It is well known that ovarian function is regulated by hypothalamic–pituitary–ovarian hormones. However, although several histological studies have described the autonomic innervation of the ovary, the involvement of these autonomic nerves in ovarian function is unclear. Recently, it has been shown that both the superior ovarian nerve (SON) and the ovarian nerve plexus (ONP) induce vasoconstrictor activity by activation of alpha 1-adrenoceptors, whereas the SON, but not the ONP, inhibits ovarian estradiol secretion by activation of alpha 2-adrenoceptors. Furthermore, reflex activation of these ovarian nerves by noxious cutaneous stimulation of the rat hindpaw results in ovarian vasoconstriction and inhibition of estradiol secretion. Thus, in addition to long-term regulation of ovarian function by hormones, ovarian autonomic innervation may be involved in rapid regulation of ovarian function by responding to either internal or external environmental changes. Many studies have examined hypothalamic and pituitary hormonal regulation of such ovarian functions as ovulation and secretion of ovarian hormones , 2. HoweThe activity of autonomic efferent nerves and function of the target organs are regulated reflexly by somatic afferent stimulation, for example noxious mechanical stimulation of the skin. The neural mechanism of reflex responses in the sympathetic and parasympathetic nervous systems produced by noxious somatic afferent stimulation has been described for anesthetized animals, for which emotional factors are eliminated , 10. ForConcerning neural regulation of the female reproductive organs by autonomic nerves, some physiological functions have been demonstrated for afferents –16 and eRecently, a function of autonomic efferents innervating the ovary in rats has been demonstrated. These sympathetic efferents have vasoconstrictor activity in regulating ovarian blood flow and inhibitory activity in ovarian estradiol secretion –27. FurtHerein, we review the autonomic nervous regulation of ovarian function in anesthetized rats by noxious somatic afferent stimulation.Histological studies have revealed the distribution and innervation of sympathetic (splanchnic) and parasympathetic  nerves of the rat ovary, including both afferents and efferents –8. SympaThe ovary receives blood from the ovarian and uterine arteries , 33. TheFigure Reynolds and Ford  describeIn anesthetized rats, we examined the effects of NA and electrical stimulation of the autonomic nerves to the ovary on the diameter of ovarian arterioles, by use of digital video microscopy, and on ovarian blood flow, by use of laser Doppler flowmetry , 22. NA These results suggest that the activation of sympathetic nerves to the ovary (both the ONP and the SON) and NA, a sympathetic neurotransmitter, induce vasoconstriction of ovarian arterioles, thereby reducing blood supply to the ovaries.We examined the effects of electrical stimulation of the SON and the ONP on the rate of secretion of estradiol by the ovary in rats . The ratThe SON or ONP, ipsilateral to the ovary from which ovarian venous blood was collected, was stimulated electrically at an intensity which was supramaximum for C-fibers. Stimulation of either the SON or the ONP produced a decrease in ovarian venous plasma flow rate Fig. c, d. DurFurthermore, it was shown that the reduction of estradiol secretion during SON stimulation was blocked by an alpha 2-adrenoceptor antagonist (yohimbine) but was not affected by an alpha 1-adrenoceptor antagonist (prazosin) or a beta-adrenoceptor antagonist (propranolol) . On the Estradiol is synthesized from testosterone by aromatization in the ovary , 41. RecOther examples of adrenergic innervation of endocrine secretory structures are found in the kidney  and pineEffects of noxious mechanical stimulation of a hindpaw (pinching stimulation) on ovarian blood flow, ovarian sympathetic nerve (ONP) activity, and MAP have been examined in anesthetized rats , 20. PinAfter the ovarian sympathetic nerves (ONP and SON) were severed, pinching of a hindpaw was repeated. The MAP increased in the same way as before severing the ovarian sympathetic nerves Fig. e whereasAfter spinal transection at the third thoracic segment, the responses to hindpaw stimulation of MAP, ovarian sympathetic nerve activity, and ovarian blood flow were nearly abolished. These results indicate that, in central nervous system-intact rats, hindpaw afferents contribute to a supraspinal reflex pathway to the ovarian sympathetic nerves innervating ovarian blood vessels Fig. a.It has been shown for anesthetized rats that noxious mechanical stimulation (pinching) of a hindpaw for 5 min produced an increase in SON activity and a decrease in the rate of secretion of estradiol by the ovary Fig. a, c 28]28]. The Pinching stimulation of a hindpaw for 5 min reduced the rate of estradiol secretion by the ovary, but did not change the plasma estradiol concentration in systemic blood . The cliWe have reviewed several interesting results from recent studies. First, ovarian blood flow and ovarian estradiol secretion are controlled independently by sympathetic adrenergic innervation. Of the two pathways of sympathetic nerves to the ovary (SON and ONP), stimulation of either reduces ovarian blood flow via activation of alpha 1-adrenoceptors, whereas stimulation of the SON, but not the ONP, reduces estradiol secretion via activation of alpha 2-adrenoceptors. Second, reflex activation of ovarian sympathetic nerves by noxious cutaneous stimulation causes ovarian vasoconstriction and inhibition of ovarian estradiol secretion. The ovarian vasoconstrictive response is produced via reflex activation of the SON and ONP, whereas inhibition of ovarian estradiol secretion occurs as a result of reflex activation of the SON only Fig. . These r


Obesity results from imbalance between food intake and energy expenditure. The energy homeostasis is regulated by hypothalamic neurons that receive different neural, hormonal and metabolic signals. Insulin is one of the main hormones that regulate energy homeostasis and acts through a cascade of intracellular signaling that depends on the activation of several proteins, such as Akt. Our hypothesis is that PH domain and Leucine rich repeat Protein Phosphatase 1 (PHLPP1) inhibits Akt activity by dephosphorylating serine 473 residues, impairing the insulin action in the hypothalamus. However, PHLPP1 expression and the role played by it on the hypothalamus of diet induced obesity (DIO) animals are not fully understood.To investigate PHLPP1 protein expression in the hypothalamus of DIO rats and to assess whether the PHLPP1 silencing improves insulin action and decreases body adiposity.Eight-week-old male Wistar rats were distributed into two groups. One group was fed with standard diet and the other group with high-fat diet, during eight weeks. After this period, the hypothalamus of Chow and DIO animals were dissected for analysis of PHLPP1 protein expression. In another experiment, DIO animals were cannulated in the lateral ventricle and received intracerebroventricular (ICV) treatment to silence PHLPP1 expression, or its control, scramble (siRNA-SCR), during 7 days. Body weight and food intake were measured daily. On day 8, fasted animals received an insulin or saline ICV injection and their hypothalamus was extracted after 15 min to evaluate the PHLPP1 expression and insulin pathway proteins. Retroperitoneal and epididymal adipose depots were dissected and weighted.Initially, DIO animals showed increased protein expression of PHLPP1 in hypothalamus compared to the Chow group. After 7 days of ICV treatment with siRNA-PHLPP1, DIO rats showed reduced expression of this phosphatase and led to greater weight loss and reduction in adiposity, epipydimal and retroperitoneal. Interestingly, hypothalamic PHLPP1 inhibition restored the phosphorylation of Akt in the hypothalamus. Conversely, it was not observed significant changes in food intake and fasting glucose.Hypothalamic PHLPP1 silencing in DIO animals restored, at least in part, the insulin signaling, promoting reduction in body weight and adiposity.


Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels comprise a family of cation channels activated by hyperpolarized membrane potentials and stimulated by intracellular cyclic nucleotides. The four members of this family, HCN1–4, show distinct biophysical properties which are most evident in the kinetics of activation and deactivation, the sensitivity towards cyclic nucleotides and the modulation by tyrosine phosphorylation. The four isoforms are differentially expressed in various excitable tissues. This review will mainly focus on recent insights into the functional role of the channels apart from their classic role as pacemakers. The importance of HCN channels in the cardiac ventricle and ventricular hypertrophy will be discussed. In addition, their functional significance in the peripheral nervous system and nociception will be examined. The data, which are mainly derived from studies using transgenic mice, suggest that HCN channels contribute significantly to cellular excitability in these tissues. Remarkably, the impact of the channels is clearly more pronounced in pathophysiological states including ventricular hypertrophy as well as neural inflammation and neuropathy suggesting that HCN channels may constitute promising drug targets in the treatment of these conditions. This perspective as well as the current therapeutic use of HCN blockers will also be addressed. If in the heart and Ih in the brain [The ion channel family of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels comprises four members termed HCN1–4. Upon hyperpolarization, all four isoforms generate a characteristic inward current which shows the typical properties of the conductance coined he brain ,3,4. NotA second key difference between the isoforms is the differential modulation of HCN channels by cyclic nucleotides. Intracellular cAMP positively modulates the gating of the HCN2 and HCN4 isoforms resulting in a shift of the activation curve by 15–20 mV in the positive direction. In contrast, HCN1 is only slightly shifted (shift by around +5 mV) and HCN3 is not modulated by cyclic nucleotides. cAMP binds to the cyclic nucleotide-binding domain (CNBD) in the carboxy-terminus of the channels. In the cAMP-unbound state, the CNBD acts as an autoinhibitory domain which constrains gating to more hyperpolarized potentials . Binding2) [2 is independent of cyclic nucleotides and, in contrast to cAMP, is equally effective in all four isoforms [2 arises, at least in part, from stabilization of the activated state of the voltage sensor [Another factor shifting channel activation towards positive potentials to a similar degree as cyclic nucleotides are phosphoinositides including phosphatidylinositol-4,5-bisphosphate (PIP2) ,8. The misoforms . Experime sensor .An independent mechanism regulating channel activation is tyrosine phosphorylation by Src kinase ,12,13,14If in these cells [The expression profile of the isoforms is markedly different and strongly depends on the tissue and cell type analyzed. In addition, this tissue-specific expression pattern may vary among different species. Recent studies have shown that the expression pattern in a given cell type is often broader than initially thought. A prime example is sinoatrial node cells where HCN4 has been determined as the principal HCN isoform. However, we, and others, could demonstrate a significant expression of HCN1 and also a small amount of HCN2 beneath HCN4 in murine sinoatrial cells at the protein level indicating that the three subunits together contribute to se cells ,16. HCN2se cells  and in ase cells  has beenin vivo is lacking.The regional expression in the nervous system shows characteristic differences between the four isoforms ,19. HCN2The role of HCN channels in mammalian physiology has been examined intensively by the generation and analysis of HCN-deficient mice. All four isoforms have been mutated by using global and/or conditional gene deletion approaches. We will first present a brief overview of the published HCN-knockout lines before covering recent work regarding the role of the channels in the cardiac ventricle and dorsal root ganglion cells.Mice lacking HCN1 globally are viable and do not display an overt phenotype. However, these animals are defective in the learning of fast motor tasks due to a malfunctioning of cerebellar Purkinje cells . HCN1-knapathetic carries an insertion in the coding region of the CNBD of HCN2 resulting in a premature stop codon and lack of the truncated HCN2 protein in the brain [2+ channels and thereby promotes low-threshold burst firing in response to depolarizing inputs.In contrast to the lack of HCN1, global deletion of HCN2 results in a pronounced neuronal phenotype characterized by absence epilepsy, tremor, hypoactivity and ataxia . In addihe brain . As expehe brain ,29,30. TMice with a disruption of the HCN3 isoform do not show visible behavioral and physical abnormalities. However, these mutants display an acceleration of the late repolarization phase of the cardiac action potential in epicardial ventricular myocytes demonstrating that HCN3 contributes to the action potential in these cells   depolarization phase of the ventricular action potential rather than repolarization. However, the involvement of If to ventricular repolarization is explainable by the very slow deactivation kinetic of HCN channels. Because of the relatively fast ventricular cycle length a significant fraction of HCN channels remains activated during the entire cardiac action potential [If current is increased and simultaneously outward potassium currents are reduced. Our ventricular HCN2/4 knockout model identified the enhanced If as an important contributor to the typical electrophysiological alterations observed in the hypertrophic heart including prolonged ventricular action potentials and lengthened QT (time between start of the Q and end of the T wave) intervals [Recently we addressed this issue directly by analyzing pro-arrhythmic parameters in ertrophy . The comotential . This waotential ,47,48,49ntervals . Considentervals ,52.et al. who analyzed the effect of ivabradine, an HCN channel blocker, on survival and rhythmicity in a transgenic mouse model of dilated cardiomyopathy [If and other fetal ion channels and developed progressive cardiomyopathy accompanied by ventricular tachyarrhythmias and sudden arrhythmic death. The survival rate of dnNRSF-Tg animals significantly improved when ivabradine was administrated at low doses which did not affect heart rate. Additionally, in isolated isoproterenol-stimulated transgenic ventricular myocytes ivabradine suppressed spontaneous activity triggered by early after-depolarizations. Interestingly, both the ventricular specific block of HCN channels [This suggestion is further supported by a recent work of Kuwabara myopathy . In thismyopathy . In conschannels  as well channels  was withchannels ,58,59,60If is significantly increased. In a thorough analysis of the cardiac phenotype of HCN3 knockout mice, Fenske et al. found that the lack of HCN3 enhances the repolarization of epicardial cardiomyocytes, which was the first report of a significant involvement of If channels to ventricular excitability in the adult and healthy heart [Remarkably, this concept is not limited to conditions of ventricular hypertrophy where ventricular hy heart ,49.Ih was first discovered by Mayer and Westbrook in a study of mechanisms of anomalous rectification in the embryonic mouse dorsal root ganglion (DRG) [If identified in sinoatrial node cells a few years before [Ih was most prominent in large and medium sized neurons, whereas only half of small sized DRG neurons displayed a functional Ih current [Ih in large and medium sized DRG neurons as compared to the characteristics of the current in small diameter neurons [Ih in most large neurons but left slowly activating cAMP-sensitive Ih, preferably present in smaller DRG neurons unaffected [In neurons, on (DRG) . They des before ,64. Late current . Among t current ,67,68,69 current ,72,73,74 neurons ,75,76. Taffected .Ih for sensory function is unknown. The results of a huge number of animal studies analyzing physiological sensation in vivo by the use of HCN blockers or mouse mutants argue against an obvious function of Ih in healthy sensory neurons. In addition, the wide clinical use of the Ih blocker ivabradine for the treatment of stable angina and heart failure is not associated with peripheral sensory side effects such as dysesthesias and paresthesias. It seems that HCN channel function in the peripheral sensory system is limited to pathological pain conditions, which might make these channels an attractive target for the treatment of pain disorders. Several studies reported enhanced HCN channel expression and/or Ih activity in the peripheral pain pathway following neuronal damage [Despite the relatively high and broad expression in sensory neurons, the physiological role of l damage ,73,78 orl damage ,69,74,79Ih in the pain sensing pathway. This is plausible because HCN channels conduct an excitatory mixed inward current which is typically activated near the neuronal resting membrane potential. A pronounced HCN activity in sensory neurons following nerve injury or inflammation may shift membrane potential to more depolarized values. This lowers the activation threshold of nociceptors and facilitates spontaneous and/or neuronal repetitive activity in line with the increased sensitization to painful stimuli. Such an increased functional importance of Ih may mechanistically be attributed to enhanced gene expression [2 interaction [These observations have raised the hypothesis that nociceptor hyperexcitability, which is associated with hyperalgesia and allodynia, is attributed to an increased activity of pression ,74,79,80eraction , channeleraction ,82 or toeraction ,84,85.Ih to tactile allodynia and thermal hyperalgesia was first demonstrated by studying the analgesic effect of the unselective HCN channel blocker ZD7288 in various neuropathic pain models including the CCI model (chronic constriction injury), the Chung model  and the Seltzer model  [A significant involvement of  injury) ,73,86,87Ih to neuropathic pain and suggested a pivotal role for HCN2 [et al., demonstrated that conditional deletion of HCN2 abolished pain behavior in the CCI model [p < 0.01, V1.8-expressing neurons (snsHCN2KO) developed no significant hyperalgesic behavior as compared to baseline values. Similar findings were observed with mechanical stimulation. After induction of neuropathy, tactile hypersensitivity developed equally for the first three days in controls and knockouts  signaling . NeitherIh contributes to neuronal sensitization since deletion of HCN2 as well as administration of ZD7288 reversed tactile hypersensitivity in CFA-inflamed animals [The concept of increased cAMP levels as a precondition for HCN-mediated sensitization applies to acute inflammatory states, but may not be relevant for chronic inflammatory pain conditions where transcriptional regulatory processes are more important. This fits well to the observed up-regulation of HCN2 protein expression in small DRG neurons and in the spinal dorsal horn after chronic inflammation following peripheral injection of complete Freunds adjuvant (CFA) ,72,74,79 animals ,74.The neuronal site of HCN action was studied by analyzing pain behavior in controls and conditional HCN2 mutants (snsHCN2KO) after local administration of ZD7288. The region-specific block at peripheral nociceptors as well as at spinal terminals reduced mechanically evoked pain in controls, but was without any effect in snsHCN2KOs . This suIvabradine is the first and to date the only HCN blocker introduced into clinical therapy. In