The most conspicuous function of plastids is the oxygenic photosynthesis of chloroplasts, yet plastids are super-factories that produce a plethora of compounds that are indispensable for proper plant physiology and development. Given their origins as free-living prokaryotes, it is not surprising that plastids possess their own genomes whose expression is essential to plastid function. This semi-autonomous character of plastids requires the existence of sophisticated regulatory mechanisms that provide reliable communication between them and other cellular compartments. Such intracellular signaling is necessary for coordinating whole-cell responses to constantly varying environmental cues and cellular metabolic needs. This is achieved by plastids acting as receivers and transmitters of specific signals that coordinate expression of the nuclear and plastid genomes according to particular needs. In this review we will consider the so-called retrograde signaling occurring between plastids and nuclei, and between plastids and other organelles. Another important role of the plastid we will discuss is the involvement of plastid signaling in biotic and abiotic stress that, in addition to influencing retrograde signaling, has direct effects on several cellular compartments including the cell wall. We will also review recent evidence pointing to an intriguing function of chloroplasts in regulating intercellular symplasmic transport. Finally, we consider an intriguing yet less widely known aspect of plant biology, chloroplast signaling from the perspective of the entire plant. Thus, accumulating evidence highlights that chloroplasts, with their complex signaling pathways, provide a mechanism for exquisite regulation of plant development, metabolism and responses to the environment. As chloroplast processes are targeted for engineering for improved productivity the effect of such modifications on chloroplast signaling will have to be carefully considered in order to avoid unintended consequences on plant growth and development. According to the endosymbiotic theory plastids originated from free-living cyanobacteria that were engulfed by early eukaryotic cells. These cyanobacteria were retained by their hosts and have co-evolved with their host cells over 1.5 billion years to become an integral part of the modern plant cell . It is aThe most conspicuous function of modern plastids is the sophisticated oxygenic photosynthesis performed by chloroplasts. However, plastids perform many other functions that are critical for proper plant development and physiology including the synthesis of amino acids, nucleotides and fatty acids, production of phytohormones, some vitamins and a multitude of secondary metabolites, as well nitrogen and sulfur assimilation. Many chloroplast secondary metabolites, besides being necessary for basic plant metabolic functions, are also important for interaction with the environment, as they function in plant defense against pathogen ingress and plant adaptation to stresses including heat, drought and high light. Thus, chloroplasts act as a hub in the cellular response to signals, generating a variety of signals that coordinate a fine-tuned and appropriate response to any given situation .Figure 1A). An often over-looked aspect of cell biology is the physical interaction between organelles for coordination of signaling and metabolism . This area is beginning to receive attention and we will examine these findings as they relate to chloroplasts and their roles in signaling. Given the dizzying array of signals that chloroplasts respond to and produce it is not surprising that there is considerable crosstalk between signaling pathways. This is particularly evident during responses to biotic and abiotic stress  albostrians chloroplast ribosome-deficient mutant had severely decreased chloroplast protein synthesis, in conjunction with depressed expression of nucleus-encoded chloroplast genes. This revolutionary discovery was in opposition to Ellis’ “cytoplasmic control principle” that posited control of organellar protein synthesis by cytoplasmic components  mutants escaped the pattern of suppressed PhANG expression despite defective chloroplast physiology or inhibited biogenesis. There are numerous excellent reviews of the role of guns in retrograde signaling , the gun2-6 mutants are associated with tetrapyrroles synthesis -induced signaling and feedback regulated 5-aminolevulinic acid (ALA) synthesis .The detailed analysis of t stress . It has Arabidopsis constitutively expressing HPL (ceh1) mutant displays enhanced expression of hydroperoxide lyase (HPL), a stress-inducible nuclear gene encoding a plastid-localized protein of the oxylipin pathway. The ceh1 mutation disrupted a plastid-localized enzyme (HDS) that catalyzes conversion of methylerythritol cyclodiphosphate (MEcPP) to hydroxymethylbutenyl diphosphate -biosynthetic pathway) and HPL, but not ALLEN OXIDE SYNTHASE . HDS-depleted plants with increased levels of MEcPP accumulated SA and displayed increased resistance to infection by biotrophic pathogens. Importantly, elevated MEcPP levels and increased expression of HPL are observed upon both wounding and high light treatment, demonstrating involvement of MEcPP in a retrograde pathway involved in abiotic stresses  distinct from the gun signaling pathway. Therefore MEcPP is a retrograde signal inducing targeted stress responses. The proposed mechanism of MEcPP action involves direct modification of chromatin remodeling by disruption of DNA-histone interactions .Isoprenoid metabolism is a major biosynthetic pathway in plants . The Arae HMBPP; . The abs1O2) generated by the PSII reaction center  mutant (S-transferase (GST) genes and 12 UDP-glycosyltransferases . Both groups of genes are involved in detoxification processes conferring tolerance to singlet oxygen in Chlaydomonas reinhardtii . β-cyclocitral’s accumulation in carotene . Consist) mutant . Using qnhardtii . The pronhardtii . Finallynhardtii , 2013. Vtabolism . The abcArabidopsis ζ-carotene desaturase mutant (zds/clb5/spc1/pde181) displaying arrested chloroplast biogenesis at a very early stage of development led to the conclusion that the accumulation of an uncharacterized apocarotenoid can act as a retrograde signal . Accumulation of this putative cleavage product was shown to modulate expression of many nuclear genes required for leaf development leading to a severe phenotype that included arrested chloroplast development and leaves with defective adaxial-abaxial patterning. Since neither ROS nor ABA nor strigolactone signaling pathways were responsible for the observed phenotypes, it was concluded that the putative phytofluene or ζ-carotene –derived apocaroteniod is part of a novel retrograde signaling pathway. Interestingly, the observed defects were restricted to primary leaves, underscoring the differences in developmental regulation between plastids in different organs  development . Reducedormation . Extensifunction . Intrigufunction .It is a broadly accepted paradigm that most nucleus-encoded chloroplast proteins reach this compartment due to the transit peptides located at their N-termini. Cleavage of the signal peptides after entry in to the chloroplast generates the functional chloroplast proteins. However, accumulating data show that some chloroplast proteins also act in the nucleus. While it is tempting to speculate that this is attributable to two-way protein movement between chloroplasts and cytoplasm, this has not been unambiguously demonstrated. However, a physiological function for this chloroplast-nucleus dual localization is apparent and it seems to be indispensable for proper plant response to pathogen attack and abiotic stress, and emphasizes the function of chloroplasts as signaling compartments.Figure 3). The N-terminus of PTM contains a DNA-binding homeodomain box, a different transcription factors (DDT) domain and a plant homeodomain (PHD). Interestingly, a shorter variant of this protein, lacking the transmembrane domains, was detected in nuclear fractions. Notably, increased amounts of the shorter PTM variant were detected upon treatment with either norflurazon or lincomycin, and on exposure to high light. Through the application of protease inhibitors, it was demonstrated that the shorter form of PTM was the result of serine protease activity  was shown to provide a physical link in signaling between chloroplasts and nucleus to regulate gene expression . This meactivity . AccordiPR (PATHOGENESIS RELATED) genes were upregulated under normal growth conditions . WHIRLY1 is part of the transcriptionally active chromosome in plastids, and interestingly, another component of this complex, pTAC12 (HEMERA), also showed dual localization to chloroplasts and nuclei with unchanged molecular mass, excluding any proteolytic modification . Such direct contact would also provide routes for inter-organelle signaling, although evidence for this is still limited. Thus, chloroplasts, besides producing signals that may travel long distances from plastids to their targets, may also communicate directly with other organelles through physical contacts. Here we consider evidence for chloroplast-organelle contacts and possible roles for these contacts in signaling.It has long been appreciated that there is metabolic crosstalk between organelles. Metabolic pathways often involve multiple organelles and metabolic intermediates may be transported across membranes by diffusion or by specialized transporters in an energy-dependent manner after traversing the cytosol. However, there is emerging evidence that direct physical contact between organelles may provide a major route for metabolic exchange  gene from the GV3101 plasmid into LBA4404 led to the induction of stromules and other cellular changes typically observed on GV3101 infiltration. Indeed, direct application of cytokinin to leaves produced phenotypes similar to those obtained with GV3101. Thus, cytokinin may mediate the production of stromules and chloroplast movements during some plant–pathogen interactions.Stromules and stromule-nucleus contacts may have important roles in host–pathogen interactions. The induction of stromules and remobilization of chloroplasts to surround nuclei was observed in efaciens . In addiNicotiana N RECEPTOR INTERACTING PROTEIN (NRIP)1 is a chloroplast rhodanese sulphurtransferase that is required for effector triggered immunity (ETI) against TMV mediated by the N innate immune receptor . The interaction with N in the nucleus and cytoplasm is necessary for ETI. These results raised the intriguing questions of whether NRIP1was remobilized from the chloroplast and how this could be accomplished. Recent data suggest that stromules may provide a route for NRIP to traffic from the chloroplast to the nucleus. Co-expression of N and p50 induced formation of stroumles during HR-PCD associated with this interaction . Similarly, stromule induction was observed during