Physical activity is key to improve the prognosis of chronic obstructive pulmonary disease (COPD). To help to tailor future interventions we aimed to identify the baseline characteristics of COPD patients which predict 12-month completion and response to a behavioral physical activity intervention.This is a 12-month cohort study of the intervention arm of the Urban Training randomized controlled trial (NCT01897298), an intervention proven to be efficacious to increase physical activity. We considered baseline sociodemographic, interpersonal, environmental, clinical and psychological characteristics as potential determinants of completion and response. We defined completion as attending the 12-month study visit. Among completers, we defined response as increasing physical activity ≥1100 steps/day from baseline to 12 months, measured by accelerometer. We estimated the factors independently for completion and response using multivariable logistic regression models.Of a total of 202 patients (m (SD) 69 (9) years, 84% male), 132 (65%) completed the study. Among those, 37 (28%) qualified as responders. Higher numbers of baseline steps/day  and living with a partner  were related to a higher probability of completion while more neighborhood vulnerability  was related to a lower probability. Among the completers, working  and having an endocrino-metabolic disease  were related to a higher probability of response while unwillingness to follow the intervention  was related to a lower probability.This study found that 12-month completion of a behavioral physical activity intervention was generally determined by previous physical activity habits as well as interpersonal and environmental physical activity facilitators while response was related to diverse factors thought to modify the individual motivation to change to an active lifestyle. Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide . Over thpost hoc or secondary analyses. Altenburg et al showed that only patients with baseline physical activity <10,000 steps/day maintained a significant long-term effect at 15 months of a 12-week physical activity counselling program [Recent efforts have succeeded in designing and testing interventions that increased physical activity in COPD patients either at short –8 or lon program . Moy et  program . OverallAn important characteristic of the mentioned trials is that they were limited to COPD patients with reduced functional exercise capacity  test ≈ 450m ,11 or beAccording to the World Health Organization (WHO) strategy for chronic medical conditions, the outcomes of health promotion and secondary prevention interventions strongly depend on the involvement of patients and their families . Thus, tThe aim of our study was to identify which sociodemographic, interpersonal, environmental, clinical, and psychological baseline characteristics were associated with 12-month completion and response to a behavioral physical activity intervention in COPD patients, using data from the intervention arm of the randomized controlled Urban Training trial NCT01897298) mostly involving COPD patients from primary care. This intervention has previously been reported to be efficacious to increase physical activity in the per protocol population but ineffective in the full population [7298 most1) to forced vital capacity (FVC) ratio <0.70) [We performed a cohort study of 12-month follow-up of the intervention group participating in the Urban Training trial behavioral physical activity intervention in COPD patients. This was a prospective, multicenter, parallel-group, randomized controlled trial, registered at clinicaltrials.gov NCT01897298) and previously described in detail [7298 and o <0.70)  from 33 rd, 2011) and all participants provided written informed consent.The Urban Training trial was approved by the Ethics Committees of all participating institutions  a pedometer and a personalised calendar to monitor and note their physical activity and keep motivation; (4) the project website [phone text messages every 2 weeks with educational or motivational messages and the European Lung Foundation information brochure [walking group once per month; and (6) a support phone number available during the 12-month study period.The Urban Training intervention included six elements aiming to elicit a behavioral change and to maintain it during the 12 months of the study: (1) a nterview , followinterview , with upnterview  Urban Tr website  and phonbrochure ; (5) a gsociodemographic variables: age, sex, smoking and socioeconomic status; (ii) interpersonal variables: living with a partner , grandparenting and working status ; (iii) environmental variables: urban vulnerability index, a measure of socioeconomic status at the census tract level (median area of 0.26 km2) that combines demographic, economic, residential and subjective indicators, and ranges from 0 (lowest) to 1 (highest level of neighborhood vulnerability) [clinical variables: forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) by forced spirometry after bronchodilator, functional exercise capacity measured by the 6-min walking distance (6MWD) test, the modified Medical Research Council dyspnea scale (mMRC), the number of severe COPD exacerbations  in the 12 months prior to recruitment and during follow-up, body mass index (BMI) and fat free mass index (FFMI) by physical examination and bioelectrical impedance, physician diagnosed comorbidities categorized according to the International Classification of Diseases, Tenth Revision ICD-10  and physical activity variables: steps/day and time spent on moderate to vigorous physical activity  measured by the Dynaport accelerometer  previously validated for COPD [psychological variables: the Hospital Anxiety and Depression scale (HADs), unwillingness to follow the physical activity intervention  , and seafor COPD ,25, defifor COPD ; and (v)uctions’ ,18 and sWe defined study completion as participation in the final visit (12 months after the baseline visit). Among patients who completed the study, we defined intervention response as a positive change in mean daily step count between baseline (visit 2) and 12 months (visit 4) equal or higher than the minimal important difference (MID) . Steps M1, FVC, 6MWD, and steps/day at baseline and resulted in a range of statistical power between 93% to 98% for study completion and 79% to 90% for response, using unpaired t-test (p<0.05) and assuming a relation of loss-to follow-up to completion and responders to non-responders of 1:2.The available sample size (n = 202) was fixed by the primary objectives of the Urban Training study, therefore we calculated the statistical power to answer the current research question. Calculations were performed for a difference in age, FEVWe assessed the presence and patterns of missing data. Due to the small proportion of missings , main analysis was conducted using a complete case strategy and we reported missing data in the Table footnotes. As a sensitivity analysis, to account for the possibility of bias due to missing data, we used multiple imputation (20 imputed datasets) with the method of chained equations  assumingTo analyze determinants of study completion and response to intervention, we first compared the individual’s characteristics  at baseline between patients lost to follow-up and completers, and between non-responders and responders using unpaired Student’s t-test, Wilcoxon rank-sum, Chi square or Fisher’s tests, depending on the variable distribution. Second, we built two multivariable logistic regression models (one for study completion and one for response to the intervention) including as exposures all variables that exhibited a p-value <0.2 in the bivariable analysis between lost to follow-up and completers, and non-responders and responders, respectively. Model building combined step-forward and backward algorithms, and covariates were included in the final model if: (i) they related to both the exposure and the outcome in bivariable analysis; (ii) they modified (>10% change in regression coefficient) the estimates of the remaining variables in the multivariable models; or, (iii) there was consistent evidence in the literature of their association with the outcome . To exclWe conducted several secondary analyses. First, we used 600 steps (lower limit of steps MID) as a cut-off to define response to the intervention both in bivariable and multivariable analyses. Second, we repeated the final multivariable model for response: (1) including severe acute exacerbations of COPD during follow-up in the multivariable models to test the possibility that this variable affected the risk of being a non-responder; and (2) using time in moderate to vigorous physical activity  instead of steps to define response to the intervention.All analyses were conducted with Stata 12.0 .1 56% of predicted and a mean 6MWD of 487m; 29% of patients had an mMRC dyspnea score ≥2, 9% of patients had a severe COPD exacerbation within the previous year and only 6% were participating in a pulmonary rehabilitation program at study inclusion. Patients had a large prevalence of endocrino-metabolic and cardiovascular chronic comorbid conditions and walked more than 7000 steps per day on average. At baseline, 26 patients (13%) reported unwillingness to follow the intervention, 50% of patients were in later phases of stage of change , and self-efficacy was high .Patient characteristics are shown in After 12 months, 70 patients (35%) were lost to follow-up and 132 patients (65%) completed the study (completers) .Of the broad range of variables assessed at baseline, physical activity levels, marital status, neighborhood vulnerability, unwillingness to follow the intervention and baseline 6MWD were significantly different between patients lost to follow-up and completers . In the Among patients who completed the study, 37 patients (28%) qualified as responders using as threshold 1100 steps/day . The chaActive working, diagnosis of an endocrino-metabolic disease, unwillingness to follow the physical activity intervention, and FVC at baseline were related to the response to the intervention on bivariable analysis .After mutual adjustment in a multivariable model, being an active worker  and having an endocrino-metabolic disease  were independently related to a higher probability of response while unwillingness to follow the intervention  was related to a lower probability .The LASSO approach confirmed that final models of determinants of completion and response Tables  and 4 weThis study about the determinants of 12-month completion and response to a behavioral physical activity intervention in COPD patients has found that: (1) being more physically active and living with a partner were positively associated with study completion while living in more vulnerable neighborhoods was negatively associated; (2) active working and endocrino-metabolic comorbidities were positively associated with