isolated sinoatrial node cells, ivabradine blocks HCN pacemaker currents in the low micromolar range with minimal or no off-target effects. Channel block is accomplished from the cytoplasmic side with a strong preference for the open channel state resulting in use-dependence characteristics. This may minimize the risk for severe bradycardia and improve efficacy during elevated heart rates. At therapeutic concentrations, ivabradine exclusively reduces heart rate without affecting myocardial contractility or intracardiac conduction. Since an elevated heart rate is thought to be an independent cardiovascular risk factor, drugs causing a selective reduction in heart rate should constitute an interesting therapeutic option.Ivabradine was approved for the treatment of stable angina pectoris in patients with elevated heart rate. It can be used as a single agent when beta-blockers are not tolerated or contraindicated or as add-on therapy when adequate heart rate control is not achieved. It was shown that ivabradine is as effective as the beta-blocker atenolol in its anti-ischemic and anti-anginal efficacy . Two othn = 10.917) showed that the composite endpoint was not significantly different between the ivabradine and the placebo groups. However, a subgroup analysis suggested a specific benefit for patients with heart rates over 70 bpm [n = 19.102) [Although ivabradine is effective in reducing angina symptoms, a benefit in terms of prognosis for patients with stable coronary artery disease (CAD) is questionable. The BEAUTIFUL trial investigated such a potential effect of the drug added to standard therapy . This lar 70 bpm . To corr 19.102) . In the Ivabradine is recommended for heart failure (HF) patients with reduced left ventricular ejection fraction and elevated heart rate when symptoms are still present despite standard therapy. The SHIFT study included over 6.500 HF patients with increased heart rate and was designed to determine a prognostic effect of ivabradine when added on stable contemporary background therapy . The priA progress in the therapeutic use of HCN channel blockers could be achieved with the development of subtype-selective drugs. Typical side effects of ivabradine are phosphenes , which are most probably caused by the block of HCN1 in retinal photoreceptors . A subty


There are various subgroups of patients with metastatic prostate cancer: polymetastatic, oligometastatic, or oligo-recurrent cancers whose progression follows different courses and for whom there are different treatment options. Knowledge of tumor dissemination pathways and different genetic and epigenetic tumor profiles, as well as their evolution during disease progression, along with new diagnostic and therapeutic advances has allowed us to address these situations with local ablative treatments such as stereotactic body radiation therapy or stereotactic radiosurgery. These treatments provide high rates of local control with low toxicity in metastatic spread for primary cancers including those of pulmonary, digestive, and renal origin, while these types of treatments are still emerging for cancers of prostatic origin. There are several retrospective studies showing the effectiveness of such treatments in prostate cancer metastases, which has led to the emergence of prospective studies on the issue and even some phase II studies intended to prevent or delay systemic treatments such as chemotherapy. Here we collect together and review these past experiences and the studies currently underway. These types of radiotherapy treatments redefine how we approach extracranial metastatic disease and open up new possibilities for combination therapy with new systemic treatment agents. Beginning with Halsted  in 1907 Progression describes the cause of cancer as the accumulation of acquired somatic mutations and chromosomal rearrangements which gradually add up over a long period of time, progressively converting normal epithelium into neoplastic cells which later acquire a metastatic phenotype . AccordiHowever, phenomena such as tumor mutations, adaptations, and changes in their genetic and epigenetic profiles cannot be explained by the theory of progression and so new tumor models are emerging. Based on data uncovered by the human genome project, Stephens et al.  proposedIf metastases arise from clonal expansion  clones wAnother important method of metastasis classification is Rubin’s amended TNM staging system . He suggBubendorf et al. , in a seVenous retrograde invasion through the Batson plexus, leading from the prostate to the periprostatic veins and presacral veins, column, and subsequently to the lungs is one of the main routes of dissemination in metastatic patients. Indeed, there is evidence which proves this mechanism, such as the inverse relationship between spinal and pulmonary metastases. Spinal cord involvement most often occurs in smaller tumors (4 to 6 cm), while lung involvement is more likely in large tumors (6 to 8 cm), with liver involvement usually restricted to the largest tumors (8 cm or larger), suggesting that spinal metastases precede lung and liver metastases. Further supporting this idea, a gradual decline in lumbar spinal and cervical vertebrae involvement (97% to 38%) ,20.The role of PSA (prostatic specific antigen) should be highlighted in prostate cancer: it is a highly predictive marker for bone metastases, with a positive predictive value of 74% when present at a concentration greater than 100 ng/mL and a negative predictive value of 98% when present at less than 10 ng/mL. Other serological tests, such as models based on circulating tumor cells, are still in development.CT (computed tomography) plays an important role in detecting bone (mainly blastic), lung, and liver metastases and MRI (magnetic resonance imaging) is useful in the diagnosis of liver, and especially, spinal metastases. New non-parametric techniques such as whole-body (WB) MRI and diffusion-weighted imaging (DWI) studies have proven useful for detecting lymph node, bone, and visceral metastases -26.Undoubtedly, modern techniques such as PET (positron emission tomography) and CT have the greatest versatility in terms of metastasis detection. Different radiolabels that are useful for prostate cancer diagnosis and tracking are in development, including markers of cell membrane proliferation (11C/18F-choline), fatty acid synthesis (11C- acetate), amino-acid transport and protein synthesis (11C- methionine), androgen receptor expression (18F- FDHT), sodium fluoride labeled with fluorine 18 (sodium fluoride F 18 [18F-NaF]) and osteoblast activity (18F-fluoride). In the case of the latter, there is more reported experience with 11C-choline and 18F-fluorocholine ; the maiSince the discovery of PET/CT, many authors have proposed the use of these techniques in the diagnosis and staging of prostate cancer and for restaging local and systemic relapse patients, albeit with mixed results. In order to try to unify these varied criteria and conclusions Umbehr et al.  publisheThus, when using this method in staging, high-risk patients with a Gleason score of 8–10 and elevated PSA levels (20 ng/ml or more) are the groups most suitable for radical treatment . In contCurrent guidelines, such as those from the National Comprehensive Cancer Network (NCCN)  do not iIn 2004 the University of Rochester  studied Piet Ost et al.  tried toAccording to present knowledge, the criteria for performing ablative treatment in metastatic patients, and particularly metastatic prostate cancer, patients would be: oligo-recurrent patients (with a controlled or controllable primary lesion) with five or fewer metastases  located in the bone  and takSurgical experience of prostate cancer bone metastases mainly focusses on spinal metastases, usually as a palliative treatment. Different grading systems or "scores" have been proposed to establish the indication and to select patients suitable for spinal surgery. The first relevant system was published by Tomita  and usedMany factors must be considered when selecting different treatments for palliative-care patients including the presence or absence of spinal cord compression syndrome, spinal stability, and histology ,50. HoweFollowing on from the good results from the phase II Radiation Therapy Oncology Group (RTOG) 0631 trial which assessed the feasibility and safety of spinal SRS, the phase III trial may obtain level 1 evidence for this type of treatment  by compaThere are more than 133 clinical trials exploring the role of ablative radiotherapy in different locations, 53 of them in oligometastases and almost 18 in spinal metastases. Focusing on prostate cancer treatment (discussed in greater detail in sections below) current experience suggests that single high doses may be preferable to SBRT or SRS. For example, Muacevic et al. analyzed 40 patients with prostate cancer bone metastases ; 75% of them were in a single location, and the mean tumor volume was 13 cc. Fifty-four consecutive radiosurgery sessions were administered, with an average single dose of 20.2 Gy and after a mean follow up of 14 months the actuarial local control was 95.5% .In terms of surgical salvage of prostate cancer lymph node metastases, current experience is much more limited compared to other histologies. Randomized studies like that of Verhagen et al. show that combined local surgical treatment and hormone therapy is beneficial in aggressive prostate cancer patients with nodal disease, compared with hormone-only therapy . Recent Ablative radiotherapy studies of lymph node metastases, both for prostate cancer and other histologies, are still emerging. The work of Bignardi et al., among others, which used SBRT to treat lymph node metastases with various histologies, has shown this treatment to be a safe and effective option and have laid the groundwork for the future study of prostate cancer lymph node metastasis -68.Historically there have been a number of difficulties in treating metastases with ablative radiotherapy doses: one is the dose-limit tolerance of healthy tissue to fractionation, a problem that was already successfully solved in the development of cranial radiosurgery . The assAnother difficulty is identification of patients that truly fit the criteria for oligometastatic disease rather than, for example polymetastatic or oligo-recurrent disease. In spite of this, the development of new imaging techniques such as WB-MRI and the introduction of new PET radiotracers with enhanced diffusability  has greatly improved our ability to adequately select patients.However, the greatest difficulty, and one that is inherent to all ablative treatments, is that different cohorts of oligometastatic sub-types must be taken into account. A patient who is oligometastatic on diagnosis is not the same as one who is oligometastatic after cytoreductive (oligoprogressive) curative locoregional treatment, as they would probably have different prognoses which would require different therapeutic approaches. Therefore it is essential to define the true oligometastatic stage, and as such various theories have been proposed.Oligometastasis is generally thought of as a situation in which there is a long interval between primary disease treatment and the occurrence of a metastasis, which would have a high percentage growth rate compared to the primary tumor. Indeed, as Withers and Lee  state, tThus we can formulate the following hypothesis: patient subgroups with a true oligometastatic disease phenotype can benefit from high-dose ablative therapy which could change their prognosis, and therefore they can receive treatment beyond merely palliative care.The foundations of these new techniques can be defined based on three principles: high-dose, high-precision, and high conformation of the dose distribution to the target volume.Several reports, such as those by Greco et al.  have demCell apoptosis can be initiated by DNA damage induced by stereotactic ablative radiotherapy (SABR) which causes positive p53 tumor suppressor gene regulation, and may also trigger lipid cell membrane damage. This damage is capable of inducing the formation of ceramides which activate the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling pathway which in turn can upregulate protein kinase R (PKR) expression, potentially inducing major histocompatibility complex (MHC) and cytokine expression via NF-kB, resulting in cellular expression of MHC class I, adhesion molecules, co-stimulatory molecules, heat shock proteins, inflammatory mediators, immunomodulatory cytokines, and cell death receptors . This tyPrecise conformation treatment is required in the use of SABR because there is a high dose gradient between the target volume and the dose-limiting organs at risk. Ablative radiotherapy techniques require precise treatment conformation to maintain correct patient immobilization, accomplished by using one of several commercial immobilization systems which promote patient stability and comfort. It is also very important that these systems are compatible with MRI and PET/CT, given that they may be used for fusion imaging with the radiotherapy planning CT to generate the treatment position. Thus, most radiotherapy planning systems allow either rigid or deformable registration image fusion with other studies. There are currently two trends for ablative vertebral treatments: the use either of fusion imaging with MRI to delineate the spinal cord or a myelogram during the planning CT . In contAnother key aspect of conformational high-dose radiotherapy is the development of intensity modulated radiotherapy (IMRT), and more recently, volumetric radiotherapy or arc radiotherapy (VMAT and Rapid Arc)  which alThe fundamental tool used to give high precision in radiotherapy treatments is the use of image guided radiation therapy (IGRT)  which deThere are several documented experiences that focus on radiation treatment of prostate cancer oligometastases and although most of them focus on general oligometastasis treatment or on spinal or nodal metastases, some prostatic-origin cases are included because it is a common presentation .Using 3D dynamic IMRT techniques, Schick et al.  publisheAnother study using IMRT techniques and conventional fractionation, Würschmidt et al.  analyzedFocusing on ablative radiotherapy schedules in patients with a local relapse, Casamassina et al.  analyzedMedian follow-up was 16.9 months. Acute urinary toxicity was described in 8 patients (in three cases grade 1 and in five cases grade 2) and rectal toxicity presented only in one case (grade 1). Late-onset urinary toxicity occurred in five cases (in three cases grade 3 and in two cases grade 2) and one case each of grade 1 and grade 2 late-onset rectal toxicity was described. A biochemical response was observed in 32 of 38 evaluable lesions, PSA stabilization was observed in four lesions, and in two cases PSA progression was reported. PFS was 42.6% at 30 months and there was disease progression in 14 lesions ; progression was observed in only three cases. At the time of analysis, 19 patients were alive without evidence of disease and 15 were alive with disease. In the case of nodal metastases, at a median follow-up of 21.9 months 16 cases presented solely nodal relapse, 10 had a complete response, there was one case of stable disease, and five cases of disease progression, as assessed by choline-PET/CT. At the level of PSA response, 12 complete responses, one partial response, two cases of stable disease, and one case of disease progression were reported. With regard to the three metastases, at a median follow-up of 13.7 months, there was one case of radiological progression outside the treatment field, one case of disease progression due to a rise in PSA, and one complete response. The median PFS time was 11 months (range: 6–16 m) and no cases of toxicity were reported.Using single doses of SRS with a Cyberknife® Muacevic et al. publisheWith respect to the management of oligometastatic patients using ablative radiotherapy to delay the onset of hormone therapy, Berkovic et al.  publisheRecently, Picchio et al.  publisheHospital Provincial de Castellón (Spain) and sponsored by the Spanish research group in radiation oncology (GICOR), the Spanish Society of Radiation Oncology (SEOR), and the Spanish SBRT group (SBRT-SG) is seeking to uncover any association between hormone therapy and SBRT for these oligometastases and whether these treatments can increase local control, PFS, and chemotherapy-free survival. Another phase II prospective clinical trial from the University of Florida is exploring the efficacy and safety of SBRT in patients with metastatic prostate cancer, either with or without an active primary tumor, in two subgroups: castration-resistant and hormone-receptive patients [In summary, the major schemes used for treating prostate cancer oligometastases are all based on SBRT, with an average dose range of one fraction of 20 Gy to three fractions of 10 Gy. Other schemes used are 5–6 fractions of 5 or 6 Gy and 10 fractions of 5 Gy, with no discernable difference between the two. Concepts such as the logistics of radiation oncology services and the possible advantage of hypofractionation doses versus high single doses should be considered in future studies   and their expression patterns, meaning that tumor biopsies can be used to identify poor-prognosis oligometastatic patient subgroups who may benefit from systemic treatments, before or after the discovery of new metastases. Furthermore, future ablative radiotherapy treatments may also be administered in combination with bisphosphonates and denosumab as well as hormone therapy. Moreover, it are expected to the new anti-androgens  and target therapy drugs may promote greater control of systemic disease by acting on micrometastases while radiation acts on macroscopic disease ,97 “Sea”.Studies were identified in July, 2014, by searches of PubMed, www.clinicaltrials.gov and Web of Knowledge , and by review of cited papers in selected articles. We identified 435 papers. We assessed retrospective and prospective studies published in English. We excluded duplicates or studies reporting a mixture of hystologies, or non oligometastatic disease. Studies were eligible if the dose per fraction was 6 Gy or higher for a total dose of more than 24 Gy, or 5 Gy per fraction for a total dose of more than 45 Gy. Studies were excluded if fewer than seven patients were reported or if median follow-up was less than 12 months. 8 studies in oligometastatic prostate cancer met the eligibility criteria for this Review, and 5 focused in SBRT/SRS.More clinical and molecular markers which can classify patients with stage IV cancers are becoming available.Different approaches must be taken for patients with polymetastatic, oligometastatic, or oligo-recurrent disease because these pathologies are different entities.SBRT and SRS are now alternative treatment options for oligometastatic patients with various diseases such as colorectal or lung cancer, and there is evidence from different studies that suggests that these treatments may also be a viable alternative for prostate cancer patients.The main conclusions that can be drawn from the evidence presented here can be summarized in the following points:Several prospective ongoing trials may help to clarify the role of ablative therapy in the face of increasing hormone-free and chemotherapy-free survival rates in oligo-recurrent prostate cancer patients.


Arabidopsis, ambient temperatures that are high, but not so high as to induce a heat stress response, confer morphological changes that resemble the shade avoidance syndrome. Similarly, these high but not stressful temperatures can accelerate flowering under short day conditions as effectively as exposure to long days. Photoperiodic flowering entails a series of external coincidences, in which environmental cycles of light and dark must coincide with an internal cycle in gene expression established by the endogenous circadian clock. It is evident that a similar model of external coincidence applies to the effects of elevated ambient temperature on both vegetative morphology and the vegetative to reproductive transition. Further study is imperative, because global warming is predicted to have major effects on the performance and distribution of wild species and strong adverse effects on crop yields. It is critical to understand temperature perception and response at a mechanistic level and to integrate this knowledge with our understanding of other environmental responses, including biotic and abiotic stresses, in order to improve crop production sufficiently to sustainably feed an expanding world population.Plant development is exquisitely sensitive to the environment. Light quantity, quality, and duration (photoperiod) have profound effects on vegetative morphology and flowering time. Recent studies have demonstrated that ambient temperature is a similarly potent stimulus influencing morphology and flowering. In  Plant development is highly sensitive to the environment. For example, light dramatically affects plant morphology . When grArabidopsis, growth at 27°C results in elongated hypocotyls and petioles and other morphological changes that are reminiscent of the response to shade. In addition, elevated temperature accelerates flowering, especially in short days that are non-inductive in Arabidopsis grown at lower ambient temperatures .Plant morphology and reproductive development are also strongly influenced by temperature . AmbientArabidopsis, where our mechanistic understanding is greatest.In this mini review, we will consider the similarities and differences in the thermoresponsiveness of growth and flowering, with an emphasis on One of the first manifestations of thermomorphogenesis, the suite of responses in growth to elevated temperature, is increased elongation of the hypocotyl. The similarity of thermomorphogenesis to responses to shading suggested common underlying mechanisms. PHYTOCHROME INTERACTING FACTOR4 (PIF4) and PIF5, basic helix-loop-helix (bHLH) transcription factors that are key components of phytochrome signaling with central roles in photomorphogenesis , also plPIF4 and PIF5 transcription and mRNA accumulation are regulated by the circadian clock (PIF4 transcription is repressed by ELONGATED HYPOCOTYL5 (HY5) and the evening complex (EC) as well as by additional transcriptional repressors  and LUX ARRHYTHMO (LUX), as well as PHYB. The elf3-1 loss of function mutant exhibits enhanced growth under control temperatures and does not increase growth at high temperature, coinciding with elevated PIF4 levels under both conditions. These mutants also lose the high temperature induction of LUX expression suggesting that ELF3 is required for this rapid thermoresponsiveness  population revealed that the Bay-0 ELF3 allele confers longer period and greater response to shade than the Sha allele  mapping with siveness . Naturala allele . Loss ofnditions .Arabidopsis has at least four flowering pathways: autonomous, vernalization, gibberellic acid (GA), and photoperiodic , an inducer of the flowering repressor, FLOWERING LOCUS C (FLC). FLC complexes with SHORT VEGETATIVE PHASE (SVP) to form a potent transcriptional repressor of floral inducers, including FLOWERING LOCUS T (FT), FD, and SUPPRESSOR OF CONSTANS 1 (SOC1)   cold. Loss of function of either FRI or FLC eliminates the vernalization requirement, permitting accelerated flowering