ETI initiated in response to bacterial pathogens, and on treatment with the defense-related signaling molecules SA and the ROS H2O2. Through correlative EM-fluorescence microscopy, the authors provide convincing evidence that stromules contact the nucleus during N-mediated defense. In elegant experiments that fuse a nuclear exclusion sequence (NES) to NRIP, the authors provide quantitative evidence that NRIP1 is indeed trafficked from the chloroplast to the nucleus. H2O2 also traffics to the nucleus via stromules. In plants overexpressing chloroplast outer membrane protein CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) stromule formation is abolished, suggesting that this membrane plays a role in stromule formation. This was confirmed in chup mutants and knockdown plants, where constitutive stromule formation was observed. The importance of stromules to HR-PCD and the defense response was underscored by the accelerated HR-PCD observed in those plants. These results are suggestive of a role for stromules in intracellular trafficking and possibly signaling during plant–pathogen interactions, and these possibilities warrant further examination. It will be exciting to test whether plastids and/or stromules are able to create hemifusion membranes with the nuclear membrane and whether other signaling molecules or metabolites use this route for chloroplast signaling.The question of why stromule induction and chloroplast-nucleus associations occur during plant–pathogen interactions has recently been addressed. receptor . In the N. benthamiana by virus induced gene silencing (VIGS) led to decreased intercellular trafficking of AbMV while viral replication was unaffected, suggesting that the chloroplast chaperone and stromules may have a role in viral trafficking that is independent of viral replication and accumulation  was identified as interacting with the AbMV movement protein MP; . In uninmulation . Indeed,mulation . AbMV ismulation ; therefoRESISTANCE TO POWDERY MILDEW (RPW)8.2 confers broad resistance to fungi that cause powdery mildew. RPW8.2 localizes to the extrahaustorial membrane (EHM) during fungal penetration of the host cell . Peroxisomes are able to adjust their complement of enzymes in response to changes in environmental and developmental signals  mutant also provides support for signaling between chloroplasts and peroxisomes. In seedlings, the sco3-1 mutation interrupted chloroplast biogenesis, decreased chlorophyll accumulation, and disrupted thylakoids and, as a consequence, photosynthesis , a nucleus-encoded mitochondrial gene, seems to be regulated by chloroplasts as increased expression of Arabidopsis and soybean AOX has been observed upon high light treatment  suite of mitochondria-associated genes was regulated in response to chloroplast perturbations that included increased ROS and NO production  mutant deficient in a nucleus-localized cyclin-dependent kinase E1 (CDKE1) that is a prerequisite for AOX induction, indicates it is another nucleus-localized sensor integrating mitochondrial and chloroplast retrograde signals (rao1(cdke1) mutant was unable to induce AOX1A expression upon application of antimycin A or DCMU . Further, the mutant displayed the gun phenotype upon induction of redox stress originating specifically from chloroplast photoelectron transport.Several possible routes for communication between chloroplasts and mitochondria are proposed. There is coordinated expression of nuclear genes encoding chloroplast and mitochondria proteins, and it is therefore likely that some of the chloroplast signaling to mitochondria is accomplished by modulating nuclear gene expression. Indeed, expression of the oduction . These M signals . Moreovenication . Analysi signals . Unlike Physcomitrella patens . It seems that the formation of this tri-organellar unit is regulated, with the chloroplast-peroxisome association being established first and then recruiting mitochondria  as well as redox signals are critical components of the plant stress response. Therefore chloroplast signaling is indispensable for plant survival of abiotic and biotic stress. The roles of all these chloroplast-associated molecules in coping with biotic and abiotic stress have been extensively examined and several excellent reviews are available .ics1 ics2 double mutant has revealed that there are other cellular sources of SA . CAS, and thus CA2+, regulates chloroplast SA biosynthesis and plants depleted of CAS failed to induce SA production in response to pathogen infection. In addition, expression of several nuclear defense-related genes was shown to be dependent on CAS, and the pattern of gene regulation was most similar to that observed in response to 1O2.Salicylic acid is important for basal defense as well as for ETI , and apptor CAS; . CAS medArabidopsis pdlp5 mutants have decreased resistance to bacterial pathogens. These results clearly demonstrate crosstalk between SA signaling and plasmodesmata, and illustrate how chloroplast signals can act to regulate intercellular trafficking via plasmodesmata (discussed below). It will be interesting to see if similar mechanisms are deployed for viral resistance since viruses use plasmodesmata for intercellular trafficking.Interestingly, exogenous application of SA induces closure of plasmodesmata . This is3H]-SA) suggested that NPR1 did not bind SA , an SA analog, the Popescu lab identified numerous chloroplast-localized proteins with roles in photosynthesis and oxidative phosphorylation as proteins interacting with AzSA (At5g10540 (TOP2), both of unknown cellular function. TOP1-GFP fusions localized to chloroplasts and TOP2 is likely cytosolic. Interestingly, top1 top2 double mutants had compromised ETI and PCD to bacterial pathogens. Further, TOP1 and TOP2 dimerize in an SA- and redox-dependent manner -7-iso-JA-Ile -12-oxophytodienoic acid (OPDA). OPDA is then translocated to the peroxisome, where its hydrocarbon chain is shortened by β-oxidation. It is not clear how OPDA is transported to the peroxisomes, but the process must be tightly regulated, given that OPDA itself is able to act as a signaling molecule. Indeed, OPDA has been implicated in tendril coiling  on exogenous application of SA  to produce the C15 compound xanthoxin, that is transported from the plastids into the cytosol by an unknown mechanism, where it will be converted into ABA , hydroxyl, alkoxyl (.RO), and peroxyl radicals as well as non-radical molecules like hydrogen peroxide (H2O2) and singlet oxygen (1O2). It is well established that while large amounts of ROS are damaging, small amounts act as signaling molecules  mutant have shed light on 1O2 signaling  and EX2 proteins  is a small gaseous molecule whose role in signaling in plant and non-plant systems is well established. While NO is an important second messenger in plants, major aspects of NO synthesis and action remain undiscovered. NO has critical roles in normal plant physiology including seed dormancy, germination, the floral transition, and during stress responses . In planL-arginine to NO  that oxidize ne to NO . Curiousne to NO . In planne to NO . Anotherne to NO . Nonethels of NO .-) RNS formed on reaction of NO with O2 . Several of these targets are components of PET, suggesting that photosynthesis and chloroplast function are important for defense against pathogens. A series of experiments aimed at addressing the role of chloroplasts beyond phytohormone production in plant defense has been reported. VIGS of two genes encoding components of the PSII oxygen-evolving complex, PsbQ and PsbO, resulted in a three to four-fold increase in the number of Turnip mosaic virus (TuMV) infection foci  mutants , and gat1 roots accumulate higher levels of ROS than do wild-type roots. In addition, callose was found to accumulate at plasmodesmata in gat1 roots. Similar observations have been made for the other gat mutants  mutants were identified by screening embryonically lethal mutants and monitoring their ability to traffic fluorescent dyes between cells. ISE1 and ISE2 have been mapped and cloned . Studies in the algae Chara australis also suggest a relationship between chloroplast and cell wall structure  seem to represent a fascinating signaling system that enables a plant to communicate with not only itself , and with the entire ecosystem that includes plant–plant, plant–animal/insect interaction, but could potentially also impact atmospheric chemistry .2, producing O3 , synthesized in the LOX-pathway that consists of C6 and C9 aldehydes, alcohols and their acetate esters, that are responsible for the ‘mowed grass odor’ . These vucing O3 . Therefoucing O3 .Chloroplast-produced BVOCs have other ecological functions that are a consequence of their signaling propensity. One of these is the so-called priming effect that leads to enhanced and more effective defense response to pathogen or insect attack in plants pretreated with volatiles . AlthougThe molecular mechanism of volatile signaling is far from understood, however, an attractive scenario of volatile-driven epigenetic modification through DNA methylation has been demonstrated as a very reliable strategy for priming defense induction. Moreover, since the JA-signaling pathway is triggered upon herbivore attack (wounding), it is expected that induced JA formation as a downstream effect would be relevant to the HIPV-primed defense responses . HoweverAnother intriguing aspect of chloroplast originating volatiles is that their release from attacked plants can serve as attractants of the offending herbivore’s natural biological enemies, a phenomenon known as tritrophic chemical communication or tritrophic interaction . Importaper se, but a single protein in the context of an entire cell, tissue, organ, individual plant and ultimately the entire ecosystem biology. Current progress in studying chloroplast biology shows their function goes far beyond photosynthesis and includes all aspects of plant biology, making these organelles an integral part and a full-fledged player in the cell. Understanding the signaling pathways chloroplasts and other plastids use to communicate with the rest of the plant cell under normal and stress conditions will provide new ideas for developing powerful strategies for engineering improved plants. Moreover, a thorough knowledge of chloroplast signaling will also allow the design of rational approaches that could minimize if not avoid unintended crosstalk and undesirable outcomes.The era of transcriptomics initiated by the microarray platform and continued by Next-generation sequencing technology, together with metabolomics and proteomic approach provided by mass-spectrometry, established new possibilities and new directions in addressing questions in biology. Inevitably, due to these two strategies contemporary plant biology is progressing toward a more holistic view, which translates into studying not a single protein 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.