intervention response while unwillingness to follow the intervention was negatively associated; (3) most clinical and functional characteristics of COPD previously reported to be relevant for physical activity and specifically interventions‘ completion or response were not associated with these outcomes in our sample population; and (4) predictors of study completion were different from predictors of intervention response.Our study shows that patients who were more physically active at baseline were more likely to complete the study. We hypothesize that physically active patients already had established a habit around regular physical activity, which acted as a facilitator for participation so that these patients were willing and able to complete the study. Indeed, continuation of an active lifestyle from the past has been reported as a reason to be physically active in an earlier mixed-methods study which combined qualitative and quantitative approaches . Thus, wseeing others active‘ or ‘neighborhood walkability‘), has been recognized within the ecological model of the determinants of physical activity in the general population [Living with a partner or in a less vulnerable neighborhood were further predictors of study completion. The role of interpersonal factors such as social support from family, or environmental factors , which could have increased their capacities to engage in the intervention. However, in our study age was not related with response , psychological factors , the season of inclusion, or the baseline levels of physical activity. Whether their lack of association with intervention response may be due to the fact that other studies recruited patients in more advanced stages of disease needs to be confirmed in further clinical trials.There was a number of variables we had expected to be associated with the intervention response, based on the results of previous studies ,11,13, oOur study has implications for future research, clinical management and public health and policies. First, the response to the Urban Training intervention  has beeA major strength of our study lies within the fact that the Urban Training intervention was administered to COPD patients from hospital and primary care settings. The findings therefore reflect more closely than previous studies  what can be expected when deploying a physical activity intervention at the population level. Moreover, the study design allowed us to assess the determinants of a long-term effect. These are more likely to be the determinants of a real behavior change as compared to the short-term effects shown in literature. Finally, the broad number of variables included in our study reflect well the various domains of the determinants of physical activity within the ecological framework .elderly inhabitants of Mediterranean cities’ [As potential limitation we have to acknowledge the small sample size that did not allow testing the potential association of some rare factors (e.g. less frequent comorbidities) with completion or response. Although we included a broad range of variables, some variables related to physical activity limitation in COPD patients such as reduced muscle strength or impaired lung volumes were missing. Finally, our study population reflects the behavior of a population from a specific geographic area (i.e. ‘ cities’ ) and incAmong a broad range of potential predictors of 12-month completion or response to a behavioral physical activity intervention, this study found that completion was generally determined by previous physical activity habits as well as interpersonal and environmental physical activity facilitators while response was related to diverse factors thought to modify the individual motivation to change to an active lifestyle. These results support a look beyond the traditional clinical and functional variables and the consideration of psychological, interpersonal and environmental factors related to habits and motivation to optimize the outcome of physical activity interventions in COPD.S1 File(PDF)Click here for additional data file.


The need to diagnose RV dysfunction is evident. Because of itswide availability, echocardiography is the most frequently used imaging test in clinicalpractice to assess RV size and function. That assessment can be hindered by the complexRV anatomy; thus, important international societies of cardiovascular imaging haverecommended the routine and systematic addition of several echocardiographicmeasurements and techniques.3 That approach includes conventionalparameters, such as RV basal diameter  and tricuspid annularplane systolic excursion , as well as advancedparameters, such as the s wave of the RV free wall on tissue Doppler, ejection fraction on 3D echocardiography  and longitudinal strain of the RV free wall .The importance of the right ventricle in cardiovascular physiology has beenunderestimated for decades. Previously considered a mere conduit, the right ventricle iscurrently known to play a major role in maintaining global cardiac function intact. Inparallel, right ventricular (RV) systolic function has been shown to be an essentialdeterminant of clinical outcomes in several scenarios,1 Right ventricular longitudinal strain is a parameter lessdependent on the angle, with less intra- and interobserver variability, that canapparently detect early RV dysfunction. Its drawbacks include the high dependence onimage quality and the variability of the software of the equipment available in themarket.3 Recently aninternational consensus has been reached to standardize the use of 2D-STE to obtain RVstrain.4 The specific use ofright-ventricle-focused apical 4-chamber view is recommended for correct strainmeasurement. Extreme care should be taken to define the region of interest (ROI) of theendocardial border (suggested ROI: 5 mm), because of the RV shape and thin walls. Thepericardium should be excluded from the analysis, because of the risk of strainunderestimation.In this scenario, strain (systolic shortening percentage) and strain rate (shorteningrate), calculated by speckle tracking on two-dimensional echocardiography (2D speckletracking or 2D-STE), emerge as alternatives in the RV systolic function analysis. Thelongitudinal strain of the RV free wall, excluding the ventricular septum, showedprognostic value in patients with signs and symptoms of cardiopulmonary disease, such asheart failure, myocardial infarction, pulmonary hypertension, congenital heart diseases,RV arrhythmogenic cardiomyopathy and amyloidosis.5 published inthis issue of the Arquivos Brasileiros de Cardiologia, shows that theassessment of strain and strain rate by use of 2D-STE can detect early RV dysfunction inindividuals with systemic lupus erythematosus (SLE) associated with mild and subclinicalpulmonary hypertension [PH: systolic pulmonary artery pressure (SPAP) between 30 and 50mmHg]. It is worth noting that, considering the other conventional and nonconventionalparameters of RV size and systolic function, RV dysfunction was only diagnosed inindividuals with moderate to severe PH (SPAP ≥ 50 mmHg). It is necessary toacknowledge the little methodological limitation in estimating right atrial pressure, towhich only two values were attributed (8 or 15 mmHg) in the dynamic analysis of theinferior vena cava. This might have overestimated the SPAP in some patients, but thatbias does not invalidate the results of that study. Another study has reported that thesurvival rates of patients with SLE who develop PH seem lower than those of individualswith primary PH.6 The findings of thestudy by Luo et al.5 allow us tospeculate that RV dysfunction is the mediator of the high mortality risk in that groupof individuals. Those findings suggest that the use of strain to analyze RV systolicfunction in SLE can select patients in a subclinical phase who require carefulsurveillance and early therapy to prevent the development of RV failure andcardiovascular complications. Further studies are necessary to deepen thepathophysiological knowledge of RV dysfunction in the clinical context of SLE and toassess the role of intervention strategies to reduce mortality.The study by Luo et al.,


The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg In this case, the change in the relative orientation of the CBS motifs appears to be triggered by the electrostatic attraction between the surfaces of the two interacting proteins . The aut2+ atoms to the CBS module of the bacterial channel MgtE [2+ ions, the interfacial helices from complementary CBS2 motifs  remain distant from each other due to the electrostatic repulsion exerted by acidic residues located at their interfacial α-helices  the presence of a transmembrane domain potentially capable of passing metal ions through the cell membranes; (2) the presence of a regulatory Bateman domain directly connected to the TMD in a similar fashion to other transport proteins such as MgtE, CorC, or the CLC family of chloride channels; (3) the ATP- and Mg2+-binding capacity of the Bateman module, and the conformational changes associated to the interaction with these ligands, formerly observed in other channels; (4) the presence of a C-terminal CNBH domain, whose removal impairs Mg2+ extrusion; (5) the structural role exerted by hypomagnesemia causing mutations, such as CNNM2 T568I, which impair Mg2+ extrusion at the DCT; and (6) the lack of identified interacting transporters that could potentially be regulated by CNNMS, thus positioning the latter as simple modulators of their activity. Evidence not supporting the activity of CNNMs as direct transporters includes (1) the scarce number of Mg2+ binding sites located in the regulatory Bateman module, (2) the moderate number of transmembrane α-helices in the DUF21 domain, and (3) the low oligomerization degree (a dimer) in comparison with other channels and transporters.This review summarized the current three-dimensional knowledge about the CNNM family of magnesium homeostatic factors. Their exact role remains unclear ,23,25,822+-binding mode. MgtE binds Mg2+ in an ATP-dependent manner, whereas CNNM2 only interacts with ATP at a certain (mM) Mg2+ concentration. These striking features make it tempting to speculate that these opposing affinities for the two ligands could be related to the inverse abilities of these homeostatic factors to transport magnesium ions in opposite directions across cell membranes, thus supporting the direct role of CNNMs in Mg2+ extrusion. The behavior of the MgtE channel and CNNM2 are opposite in terms of ATP- and MgThe recent structural advances have contributed to our early understanding of the mechanisms underlying the CNNMs function, as well as their interaction with different molecules. Answering the still significant number of unanswered questions will help our ability to modulate the activity of these relevant machineries and use them as promising targets to treat Mg-related disorders and several types of cancer.