and a summer annual lifecycle, whereas accessions with functional FRI and FLC genes have a vernalization requirement and a winter annual habit  . The expal habit . A seconal habit . Like FLal habit .Arabidopsis indicate a complex architecture of natural variation in thermal responses (The transition to flowering is influenced by moderate changes in ambient temperature. Genome-wide association (GWAS) and QTL studies in esponses . A growtesponses . Althougesponses .FT promoter is decreased at higher temperatures, permitting promoter binding by PIF4  and PHOTOPERIOD-INSENSITIVE EARLY FLOWERING1 (PIE1) . H2A.Z i by PIF4 . PIF4, ieratures . Similarperature .FT and its paralog TWIN SISTER OF FT (TSF) in response to warm nights and independently of FT during warm days  loss of function mutants only partially suppress the early flowering and elevated FT expression at high temperature persists in the arp6 mutant . SVP forms repressor complexes with MADS box transcription factors related to FLC: FLM and MAF . The circadian clock imposes a circadian oscillation on SVP expression, linking thermosensitivity to circadian cycling  but not in the phloem companion cells prevented GA-induced flowering, indicating that the GA acts at the SAM, consistent with its independence from the PIFs, which induce FT and TSF in phloem companion cells. The action of GA at the SAM, at least in part, involves the induction of the floral inducers SPL3 and SPL5  or exon 3 (FLM-δ); FLM-β binds DNA but FLM-δ does not  and FT, via an external coincidence mechanism in which the external stimulus, light, must coincide with an inductive window that is restricted (gated) by the circadian clock . The folCYCLING DOF FACTOR (CDF) genes whose protein products repress CO transcription. The CDF proteins are targeted for degradation by a SCF complex containing FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1), and GIGANTEA (GI) . Both FKF1 and GI exhibit circadian cycling in protein abundance. In short days, GI protein abundance peaks at dusk while FKF1 protein peaks after dark. This leads to the formation of the FKF1-GI complex in the dark. Thus, CO transcription is repressed until about dusk and CO mRNA accumulates after dusk. CO protein is unstable in the dark so, in short days, CO protein fails to accumulate and FT transcription is not induced.The circadian clock drives morning-specific expression of several GI expression is delayed and coincides with that of FKF1 in late afternoon. FKF1 is a blue-light photoreceptor, and the interaction of FKF1 with GI is enhanced by blue light. This is a second example of external coincidence, when the peaks of FKF1 and GI proteins coincide in the light to allow the formation of the FKF1-GI complex to degrade the CDFs in the late afternoon. As a consequence, transcriptional repression of CO is relieved in the afternoon of long days and CO mRNA accumulates in the light, which permits the stabilization of nascent CO protein and activation of FT transcription.In long days the phase of peak FLOWERING LOCUS T transcription is also induced independently of CO. Several CRY2-INTERACTING bHLH (CIB) transcription factors accumulate in long days to stimulate FT transcription. The CIBs are activated in the afternoon by blue-light dependent interaction with CRY2 , each of which has dramatic effects on vegetative morphology and the developmental transition from vegetative to reproductive growth . TemperaArabidopsis as guides.It is well-established that there is heterogeneity among plant species in terms of clock function and its relationship to flowering time , so extrAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While additional research is needed, a number of large epidemiological studies show an association between circadian disruption and metabolic disorders. Specifically, obesity, insulin resistance, cardiovascular disease, and other signs of metabolic syndrome all have been linked to circadian disruption in humans. Studies in other species support this association and generally reveal that feeding that is not in phase with the external light/dark cycle, as often occurs with night or rotating shift workers, is disadvantageous in terms of energy balance. As food is a strong driver of circadian rhythms in the periphery, understanding how nutrient metabolism drives clocks across the body is important for dissecting out why circadian misalignment may produce such metabolic effects. A number of circadian clock proteins as well as their accessory proteins (such as nuclear receptors) are highly sensitive to nutrient metabolism. Macronutrients and micronutrients can function as zeitgebers for the clock in a tissue-specific way and can thus impair synchrony between clocks across the body, or potentially restore synchrony in the case of circadian misalignment. Circadian nuclear receptors are particularly sensitive to nutrient metabolism and can alter tissue-specific rhythms in response to changes in the diet. Finally, SNPs in human clock genes appear to be correlated with diet-specific responses and along with chronotype eventually may provide valuable information from a clinical perspective on how to use diet and nutrition to treat metabolic disorders.This article presents a background of the circadian clock components and their interrelated metabolic and transcriptional feedback loops, followed by a review of some recent studies in humans and rodents that address the effects of nutrient metabolism on the circadian clock and vice versa. We focus on studies in which results suggest that nutrients provide an opportunity to restore or, alternatively, can destroy synchrony between peripheral clocks and the central pacemaker in the brain as well as between peripheral clocks themselves. In addition, we review several studies looking at clock gene SNPs in humans and the metabolic phenotypes or tendencies associated with particular clock gene mutations.Targeted use of specific nutrients based on chronotype has the potential for immense clinical utility in the future. Macronutrients and micronutrients have the ability to function as zeitgebers for the clock by activating or modulating specific clock proteins or accessory proteins (such as nuclear receptors). Circadian clock control by nutrients can be tissue-specific. With a better understanding of the mechanisms that support nutrient-induced circadian control in specific tissues, human chronotype and SNP information might eventually be used to tailor nutritional regimens for metabolic disease treatment and thus be an important part of personalized medicine's future. While this association seems obvious, less obvious is that our endogenous circadian clocks may reflect what we eat. In fact, our ability to adjust to jet lag, recover from a sleepless night, or respond to and metabolize medicines prescribed may heavily depend on what we eat and when we eat it. Because evidence to date strongly links our internal clock to metabolism and metabolic health, the effect of nutrient intake on our internal 24-h rhythms has taken a spotlight in the field of metabolism research.Circadian oscillations are naturally recurring rhythms with a periodicity of approximately twenty-four hours. Most organisms display biological circadian rhythms and in humans, they are fundamental to physiology and behavior. The light–dark cycle is considered one of the most potent zeitgebers (or “time-giver”) driving behavioral preferences and almost all organisms studied to date respond to this circadian cue. Animal studies indicate that other cues, such as food, also drive our internal clocks to a significant extent. Fundamentally, as a consequence of the Earth's rotation on its axis, seasonal and daily environmental changes occur to which organisms must adapt at the metabolic level. Diurnal species, such as humans, carry out their daily activity during the light cycle, while nocturnal species are active during the dark cycle. This activity-rest cycle requires metabolic and physiological adaptation, producing rhythms in processes as disparate as blood pressure, body temperature, cardiovascular efficiency, muscle strength, hormonal secretion in blood, cognitive ability, etc. 2Circadian rhythms are supported at the cellular level by a wide range of complex molecular pathways and specific oscillatory enzymes. Nonetheless, from a basic point of view, a circadian clock system is shared among species worldwide Per) and Cryptochrome (Cry) genes, which ultimately reach critical protein concentrations, dimerize, and inhibit the subsequent activity of the CLOCK:BMAL1 heterodimer in the nucleus Rorα) and the nuclear receptor subfamily 1, group D (Nr1d1), also known as reverse erythroblastosis virus alpha (Rev-erbα), its own activator and repressor, respectively. These important nuclear receptors both compete for a binding site within the response element (RORE) into the Bmal1 promoter, generating another loop of regulation The core circadian clock system in mammals depends on a central clock located in the hypothalamic suprachiasmatic nucleus (SCN), and on “peripheral” clocks spread throughout the anatomy 3Alas1), plasminogen activator inhibitor-1 (Pai-1) or thyroid hormone receptor alpha (Trα), which play important output roles in heme biosynthesis and vascular or cardiovascular function, respectively (reviewed in Refs. Tef) and hepatic leukemia factor (Hlf), which have important regulatory functions activating downstream metabolic target genes through direct binding to D-boxes Dpb binds to D-elements in the promoter of Per1The number of clock-controlled genes  is extensive. Therefore, we propose a classification of these genes according to their bidirectional clock regulatory functions at the level of interaction with CLOCK and BMAL1 at their cognate E-box target sites . Listed Pparα), nicotinamide phosphoribosyltransferase (Nampt), Dec1, Dec2, estrogen-related receptor alpha (Errα) and proper homeobox 1 (Prox1).Alternatively, there is a group of metabolic CLOCK:BMAL1 target genes that possess direct regulatory feedback properties via one or more of the loops described in Bmal1 expression and thus functions as a bidirectional clock regulatory protein by binding to a PPARα response element (PPRE) located in the Bmal1 promoter. BMAL1, in turn, is an upstream regulator of Pparα gene expression, producing an additional regulatory feedback loop for peripheral clocks  + is a key molecule in metabolism, activating a number of enzymes and transcriptional factors involved in multiple metabolic pathways. The NAD+-dependent sirtuin deacetylase, SIRT1, is one such factor, which binds directly to CLOCK:BMAL1 and affects its transactivating activity. Thus, BMAL1 activation of Nampt generates an additional negative feedback loop , phosphoenolpyruvate carboxykinase (Pepck), the nuclear receptor heme binding protein (Shp) and nuclear factor interleukin 3 (Nfil3)  through RORE Although Approximately half of the rhythmic proteins identified in the mouse liver cannot be explained by the rhythmicity of mRNAs, suggesting that translation and/or protein stability might play a pivotal role in controlling rhythmic protein accumulation In addition to acetylation, BMAL1 can also be phosphorylated by protein kinase C alpha (PRKCA), following stimulation by Receptor of activated protein kinase C1 (RACK1). The BMAL1-RACK1-PRKCA complex is rhythmically assembled during the negative phase of the molecular circadian cycle, resulting in an inhibition of CLOCK:BMAL1 transcriptional activity Interestingly, some part of rhythmically expressed metabolic genes appear to oscillate independently of the TTFL, and have no apparent CLOCK:BMAL1 binding site, suggesting the importance of indirect transcriptional and posttranscriptional