In the face of excellent survival rates for pediatric and adolescent cancer, preserving the opportunity to have biological children is an important component of long term quality of life. Yet, modern chemotherapeutic regimens continue to pose a threat to fertility. The only fertility preservation methods available to pre-pubertal children of both genders is cryopreservation of gonadal tissue, a highly experimental intervention, or shielding/re-location of reproductive tissue in the setting of radiation. These techniques are available in the post pubertal population as well, but post pubertal patients also have the option for cryopreservation of gametes, a process that is much simpler in males than females. For this reason, prior to the initiation of therapy, sperm banking should be considered standard of care for males, while consideration of embryo or oocyte cryopreservation should be limited to those females at risk of developing ovarian failure. Attention to reproductive health and fertility preservation should continue after the completion of therapy. Establishing programs that streamline access to current fertility preservation techniques will assist in ensuring that all eligible patients can avail themselves of current options. The refinement of therapeutic interventions and supportive care has resulted in cure rates upwards of 80% in children and adolescents diagnosed with cancer, with subsequent estimates that 1 in 530 young adults between the ages of 20 and 39 is a survivor of a childhood cancer .vs. 57% respectively for males and 50% vs. 45% for females. In addition, while males who had discussions about fertility were satisfied with the content of those discussions, females were not [Newly diagnosed patients, survivors of childhood and adolescent cancer, and their families have identified that the retention of fertility is an important aspect of their lives ,6, and nwere not .Studies of health care professionals have also suggested that risks for infertility are not routinely discussed . ReasonsProviding fertility preservation support in this population requires prior knowledge of who is at risk and what options are available and having an infrastructure in place for patient counseling and referral. Institutional programs that systematically address this issue have begun to be implemented  in an efParamount in importance when considering infertility risks in females is that their full reserve of oocytes are present at birth and are depleted over time. At the onset of puberty, approximately 400,000 follicles remain within the ovaries. Through menstruation, atresia and apoptosis a progressive decline in ovarian primordial follicles continues during a healthy woman’s reproductive lifespan. Since fertility is related to the number of oocytes present at a given time, fertility begins to wane when a woman reaches her late 20s, drops more significantly in the mid to late 30’s and is no longer a possibility following menopause .2, cyclophosphamide >7.5 g/m2, or conditioning for transplant, prepubertal gonadal irradiation of ≥10 Gy, pubertal gonadal irradiation of ≥ 5 Gy, and alkylators in combination with irradiation of the abdomen/pelvis, lumbar or sacral spine, or total body irradiation (TBI) as risk factors for hypogonadism [Cancer directed treatment can cause depletion of the existing oocyte reserve. Alkylating agents, particularly cyclophosphamide and procarbazine , and heagonadism . There igonadism .The degree to which treatment depletes the ovarian reserve contributes to the length of the reproductive window that remains for a given patient. If the treatment related depletion approaches the threshold for menopause this is referred to as acute ovarian failure (AOF). If the reserve is diminished, but not exhausted from treatment, then the individual may remain fertile following therapy but have an overall shortened reproductive lifespan, referred to as premature menopause (PM). With the exception of patients who receive very high dose chemotherapy as part of conditioning regimens for stem cell transplants, particularly in conjunction with radiation, young female cancer patients generally have a greater risk of developing PM than AOF. The Childhood Cancer Survivor Study (CCSS) identified AOF, assessed as self-reported amenorrhea, in 6.3% of survivors surveyed, the majority of whom had received abdominal or pelvic radiation . In the Radiation therapy may also impact fertility by causing damage to the uterus thereby limiting the ability of a survivor to carry a pregnancy to term. Lower birth weight , stillbiin vitro with partner or donor sperm and then cryopreserved. When fertility is desired in the future the oocytes or embryos are thawed. Embryos are placed through the cervix into the uterus, either of the patient themselves or of a gestational surrogate, if the patient’s uterus is compromised. IVF with frozen embryos has been widely studied and evaluated in the general population; live birth rates per embryo transferred are estimated at 30%–40% [Embryo and oocyte cryopreservation are both considered non-experimental options for fertility preservation in post pubertal females, with oocyte cryopreservation having achieved this designation by the ASRM in 2012 . This ch 30%–40% , rates s 30%–40%  in the U 30%–40%  in the U 30%–40% . Improve 30%–40% . Data onWhile embryo and oocyte cryopreservation is technically possible as a means of preserving fertility for post pubertal females, a number of practical limitations exist. Of concern to both patients and practitioners is the need to delay the start of chemotherapy to allow for controlled ovarian stimulation of the ovaries. Recently protocols have begun to be developed where stimulation begins in the luteal  of the menstrual cycle  and may The use of GnRH analogues (both agonists and antagonists) to lessen the effect of chemotherapy on follicular depletion has long held appeal as this is much less invasive compared to the cryopreservation of reproductive tissue . Most eai.e., at the site of the ovaries, or heterotopically, i.e., at another location. Once transplanted the follicles within the ovary have the potential to mature when appropriately stimulated. Approximately 30 live births have been reported utilizing this procedure of removal and orthotopic re-transplantation in individuals who were post-pubertal at the time of retrieval [Ovarian tissue cryopreservation involves surgically removing all or a part of the cortex of the ovary, which contains thousands of primordial follicles. The resected tissue is cut into strips and cryopreserved. Because the process does not require hormonal stimulation it is the only fertility preservation technique that is available to pre-pubertal girls or females in whom initiation of treatment cannot be delayed ,51. Folletrieval . No birtin vitro. The ability to reliably do this would allow much greater control of the maturation process and precludes the need to transplant tissue back into the body [At this time ovarian tissue cryopreservation remains highly experimental without reliable means of ensuring maturation of the follicles and as such should be offered within the context of an IRB approved protocol. In addition, there are a number of challenges that exist. Obtaining tissue requires a surgical procedure with anesthesia, although ideally this could be coordinated with other procedures required for evaluation or treatment . A signithe body . This isthe body .Oophoropexy, the relocation of the ovaries outside of the radiation field, may mitigate ovarian damage, although radiation scatter can still cause follicle depletion . Shieldiet al. examined how frequently physicians of children and adolescents diagnosed with cancer discussed fertility preservation and how often referrals were made. Prior to therapy a discussion on risk for fertility was had with 86% of post pubertal females, fertility preserving techniques were discussed with 13% of females and 4 patients (1%) were referred to a fertility specialist [et al. offers some insight into how the physician/patient interaction may affect decision making and referrals. Fifteen medical professionals and 18 female cancer patients aged 17–45 were interviewed. Medical professionals were less likely to have detailed discussions with women than with men and took many more individual factors  into account when deciding how much information to provide. Women felt that pursuing fertility preservation options was discouraged and that conversations strongly emphasized the risk of delaying therapy. They perceived that individual factors were being taken into account in terms of how much information they were given, and they felt that they were not given the opportunity to have an adequate discussion to help make their decision [Making decisions about fertility preservation requires sufficient information and the time to weigh the benefits and limitations of the proposed procedures. As noted above discussions between medical teams and patients/parents are still not routine practices. Anderson ecialist . Low levdecision . This dadecision , particuAfter therapy has been completed patients exposed to potentially gonadotoxic therapies should have their ovarian function monitored. Traditionally, assessment of diminished ovarian function has relied upon sustained cessation or irregularities in the menstrual cycle as well as persistent elevations in follicle stimulating hormone (FSH) indicating proximity or achievement of menopause, with accompanying infertility. In this setting, further evaluation by an endocrinologist is warranted, particularly as early ovarian failure may increase the risk for the development of osteoporosis and cardiovascular disease . PatientAmenorrhea, both during and after therapy, is not uncommon in post-pubertal females. Those females who resume regular menstrual cycles following the completion of cancer directed therapy may still be at risk of developing premature menopause. Increasingly Anti-Mullerian hormone (AMH), which can be measured in the serum, has been utilized to provide earlier information about ovarian reserve ,60. CrosIt is encouraging to note that, even in the setting of diminished ovarian reserve  or cliniSpermatogenesis, the maturation of spermatogonia from spermatogonial stem cells (SSC), begins in puberty and then continues through a male’s lifetime, as the SSC are also self-renewing. The process, which takes approximately 74 days, is regulated by hormones secreted from the hypothalamic-pituitary-testicular axis. Therefore, unlike females, males do not face a defined reproductive window unless they sustain a very significant insult to the population of SSCs. While sperm quality may change over time, males continue to be able to have biologic children well into older adulthood .i.e., the ability to maintain an erection and ejaculate, can also be affected by chemotherapy and can contribute to the ability for males to father children through intercourse. Finally, surgical interventions that result in disruption of the anatomy or nerves supplying the reproductive organs may limit unassisted reproduction.As rapidly differentiating cells, spermatogonia are particularly sensitive to chemotherapy and radiation. Post pubertal males commonly experience temporary azoospermia during therapy that may persist for a number of years post therapy . Since S2, cyclophosphamide ≥60 g/m2, multiple alkylating agents, any alkylator combined with testicular irradiation, pelvic irradiation or TBI, and radiation doses in the range of 3–6 Gy as risks for the development of oligospermia/azoospermia [Alkylating agents such as cyclophosphamide, ifosfamide, procarbazine, and busulfan as well as cisplatin have been shown to be highly gonadotoxic, in a dose dependent fashion . The tesospermia . Prior tospermia  and testospermia .Preservation of fertility in post pubertal males is reliably accomplished by cryopreserving sperm prior to the onset of gonadotoxic therapy. This timing is important as there is the possibility that chromosomal damage can occur when the collected sperm has been exposed to gonadotoxic therapy . The mosLimitations to this form of cryopreservation are related primarily to an inability to masturbate, whether secondary to age, illness, discomfort  or cultuAlternative approaches exist when masturbation is not possible. Electroejaculation (EEJ) involves the placement of a transrectal probe while the patient is under general anesthesia. Electrical stimulation is applied until ejaculation occurs and sperm is collected . Sperm rIdeally, the development of a relationship between an oncology program and a designated sperm bank can streamline the process for a given patient as time constraints related to starting therapy are often cited as an obstacle