Heat shock proteins (HSPs), a large group of highly evolutionary conserved proteins, are considered to be main elements of the cellular proteoprotection system. HSPs are encoded by genes activated during the exposure of cells to proteotoxic factors, as well as by genes that are expressed constitutively under physiological conditions. HSPs, having properties of molecular chaperones, are involved in controlling/modulation of multiple cellular and physiological processes. In the presented review, we summarize the current knowledge on HSPs in the biology of epidermis, the outer skin layer composed of stratified squamous epithelium. This tissue has a vital barrier function preventing from dehydratation due to passive diffusion of water out of the skin, and protecting from infection and other environmental insults. We focused on HSPB1 (HSP27), HSPA1 (HSP70), HSPA2, and HSPC (HSP90), because only these HSPs have been studied in the context of physiology and pathophysiology of the epidermis. The analysis of literature data shows that HSPB1 plays a role in the regulation of final steps of keratinization; HSPA1 is involved in the cytoprotection, HSPA2 contributes to the early steps of keratinocyte differentiation, while HSPC is essential in the re-epithelialization process. Since HSPs have diverse functions in various types of somatic tissues, in spite of multiple investigations, open questions still remain about detailed roles of a particular HSP isoform in the biology of epidermal keratinocytes. Heat shock proteins (HSPs) are principally referred to as molecular chaperones. Having diverse intracellular localization and functional differentiation, HSPs are involved in folding newly synthesized proteins, assembly of protein complexes, intracellular transport of proteins, or prevention of denaturation and/or unspecific aggregation of improperly folded proteins both under physiological and stress conditions. Through chaperoning multiple client proteins, HSPs participate in modulation of signal transduction including immunological responses, while by cooperation with the ubiquitination system, participate in directing irreversibly damaged proteins into proteasomal degradation Saibil . The resOn the basis of molecular weight and amino acid (aa) sequence similarity, HSPs are subdivided into several families, namely HSPB , HSPA (HSP70), DNAJ (HSP40), HSPC (HSP90), and HSPH (HSP110). The first name in the above list refers to the novel nomenclature system , which undergo a tightly controlled program of terminal differentiation. During this process, the undifferentiated keratinocytes from the basal layer (the innermost layer of the epidermis) permanently withdraw from the cell cycle, change their biological activity and morphology, and move suprabasally to form Human epidermis follows a tightly regulated proliferation and differentiation program with an interplay between stem cell self-renewal and differentiation in order to maintain tissue homeostasis Fig. . DespiteAdvances in cell tracing analysis caused the EPU model to be questioned, and currently, alternative ideas were proposed to explain the maintenance of epidermal homeostasis , HSPA2, HSPB1 (HSP27), and HSPC (HSP90) in keratinocyte biology. Of all HSPs, the above-mentioned proteins have been the most intensively investigated ones in the context of epidermal homeostasis. Here, we discuss their involvement in keratinocyte cytoprotection, epidermal differentiation, and wound healing. Some aspects concerning implications of HSPs in plastic surgery, dermatology, skin aging, and immunotherapy of skin cancer have been discussed in earlier reviews Morris .The HSPB family contains several members of which some are expressed ubiquitously, and others in a cell-type specific fashion Arrigo . Among sstratum corneum, while Crowe et al.  studies   protein, one of the members of numerous DNAJ (HSP40) family. It was found that TID-1s, the shortest splicing form of DNAJA3, when complexed with MK5 kinase, inhibited HSPB1 phosphorylation and negatively affected polymerization of F-actin in HeLa cells. Oppositely to HeLa cells, psoriatic keratinocytes were negative for TID-1s and contained increased levels of phosphorylated HSPB1. In HaCaT cells, increasing TID-1s levels interfered with TNF-α induced phosphorylation of HSPB1 and polymerization of F-actin, as well as inhibited TNF-stimulated cell migration. These results suggested that the loss of the TID-1s is one of factors responsible for the development of psoriasis via mechanism partially related to enhanced phosphorylation of HSPB1   in media maintain basal-like phenotype of keratinocytes, while higher Ca2+ levels (0.5–1.2 mM) activate differentiation process. Early studies showed that HSPB1 was barely detectable in NHEK grown in low Ca2+ media, whereas a switch to high Ca2+ concentration significantly raised the intracellular level of HSPB1 and increased subpopulation of HSPB1-positive keratinocytes (Kindas-Mugge and Trautinger 1994). Ca2+-mediated increase in HSPB1 expression was confirmed in studies performed on human HaCaT  signaling cascade represent one of pathways engaged in HSPB1 phosphorylation , an upstream activator of the MAP kinase pathways  which consists mostly (96–99%) of long wave ultraviolet , and to less extent (1–6%) of short wave ultraviolet . While UVA can reach dermis, UVB is almost completely absorbed by the epidermis, and constitutes a main environmental factor damaging keratinocyte DNA. UVC (100–290 nm), the third component of solar radiation, is entirely absorbed by the atmosphere; thus, no significant irradiation of the skin results from natural sources. Most harmful effect of phototoxicity is a development of skin cancer  —suppressing the process in keratinocytes while activating in DFs  in somatic cells under physiological conditions, but its levels can be highly elevated by HS and other environmental stresses as well as under various pathological conditions, particularly in tumors  with its receptor present on keratinocytes  of HSPA1 expression in keratinocytes was suggested to depend on differentiation stage, but still it is unknown whether the protein is causally involved in the differentiation process. HSPA1 level was found relatively low in NHEK (3rd-passage) and HaCaT cells grown in low CaMolecular mechanisms supporting constitutive expression of HSPA1 in normal keratinocytes are not known, and available data did not confirm the central role of HSF1 transcription factor. Although NHEK and HaCaT cells contain significant levels of HSF1  revealed different kinetics of various HSPs induction. For example, the expression of HSPA1 raised already at 4 h after HS, while the maximal levels of HSPB1 occurred 20 h later  generated by laser pulse. Most NHEK survived temperature 60 oC for as short a time as 1 s, or 55 oC for 30 s, notably the consecutive short heat pulses induced thermotolerance  severe HS , two potential photoprotector molecules (porfirin-334 and shinorine) isolated from red alga Alkanna tinctoria of the Boraginaceae family  in HSPA1 knockout, but not in wt mice. At 24 h after irradiation, the entire epidermis of HSPA1 null mice was necrotic. The authors suggest that the lack of HSPA1 had a major negative impact of the DNA repair process  and HSPA1 knock-out mice was similar, a massive dying of keratinocytes in the basal layer of epidermis was detected 12 and 24 h after the UVB irradiation -1,2-ethanediamin  leaving undamaged tissues between them , beam power, and parameters of the treatment modality used. Non-ablative treatment induces thermolysis of dermis as well as lower epidermal strata leaving stratum spinosum and stratum basale, and improved the process of wound healing  onto murine skin reduced skin inflammation in experimental psoriatic-like lesions induced by imiquimol , onto immunocompromised mice revealed that HSPA1 accumulates in keratinocytes in the close vicinity of immune cells, in particular dendritic cells expressing the LRP-1/CD91 receptor. It was thus proposed that eHSPA1 released from psoriatic keratinocytes, via interacting with LRP-1/CD91 receptor present on dendritic cells, can activate pro-inflammatory responses , an IL-7-like chemokine/cytokine produced by keratinocytes. The release of TSLP was found to be suppressed in HaCaT cells exposed to HS or in keratinocytes incubated with exogenous recombinant HSPA1. This inhibitory action of HSPA1 on TSLP secretion was mediated by inhibition of NF-κB activation. These observations suggested that exaggerated secretion of eHSPA1 from keratinocytes could be a potential component of AD therapy  to its rodent counterparts  of HSPA2 in murine and human skin can be distinct. Analysis of transgenic mice bearing GFP reporter gene under the control of the promoter of the rat HSPC1 (HSP90AA1), and HSPC3 (HSP90AB1) genes, respectively  and HSPC5 (TRAP-1) proteins group into the HSPC (HSP90) family. In mammals, HSPC1 and HSPC3 are two homologous isoforms sharing approx. 