regulation 4in vivo or are direct targets of one or more of the TTFL proteins in vivoSometimes, tissues use tissue-specific mechanisms to carry out metabolic oscillations in response to environmental demands + levels and can bind to the CLOCK:BMAL1 heterodimer directly to modulate target gene expression +-sensitive sirtuins proteins provide an example of a somewhat ubiquitous method by which cells can fine-tune their clock based on the energy state of the cell.SIRT1 is a key regulator of several metabolic processes including gluconeogenesis, lipid metabolism, and insulin sensitivity Another key mechanism by which nutrients and energy status help the clock keep time is via AMPK. AMPK detects changes in cellular AMP:ATP ratios during the rest and activity cycle. As a detector of this ratio, it promotes the rhythmic activation of metabolic pathways in response to increasing AMP levels. The molecular targets of AMPK cover a wide range of metabolic processes like glucose uptake, glycogen and protein synthesis, glycolysis, gluconeogenesis and fatty acid synthesis or oxidation + and AMP influence the clock in very direct ways. For example, the DNA-binding activity of the CLOCK:BMAL1 and NPAS2:BMAL1 heterodimers themselves are regulated by the redox state of NAD+ cofactors. The reduced forms NAD(H) and NADP(H) strongly enhance DNA binding, while the oxidized forms NAD+ and NADP+, inhibit it +, NAD(H), and AMP so that when the external energy contribution is low as under fasting or exercise situations, AMPK is activated. AMPK induces CRY and CKIɛ phosphorylation  and indirectly activates SIRT1 via increases in NAD+ levels +-dependent sirtuin activity, NAM levels increase progressively, inhibiting in turn NAD+ formation and therefore, SIRT1 activity +/NAD(P)H balance depends on the metabolic cell state and the metabolic cell state depends on the balance between energy intake and energy demands. In summary, the entrainment of the TTFL in a particular cell depends directly on the redox state of the cell NAD5Srebp-1c and Ppara in mouse liver Per2 and Dec1 expression in the rodent liver. One hour following food intake initiation, the Per1, Per2 and Dec1 mRNA levels increase, while Rev-erb-α mRNA levels decrease Per1, Per2, Dec1 and Rev-erb-α relative expression not only in liver but other insulin-sensitive tissues like the muscle and the white adipose tissue, an effect which is blocked after the addition of specific inhibitors for MAPK and PI3K Many studies have reported the impact of the fasting–feeding cycle on the circadian system through different designs of limited food availability in a period of 24 h  levels, and TEF and nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (NFKB1a) and ACOX3  changes The human clock also appears to be sensitive to dietary manipulations at the level of macronutrient content. In a study designed to test the impact of a low carbohydrate/high fat diet vs. a low fat/high carbohydrate diet on the human clock, it was found that a switch from a low carbohydrate/high fat diet to a high fat/low carbohydrate diet produced a phase delay in salivary cortisol levels , and altered gene expression peripherally, as measured by gene expression in circulating monocytes Glucose metabolism is highly circadian circadiomics.igbuci.edu).The effect of sugar and other nutrient metabolism on the circadian oscillator (and vice versa) has been well studied in plants  + levels in liver. However, the zeitgeber time of proanthocyanidin administration controls the precise effect of this polyphenol on the molecular clock in vivoProcanthocyanidins (PAs) are the most consumed polyphenols in human diet based on their widespread presence in vegetables, fruits, cacao, nuts and some beverages like red wine or tea in vivo. Studies beginning to look carefully at this distinction clearly provide examples of specific nutrients that profoundly alter the clock machinery directly. For example, recent discoveries include the effect of polyamines on the circadian clock. In this case, rather than directly modifying the CLOCK:BMAL1 heterodimer, polyamines directly affect the efficiency of the PER2:CRY1 interaction Most of the studies to this point show the response of the clock to diets of largely different macronutrient composition. Yet, unclear is whether the response of the circadian clock to a specific diet is due to particular signaling pathways (i.e. impaired insulin sensitivity in the context of a high fat diet) vs. specific metabolites or micronutrients within the diets affecting the clock components more directly. Based on emerging data addressing this question, both indirect and direct mechanisms likely are responsible for clock manipulation 6As substantiated by numerous human and rodent studies, synchronicity between clocks appears to be important for metabolic health. Examples of this are found in studies showing the strong association of night shift or rotating shift working humans with increased adiposity, type-2 diabetes, and even cardiovascular disease  and non-esterified polyunsaturated fatty acids is disturbed when the adipose clock is impaired, a situation which results in altered circadian energy intake and obesity in mice Studies are starting to address this complex question by asking what circadian signals arise from one tissue that assist in entraining or maintaining circadian rhythms in other tissues. For example, phosphatidylcholine 18:0/18:1 (PCI) has been identified in mouse serum to undergo a pronounced PPAR δ-controlled rhythmicity, which is essential for PPARα-mediated fatty acid uptake by the muscle de novo rhythmicity in a number of metabolic pathways One important discovery regarding the effect of nutrients on tissue-specific clock response is that in specific circumstances  a high fat diet can induce obesity simply by disrupting the circadian rhythmicity of energy intake, inducing increased energy intake during the normal resting phase 7Much of the molecular glue connecting circadian rhythmicity and metabolism comes in the form of nuclear receptors (NR), which connect endocrine and circadian physiology. At the level of gene expression, over half of the 49 mouse nuclear receptors display rhythmic mRNA accumulation Natural ligands have been discovered for roughly half of the nuclear receptor superfamily, the majority being lipidic structures 8While new studies considering the role of the human diet and nutrition on our circadian clocks (and vice versa) are emerging, these data lag far behind the numerous rodent studies demonstrating a direct role for nutrients in circadian biology. Since the introduction of artificial light and nighttime work, serious health consequences have been reported for those who sleep less, work night or rotating shifts, or have social jetlag resulting from work or social schedules that are not in sync with the external light/dark cycle. Association studies have revealed that such circadian misalignment is linked with impaired glucose tolerance, reduced insulin responsiveness following glucose challenge, increased body mass index, decreased levels of leptin, and increased levels of ghrelin While additional studies are needed, one way to include behavioral circadian predisposition into personalized medicine may be to ascertain the circadian chronotype  of patients, which may predict the effectiveness of a given treatment. Humans display great circadian variability at the behavioral level with some individuals having more ‘eveningness’ (a delayed sleep onset) compared to those with more ‘morningness’. To determine chronotype, questionnaires are often used, the most common one being the ‘Morningness–eveningness questionnaire’ of Horne-Östberg  PER2, CRY1, REV-ERBα and CLOCK have been associated with metabolic disturbances. Specifically, PER2 SNPs rs2304672C>G and rs4663302C>T have been associated with abdominal obesity. Interestingly, rs2304672 C>G minor allele carriers have a greater probability of displaying extreme snacking, experiencing stress with dieting, eating when bored, skipping breakfast and dropping out of a dietary treatment of obesity than non-carriers, while frequency of the rs4663307 minor allele is increased in withdrawers than in those subjects who successfully completed treatment CRY1 rs2287161 polymorphism have been associated with an increase in carbohydrate intake (%of energy intake), HOMA-IR and fasting insulin A defined chronotype in humans is ultimately based on the same circadian clock mechanisms identified in cells and tissues of other mammals. For example, the circadian expression pattern of a majority of clock genes has been observed in RNA samples from human peripheral tissues including the skin, oral mucosa, colonocytes, white blood cells, peripheral blood mononuclear cells, pineal gland tissue and visceral and subcutaneous white adipose tissue CLOCK gene SNPs have been the most comprehensively studies. Several of these SNPs are associated with obesity and individual components of the metabolic syndrome. SNP rs4580704 is associated with reduced BMI and compared to non-carriers, minor allele carriers have a 31% lower risk of diabetes, 45% lower risk of having hypertension, lower postprandial triglyceridemia after a fat-loading test, lower intake of fat, and lower plasma glucose concentrations and HOMA values when MUFA intake is the same or more than 13.2% of energy Out of the identified SNPs in clock genes, the Understanding one's genetic or epigenetic predisposition for circadian disruption may be one of the key aspects of personalized medicine in the future. Several studies have begun laying the groundwork for this. Specifically, a better understanding of the internal clock in humans at the level of circulating metabolites is gaining ground, with efforts to identify internal body time 9While the model of personalized medicine, the idea of customizing healthcare based on parameters such as the genetic and behavioral profiles of an individual, once seemed a thing of the future as it related to nutrient input, some key studies already show its tremendous promise. For example, machine-learning algorithms can use blood parameters, nutrient input choices and gut microbiota composition to help predict glycemic responses to a meal Already, practical applications for the general public are being developed based on decades of research on the internal clock and its molecular underpinnings. For example, the Entrain app was developed based on the heavy dependence of our internal clock on the light/dark cycle. This app was designed to assist with optimal circadian reentrainment to a new light/dark cycle using mathematical models based on and incorporating thousands of different circadian misalignment possibilities In summary, what is clear is that circadian rhythms are important for energy balance at the cellular and organismic level. Importantly, feeding is a critical modulator of the internal clock and, in addition to affecting synchronicity between the central pacemaker and metabolically active peripheral tissues, it likely controls the extent to which peripheral tissues are in phase with one another. With this in mind, several questions remain: 1) how does synchronization across tissues take place, 2) what are the specific nutrients that modulate tissue-specific rhythms in a way that preserve synchronization across the body, and 3) can we use nutritional input more effectively in dealing with metabolic disorders arising from circadian misalignment? The complexity of the interplay between nutrient metabolism and circadian rhythmicity remains, but understanding this dialog more fully is likely to eventually produce great benefits in terms of metabolic disease prevention and treatment as it relates to circadian misalignment.