to sperm banking . With a in vitro so that they could be used with assisted reproduction techniques and to evaluate the acceptability, feasibility and safety of testicular tissue biopsies in young, newly diagnosed pediatric cancer patients [Sperm banking is not possible for pre-pubertal boys as they cannot yet produce mature spermatozoa. Cryopreservation of testicular tissue that contains SSC offers an experimental option for preservation of fertility in pre-pubertal boys and post-pubertal males who cannot produce a sperm sample . Currentpatients .The testes should be shielded during radiation therapy to try to minimize the exposure to scatter radiation. Consideration can also be given to moving the testes out of the radiation field. At this time, there are no medications shown to protect males from gonadotoxicity .et al. referenced above both males and medical professionals recalled that fertility conversations at diagnosis generally encouraged sperm banking, even if the face of low risk for infertility, and was presented as a straight forward intervention, regardless of individual demographic factors [As stated in the introduction, males are often more satisfied with the information that they receive about fertility preservation prior to the initiation of therapy and much higher referral rates occur than for females . In the  factors . Despite factors . Charact factors . Oncolog factors . Deterre factors . The latet al. studied the factors involved in making a decision to cryopreserve testicular tissue among 74 patients, 12 years old or younger, and their parents who were approached to undergo this procedure. Fifty seven families consented and 48 boys had a biopsy performed. The researchers found that although diagnosis was a stressful time, they were able to consider the experimental nature of the procedure. Of note, those that did consent to biopsy felt less overwhelmed than those that refused. In those families where a biopsy was performed there was an expression of hopefulness for future scientific advances, a stronger desire to maintain the capacity for biologic offspring for their son and a hope to minimize distress if infertility did occur. For those that chose not to consent concern about the risk of the biopsy was cited as a reason [With regard to testicular tissue banking, Ginsberg a reason .Predictions about which patients will become infertile following gonadotoxic therapy remains imprecise and exposed males may still retain sufficient fertility to sire a child through sexual intercourse . The CCSin vitro fertilization (IVF) with or without ICSI. Pregnancy rates using this methodology are similar to those in the general population [Assisted reproduction offers options for biologic children for men with very low sperm counts (oligospermia) via a technique known as intracytoplasmic sperm injection (ICSI), the injection of a single sperm into an oocyte. ICSI greatly improves the likelihood that a small number of viable sperm retrieved pre or post therapy can be used for fertilization. In survivors, banked sperm can be thawed for use with pulation , and thepulation ,97. Yet,pulation . When viWith excellent survival rates and advances in the field of reproductive endocrinology, discussing risks for infertility and fertility preservation options with pediatric and adolescent patients diagnosed with cancer at diagnosis is an important component of comprehensive care. Even though such discussions are limited by inexact estimates of risks and reproductive technologies, early discussions allow patients and families to engage in decisions and may decrease the potential for regret later on. Discussion of fertility preservation should not be limited to diagnosis; survivorship is also an important time for patients to be aware of their fertility status and choices. For girls it is particularly important to monitor ovarian reserve as they have the potential to avail themselves of fertility preservation interventions post therapy. Algorithms for consideration of fertility preservation practices are provided for females and males in Despite an acknowledgement that discussions of fertility are important they do not occur routinely, yet these discussions are vital to help patients and families make decisions about fertility preservation. Understanding the risks for gonadal toxicity and options for fertility preservation are only the first steps to improving the likelihood that these discussions take place. Developing routine institutional practices and infrastructure is necessary given the time constraints and multiple stressors that exist at the time of diagnosis. The type of program that suits an individual institution may vary depending on the size and complexity of the practice but needs to include a mechanism that triggers identification of the target population, provides patients and families with information and facilitates access to services. The ability to consistently provide patients and families with up to date information in a timely fashion is likely to be beyond the scope of an individual physician. Identifying or creating reliable and relevant supplemental information, or directing patients to cancer focused websites is critical as information available by a patient initiated search may be inadequate and confusing especially at the time of a new diagnosis . Develop


Human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL) and a number of lymphocyte-mediated inflammatory conditions including HTLV-1–associated myelopathy/tropical spastic paraparesis. Development of animal models to study the pathogenesis of HTLV-1–associated diseases has been problematic and the mechanisms driving disease remain poorly understood. Here we report our findings of HTLV-1 infection in humanized nonobese diabetic (NOD) severe combined immunodeficiency (SCID) common gamma chain knockout (NSG) mice that have been implanted with fetal bone marrow-liver-thymus tissue (BLT). BLT mice were injected with lethally irradiated CD4+ HTLV-1 producing cells. Blood samples were collected every four weeks to monitor CD4 and CD8 phenotypes and DNA isolation. Concomitant with an increase in the number of CD4+CD25+ T-lymphocytes in exposed animals compared to controls, we observed a significant increase in the HTLV-1 viral DNA load. Surprisingly, unlike what we observed in macaques, HTLV-1 virus mutated to knockout Orf-I protein expression was able to infect BLT mice and high viral DNA loads were detected. However, after sequencing the orf-I region of proviral DNA from infected animals, we found reversion of the point mutation to wild type. This was observed as early as four weeks after exposure, indicating in vivo viral replication and selection for Orf-I expression. Thus, the BLT mouse model successfully recapitulates HTLV-1 infection and may serve as an important tool for investigating in vivo mechanisms of HTLV-1 disease pathology and in evaluating drugs and treatment strategies.


Recent trends show that Internet traffic is increasingly dominated by content, which is accompanied by the exponential growth of traffic. To cope with this phenomena, network caching is introduced to utilize the storage capacity of diverse network devices. In this paper, we first summarize four basic caching placement strategies, i.e., local caching, Device-to-Device (D2D) caching, Small cell Base Station (SBS) caching and Macrocell Base Station (MBS) caching. However, studies show that so far, much of the research has ignored the impact of user mobility. Therefore, taking the effect of the user mobility into consideration, we proposes a joint mobility-aware caching and SBS density placement scheme (MS caching). In addition, differences and relationships between caching and computation offloading are discussed. We present a design of a hybrid computation offloading and support it with experimental results, which demonstrate improved performance in terms of energy cost. Finally, we discuss the design of an incentive mechanism by considering network dynamics, differentiated user’s quality of experience (QoE) and the heterogeneity of mobile terminals in terms of caching and computing capabilities. The ever-growing number of smart phones is producing explosive amounts of traffic in order to support a wide plethora of multimedia services. A recent Cisco report estimates that global mobile traffic will exceed 24.3 exabytes monthly in 2019 [Furthermore, alongside the use of diverse network resources ,6 in terTo illustrate, viral on-line videos are the kind of content that mobile user repeatedly access, which leads us to an assumption that this content could be cached and shared at the edge of the network ,17,18. TThe problem of caching placement to maximize the probability that the user can access content in a wireless system where both SBSs  and userWe propose a novel caching placement strategy named MS caching. Then, we discuss the impact of the user mobility and the density of SBS on the content caching.We discuss the differences and relationships between caching and computation offloading and present a hybrid computation offloading based on MBS computation offloading, SBS computation offloading and D2D computation offloading.Considering the selfishness of mobile users, we suggest an incentive design based on network dynamics, differentiated user’s QoE, and the heterogeneity of user terminals in terms of caching and computing.In this paper, we investigate the impact of the user mobility on the performance of caching and computation offloading in 5G ultra-dense cellular networks. Then, we propose a joint Mobility-aware and SBS density caching placement scheme (MS-caching), taking into account the impact of user mobility and SBS distribution on the caching placement. Moreover, we addressed the SBS and mobile devices’ computing power, and we summarize four computation offloading modes in 5G ultra-dense cellular networks and propose a hybrid computation offloading strategy. Finally, we discuss an incentive mechanism to encourage content sharing and computation offloading between users with heterogeneous mobile devices. In summary, the contributions of this article include:The remainder of this article is organized as follows. In l files γ. i.e.,γ stands for the uneven distribution of popularity in these content. As shown in Local caching: When the user requests content, he or she will firstly examine whether or not such content is cached locally. Once such content is confirmed in the local storage, the user will get access to it without any delay.D2D caching: If the content requested by the user is not cached locally, the user will seek such content among the devices within the range of D2D communications. If there exists one user caching such content, the content will be transmitted to the target user via D2D communications.SBS caching: Besides D2D caching, if the required content is cached by one SBS, it will be transmitted to the user by the SBS.MBS caching: If the content requested by the user cannot be accessed in the aforementioned ways, such a request will be forwarded to the MBS, and the content will be delivered to the user by cellular network connection.In this section, we present the strategy of caching placement by considering the user mobility and SBS density. We assume that each user will randomly request files from one content library containing Rachel obtains the requested content by one of the means mentioned above when she moves to different locations starting from time Given the example in R denote the transmission radius of the SBS; ρ. In terms of cache placement on the SBS, we can describe it as follows: set Now, let us look at the SBS cache placement. Let model in ,31, the Thus, the total probability that a user can get the content from the SBS becomes:If we maximize the probability that the user obtains the content requested under the condition of the storage capacity of SBS, the SBS density-aware caching placement can be obtained as follows:t. Following the model in [i and the mobile device j. Let j caches content i obtains the content f via D2D can be calculated as follows:For user terminals, we assume that there are model in , the intThus, the total probability for the user to get the the content through D2D communication becomes:If we maximize the probability that the user obtains the content requested under the condition of the storage capacity of mobile devices, the optimal mobility-aware caching placement can be obtained as follows:Through joint optimization of popular caching [random caching [Popular caching: The popular caching strategies on SBSs and on mobile devices of users are as follows: (1) caching strategy on SBSs: most popular content should be stored on each SBS; (2) caching strategy on mobile devices: most popular content should be cached on each mobile device.Random caching: The random caching caching strategies on SBSs and on mobile devices of users are as follows: (1) caching strategy on SBSs: content should be stored at random on each SBS; (2) caching strategy on mobile devices: content should be cached at random on each mobile device.In this subsection, we evaluate the probability that users can get contents via simulation results. We compare MS caching with two different caching strategies:  caching  and rand caching .PopularSBS density-aware caching placement: We have provided the relationship between the SBS density and the probability that the user can obtain the requested content. The SBS density-aware caching placement is compared to the popular caching strategy and the random caching strategy, as shown in Mobility-aware caching placement: The user’s mobility is closely related to the probability for the user to access the content. The λ is the average contact rate of user devices. Similarly, with the analysis of SBS-assisted caching placement, MS caching placement: If we take into account the user mobility and the SBS density, a more advanced cache strategy named MS caching placement can be designed as demonstrated. In As for the simulation settings, for simplicity, assume the content size is the same and the value is ectively . The sys,1/1088) . The cacIn Rachel sends a request to a file K deadline Rachel to meet another user . After a short while, Tommy moves to the D2D communication range and sends the file K to Rachel. In the opposite case that Rachel still fails to obtain the requested content while the deadline is soon to expire, the MBS will still utilize the traditional SBS caching.Based on the above discussions, here, we provide an example of content caching when user’s mobility and SBS density are considered. As shown in Mobile cloud computing has been widely studied. Traditional mobile cloud architecture is based on a centralized cloud. For example, in  a cloud-In this section, we discuss the essential similarities and differences between caching and computation offloading. Content caching is generally provided by the server where the requested content originates from; content is cached during non-peak periods at the MBS, the SBS or the user terminal in order to save the bandwidth in critical time. During “rush hour”, corresponding contents are preferred to deliver to the user via SBS or other user terminal. Furthermore caching and computation offloading are correlative; for example, when the user requests for popular videos, the user terminal or SBS will transmit such content to the user, but the content is found not satisfactory in terms of video quality or the format specially required by the user. The user needs to transcode the original format to the one that satisfies the user. Thus, the task will be offloaded to SBS and/or other user terminals to speedup the computation. MBS computation offloading [floading : A user SBS computation offloading [floading : The comD2D computation offloading [floading : A user In this section, we have summarized the methods of mobile-edge computing offloading assisted by MBS, SBS and the user terminal ,38,39. Thybrid computation offloading. In the context of the a computation offloading, we name the user terminal that has been assigned the computation task as a computation node and the user terminal processing the computation task as a service node. When the computation node and the service node are within range of the D2D communication, the computation node offloads the computational task to the service node. After a period of time, the service node finishes the assigned task; at this moment, the computation node and service node are possibly out of the range of D2D communication because of user mobility. Thus, in some cases, the service node might be required to cache the computing results for a long time until it again comes into the vicinity of the computation node. On the other hand, if a higher storage capacity and a larger transmission radius of the SBS are available, the computing results can be returned back to the computation node in three manners after the computational task is processed at the service node:D2D computing result feedback: After the computational task is processed at the service node, the computing results will be returned directly back to the computation node if the service node and the computation node are still within the range of the D2D communication.SBS computing result feedback: After the computational task is completed at the service node, the service node will offload the computing results onto the SBS if the computation node is out of the range of the D2D communication. Then, the SBS will transmit the results to the user if it is within the communication range with the computation node.MBS computing result feedback: When the result of the computing task has not been transferred to the user before the deadline, namely when the user and the SBS are still not within the communication range, the SBS will upload the results to the MBS, and then, the final results will be passed back to the user.There are some advantages and disadvantages to the the above methods. The MBS computation offloading brings the highest communication cost, but provides the largest coverage . The D2DRachel  first divides the computation task into three sub-tasks. Within her D2D communication range, there are three users that can work as service nodes, i.e., Tommy, Eva and Suri. Then, Rachel offloads the three sub-tasks to them via D2D links. When a service node  finishes the computation sub-task, it possibly loses D2D connections with Rachel due to the user mobility. Tommy’ sub-task completion, Tommy is still within Rachel’s D2D communication range, and the D2D computing result feedback is used. When Eva’s sub-task is completed, Eva cannot connect with Rachel via the D2D link; however, an SBS between Eva and Rachel is available. Then, the SBS computing result feedback is used. The worst case is the MBS computation result feedback. Given Suri as an example, he moves far away, and the cellular network link is the only way to feed back the computation result. Based on the above discussion, we can see that the hybrid computation offloading achieves a flexible tradeoff among D2D computation offloading, SBS computation offloading and MBS computation offloading.As shown in Q, which can be decomposited into n sub-tasks. That is: h denote the channel gain and B is the channel bandwidth. Likewise, the channel capacity of the user terminal, SBS and D2D can be obtained We consider four kinds of energy consumptions corresponding to four operations during mobile edge computation, i.e., local computing, mobile offloading, edge cloud computing and downloading of computation results from the edge cloud to mobiles. Here, we mainly consider the energy consumption for the mobile terminal. For the four kinds of computation offloading, they have the same local energy consumption. Additionally, edge cloud computing and downloading of computation results do not consume user terminal’s energy. Thus, the major energy consumption of the task is up to mobile offloading. We consider that a user has computation task rding to ,39, we sλ is equal to 0.00011. However, the energy cost increases again when the mobility is too high. This is because the contact time of the D2D connection is too short, which easily causes the failure of the computation result feedback. In comparison, the hybrid computing offloading combines the advantages of the other three computation offloading schemes and produces the lowest energy cost.In As already mentioned, the main target of caching and computation offloading in 5G ultra-dense cellular networks is to reduce traffic load and encourages the D2D communications among users. However, the intrinsic selfish feature of user terminals constitutes the biggest obstacle for content caching and computation offloading in practical situations. For example, most users intend to store their favorite files, which at the same time might be also cached by many other users. This fact could result in replicated caching and insufficient use of the accumulative storage space of the network nodes cumulatively. As for computing, most users like to count on others to help them to execute the computation tasks while being reluctant to share computing capacity with others.In order to solve the problem, this paper designs an incentive mechanism based on the following three kinds of heterogeneities: (1) the heterogeneity of the user devices, namely each user terminal’s storage and computing capabilities are different, which makes some users willing to cache contents and earn incentives through content sharing, while other users prefer to provide computing service to others, and the earned incentive can be used to request cached contents; (2) the heterogeneity of user requirements in terms of user’ QoE, namely each user’s demand for computing and caching and his/her preference for content are different; (3) the heterogeneity of network conditions, namely the user mobility within the region and the density of the SBSs are different.Bob’s mobile phone has large storage capacity to cache more popular content, but its computing capacity is relatively weak; whereas, user Suri’s mobile phone has great computing capacity, but weaker storage capacity. When both are within the range of the D2D communication, user Bob may offload some content as requested by user Suri and then get some incentive when the content is sent to Suri. Thereby, user Bob may offload computational tasks to be processed onto user Suri, and user Suri can get some incentive, which can pay for the “debt” for getting the content. Therefore, an incentive balance of content is achieved and replaced by computation. We introduce an incentive mechanism to encourage various users with heterogeneous mobile devices to exchange favors of content sharing and computation offloading.Similar to the incentive mechanism for crowdsourcing, more incentives lead to a higher user’s QoE. There are two main methods to earn the incentive: cache content and computing tasks for others. Moreover, the two can be transformed into each other; for example, user Caching incentive: When the user B obtain content from the user A, the user B needs to pay an incentive , this incentive includes the cost of D2D communication between B and A, the cost of storing content at the expense of the content value from the perspective of the user A. Meanwhile, the user A can get these incentive. From the above, we can see that the popularity of the content of the caching incentive, downloading times of users and caching time are all related to these three aspects.Computing incentive: When the user A offloads computing tasks and transfers them to the user B and then the user B helps the user A to proceed with the calculation, the user A will pay the user B an incentive for the communication cost and the computing cost. At the same time, the user B will obtain these incentives. The costs are relatively high due to the fact that the result of computation is equivalent to the content, whose popularity is zero.Specifically, we can divide this incentive into two levels:With the increasing capabilities of mobile terminals in terms of storage and computing, caching of popular content on wireless devices enables content sharing through the D2D links. Even though various works focus on caching placement in the 5G ultra-dense cellular networks, it is still a challenging issue to jointly consider caching and computing by using the advantage of the user mobility. In this article, we proposed an MS caching placement with the use of the SBS and the user terminal while taking the effect of user mobility and SBS density into consideration for content caching. Then, we designed a hybrid computation offloading scheme to achieve flexible tradeoffs among the MBS computation offloading, the SBS computation offloading and the D2D computation offloading. Finally, we discussed an incentive design in terms of caching and computing by considering the hardware heterogeneity of the mobile devices, various user’s requirements on QoE and the heterogeneity status of the network.In future work, we will consider the social relationship of the user terminal in the D2D communication. It can be concluded from the users’ social relationship that those with social connections tend to have the same request for content; for example, one region may be divided into different groups, such as an industrial group, a tourism group, a residential group, etc. Different contents will be cached in different regions, and in the same region, the interchange of content may be better achieved.