86% of aa sequence similarity; they are encoded by Compared to other organs, the endogenous expression of HSPC in the skin is rather low, and the protein level is lower in the epidermis than in the dermis  initiates the complex, multistep healing process that includes inflammation, tissue formation through re-epithelialization, angiogenesis and matrix protein production, and finally tissue remodeling. The closure of human wounds is largely mediated by re-epithelialization, a process accompanied by acquisition of highly active keratinocyte phenotype, characterized by continuous proliferation and high migratory properties Rittie . The pooThe role of eHSPC1 in stimulating keratinocytes migration was also demonstrated on murine  in HaCaT cells blocked calcium-induced differentiation. This observation suggests that HSPC can contribute to early stages of keratinocyte differentiation (Miyoshi et al. Epidermis and keratinocytes are highly valuable models for investigating the complexity in expression and function of various HSPs. The current knowledge of molecular and cellular mechanisms underlying the processes of epidermal stratification and re-epithelialization gives opportunity to expand our understanding of HSPs involvement in the maintenance and restoration of homeostasis in adult tissue. The spatial and functional stratification of keratinocytes in the epidermis also facilitates the study of cytoprotective mechanisms in the context of tissue predisposed to frequent contact with multiple environmental stressors. Technical improvements, in particular development of three-dimensional human skin equivalents for in vitro assays, in combination with possibility of genetic manipulations of cutaneous cells enable the investigation of a particular gene in tissue formation, epidermal cells differentiation, phenotypic reprogramming, and death of keratinocytes.In spite of above-mentioned achievements, knowledge of the importance of HSPs in epidermal differentiation, wound healing, development of keratinocytic carcinoma, and non-cancerous skin diseases, as well as in cytoprotection against harmful environmental factors, is still far from being satisfactory. One of highly relevant issues, which is still not sufficiently explained in the functional context, is stratum-specific expression of HSPB1 and HSPA2 chaperones, for which no convincing confirmation of their cytoprotective role in keratinocytes has been provided. Different spatial localization of these two proteins in the epidermis seems to reflect their distinct contribution to keratinocyte differentiation. HSPA2 was shown to prevent the basal keratinocytes from premature entry into terminal differentiation process, while HSPB1 plays a role during the final steps of keratinization and formation of epidermal barrier. There is no doubt that intensive work has to be done in order to fully determine function of these chaperone proteins in the biology of epidermis.In contrast, stress-inducible HSPA1 is nonselective for epidermal strata, shows no relationship with keratinocyte differentiation, but it can ensure quick and effective protection against various environmental stressors. Taking into account that HSPA1 can be secreted from keratinocytes, the further research on the impact of eHSPA1 in pathophysiology of inflammatory skin disorders deserves special attention.With regard to HSPC proteins, it seems particularly important to notice differential expression of HSPC1 and HSPC3 isoforms in the normal and pathological skin. Although there are some hints that HSPC can participate in early steps of keratinocyte differentiation, the most firmly accepted knowledge concerns the regulatory role of eHSPC1 in keratinocyte and DFs migration during wound healing.In summary, the studies performed so far indicate that HSPB1, HSPA1, HSPA2, and HSPC in keratinocytes contribute both to the maintenance of epidermal homeostasis as well as to skin pathophysiology, although the molecular mechanisms behind these functions are poorly understood. Thus, it seems justified to intensify studies aimed at determining how changes in HSPs expression, alterations in their posttranslational modifications, or secretion rate would influence the skin homeostasis and impact on etiopathogenesis of cutaneous diseases. Such knowledge would be highly valuable for better understanding and treatment of skin diseases and wound healing.


Feeding schedules entrain circadian clocks in multiple brain regions and most peripheral organs and tissues, thereby synchronizing daily rhythms of foraging behavior and physiology with times of day when food is most likely to be found. Entrainment of peripheral clocks to mealtime is accomplished by multiple feeding-related signals, including absorbed nutrients and metabolic hormones, acting in parallel or in series in a tissue-specific fashion. Less is known about the signals that synchronize circadian clocks in the brain with feeding time, some of which are presumed to generate the circadian rhythms of food-anticipatory activity that emerge when food is restricted to a fixed daily mealtime. In this commentary, I consider the possibility that food-anticipatory activity rhythms are driven or entrained by circulating ghrelin, ketone bodies or insulin. While evidence supports the potential of these signals to participate in the induction or amount of food-anticipatory behavior, it falls short of establishing either a necessary or sufficient role or accounting for circadian properties of anticipatory rhythms. The availability of multiple, circulating signals by which circadian oscillators in many brain regions might entrain to mealtime has supported a view that food-anticipatory rhythms of behavior are mediated by a broadly distributed system of clocks. The evidence, however, does not rule out the possibility that multiple peripheral and central food-entrained oscillators and feeding-related signals converge on circadian oscillators in a defined location which ultimately set the phase and gate the expression of anticipatory activity rhythms. A candidate location is the dorsal striatum, a core component of the neural system which mediates reward, motivation and action and which contains circadian oscillators entrainable by food and dopaminergic drugs. Systemic metabolic signals, such as ghrelin, ketones and insulin, may participate in circadian food anticipation to the extent that they modulate dopamine afferents to circadian clocks in this area. In this article, I describe how regularly scheduled meals induce daily rhythms of food-anticipatory behavior by entrainment of circadian clocks. I then consider evidence that these clocks may be located in peripheral organs, such as the stomach, liver or pancreas, or may be entrained by metabolic hormones (ghrelin and insulin) or fuels (ketone bodies) released by these tissues under control of circadian clock genes or in direct response to daily cycles of feeding and fasting. A primary objective is to evaluate how well the evidence accounts for established circadian properties of food-anticipatory rhythms. In reviewing relevant studies, I focus on those experiments that have behavioral endpoints, as these must stand on their own merits, independent of companion experiments that have cellular or molecular endpoints. I conclude with a reminder that animals can anticipate daily rewards other than food, including water, salt, sex and psychomotor stimulants. Evidence for food- and dopamine (DA)-entrainable circadian clocks in the brain’s reward system suggests a common mechanism for anticipation of any salient reward stimulus that recurs at predictable times of day.1. In mammals, the clock genesBmal1 andClock encode proteins that form dimers that bind to enhancer boxes and drive expression of other clock genes, includingPeriod ,Cryptochrome (Cry1 andCry2),Reverbα andRor genes. PER and CRY proteins accumulate in the cytoplasm, form dimers, translocate, interfere with BMAL1-CLOCK binding and thereby terminate their own expression.Bmal1 expression is activated by RORs and repressed by REV-ERBs. Post-translational modifications target PER and CRY proteins for removal, permitting BMAL1-CLOCK to initiate the next cycle of transcription. Post-translational modifications also regulate the period of the cycle2. Clock proteins act as transcription factors for other clock-controlled output genes and thereby propagate circadian cycles through the genome and the functions of the cell4. Cellular inputs that acutely alter clock gene expression or proteins can shift clock phase and enable entrainment to periodic environmental stimuli (Zeitgebers), such as the light–dark (LD) cycle5. The specific clock genes differ across life kingdoms, but the autoregulatory TTFL is a common mechanism. The discovery of clock genes and the TTFL in the fruit fly was celebrated by a Nobel Prize in 20177.Circadian (~24-hour) rhythms and the clocks that time them are a biosignature of life on earth, an adaptation that enables most organisms to anticipate the regular alternation between day and night and to segregate cellular, systems and behavioral functions in accordance with a diurnal or nocturnal temporal niche. The mechanism of the circadian clock is intracellular and employs interlocking transcription-translation feedback loops (TTFLs) through which the expression of clock genes is activated or repressed by clock proteins8. Clock genes also exhibit circadian expression in other brain regions and in most organs and cell tissues outside of the brain. Bioluminescent gene reporter assays show that clock gene cycling in the SCN and in most non-neural cells and tissues can persist for many cycles, if not indefinitely,in vitro14. This has also been established for some other brain regions. Mammalian circadian rhythms therefore reflect the operation of a distributed, multioscillatory circadian system, in which a master clock in the SCN is entrained by LD cycles and drives daily rhythms either directly or by coordinating circadian clocks that