SOD1), TAR DNA-binding protein 43 (TARDBP/TDP-43) and C9orf72, have been identified in subsets of familial and sporadic patients. Key associated molecular and neuropathological features include ubiquitinated TDP-43 inclusions, stress granules, aggregated dipeptide proteins from mutant C9orf72 transcripts, altered mitochondrial ultrastructure, dysregulated calcium homeostasis, oxidative and endoplasmic reticulum (ER) stress, and an unfolded protein response (UPR). Such impairments have been documented in ALS animal models; however, whether these mechanisms are initiating factors or later consequential events leading to MN vulnerability in ALS patients is debatable. Human induced pluripotent stem cells (iPSCs) are a valuable tool that could resolve this “chicken or egg” causality dilemma. Relevant systems for probing pathophysiologically affected cells from large numbers of ALS patients and discovering phenotypic disease signatures of early MN susceptibility are described. Performing unbiased ‘OMICS and high-throughput screening in relevant neural cells from a cohort of ALS patient iPSCs, and rescuing mitochondrial and ER stress impairments, can identify targeted therapeutics for increasing MN longevity in ALS.Amyotrophic lateral sclerosis (ALS) is a largely sporadic progressive neurodegenerative disease affecting upper and lower motoneurons (MNs) whose specific etiology is incompletely understood. Mutations in superoxide dismutase-1 ( Amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease, is one of the most common neurodegenerative disorders and adult-onset motoneuron disease (MND), with a global incidence of 2.5 cases per 100,000 individuals each year, and is classified as a rare disease. The disease is primarily characterized by a relentless degeneration of upper motoneurons (UMNs) in the cerebral cortex and lower motoneurons (LMNs) in the brain stem and spinal cord, causes muscle weakness, progressive paralysis, speech and swallowing disabilities, and ultimately leads to death by respiratory failure , fundamental advances in our understanding of the disease have been made with regard to biological, genetic and clinical processes  repeats. C9orf72 encodes for a protein with two different isoforms: (i) a mainly diffuse in the cytoplasm localized C9-L and (ii) a nuclear membrane localized C9-S  DNA-binding protein  imbalance, and axonal transport defects  and act as gatekeepers in intrinsic apoptotic pathways. Mitochondrial dysfunction can lead to oxidative stress, failure of cellular bioenergetics and ultimately to cell death Figure . Thus, ade novo risk genes and pathways, i.e., TBK1  to recapitulate disease phenotypes we will outline some of the observed phenotypes and to what extent they resemble hallmarks of disease progression observed in the clinic.The risk of developing ALS peaks between 50–75 years of age, which is in contrast to other neurodegenerative diseases such as Parkinson’s disease (PD) or Alzheimer’s disease (AD), thus emphasizing that it is not necessarily a disease of aging, but that age is one of the chief risk factors. In a majority of patients, ALS manifests as asymmetric, painless weakness in a limb, referred to as spinal onset. About 20% of patients present with symptoms of weakness in bulbar muscles, difficulties swallowing (dysphagia) and speech impairment, referred to as bulbar onset. In about 3–5% of patients, ALS is characterized by onset affecting the respiratory system, with shortness of breath and difficulty breathing (orthopnea and dyspnea). While these symptoms of onset can be overlapping, the average survival is 2–5 years, with bulbar and respiratory onsets having a worse prognosis  innervate extrafusal muscle fibers, the most abundant type of muscle fiber and the type being involved in muscle contraction. Beta MNs (βMNs) innervate intrafusal fibers of muscle spindles with collateral extensions to extrafusal slow twitch fibers. Gamma MNs (γMNs) innervate intrafusal muscle fibers and together with sensory afferents, compose a muscle spindle and are part of proprioception, the sensing of body positioning. According to the contractile properties of the motor units (MU) they form with their target muscles, αMNs can be further classified into fast-twitch fatigable (FF), fast-twitch fatigue-resistant (FR) and slow-twitch fatigue-resistant  The “dying forward” hypothesis suggests that events within the MN are the primary determinant initiating MN damage, while alterations in closely associated non-neuronal astrocytes, Schwan cells, microglia and skeletal muscle cells only contribute to the disease progression; (II) In a counter-proposed “dying backward” mechanism, ALS is considered to begin within the muscle cells and/or neuromuscular junction as a result of one or more neurotrophic factors being released by postsynaptic cells. When retrogradely transported via the presynaptic axon to the cell body those factors may initiate MN damage and degradation , for instance, have been found in spinal MNs of human sALS autopsy cases  microglia, the major resident immune cells, constantly survey the microenvironment and produce factors that influence other glial cells such as astrocytes. While microglia remain in a deactivated state during physiological conditions Streit,  they swiSigns of neuroinflammation can be readily observed in pathologically affected areas in the brain and spinal cord in both human ALS patients as well as mouse models of the disease. Characteristic for ALS are gliosis and accumulation of large numbers of activated microglia and astrocytes marked by elevated production of inflammatory mediators , proinflammatory cytokines  and potentially cytotoxic molecules such as ROS. For instance, major histocompatibility complex molecules and complement receptors were found highly expressed by activated microglia in the primary motor cortex and the anterior horn of the spinal cord in ALS patients  has been associated with ALS-associated MN death, as nerve growth factor (NGF) secreted by mSOD1 astrocytes induced death of p75NTR expressing MNs via the formation of NO and peroxide  confirmed the progressive decrease in the number of OGs as a function of disease progression in SOD1G93A mice , a pro-fusion factor, dynamin related protein 1  excessive ROS leads to lipid peroxidation interfering with mitochondrial membrane integrity; (ii) a plethora of mitochondrial proteins, such as essential proteins of the ETC complexes, contain highly oxidizable iron-sulfur clusters; and (iii) due to the lack of protective histone complexes mitochondrial DNA is far more susceptible to mutations in organelle encoded proteins  The inositol-requiring transmembrane kinase/endonuclease (IREI1), the PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). IREI1 initiates splicing of an mRNA coding for the transcription factor X-Box-Binding protein 1 (XBP1), converting it into a potent activator of multiple UPR-responsive genes, the XBP1s  subclass of 2xRBD-Gly proteins, shuttles between the nuclear and cytoplasmic compartments, binding both DNA and RNA, and is involved in exon splicing of several genes  introduction  expansion inclusions, TDP-43 pathology, and behavioral abnormalities similar to symptoms observed in C9ORF72/ALS patients  are often observed in animal models such as mice and flies. The corresponding genetic mutations in ALS patients however, display later in life with median onset age greater than 50. Many more elements of ER stress responses, i.e., the UPR, have been found to impact phenotypes of animal models and reviewed elsewhere folding and the proteostatic capacity of MNs represent a promising therapeutic target to delay the symptomatic phase of ALS. With regard to this, utilizing gene therapy or small molecule approaches to reinforce the capacity of coping with ER stress mechanisms may be an applicable means for disease intervention  of interest, and the second is to derive stem cells from patients with target diseases. The genetic modification of mouse embryonic stem cells (ESCs) by targeting specific gene loci via homologous recombination has been routinely applied in many laboratories as a first step to creating transgenic animals. Likewise, this same principle is applicable when using human embryonic stem cells  can be generated from somatic cells such as fibroblasts  translation products  providing accessibility of difficult-to-obtain CNS cell types such as MNs; (b) allowing investigations on developmental effects of any ALS-associated mutation; (c) giving options to analyze single mutant cells; (d) supplying glial cells for interaction studies in co-culture with neurons; (e) allowing prospects of ALS biomarker discovery; and (f) providing platforms for therapeutic screening strategies.A crucial step on the way to treatment for ALS is the identification of an early biomarker signature. Whether it comprises markers of neural and glial pathogenesis, mitochondrial and ER stress signals  obtaining homogenous population of neural subtypes devoid of undifferentiated progenitors; (b) prolonged time required in culture for maturation; and (c) fetal/young “age” of the different neural subtypes. Further development of cell sorting tools, fluorescent reporters, and better markers (surface/intracellular) for the human cell types is imperative. Recent advancements in genome editing and creation of CRISPR/Cas edited reporter iPSC lines will help solve some of these technical issues. The big question that remains and needs addressing is whether it is required to generate completely homogenous cellular models (neurons or glia) to mimic human ALS-in-a-dish. The answer is going to be context and question-dependent. Nevertheless, a motor neuron during development will not engender synaptic maturity without the presence of astrocytes in culture.Apart from using patient-derived iPSCs for identification of diagnostic markers, they may also bear the potential for cell-based therapies. While such approaches have been used in neurological diseases with focal pathology, i.e., PD  is a collaborative initiative with the aim to catalog cellular processes under conditions such as cell stress and disease. Funded by the National Institute of Health (NIH) the LINCS centers set out to collect large data sets of cellular events in human diseases compared to healthy control samples. Utilizing computational tools to integrate such diverse information into a comprehensive view of molecular disease events and the development of new biomarkers and therapeutics  to elicit responses related to mitochondrial and ER stress. Our understanding of the molecular circumstances of disease progression could