The gastrointestinal peptide hormones cholecystokinin and gastrin exert their biological functions via cholecystokinin receptors CCK1R and CCK2R respectively. Gastrin, a central regulator of gastric acid secretion, is involved in growth and differentiation of gastric and colonic mucosa, and there is evidence that it is pro-carcinogenic. Cholecystokinin is implicated in digestion, appetite control and body weight regulation, and may play a role in several digestive disorders.http://sblab.celldesigner.org:18080/Payao11/bin/).We performed a detailed analysis of the literature reporting experimental evidence on signaling pathways triggered by CCK1R and CCK2R, in order to create a comprehensive map of gastrin and cholecystokinin-mediated intracellular signaling cascades. The resulting signaling map captures 413 reactions involving 530 molecular species, and incorporates the currently available knowledge into one integrated signaling network. The decomposition of the signaling map into sub-networks revealed 18 modules that represent higher-level structures of the signaling map. These modules allow a more compact mapping of intracellular signaling reactions to known cell behavioral outcomes such as proliferation, migration and apoptosis. The integration of large-scale protein-protein interaction data to this literature-based signaling map in combination with topological analyses allowed us to identify 70 proteins able to increase the compactness of the map. These proteins represent experimentally testable hypotheses for gaining new knowledge on gastrin- and cholecystokinin receptor signaling. The CCKR map is freely available both in a downloadable, machine-readable SBML-compatible format and as a web resource through PAYAO  contains supplementary material, which is available to authorized users. Helicobacter pylori infection [Gastrin and cholecystokinin (CCK) are gastrointestinal peptide hormones that share a common C-terminal pentapeptide amide . Gastrinnfection , 13. In Information concerning intracellular signaling is commonly retrieved from databases such as Reactome  and KEGGIn the past decade, several manually constructed maps of signaling events have been published –25, eachPartitioning the total CCKR signaling map into sub-networks using the BiNoM tool  resultedi).http://www.genomatix.de/solutions/genomatix-software-suite.html) [http://www.ihop-net.org/UniPub/iHOP/) [evidence that the reported signaling event is mediated by the specific interaction of CCK or gastrin (G-17) with their receptors CCK1R and CCK2R.signaling information to allow for linkage of a new CCKR map component to its upstream and/or downstream regulators in CCKR signaling.Knowledge encoded in the CCKR map was obtained from scientific publications that were identified by searching for different combinations of cholecystokinin (CCK)/CCK1R and gastrin (G-17)/CCK2R in PubMed or through various literature mining tools, e.g. LitInspector (te.html)  and iHOPb/iHOP/) . We adopii).CellDesigner species and reaction “note” features were used to record PubMed IDs (PMID), cell-type specific information for each reaction, and the interacting components in the CCKR map.iii).http://www.payaologue.org) [http://sblab.celldesigner.org:18080/Payao11/bin/). The CCKR map is also available in the SBML data exchange format  , enablingue.org)   standard for process diagrams  and the We welcome comments and tags from a community of curators to continue increasing the quality of this CCKR signaling map and will keep it up to date with our growing biological understanding.k, and the node degrees of all nodes in the network represent the ‘degree distribution’ of the network. Nodes with degree k > 5 were termed ‘hubs’.Network topology studies were performed by importing the CellDesigner-generated SBML file of the CCKR map to Cytoscape version 2.8  using thWe used the ‘prune the graph’ function of BiNoM to automatically separate the strongly connected component (SCC), i.e. the central cyclic motif of the map, from more loosely connected upstream and downstream species. The SCC was further decomposed into smallest sub-networks with the function ‘extract material components’. Next, some manual curation work was performed: sub-networks with 50 % or more overlapping nodes were clustered together while the large sub-networks were decomposed further until each sub-network contained a unique central cyclic motif. Any unconnected nodes were removed from the sub-networks, yielding a total of 18 sub-networks or modules. Next, these 18 modules were merged together and the resulting network was compared with the initial map to check for completeness in terms of any missing interactions or nodes. The complete map and its 18 decomposed modules are available as a Cytoscape session file , and filtered for binary physical interactions based on PSI-MI controlled vocabulary experimental method descriptions, following the procedure in  , and based on experiments performed in 37 different cell lines representing a wide array of cell types , describing a total of 530 molecular species and 431 reactions experimentally documented to play a role in CCKR signaling. The reactions include molecular state transitions , transport, heterodimer associations/dissociations as well as transcriptional regulation of target genes by transcription factors, which are reported to be associated with the regulation of gastrointestinal hormone responsive biological processes , PKC dependent phosphorylation of adaptor protein SHC - transforming protein C, and its association with GRB2/SOS1 leading to activation of the HRAS/RAF/MAPK1/3 cascade [Downstream signaling mechanisms shared by CCK1R and CCK2R include the trimeric guanine nucleotide binding alpha q protein  [13) [2+ signaling pathways by opening ryanodine receptors and two-pore channels that release calcium from the endoplasmic reticulum and endolysosomes, respectively [2+/calmodulin (CaM) dependent activation of CaMKIV and calcineurin (CaN) – NFAT1 signaling pathways [Two trimeric G-proteins appear to be regulated only by CCK1R. One is G alpha S (Gαs) , which ls) [13) , involves) [13) . The NOSs) [13) , 51 reguectively –54. Othepathways –57.CCK2R activates EGFR via PKC activated MMP3, which cleaves membrane-attached pro-HBEGF into mature HBEGF , 59. PKCThe complete CCKR signaling map Fig.  is very Depending on cell type and the state of cells, gastrin can induce different cellular outcomes, such as proliferation, migration and apoptosis. In order to investigate what is known about the involvement of each of the signaling pathway modules in these processes, we checked the scientific literature that we have used to build the CCKR map. We chose to focus on gastrin-mediated effects, because molecular mechanism studies of gastrin-regulated cellular responses are more abundantly reported in literature. Specifically, we recorded all instances where experimental evidence was reported indicating that the function of a specific signaling component had an effect on the cellular outcome in response to gastrin treatment. Based on this literature survey, involvement in cellular outcomes was indicated in Fig. Molecular mechanisms underlying gastrin-mediated proliferation involve regulation of protein synthesis and cell cycle. Protein synthesis is stimulated via the AKT1-module component mTOR triggering p70 S6 kinase , 70. GasGastrin promotes cell migration by activating transcription of MMP7 and MMP9 , 78 via BIRC2 and BIRC3 target genes, which can be activated either directly by PKC or independently of PKC through the Rho GTPase module. Likewise, the AKT1-involvement in regulation of the BCL-module can be mediated by PKC-dependent mechanisms or independently of PKC by the CCK2R - Rho GTPase pathway. Activation of AP1, on the other hand, seems to be strictly dependent on PKC that mediates its effect via either RAF1-MAPK1/3 or SRC-MAP3K11 cascades.Apoptosis is inhibited by gastrin via several mechanisms including BCL-mediated repression of pro-apoptotic caspases and AP1-activated expression of Clusterin , 81, 82.We note that the AKT1-module both inhibits the RAF1-MAPK1/3-route to AP1-activation and enhances Rho GTPase activation of the BCL-module. Thus, AKT1 can potentially promote BCL-module apoptosis-regulating mechanisms and at the same time block MAPK1/3-mediated AP1-activation. In the latter configuration the cell would rely on MAP3K11 to bypass the inhibitory effect of AKT1 on AP1-mediated regulation of gene expression.The comprehensive CCKR signaling map Fig.  has beenWe identified 4119 proteins interacting with at least one CCKR signaling protein  that enhance hydrolyzation of the bound GTP. In gastrin-mediated signaling deactivation of RHOA and RAC1 is effectuated by GAPs, ARHGAP4 and RGS2, respectively.Central to the Rho GTPase module Fig.  is the aEight of the ten PathExpand protein interactors Fig.  of Rho GThe PathExpand interactor BNIP1 (BCL2/adenovirus E1B 19 kDa interacting protein 1) interacts with two downstream BCL module components BCL2L1 and BCL2, both involved in effectuating the gastrin anti-apoptotic signal. Since BNIP1 is known to be involved in SNARE vesicular transport, our findings indicate that it may be an interesting candidate to follow up with investigating a potential role of vesicular transport in modulating BCL-linked signaling mechanisms in the gastrin signaling pathway. The PathExpand interactor programmed cell death 11 (PDCD11) interacting with the two subunits of NFκB transcription factor (NFκB1 and TF65) is known to be involved in maturation of ribosomal RNA but may also play a role in mRNA processing. Our results identify PDCD11 as a potential candidate to follow up in experimental analysis of molecular mechanisms involved in NFKB-mediated gene regulation in cellular responses to gastrin.et al. [In the work presented here, we set out to build a comprehensive and well-annotated molecular interaction map to aid future studies involving gastrin or CCK. A mechanistic understanding of CCK1R and CCK2R signaling networks is essential for experiment design and data interpretation in biological systems involving gastrin- and CCK-regulated processes. Moreover, it may enhance the identification of therapeutic chemicals able to target disease, by using the map as a functional interaction diagram with components that modeling indicates as prime targets for perturbation, as described by Lee et al. , who sucet al. , 85, 86,et al.  as well Some pathway proteins and reactions of the CCKR signaling map are also described in the Reactome  and KEGGWe enhance the applicability of the map for hypothesis generation by two central strategies. First, we provide a computationally modularized version of topologically and functionally connected meta-nodes. This modular view simplifies the complete CCKR map and provides for an improved, higher level comprehension of pathway regulatory aspects concerning cell fate decisions related to proliferation, migration and apoptosis. Secondly, we take advantage of public large scale PPI knowledge to predict new potential regulators of CCKR signaling, including 70 interactors that significantly enhance the compactness of the CCKR map , throughCompared to other recently published computational approaches for high-throughput hypothesis generation , 89, 90,Our work demonstrates how publicly available tools can aid in the manual curation of an extensive network topology, in order to build a foundation for a systems understanding of CCKR-mediated cellular responses. Furthermore, the integration of a comprehensive model of complex biological networks with genome scale data can provide new knowledge and hypotheses on molecular mechanisms underlying cellular processes. Further development of the resources presented here should be of high interest in translational research aimed at identifying new targets and biomarkers for treatment and diagnostics of gastrin- and/or cholecystokinin-related disease, including cancer.