control local functions in other brain regions and organs.In mammals, clock genes exhibit robust circadian cycling in the suprachiasmatic nucleus (SCN), a retinorecipient cell cluster in the basomedial hypothalamus that is essential for entrainment to LD cycles and for circadian organization  in constant environmental conditions15. Cell groups in these target areas control autonomic and endocrine functions and link the SCN pacemaker to physiology and behavior16. Circadian cycling is further propagated and reinforced by the consequences of behavior. The daily rhythm of feeding and fasting appears to be of special significance17. Circadian clocks in most peripheral organs can be shifted by one or more stimuli associated with feeding rhythms or food intake, including glucose18, cell metabolic sensors  and metabolic and gut hormones . Some peripheral clock inputs may be universal, and others tissue-specific.SCN outputs have a relatively restricted, predominantly hypothalamic, projection field31. The circadian physiological program is therefore flexible to the extent that it can accommodate even a reversed feeding cycle without forcing the SCN pacemaker out of phase with the LD cycle, thereby potentially preserving SCN-based encoding of daylength32.The distributed design of this system is clearly adaptive; retinal inputs entrain the SCN pacemaker to local time, the SCN determines when animals will eat (assuming food availability is unrestricted), and food intake controls peripheral clocks, ensuring that the organism is physiologically prepared to ingest, digest, absorb, distribute and store nutrients during its waking hours. An interesting feature of this design is that if food availability is restricted to a particular time of day or night, peripheral clocks shift their phase to preserve alignment with mealtime, whereas the SCN pacemaker remains synchronized to LDFor circadian reprogramming of physiology to be useful, mealtime must also control behavior, so that animals are awake and motivated to seek and ingest food when it is most likely to be found. If the SCN pacemaker controls the sleep–wake cycle but does not appreciably change its phase in LD despite inversion of the feeding cycle, then it should oppose foraging activity. There are two possible solutions: either SCN outputs representing circadian phase can be used as discriminative cues that acquire incentive properties when regularly paired with food (an interoceptive “bell” for Pavlov’s hungry dog) or there is another timing device that regulates foraging activity and supersedes SCN output when the two devices send conflicting messages.33. He called this food anticipation. The activity rhythm persisted  for a few days of total food deprivation, suggesting that anticipation was timed in a “clock-like” fashion and was not merely a reflection of accumulating hunger since the last feeding. Decades later, biopsychologist Robert C. Bolles and colleagues demonstrated that the anticipation rhythm also emerges in rats entrained to LD and fed in the middle of either the day or the night. The authors confirmed that the rhythm persists during total food deprivation36, and most importantly, showed that the anticipation rhythm does not emerge if the feeding schedule is very different from 24 hours 36. The mechanism was inherently “bound up with the 24-hour cycle”. Two groups39 then showed that the mechanism did not require the SCN pacemaker; rats made arrhythmic by SCN ablation, and maintained in constant dark or light, exhibited robust daily rhythms of food-anticipatory activity (FAA) if fed once daily. Analysis of the formal properties of the rhythm were consistent with control by a circadian clock entrained by food, a so-called “food-entrainable oscillator” (FEO); the rhythm had circadian “limits to entrainment”, shifted gradually rather than immediately following meal shifts, and persisted for as many 24-hour cycles of food deprivation as could be safely tested44. Bolles and Moot had also shown that rats could anticipate two daily meals separated by 6 hours34; Stephan confirmed this in SCN-ablated rats and provided suggestive evidence for concurrent anticipation of two daily meals: one recurring every 24 hours and the other every 25 hours45. This is not unlike activity rhythms driven by the SCN pacemaker, which can also split into two oscillating components that, under some conditions, can free-run separately and may normally couple to sunset and sunrise (communicating daylength by their phase angle difference)47.Long before the word “circadian” was coined and the concept of the circadian clock was firmly established, the biopsychologist Curt P. Richter showed that rats maintained in constant light for many days and fed for only 25 minutes at a fixed time each day exhibited a daily rhythm of locomotor activity that rose to a peak at the scheduled mealtime51. Similar phenotypes may follow lesions or KOs targeting brainstem and forebrain areas54.These results have been widely interpreted as evidence that mammalian behavior is regulated by two circadian clocks—one (the SCN) specialized for entrainment to LD cycles and the other (somewhere else) for entrainment to daily feeding cycles—and that both clocks are composed of multiple clock cells that can be configured into at least two stable, entrainable groups. Given that the SCN is in the hypothalamus and receives light information directly from the retina, a reasonable  working hypothesis is that FEOs for behavior would also be hypothalamic, but located within those cell groups that respond to peripheral metabolic signals and regulate feeding and metabolism. Some hypothalamic lesions and gene knockouts (KOs), targeting defined brain regions, cell types, or receptors in the hypothalamus, are associated with an altered food anticipation phenotype, most often characterized by a lower-level or slower emergence of anticipatory activity or, in some cases, by enhanced anticipation57. In his first report on food-anticipatory rhythms, Stephan37 cautioned that “if many oscillators exist which are entrainable by food restriction schedules, it may not be possible to abolish anticipatory activity by selective removal, or interference with, specific organ systems”. That may prove to be true. But the absence of evidence for a defined area analogous to the SCN is not yet sufficiently exhaustive to constitute evidence of absence.Interpreting these phenotypes is challenging. The site of action could be upstream or downstream from the clock mechanism, as many factors  determine the level of activity, independent of circadian timing. A fair summary is that there is no agreement on a single locus in the brain where FEOs necessary and sufficient for food-anticipatory rhythms reside. A default position is that many loci are capable of driving FAAorgan rather thanbrain, Stephan intended to include peripheral, non-neural tissues as potential mediators of behavioral rhythms. In the very first report of food anticipation, Richter33 included data on gastric contractions and proposed that “anticipation may be explained by the clock-like functioning of the (stomach)”. In that tradition, Stephan and colleagues targeted the duodenum, autonomic nerves and first-order brainstem nuclei as potential sites of FEOs driving behavior. These efforts were instructive and necessary but ultimately not successful48. The subsequent discovery that bona fide circadian FEOs (cells that express clock gene rhythms that are entrained by daily feeding schedules) are found throughout the body17, combined with a lack of success in defining critical neural loci, has renewed interest in peripheral organs and their outputs as sources of timing information for food-anticipatory behavior.By using the word58. Gastric ghrelin secretion is suppressed by food intake and stimulated by fasting and, in its acylated form, binds to the growth hormone secretagogue receptor 1a on cells located in the hypothalamus, substantia nigra, and other brain sites59. It is weakly orexigenic following systemic injection60. In mice, oxyntic cells express circadian cycles of clock gene expression synchronized to mealtime61; ghrelin-secreting cells are therefore FEOs. If these cells, via acyl-ghrelin, provide time signals critical for food anticipation, then ghrelin ligand or receptor KO should eliminate food anticipation. Several groups have conducted this experiment, and the results range from a reduction in the duration of food anticipation61, to a reduced amount of anticipatory activity with no change in duration62, to no significant effect64. The varying results across studies have not been reconciled but could relate to differences in behavioral measures  or other procedural variables. Collectively, the results indicate that ghrelin derived from gastric FEOs is not required for FAA.Richter would no doubt have been excited by the discovery of ghrelin, a hormone derived from preproghrelin secreted by gastric oxyntic cellsad libitum food access. Once established, FAA rises and falls with expected mealtime, across multiple days of fasting42. This is a critical, defining attribute of a clock-controlled process. Plasma acyl-ghrelin does not; it rises prior to the next mealtime and then gradually decreases without further cycling66. When food is providedad libitum, rats and mice previously fed in the middle of the light period revert to nocturnal feeding, and daytime FAA rapidly dissipates. Within a few days, the gastric clock has also reset to a