be dramatically improved by the generation of quantitative molecular phenotypes and cell signatures of human neurons and glia, providing rational intervention points.2+ levels observed during disease progression is a common link to both mitochondrial and ER stress, as both organelles finely tune Ca2+ homeostasis. Particularly, the latter one provides putative insights into MN susceptibility through mitochondrial stress leading to increased local Ca2+ levels and ultimately functional alteration of ALS-critical proteins; given that Ca2+ itself is typically found to affect ALS-associated proteins and processes.More than a century ago, Jean-Martin Charcot first described a disease entity of broad neurodegeneration known as ALS. Since then multiple molecular events associated with ALS disease progression have been identified, each of these working in concert leading to disease onset: (1) Developmental defects caused by genetic mutations making certain MN subtypes susceptible to aberrant maturation during CNS development could lead to early degeneration in adult life; (2) An inert susceptibility of mitochondria, provoked by an inability to balance ROS, making MNs more prone to cell stress and degeneration; (3) Misfolded proteins and protein-RNA aggregates triggering the ER UPR stress responses leading to initiation of MN apoptosis; and (4) The dysregulation of CaThe post-mitotic MNs, however, are not the only cell types affected by the pathological processes and mutant genes involved in ALS. Glial cells, in fact, are functionally impaired, as shown in animal and stem cell models alike, and these ultimately impact MNs as well. Animal models and multidimensional “organs-on-a-chip” will be crucial when future studies are required to dissect CNS cell-cell interactions. However, animal models alone will not enable us to decipher the full complexity of an ALS patient’s phenotype. Years of research with animal models have led to mostly ineffective treatments for ALS. In this regard human models utilizing patient iPS cell technology continue to provide a wealth of additional insight into the relevant disease phenotype in pathophysiologically affected human cells. The logical next step will be to use both animal and cell models synergistically and coordinate collaborative efforts of large consortium studies such as the NeuroLINCS initiative. By utilizing ‘OMICS, longitudinal imaging, big data and machine-learning approaches on large cohort of ALS patient iPSC-derivatives harboring both familial and sporadic forms of the disease we may be able to discover central ALS-specific mechanistic signatures beyond the phenotype of an individual ALS mutation, and rather also across sporadic disease that affects almost 90% of the patients. Such an approach promises discovery of key biomarkers and pathways that shift subtype-specific motor neurons and glial cells out of balance. Whether iPS cell technology can fulfil such commitments remains to be determined.2+ deregulation and apoptotic signaling. Long-term strategies will require a detailed understanding of early signs of ALS initiation and the ability to repair and regenerate at least MN sets crucial for survival and extending life span. Recent reports , National Center for Advancing Translational Sciences (NCATS), Grant UL1TR000124 (DS). DS is also supported by funds from National Institute of Neurological Disorders and Stroke (NINDS) grant U54NS091046. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.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.


Cosmetics/cosmeceuticals agents always are needed to be evaluated for their detrimental effects on the skin. Nowadays, the therapeutic potential of stem cells has and fibroblasts have increased the hope for a successful cell therapy in aesthetic medicine. Stem cells are unspecialized cells capable of renewing themselves through cell division without limit as long as the person is still alive. Each new cell has the potential either to remain a stem cell or become another type of cell.2In a healthy individual, skin integrity is supported by epidermal stem cells that self-renew and generate daughter cells undergoing terminal differentiation. In addition to accumulation of senescence markers in aged skin, epidermal stem cells are maintained at normal levels throughout life. So, skin ageing is provided by an impair in stem cell mobilization or a reduction in the number of stem cells being capable to respond to proliferative signals. The self-renewal and multi-lineage differentiation of skin stem cells make these cells attractive for ageing process studies but also for regenerative medicine, tissue repair, gene therapy and cell-based therapy with autologous adult stem cells.3 Sakrak et al. (2012) noted that cultured fibroblasts, particularly with a dermal support, contribute to the wound healing process; reduce the contraction of the wound; and support collagen synthesis and neovascularization.4 Many potential mechanisms exist to impair healing. One contributory mechanism may be inhibition of fibroblast proliferation and induction of a stress-induced premature senescence phenotype by the continuing inflammation found in chronic wounds. They showed that recognition of the role of fibroblast senescence in wound chronicity may allow for identification of those wounds that will respond positively to these products.5Healing in skin wound demonstrates an extraordinary mechanism of cascading cellular functions. Migration of fibroblasts along with the fibrin network and start of reepithelialization from the wound edges, angiogenesis and neovascularization get activated by capillary sprouting.6 The ADSC and its secretory factors were demonstrated to be effective for UVB-induced wrinkles too, and the antiwrinkle effect was mainly mediated by reducing UVB-induced apoptosis and stimulating collagen synthesis of HDF.7 Cell therapy for facial anti-aging in clinical experience was introduced including cultured gingival fibroblasts injection lasting for at least one year, making it a good option for patients.8 Park et al. (2008) demonstrated that ADSCs and their secretory factors showed promise for application in cosmetic dermatology, especially in the treatment of skin aging too.9Adipose-derived stem cells (ADSC) were shown to have relative advantages in accessibility and abundance compared to other kinds of stem cells in treatment of many dermatologic diseases. Subcutaneous injection of ADSC could significantly increase the collagen synthesis in hairless mice, and dermal thickness, collagen density and fibroblast number, angiogenesis, procollagen type I protein and mRNA expression also increased. They recommended that ADSC therapy may be useful in ageing skin as ADSC have antioxidant, whitening and wound-healing effects in the skin by secretion of growth factors and by activating fibroblasts.10 Autologous fibroblasts grown in culture (azficel-T) were shown to correct the appearance of aging and wrinkles by replacing lost dermal constituents showing that autologous cell therapy can mark the beginning of a new phase in aesthetic therapy.11 Eca et al. (2012) showed that injection of skin fibroblasts cultivated in medium supplemented with human serum was a viable method and had no side effects. Four injections at 15-day intervals containing 6.4×106 fibroblasts/mL could significantly improve periorbital skin flaccidity.12 In a study by Khodadadi et al. (2010) on 10 patients with stable vitiligo, it was shown that intra-epidermal injection of dissociated autologous epidermal cells could improve the patches in 80% of subjects.13In aging, loss of collagen and elastin are the important visible processes. Schmidt (2011) reported that the US Food and Drug Administration (FDA) approved laViv (azficel-T), a first-in-class personalized cell therapy to remove fine wrinkles or nasolabial folds around the nose and mouth.14 Fibroblasts have a crucial role in wound healing. They produce matrix metalloproteinases and plasmin too. Its synthesis is increased in remodeling of an injured area after a wound15,16 and also its production has an increasing trend in fibrotic diseases17,18 while has a decreasing trend during aging and after a sun exposure.19Dermal fibroblasts are responsible for synthesizing and organizing the dermis with three layers of (i) epidermis containing keratinocytes, melanocytes, and Langherans cells; (ii) dermis that is populated with fibroblasts, vessels and dendritic cells; and (iii) subcutaneous tissue. A basement membrane separates epidermis from dermis by composed of collagens and laminins, synthesized by fibroblasts and keratinocytes. Type I collagen is the most protein in the dermis produced by fibroblasts, synthesizing other collagens , elastin, proteoglycans, and fibronectin too. The half-life of type I collagen in human skin was shown to be greater than 1 year.20 Normal human dermal fibroblast cultures can be divided into three phases of (i) primary cultures established by enzymatic digestion of the dermis, or by outgrowth of fibroblasts from explanted tissue pieces; (ii) secondary cultures as actively proliferating cells, provided from passage and expansion of primary cultures and (iii) terminal cultures eventually reaching a state of replicative senescence to be aging at the cellular level.21.22 Cryogenic preservation of low passage-number fibroblasts is useful to maintain reserves of cells for further therapeutic measures. Therefore, it is easy to rapidly build a bank of fibroblasts from a limited number of skin samples. Culture of skin fibroblasts in collagen gels was first described by Bell et al.23 The collagen gel serves as a stimulus to modulate fibroblast behavior.24 In monolayer, fibroblasts are flat, spindle-shaped, and organized in parallel arrays, but in a three-dimensional collagen gel, they are elongated and have several dendrites. Fibroblasts in collagen gels usually proliferate slower than in monolayer cultures.25 They are cultured in Dulbecco’s MEM supplemented with non-essential amino acids and 10% fetal bovine serum (DMEM-FBS). Cell growth is undertaken at 37◦C in 5% CO2 and 95% air. Fibroblasts are subcultured using trypsin/EDTA as required, and are used at passage 3 to 5.26 Therefore, it seems that autologous fibroblasts are good sources to correct the appearance of aging and wrinkles by replacing lost dermal constituents and have a crucial role in healing showing that the autologous cell therapy can be a new phase in aesthetic therapy with no side effects for the patient.For decades, skin organ culture has successfully been used to study skin ex vivo .The authors declare no conflict of interest.