Logistic regression analysis was performed to identify independent risk factors for the infection. Assessments were carried out on 246 cases aged 17–60 and 776 controls aged 17–59, who were matched based on their ages, gender, and date and center of donation. Logistic analysis showed low income , low educational level , being born in the cities of either Mashhad  or Neyshabour , and a history of blood transfusion  or non-IV drug abuse  were significant predictors for infection with HTLV-1. Lack of variability or small sample size could be reasons of failure to detect some well-known risk factors for HTLV-1 infection, such as prolonged breastfeeding and sexual promiscuity. Pre-donation screening of possible risk factors for transfusion-transmissible infections should also be considered as an important issue, however, a revision of the screening criteria such as a history of transfusion for more than one year prior to donation is strongly recommended.Human T-cell lymphotropic virus type 1 (HTLV-1) infection is an endemic condition in Northeast Iran and, as such, identification of risk factors associated with the infection in this region seems to be a necessity. All the possible risk factors for HTLV-1 seropositivity among first-time blood donors were evaluated in Mashhad, Iran, during the period of 2011–2012. Blood donation volunteers were interviewed for demographic data, medical history, and behavioral characteristics and the frequencies of risk factors were compared between HTLV-1 positive (case) and HTLV-1 negative (control) donors. The data was analyzed using Chi square and  Human T-cell lymphotropic virus type 1 (HTLV-1) was the first human retrovirus to have been discovered in 1980 . HTLV-1 etc. and screening for blood-borne agents including HBV, HCV, HIV, HTLV, and Treponema pallidum with sensitive and accurate assays [The infection can be transmitted through the transfusion of contaminated blood or blood products, unprotected sexual contact, sharing of contaminated syringes and other instruments, or via transmission from mother to child ,6. Risk e assays . The fire assays . After te assays . Howevere assays ,10. Moree assays ,12,13,14To our knowledge, no survey has been performed to determine the risk factors for HTLV-1 infection in Iran. Therefore, in the current study the frequency of associated risk factors for HTLV-1 infection was investigated among blood donors in Mashhad, Northeastern Iran. Identification of individuals with these factors and excluding them from blood donation would result in a reduction of transfusion-transmitted cases of the infection.Treponema pallidum. Blood samples from the control group showed no reactivity to the abovementioned transfusion-transmitted agents.This case-control epidemiological study was conducted among first-time blood volunteers who had been referred to blood transfusion centers of Mashhad, Iran between September 2011 and August 2013. A total of 54,436 individuals donated blood, of which 321 individuals  had HTLV-1 infection based on screening and subsequent confirmatory test results. The cases included 316 blood donation volunteers from Mashhad city with confirmed HTLV-1 seropositivity and the controls were selected randomly from Mashhadi donors who had shown no reactivity for HTLV-1 antibodies in screening tests. Four controls were individually matched to each case on the basis of their ages (±2 years), gender, and date and center of donation. ELISA method  was used as a primary detection tool of HTLV-1 antibodies and the positive results were confirmed via a Western blot analysis . All blood donors were routinely visited by the physician of the blood transfusion center before donation and checked for the presence of any blood-borne infections such as HBV, HCV, HIV, HTLV, and etc.), dentistry procedures with bleeding , suturing, acupuncture, needlestick injuries , history of risky behaviors such as tattooing, cupping, tatbir , body piercing, unsafe sexual contact, drug abuse, and imprisonment.Participants were interviewed by trained research assistants using a questionnaire on demographic and socio-economic characteristics, such as age, gender, marital status, education, income, birth place, family size, weight and height, duration of breastfeeding, medical histories including sexually-transmitted infections (STIs), blood transfusion, hospitalization, surgery, invasive diagnostic tests  (Number: 2033-10) with regard to ethical issues. All participants in the study did so voluntarily, and signed a written consent.t-test. Logistic regression analysis was also performed to identify independent risk factors for HTLV-1 infection. Statistical significance was assumed for p values of less than 0.05.All statistical analyses were performed by SPSS version 16  using a Chi square test and p = 0.990 and p = 0.478, respectively). Among the case group, male to female ratio in participants was considerably less than that in non-participants (p = 0.004), albeit, no differences in the ratio was observed between referred and non-referred individuals from the control group (p = 0.329).From 316 cases, 246 (77.8%) individuals aged 17–60 years had been referred for consulting and were subsequently interviewed. Moreover, 1241 controls were invited to participate in the study, of which 776 (62.5%) persons aged 17–59 years, agreed to be interviewed. As p = 0.25 and p = 0.08, respectively). Cases had higher frequency of remarriage compared to controls (p = 0.003). The proportion of individuals with primary education or less in the case group (28.8%) was higher than those observed in the control group . Furthermore, patients with the infection had significantly lower incomes compared to the controls (p = 0.001). According to p < 0.0001). In addition, no significant difference was found in the marital status (p = 0.09), body mass index (p = 0.73), family size (p = 0.21), and duration of breastfeeding (p = 0.26) between the both groups.Demographic and socio-economic characteristics of donors are shown in p = 0.005). Fifteen donors, including six cases (3.7%) and nine controls (1.5%), reported blood reception in the province of Khorasan Razavi  after the routine screening for HTLV-1 infection was started in this region (p = 0.07).Medical histories of blood donors which were assumed to be associated with the risk of HTLV-1 infection are shown in p = 0.53). On the other hand, no significant differences were found in the history of hospitalization, surgery, acupuncture, suturing, invasive dental treatment, invasive diagnostic procedures, and needlestick between both groups . However, none of the subjects had stated a history of injecting drug use (IDU). Moreover, history of pre- and extra-marital sexual contact and number of lifetime sexual partners in both groups were similar (p = 0.17 and p = 0.28). On the other hand, among 22 cases with a history of pre- or extra-marital sex, 4.5% had one partner, 31.8% had two partners, and 63.6% had at least three partners. In controls with a same history, 43.6%, 27.3%, and 29.1% of 55 donors had one, two, and three or more partners, respectively (p = 0.002).Possible risky behaviors related to HTLV-1 infection are presented in All variables with a significant relation to HTLV-1 infection in univariate analysis were entered into the logistic regression model. As This study was conducted to identify possible risk factors related to HTLV-1 infection among first-time donors who referred to blood centers in Mashhad, Iran. Findings showed that the main risk factors associated with HTLV-1 infection were low educational levels, low income, being born in the cities of Mashhad and Neyshabour, and histories of blood transfusion and drug abuse.In a study conducted on Australian blood donors during 2000–2006, the prevalence of HTLV-1 seropositivity was three per 100,000 donors, of which 5% had at least one unidentified risk factor at the time of blood donation. In general, no dominant risk factors emerged, but the major identified factors related to the infection were as follows: the country of birth and parental ethnicity (24%), sex with individual from overseas (23%), at-risk household contacts (19%), tattooing or piercing (8%), surgery or endoscopy (8%), blood product transfusion (8%), and other blood contacts, such as needle sticks (4%) .In the current study, being born in the cities of Mashhad or Neyshabour showed a strong association with infection of HTLV-1. Very high prevalence of the infection in the general population of these cities had been previously reported ,4. Howevet al. and Dourado et al. could not find any significant association between literacy and income with the infection in blood donors [et al., low educational level was identified to be a main risk factor for HTLV-1 infection among blood donors in French West Indies [et al. demonstrated that low literacy was indeed an important risk factor for HTLV-1 infection [In the present study, the case group had a significant lower education and income levels compared to the controls. It seems that low education and income levels among these people would have reduced their access to health information and may increase the prevalence of risk factors related to HTLV-1 infection, such as risky sexual behaviors or contact with contaminated blood through tattooing or piercing among these individuals. In another study on the general population of Mashhad, HTLV-1 infection was not associated with income, but the highest prevalence of the infection was observed among illiterates. Nevertheless, regression analysis showed no significant association between educational level and the infection . Similard donors ,18. Convt Indies . Furthernfection .et al. could not find a significant association between the infection and breastfeeding due to very high prevalence of breastfeeding in both HTLV-1 seropositive and seronegative individuals [Breastfeeding, especially longer than six months, is one of the HTLV-1 transmission routes . The curividuals . On the ividuals .As expected, this study showed that HTLV-1 infection is significantly associated with a history of blood reception. Nevertheless, there was no significant difference between the study groups if the history was limited to reception in Khorasan Razavi blood centers after 1994 when routine screening for HTLV-1 infection began in this region. A history of blood transfusion during the last year is one of the deferral criteria for blood donations in Iran and all of the blood donors had not received any blood for at least one year before the blood donation date. In a survey in Neyshabour, the proportion of the infection among individuals with a history of the transfusion was four times higher than others ,23,24. OBased on the results of the present study, the risk of infection was not increased in individuals with a history of surgery, hospitalization, needlestick, cupping, or tattooing. Similarly, in the study conducted on the general population of Mashhad, HTLV-1 infection was not more prevalent among individuals with a history of these variables on the regression analysis . In anotet al. showed that HTLV-1 seropositive cases in Brazilian blood donors more often used non-intravenous illegal drugs (OR = 3.3), while no significant difference was observed between HTLV-1 seropositive and seronegative groups for the frequency of intravenous drugs use [This study unexpectedly identified non-IDU as a risk factor for HTLV-1 infection. The frequency of non-IDU in the case group was significantly higher than in controls . Likewise, Soares rugs use . In Iranrugs use .et al. demonstrated that a history of STIs and Chlamydia seropositivity are indeed predictive factors for HTLV-1 infection among blood donors from Guadeloupe, French West Indies [et al. showed significant correlation between having more than three partners and HTLV-1 infection among females in Guinea-Bissau, West Africa. However, they could not find a significant correlation between a history of gonorrhea and genital ulcer with HTLV-1 infection [A history of STIs during one last year is a deferral factor for blood donation in Iran, and in the current study a history of STIs among blood donors was expectedly very low as a result. A history of extramarital sexual contact was similar among cases and controls, however, among this subgroup, cases had more lifetime sexual partner than controls. In a study conducted on injecting drug users in Mashhad prison, no association was found between HTLV-1 infection and a history of STIs and the frequency of multiple sexual partners did not differ between the HTLV-1 positive and negative groups . In contt Indies . In addinfection .In summary, a history of blood transfusion, being born in the cities of Mashhad and Neyshabour, having low education and income levels, and non-IDU were significantly associated to HTLV-1 infection in first-time donors from Mashhad, Iran. Failure to detect other well-known risk factors linked to HTLV-1 infection such as breastfeeding, sexual promiscuity, and IDU could have been due, in part, to a lack of variability or the small sample size used. Various blood safety programs, such as pre-donation screening and deferral policies for blood donors with a history of possible risk factors for transfusion-transmissible infections, might also have contributed to the reduction of blood donation volunteers with these risk factors in Iran. However, a revision of the screening criteria such as a history of transfusion for more than one year prior to donation is strongly recommended.