nocturnal alignment67. If the animal is then food-deprived, activity reappears at the original scheduled mealtime. This occurs in intact rats in LD or dark–dark (DD) and in SCN-ablated rats, indicating that it is not a “memory” of SCN phase. During food deprivation, gastric clock gene rhythms (expressed by oxyntic ghrelin-secreting cells) remain nocturnally phased67. Finally, rats can anticipate two daily meals; if one is in the light period and the other at night, peripheral clocks assume an intermediate phase or maintain a nocturnal alignment, depending on the relative meal sizes, intermeal interval, and timing within the day69.A role for ghrelin in initiating food-anticipatory rhythms is also challenged by dissociations between acyl-ghrelin or the gastric clock and FAA during food deprivation and followingThese dissociations make clear that ghrelin secreted by gastric FEOs does not directly initiate FAA, but the results do not rule out a role for ghrelin as a participant in phase control of FEOs for behavior which are located elsewhere in the body or brain. This could be explored by measuring the timing of food anticipation on days after a bolus of ghrelin is administered systemically, a number of hours before or after the usual feeding time. If ghrelin can shift FEOs driving FAA, then at one or more phases of these FEOs, an artificial spike of ghrelin should induce a behavioral shift, indicated by a change in the onset of FAA the next day. Injections repeated at the same time each day might induce anticipation, especially if the animal is fasted during those days. Note that experiments that require more than one day of fasting can be carried out only by using adult rats, which, unlike mice, can be food-deprived safely for 3 to 4 days.3. The timing of these rhythms is controlled by circadian clock genes, which are food-entrainable, and also by direct effects of feeding and fasting cycles independent of clock genes25. Hepatocytes are therefore FEOs, and signals emitted by the liver could propagate food entrainment to other tissues, including the brain. Chavan and colleagues70 reported that global or liver-specificPer2 KO, but not a neuron-specificPer2 KO, eliminated daily rhythms of food anticipation in mice fed for 8 hours per day, beginning 4 hours into the daily 12-hour light period. Food anticipation was rescued in liver-specificPer2 KO mice, but not in globalPer2 KO mice, by virus-mediated constitutive overexpression ofPer2 in the liver.Per2 KO reduced expression of genes encoding enzymes important for synthesis of ketone bodies by liver cells, and plasma levels of the ketone body β-hydroxybutyrate (βOHB) were reduced during restricted feeding. Ketones provide energy for neurons during fasting, and a daily rhythm of ketone production induced by a feeding schedule could elicit food anticipation if ketone levels regulate neural output71 or clock gene cycling72. Chavan and colleagues70 confirmed that plasma βOHB increases prior to mealtime in day-fed mice and tested a role in food anticipation by using subcutaneous minipumps programmed to release βOHB prior to mealtime in liver-specific and globalPer2 KO mice. This treatment partially rescued FAA. The results were interpreted as evidence that FAA requiresPer2 expression in the liver  and in one other location  and that βOHB provides timing signals to brain circuits that drive FAA.A large portion of the transcriptome in liver cells exhibits 24 hour rhythmicity73. In the study by Chavan and colleagues, an 8-hour meal duration was used. LeSauter and colleagues74 recently showed that mice overexpressing dopamine 2 receptors (D2Rs) in the striatum fail to anticipate mealtime if food is provided for 8 hours but show robust levels of anticipation, comparable to those of wild-type mice, if the meal duration is 4 or 6 hours. Wild-type mice also showed weak anticipation to the 8-hour duration. Lack of anticipation to the 8-hour meal in D2R-overexpressing mice was attributed to a motivational rather than a clock deficit, which is consistent with other evidence for motivational deficits in these mice. This serves as a cautionary tale that if food anticipation is weak or absent at long meal durations, then the feeding window needs to be narrowed to fully interpret the results.Although the combined demonstration of loss and rescue of function makes a compelling narrative, the loss of function requires further study. To induce robust food anticipation, meal durations are conventionally set in the 4- to 6-hour range, or a limited amount of food is provided, to reduce body weight by 10 to 20%75. Therefore, circulating ketones cannot explain the timing of FAA during fasting. Nonetheless, ketones, like ghrelin, could play a role in setting the phase of FEOs in the brain. If so, acute manipulations of ketone levels, at some phases of the food anticipation rhythm, would be expected to shift its timing. Ketone pulses should also be able to shift clock gene cycles in at least some tissuesin vitro. Chavan and colleagues noted that βOHB did not inducePer2 expression in the liver, but whether it affects clock gene cycles in other tissues, including neurons, remains to be determined.The dissociations noted between FAA and circulating acyl-ghrelin levels also apply to liver ketones. During extended fasting (in rats), FAA persists with a 24-hour periodicity while plasma ketone levels rise and by the second day remain high70 ,Per2 KO alone or in combination with KO ofPer1 andPer3 did not eliminate food anticipation rhythms78. This could be attributable to differences in the mouse lines, or in some methodological details, despite what appear to be similar procedures . It would be useful to see mouse lines exchanged across labs and tested using lab-specific procedures.An unresolved issue is that, in many studies, contrary to the results reported by Chavan and others79. The pancreas thus contains FEOs and emits a hormone that reports mealtime and that has near universal access to cells in the brain and body. A recent study proposed that insulin and insulin-like growth factor-1 (IGF-1) may be universal Zeitgebers for FEOs27. If so, then the pancreas may participate in control of FEOs responsible for food-anticipatory behavioral rhythms27. The study confirmed earlier work that insulin can shift circadian clocksin vitro and extended this to anin vivo model.In vitro, high doses of insulin rapidly increased PER2 levels and shifted the rhythm of PER2::LUC expression in fibroblasts, dissociated cortical neurons, and explants of liver and kidney. In PER2::LUC mice, systemic injections of insulin and glucose induced and shifted the whole organism bioluminescence rhythm. The acute effect of insulin on PER2 levels was mediated by activation of target of rapamycin (TOR), increased phosphoinositide signaling, and microRNA downregulation27.Insulin is secreted by the pancreas in response to glucose ingestion and absorption. Clock gene rhythms in the pancreas entrain to daily mealsin vitro, insulin did not shift the SCN clock but did induce PER2 in fibroblasts lackingCry1/Cry2 orBmal1. These observations are consistent with the lack of shifting of SCN clock gene rhythms in food-restricted mice entrained to LD29 and with the failure ofCry1/Cry2 orBmal1 KO to eliminate food anticipation rhythms despite eliminating circadian rhythms when food is availablead libitum83. A number of studies have documented the resilience of circadian food anticipation to loss of clock genes in various combinations78. Crosby and colleagues27 propose that feeding cycles, via insulin, can directly drive PER2 cycling in clock gene KO mice. This potentially explains the continued appearance of FAA in clock gene KO mice.Two additional findings of note were that,Bmal1 have an expanded (unlimited?) range of entrainment82 . FAA in mice lacking all threePer genes appears to have a shortened period of oscillation and entrains best to feeding cycles normally outside of the range of entrainment 84. FAA may be weak or absent inRev-erbα KO mice85. Conceivably, FEOs in mice lackingPer genes may cycle because of effects of feeding-related signals on remaining clock genes, includingBmal1 andRev-erbα.Although the persistence of food-anticipatory rhythms in clock gene KO mice may suggest an entirely novel clock mechanism, changes in circadian properties of FAA in these models do indicate a role for canonical clock genes. For example, FAA in mice lackingin vivo”27. To test this, mice entrained to LD with food availablead libitum were released into constant light, and food was restricted to the first 8 hours of what was the previous light period, for 8 days, after which food was again providedad libitum. In constant light, LD-entrained activity rhythms free-run with a periodicity usually greater than 24 hours, as shown by thead libitum–fed control group 27. Food-restricted mice receiving BMS-754807 in drinking water or drinking water alone (vehicle group) also showed a long-period (phase-delaying) activity rhythm. Notably, both vehicle and BMS-754807–treated mice showed substantial activity immediately preceding the mealtime. One interpretation of these data is that insulin does not control the phase of FEOs that drive FAA, but it is also possible that premeal activity was part of the free-running activity rhythm and not the output of FEOs. To examine this further, the chronic BMS-754807 experiment could be repeated on mice (or rats) entrained to LD with food limited to the middle of the light