier stages of disease progression. Thus PET can be regarded as a promising tool for diagnosis of several diseases where derangements are still at the molecular level and help selecting appropriate modality of treatment. 18F reporter probes like fluoropenciclovir (FPCV), fluoroganciclovar (FGCV), etc, have been successfully deployed for imaging herpes simplex virus type 1 - thymidine kinase (HSV1-tk) reporter gene expression.Positron Emission Tomogrpahy (PET) has already been accepted as a well established noninvasive diagnostic imaging technique using positron emitting radionuclides for diagnosis, staging/restaging and monitoring of therapy response in oncology. Molecular imaging using PET tracer kinetics provides quantitative The image fusion through co-registration technique is done using dedicated software. It could enable high resolution morphological CT or MRI images to be registered on functionally informative PET images. However, the problems like motion artifact, complex realignment and computation needed for fusion of independent studies need to be extensively addressed to obtain more informative bimodal or multi-modal images. Integrated instrumentation like clinical CT/PET or, for that matter, development of MRI/PET imaging system may offer a solution to these problems; also, combined use of radionuclides and magnetic probes would permit near-simultaneous MRI and PET imaging. It is also open to choose other combinations out of optical, radionuclide, MRI and CT probes to generate multi-modal images enabling unique information relating to quantification and location of biological events/processes as well as characterization of newly developed imaging probes. The multi-modality images would substantially improve quantification and interpretation of several experimental results.The transplantation of stem cells with damaged tissue had offered tremendous potential for therapeutic applications in a wide range of disease conditions.20 includThe Positron Emission Tomography (PET), as already explained, has a unique ability to provide functional as well as metabolic information related to tumor cells. In order to effectively deploy this characteristic, Flourine-18 Fluoro-2-Deoxy Glucose (FDG) has been used to image and characterize cancer cells. In addition, the co-registration of PET and CT or MRI image provides detailed anatomical as well as functional information so as to optimize radiation therapy treatment planning. Integrated PET-CT systems enable not only precise attenuation correction but also provide fusion images. In the Indian scenario, the first PET-CT system was installed at the Tata Memorial Hospital in Mumbai in 2003 and, recently, AIIMS in New Delhi; Army Hospital, New Delhi, has also acquired this facility. The facilities are being predominantly used for oncological applications. Many more centers are in the offing of acquiring PET (CT) systems that are expected to revolutionize the use of PET (CT) in the country as far as its application in radiation oncology is concerned. PET (CT) would be of immense utility in staging, delineation of tumor volume, monitoring of treatment response and detection of recurrence of tumor in wide range of solid tumors. Even though experience related to PET (CT) application in Oncology is very limited from the Indian viewpoint, from whatever limited experience/data is available, it has been shown to improve TNM staging in patients of various cancers. Simultaneously, efficacy of radiation therapy treatment can also be evaluated by estimating Standard Uptake Value (SUV) using PET images. The differentiation between radiation necrosis and malignant metastatic disease in the treatment follow-up can be effectively predicted. In view of this, a concept of Biological Target Value (BTV) has now emerged and is expected to open up a new era of biological conformity. Hypoxic cells within the tumor can also be delineated in a better manner so as to have optimized dose escalation, further improving radiation therapy of cancer cells.The PET-CT has now been successfully deployed as a noninvasive tool for the diagnosis of pulmonary nodules to differentiate between malignant and benign lesions. The PET image generally shows intensive FDG uptake which is more than that in the blood pool activity after about 60 min of injection. The delayed images further improve the results. The benign lesions indicate declining uptake with time, whereas malignant nodules show increase in FDG uptake in time. In case of lung cancers, the PET-CT fusion images may be of immense utility for delineation of nodal disease as well as detection of distant metastasis. PET imaging can be also used as an effective diagnostic tool to rule out presence of metastasis in false positive cases.The PET imaging has assumed significant importance in accurate detection of multifocal disease as well as distant metastasis/lymph nodes in carcinoma of the breast (breast cancer). This is particularly of great clinical importance because in most of the countries, the prevalence of breast cancer in women has shown a rising trend. The PET imaging can also be effectively utilized to assess the response of adjuvant chemotherapy.The use of PET imaging in diagnosis, staging and monitoring of therapy response in cases of lymphoma patients has revolutionized the field of oncology. Additional lesions that are not detected by conventional diagnostic techniques like CT can now be delineated and therefore, the efficacy of treatment can be further optimized. In addition, the Whole Body PET imaging can help in modification of therapy in significantly large number of patients. PET images can demonstrate very high predictive value for differentiation between active tumor and fibrosis.In case of colorectal cancers, FDG PET can have a very high sensitivity for preoperative diagnosis. In addition, PET images can be used to detect recurrence of disease in the follow-up after therapy. The detection of extra hepatic metastasis can be done using PET images, thereby enabling selection of patients who may be fit cases for consideration of partial hepatectomy.In order to have optimized treatment planning, lymph node staging in head and neck cases using PET images can be more accurately and comprehensively done. A large number of undetected lesions/lymph nodes could be demonstrated in PET images. In addition, treatment response can be effectively monitored so as to institute appropriate mode of therapy in the follow-up of patients.The sensitivity for detection of primary gastric/esophageal cancer as well as metastasis is quite high from specificity viewpoint in FDG PET images. It can particularly help in distinguishing the responsive and nonresponsive disease so as to avoid unnecessary surgery in significant number of patients.FDG uptake in cases of osteosarcoma can be a good indicator for predicting tumor aggressiveness apart from its utility for enabling guided biopsy so as to select metabolically active tumor site. The ability of PET images to effectively demonstrate therapy response is an added advantage.In case of ovarian cancer, precise staging of the case is extremely important for effective management, particularly in relatively advanced disease. The sensitivity as well as specificity of PET images for detection of lymph nodes is extremely high as compared to other conventional imaging modalities.The emergence of Positron Emission Tomography (PET) has opened up a new era to demonstrate tumor physiology and anatomy. The inherent problems of intra-patient absorption as well as attenuation correction has been taken care of due to technological excellence wherein PET systems are combined with CT to not only channel fusion images but also enable attenuation correction in an approximate manner. This is to emphasize that attenuation correction factors measured by CT are only scaled up for 511 keV and PET-CT enables only segmental attenuation correction. In order to achieve precise attenuation correction, it is advisable to generate attenuation data for transmission scan using 68-Ge source instead of using integrated CT. Dedicated and self-shielded cyclotrons have been developed to provide a large number of radiopharmaceuticals for clinical applications as well as research. Molecular imaging using PET could provide quantitative estimation of biological and physiological processes in very early stages of disease progression. The future of PET imaging lies in depicting the changes at molecular level and hence in selecting appropriate mode of treatment. The application of PET in Oncology has not only altered the concepts used in treatment planning but also has registered its need for monitoring of therapy response. It therefore goes without saying that PET imaging is going to be a diagnostic tool of choice in a large number of clinical applications including Stem Cell research.