In the past, reactive oxygen and nitrogen species (RONS) were shown to cause oxidative damage to biomolecules, contributing to the development of a variety of diseases. However, recent evidence has suggested that intracellular RONS are an important component of intracellular signaling cascades. The aim of this review was to consolidate old and new ideas on the chemical, physiological and pathological role of RONS for a better understanding of their properties and specific activities. Critical consideration of the literature reveals that deleterious effects do not appear if only one primary species  is present in a biological system, even at high concentrations. The prerequisite of deleterious effects is the formation of highly reactive secondary species , emerging exclusively upon reaction with another primary species or a transition metal. The secondary species are toxic, not well controlled, causing irreversible damage to all classes of biomolecules. In contrast, primary RONS are well controlled , and their reactions with biomolecules are reversible, making them ideal for physiological/pathophysiological intracellular signaling. We assume that whether RONS have a signal transducing or damaging effect is primarily defined by their quality, being primary or secondary RONS, and only secondly by their quantity. Intracellular RONS, as well as excessive release of extracellular RONS were thought to induce deleterious effects, causing oxidative damage to different kinds of biomolecules. These processes are referred to as “oxidative stress”.Reactive oxygen and nitrogen species (RONS) include two classes of chemically-reactive molecules containing oxygen  and nitrogen . Both classes are referred to as RONS. The majority of RONS carries unpaired electrons and is called free radicals. In mammalians, a major function of specialized enzymes, such as NADPH-oxidase, myeloperoxidase and nitric oxide synthase (NOS), is the generation of RONS. The controlled generation of RONS in the extracellular space by these enzymes was developed evolutionarily as part of the innate immune system to kill bacteria. However, an overwhelming release of RONS may also induce deleterious effects, causing damage to host biological structures. Another group of enzymes release RONS intracellularly as a byproduct of metabolic processes. For instance, superoxide , cell proliferation, differentiation and migration. It took over 50 years for a clear understanding of the chemical basis of free radical/RONS biology to emerge. In the following 50 years, studies on the biological effects of free radicals with biological targets were undertaken. The aim of this review is to put together old and recent ideas on the chemical and pathophysiological role of RONS for a better understanding of their properties and specific activities. This review is predominantly based on selected reviews, elaborating on different aspects of RONS activity and thought to be a guide through a large body of literature existing on this topic.g = 2.0023 in the electron paramagnetic resonance spectrum [•OH) from hydrogen peroxide (H2O2) is catalyzed by iron ions, called the “Fenton reaction”  to another (•OH) was associated with the presence of iron ions as a catalyst . This. Thisg =iewed in ). Later,2O2 to •OH by ferrous ions. The branching chain reaction between lipid peroxides and iron ions accelerates lipid peroxidation [Another important step in understanding the biological function of RONS was the discovery of free radical chain reactions. This was done in 1935 by the Russian chemist and Nobel Prize winner Nikolai Semenov. He described four types of free radical reactions, namely initiation, propagation, branching and termination . The samIn the 1950s, researchers started to associate free radical chemistry with biomedical questions. It has been suggested that most of the damaging effects of oxygen in living systems are due to the formation of free radicals (reviewed in ). This a2•− into H2O2 , a specific enzyme catalyzing the transition of Oto H2O2 , which are also powerful oxidizing agents, participate in free radical reactions (reviewed in [•) and peroxynitrite (ONOO−) were also shown to interact with ROS, and all of these species were termed RONS. Primarily, the toxic properties of RONS were of interest. It was shown that an excessive generation of RONS damaged almost all classes of biomolecules, such as lipids [Until the mid-1970s, the literature almost exclusively refers to free radicals. Later, it became evident that not only free radicals, but also non-radical products, such as Hiewed in ). To taks lipids , proteins lipids  and DNA s lipids ). In thes lipids , reviewes lipids ). Evoluts lipids . Howevers lipids .2•−, H2O2 or NO•, in most cases reversibly react with the target molecules. NO•, for instance, reversibly binds to heme proteins, whereas O2•− reacts with proteins, changing their redox state without damage to their structure. For instance, the reaction between O2•− and cytochrome c results in the reduction of heme, which is used to detect O2•− [•OH, ONOO− and HOCl [2O2 yielding •OH; and (ii) the reaction of O2•− with NO• yielding ONOO−. Furthermore, the formation of HOCl, formed in an enzymatic reaction from H2O2 and Cl−, is associated with damage to host tissues [−, resulting in the formation of nitrated amino acids, such as nitrotyrosine. ONOO− is a secondary RONS formed in the reaction between NO• and O2•−. ONOO− and NO• may have quite opposite biological effects. For instance, NO• has an inhibitory effect on lipid peroxidation, while ONOO− activates this process . Some a tissues . Oxidatiiewed in ). Anotheiewed in ,33). Theiewed in ).2•−, NO•, H2O2) only have a weak damaging potential, whereas secondary RONS are more toxic. Primary species are well controlled by SOD, catalase and NO synthases, while secondary species are less controllable, since there is no specific enzymatic system controlling their levels.The data described above suggest that primary ROS  produce potentially dangerous H2O2. H2O2 itself is relatively inactive, but can lead to the formation of toxic •OH. In contrast, ceruloplasmin in the blood inactivates O2•−, yielding H2O [2•−. The fact that SOD, not ceruloplasmin, occurs in cells indirectly suggests that H2O2 may have a physiological function inside cells, but not in extracellular fluids. The data gathered in the last few decades suggest that primary RONS formed in mitochondria (O2•− and H2O2) and NO• are associated with intracellular signaling cascades. Since NO-mediated signaling pathways have already been extensively reviewed , canding H2O  and conteviewed  or radical scavengers has an effect on signaling pathways.Genetic manipulation of mitochondrial SOD and cytoplasmic SOD decreases the efficiency of specific signaling cascades.A number of reports suggest Oiewed in ). Eviden2•−, the primary mitochondrial ROS, was often associated with the regulation of inflammatory pathways, such as activation of the inflammasome, regulation of inflammatory cytokines synthesis and mechanisms of innate immunity. The involvement of mitochondrial O2•− in the activation of the inflammasome was suggested by Zhou et al. [2•− is involved in this signaling cascade. Another important feature of inflammation is the release of cytokines. Bulua et al. [2•−, suggesting a key role of O2•− in this signaling pathway. Weidinger et al. demonstrated that mitochondria-targeted antioxidants reduce the expression of IL-6 and iNOS in a model of systemic inflammatory response induced by LPS [2•− from mitochondria to the cytoplasm, prevented activation of NADPH-oxidase. In contrast, the deficiency of mitochondrial SOD intensified the stimulation of NADPH-oxidase, suggesting that this process is mediated by O2•−, rather than by H2O2. Applying specific mitochondrial inhibitors and direct detection of mtROS, Dikalov et al. suggested that mtROS, presumably O2•−, are able to activate NADPH-oxidase via activation of protein kinase C (PKC) [2•− orchestrate cellular ROS production upon inflammation. The same group has shown that stimulation of endothelial cells with angiotensin II elevates mitochondrial O2•− levels and simultaneously the activity of NADPH-oxidase in this non-immune cell type [2•− and decreased the activation of vascular NADPH oxidases [2•− rather than H2O2 is involved in this signaling cascade. However, in the past, it was believed that O2•− does not participate in signaling, as it cannot exit mitochondria due to its polarity. Consequently, H2O2 formed from O2•− was considered a necessary intermediate of O2•−-mediated actions. H2O2 is a nonpolar molecule and can easily diffuse through the membranes. Recently, however, the situation has shifted. O2•− has been shown to leave mitochondria via the mitochondrial permeability transition pore [2•− can be released from mitochondria by using O2•−-sensitive spin probes and electron spin resonance spectroscopy [2•− can directly contribute to intracellular signal transduction pathways.Mitochondrial Ou et al. . The auta et al.  showed td by LPS . In leukd by LPS  showed t C (PKC) . These dell type . Howeveroxidases . NADPH ooxidases , differeoxidases  and proloxidases  (revieweoxidases ). These ion pore  and anioion pore . We alsotroscopy . These d2O2 being the RONS-based messenger in intracellular signaling cascades, as well. In contrast to O2•−, H2O2 is a neutral molecule and relatively inactive. Thus, H2O2 is able to cover relatively large distances, up to several cell diameters, before it reacts with its target or is catabolized [Nevertheless, other groups propose Habolized . Therefo2O2 on intracellular signaling is supported by the following evidence:2O2 has a direct effect on signaling cascades.Exogenous H2O2 level correlates with the effectiveness of intracellular signal transduction.The HGenetic manipulation of catalase has an effect on signaling.Upregulation of MnSOD and/or Cu/ZnSOD activates signaling.In the literature, the impact of H2O2 increased the proliferation of endothelial cells in a study of Chen et al., suggesting that H2O2 directly interferes with pathways regulating proliferation [et al. [2O2 in the regulation of cell migration. Schmidt et al. [2O2 rather than O2•− as the mediator of NF-kB pathway activation. Brunelle et al. [2O2 acts as signaling molecule in the process of HIF-1 regulation. West et al. [2O2. Hoarau et al. [2O2 plays an essential role in the development of β-cells, as it activates the ERK1/2 pathway. Other studies on H2O2-mediated signaling are summarized in several reviews [Treatment with Hferation . Wang et [et al.  showed tt et al.  demonstre et al.  demonstrt et al.  showed tu et al.  demonstr reviews ,76,77.2•− or H2O2 as the major signaling molecule. Only a few studies suggest that signaling molecules may be derivatives of H2O2. Garlid et al. [2O2 as a contributor to this pathway. Others suggested that HOCl may participate in extracellular, but not in intracellular signaling, for instance by interaction with TGF-ß1 [− causes tyrosine nitration, which blocks the respective signaling cascades. Tyrosine nitration seems to have a significant impact on a number of pathways, such as MAP kinase, STAT3, ERK and PKC-mediated pathways . It iewed in ,81). The− or •OH. The secondary RONS are catalytically very active, not tightly controlled and consequently may not act as signal transducers. In contrast, primary RONS are well controlled; their reactions with targets are reversible; and they do not damage target molecules. This makes them ideal for intracellular signaling processes. Unfortunately, the majority of papers on signaling refers to RONS without specifying their types. In this review, we highlight an approach allowing one to distinguish the contribution of different RONS. This can be used to define the origin of RONS contributing to intracellular signaling cascades in future studies.We assume that whether RONS have a beneficial or deleterious effect is primarily defined by their quality, being primary or secondary RONS, and only secondly by their quantity . Therefo