period, when there can be a clear separation between food-anticipatory and nocturnal activity. If BMS-754807–treated mice exhibit a delay in the emergence of FAA or a difference in FAA duration (timing), that would suggest a role for insulin in control of the FEOs that drive behavioral anticipation. It would also be informative to determine whether the rhythm of FAA, once established, can be acutely shifted by a bolus injection of insulin and glucose outside of the usual mealtime.Crosby and colleagues establish a case for insulin signaling as a potential universal resetting stimulus for FEOs. It is therefore reasonable to infer that this would include FEOs that generate food-anticipatory behavioral rhythms. Genetic ablation of insulin and IGF-1 signaling induces profound metabolic deficits, but pharmacological block is possible using the compound BMS-754807. The authors reasoned that “If insulin and IGF-1 signaling communicates feeding time to cellular circadian clocks throughout the brain and body, then chronic application of BMS-754807 via drinking water should impair entrainment of both molecular and behavioral circadian rhythms to feed-fast cyclesad libitum. The studies highlighted here have identified specific metabolic signals  that may participate in the induction of food-anticipatory behavioral rhythms. The available evidence falls short of assigning a necessary or sufficient role for these signals on either the input side or the output side of the food anticipation timing system. It is possible that phase control of this system is multiply determined. Indeed, additional nutrient signaling pathways by which daily cycles of feeding and fasting can regulate clock gene expression have been identified in various cell types22. Synchronization of circadian clocks by food thus appears to involve multiple, tissue-specific or universal input signals acting in parallel or in series to align circadian physiology with mealtimes. Which and how many of these participate in control of behavioral rhythms remain open questions.Mealtiming exerts a profound influence on circadian clocks throughout the body and brain, some of which control behavior, ensuring that animals can exploit temporal regularities in food availability even when these conflict with a “preferred” chronotype defined when food is available87. There certainly are many FEOs, in both the brain and the body, but that does not rule out the possibility that there is a “final common” circadian clock that integrates multiple time cues and ultimately sets the phase and period of anticipatory behavioral rhythms. There is ample evidence that animals can anticipate restricted access to other appetitive stimuli, including highly palatable snacks89, water91, salt92, reproductively receptive mates93, and addictive, psychomotor stimulant drugs96. By contrast, scheduled arousal by sleep deprivation, time-restricted running-wheel access, forced running or swimming does not induce anticipatory activity98. Anticipation of water, sex and drugs has been experimentally separated from effects of these schedules on food intake, ruling out spurious food entrainment95. These appetitive stimuli share with food ingestion the ability to strongly activate DA signaling in neural circuits that mediate reward processing, reinforcement learning and habit formation99. Circadian clock genes oscillate in the major components of this system, including the dorsal and ventral striatum, and the timing of these oscillations is shifted by feeding schedules102. In the dorsal striatum, clock gene rhythms are also regulated by DA receptor signaling101. DA-deficient mice do not exhibit appetitive behavior104, but restoring DA signaling selectively in the dorsal striatum is sufficient to rescue these behaviors106, including circadian FAA54. FAA is diminished by DA antagonists108 and by DA receptor 1 KO54 and can be shifted by a DA receptor 2 agonist109. Gastric acyl-ghrelin, pancreatic insulin and hepatic ketone bodies modulate neural activity in midbrain DA neurons and thus converge on DA-receptive circadian oscillators distributed diffusively in striatal reward circuits110. Such oscillators could mediate anticipation of other rewards and, depending on how they are coupled, potentially enable anticipation of more than one daily mealtime or other reward .The availability of widely broadcast systemic time cues for food entrainment has been taken to suggest that food-anticipatory behavioral rhythms are driven by FEOs distributed across multiple brain circuits that collectively regulate appetitive behaviorBmal1,Per1/Per2 andRev-Erbα) by KOs specific to dopaminergic or DA-receptive cell populations in the striatum.The concept of a “master”, multioscillatory, DA-responsive circadian clock system for reward anticipation is consistent with the available evidence and makes testable predictions. If DA signaling sets the phase of oscillators that control FAA timing, then it should be possible to shift FAA onset by timed activation of midbrain DA neurons or DA-receptive neurons using chemogenetic or optogenetic tools. It should also be possible to reproduce FAA rhythm phenotypes  associated with global clock gene KOs  and striatal DA levels114. This has been proposed as an animal model of rapid cycling bipolar disorder114. Circadian activity rhythms induced by chronic methamphetamine share several properties with food-anticipatory circadian rhythms, including persistence in rats or mice following SCN ablation or clock gene KOs117. Ultradian rhythms in at least one species, the vole, share with food-anticipatory rhythms in rats the property of being resistant to rhythm period lengthening by chronic consumption of D2O in place of H2O119. These converging narratives invite speculation that a common pool of DA-regulated oscillators may underlie behavioral rhythmicity in the ultradian and circadian range and behavioral disorders associated with hyperdopaminergic states.The utility of a circadian system for adjusting rest–activity cycles to times of day most favorable for resource acquisition is self-evident, but it is not unreasonable to speculate that clock entrainment could support addictive behaviors. If episodes of drug, alcohol or junk food consumption are concentrated at a particular time of day , circadian entrainment processes that serve to reinforce successful daily patterns of reward acquisition could contribute to the emergence, maintenance and relapse of compulsive eating, drinking or drug taking


Chronic kidney disease (CKD) is a major public health problem worldwide and affects both elderly and young subjects. Its main consequences include the loss of renal function, leading to end-stage renal disease, an increased risk of cardiovascular disease, a significant increase in morbidity and mortality, and a decrease in health-related quality of life. This review arose in significant part from work in the authors’ laboratory, complemented by literature data, and was based on a translational approach: we studied the role of many CKD risk factors, such as hypertension, obesity, and oxidative stress/inflammation. The aim was to identify new molecular mechanisms of kidney damage to prevent it through successful behavior modifications. For this purpose, in our studies, both human and animal models were used. In the animal models, we analyzed the mechanisms of renal damage induced by hypertension (spontaneously hypertensive rats) and obesity (cafeteria diet-fed rats), showing that redox disequilibrium in plasma and tissue is extremely important in renal alteration in terms of both oxidative damage  and apoptotic pathway (intrinsic/extrinsic) activation. In hemodialysis patients, we explored the correlation between the global oxidative balance and both inflammatory markers and cardiovascular risk, showing a strong correlation between the oxidative index and the blood levels of C-reactive protein and previous cardiovascular events. This multilevel approach allowed us to individually and synergistically analyze some aspects of the complex pathogenic mechanisms of CKD in order to clarify the role of the new amplified risk factors for CKD and to prepare an effective personalized prevention plan by acting on both modifiable and nonmodifiable risk factors. Oxidative stress, a disturbance in the complex pro-/antioxidant balance, is widely recognized as a critical component of the pathogenesis and progression of chronic kidney disease (CKD) ,2. Due t2−), which is formed by the addition of one electron leak from the electron transport system during cellular respiration to the molecular oxygen. Most of the superoxide dismutates into hydrogen peroxide (H2O2) through superoxide dismutase (SOD) [2O2 is a neutral molecule because it has no unpaired electrons, but it is able to form the most reactive and dangerous radical, the hydroxyl radical (•OH), through a Fenton or Haber–Weiss reaction. Hydroxyl radicals mainly react with phospholipids in cell membranes and proteins. In activated neutrophils, in the presence of chloride and myeloperoxidase, H2O2 can be converted into hypochlorous acid that can react with DNA and produce pyrimidine oxidation products and add chloride to DNA bases [2−), forms the potent oxidant peroxynitrite (ONOO−). This compound can cause oxidative damage, nitration, and the S-nitrosylation of biomolecules, including proteins, lipids, and DNA [− has been implicated in DNA single-strand breakage, followed by poly-ADP-ribose polymerase (PARP) activation [Oxidative stress occurs when there is an imbalance between the production of free radical species and the antioxidant ability to neutralize their harmful effects . Free rase (SOD) . H2O2 isNA bases . AnotherNA bases . All iso and DNA . Nitrosativation . Exogenotivation . Endogentivation . Among ttivation .(1)Lipid peroxidation and loss of membrane fluidity: double bonds in polyunsaturated membrane lipids are vulnerable to attacks by oxygen-free radicals;(2)Protein cross-linking: free radicals promote sulfhydryl-mediated protein crosslinking, resulting in increased degradation or loss of activity;(3)DNA fragmentation;(4)Oxidative damage to carbohydrates impairs the functions of some cellular receptors, including those associated with hormonal and neurotransmitter responses.Free radicals can damage cells through several mechanisms:The overproduction of oxygen-derived free radicals has been implicated in the pathogenesis of over 200 clinical conditions . Tissue 2) or mild reduced kidney function (GFR between 60 and 89 mL/min/1.73 m2), while stages 3–5 have a severe reduction of kidney function (GFR 15–29 mL/min/1.73 m2), which leads to end-stage renal disease (ESRD).CKD is recognized as a global health problem with a high rate of morbidity and mortality and elevated healthcare costs . CKD affThe initiating causes of CKD are highly variable, since epidemiological studies have revealed that with CKD, unmodifiable and modifiable risk factors among patients can be defined . The firOver the last few years, several experimental studies have proven that oxidative imbalance is a common feature in renal diseases, representing a key process in the development and complications of CKD ,31,32. OIn particular, cardiovascular diseases are an important complication for CKD patients and represent the main causes of morbidity and mortality in these sick subjects ,41. RenaIn our laboratory, we focused on human studies regarding an evaluation of the overall redox/inflammatory state in a significant hemodialysis population, including subjects both with and without previous cardiovascular events . SeveralIn animal models, our research focused on the correlation between oxidative imbalance and kidney damage by using experimental models of hypertension and obesity . Recent CKD is a global health burden with a high economic cost to health systems, and its prevalence is expected to increase significantly in the coming years. It is necessary to carry out intervention strategies that are deliverable at scale to delay the onset/progression of CKD, and it is thus essential to have a deep understanding of all pathogenetic mechanisms. In obtaining this goal, basic and applied research and the interaction between them should be of equal importance The resulting synergy can help us to realize an effective personalized prevention plan by acting on both modifiable and nonmodifiable CKD risk factors.


XIST, NEAT1, NEAT2 (MALAT1), and GAS5. Moreover, we noted a high expression of FUS, TAF15, and EWS components of the paraspeckles, proteins that belong to the FET (previously TET) family of RNA-binding proteins and are implicated in central cellular processes such as regulation of gene expression, maintenance of genomic integrity, and mRNA/microRNA processing. We also interrogated the intra-ovarian localization of the FUS, TAF15, and EWS proteins using immunofluorescence. The presence and the dynamics of genes that encode lncRNA and paraspeckle proteins may suggest that these may mediate functions in the cyclic recruitment and differentiation of human follicles and could participate in biological processes known to be associated with lncRNAs and paraspeckle proteins, such as gene expression control, scaffold formation and epigenetic control through human follicle development. This comprehensive transcriptome analysis of lncRNAs and genes encoding paraspeckle proteins expressed in human follicles could potentially provide biomarkers of oocyte quality for the development of non-invasive tests to identify embryos with high developmental potential.Emerging evidence indicated that many long non-coding (lnc)RNAs function in multiple biological processes and dysregulation of their expression can cause diseases. Most regulatory lncRNAs interact with biological macromolecules such as DNA, RNA, and protein. LncRNAs regulate gene expression through epigenetic modification, transcription, and posttranscription, through DNA methylation, histone modification, and chromatin remodeling. Interestingly, differential lncRNA expression profiles in human oocytes and cumulus cells was recently assessed, however, lncRNAs in human follicle development has not previously been described. In this study, transcriptome dynamics in human primordial, primary and small antral follicles were interrogated and revealed information of lncRNA genes. It is known that some lncRNAs form a complex with paraspeckle proteins and therefore, we extended our transcriptional analysis to include genes encoding paraspeckle proteins. Primordial, primary follicles and small antral follicles was isolated using laser capture micro-dissection from ovarian tissue donated by three women having ovarian tissue cryopreserved before chemotherapy. After RN sequencing, a bioinformatic class comparison was performed and primordial, primary and small antral follicles were found to express several lncRNA and genes encoding paraspeckle proteins. Of particular interest, we detected the lncRNAs  NEAT1 (nuclear-enriched abundant transcript 1), which assembles various protein components, including RNA-binding proteins of the DBHS (Drosophila behavior and human splicing) family. Paraspeckles have been proposed to control of several biological processes, such as stress responses, gene expression, and cellular differentiation. Human follicle development represents a continuous cyclic process throughout the reproductive lifespan of a woman and encompasses both cell growth and differentiation. Paraspeckles are among the most recently identified nuclear bodies and were first described in 2002  is transcribed downstream of the NEAT1 gene and is found specifically associated with splicing speckles  mice fail to become pregnant despite normal ovulation, which was found to be a caused by corpus luteum dysfunction and concomitant low progesterone . Integrity of libraries was verified on library yield via KAPA qPCR measurement, and Agilent Bioanalyzer 2100 peak size with a RNA 6000 Nano Lab Chip  during different library preparation stages. Sequencing was performed on an Illumina HiSeq2000 platform  with 5 random samples per lane (AROS Applied Biotechnology).RNA was extracted from the LCM-derived samples, converted to cDNA and subjected to linear amplification [Ovation® RNA-Seq System V2 kit ]. RNA-seq libraries , and Cufflinks (2.0.2) BAM files were generated to create a list of expressed transcripts in the samples. BWA (0.6.2) was subsequently used to map all readings to the human reference genome (hg19) using the transcript list as a filter so only readings mapping to RefSeq exons . On the basis of log2 (CPM), fragments per kilobase of exon per million fragments mapped (FPKM) values were calculated, and further filtered using custom analysis in R . In silico merging of transcriptomes from three patients was performed to account for biological variance. The transcripts in the CSCEG list were ranked based on p-value, with low p-value indicating a high degree of consistency in FPKM gene expression level between patients for the given isolate type. Full lists of ncRNAs detected in human follicle development is availiable (Supplementary Table Cell Specific Consistently Expressed Genes (CSCEG) were defined as one-sample Furthermore, we generated a list of all known paraspeckle proteins based on annotated protein-coding RNAs detected Table  and non-Human ovarian cortical tissue was cut in 5 μm sections and mounted on glass slides. Dehydration and antigen retrieval was performed as described elsewhere  from the primordial and primary stage, respectively, as well as oocytes, mural granulosa cells, and theca cells from small antral follicles were collected via Laser Capture Microdissection (LCM). Each stage was isolated on the basis of stringent morphological criteria Gougeon,  Figure . PrimordEWS, HNRNPK, ZC3H6, UBAP2L, and TARDBP .The expression of 39 genes encoding paraspeckle proteins  . Although the estimated number of different types of human lncRNAs has ranged from 5,400 to 53,000 , which was likewise observed for TSIX  was detected throughout all the included stages (Resource Data), indicating the need for this paraspeckle-forming protein during human follicle development. Interestingly, several lncRNAs with no biological functions annotated were noted (Resource Data). While the expression of several lncRNA genes  was present in all samples tested, others appeared to be cell- and stage-specifically  expressed (Resource Data).ZFAS1 appears to be restricted to somatic cells, e.g., the mural granuloma cell layer and the theca cell layer in the small antral follicle (Resource Data). Interestingly, the ROR1-AS1 seems to be specific to the oocyte from small antral follicles (Resource Data). We found lncRNA Growth Arrest Specific 5 (GAS5) expressed in oocytes from primordial follicles, as well as a high expression in primordial follicles, somewhat lower in primary follicles, and in turn high in the mural granulosa cell layer and the theca cell layer from small antral follicles (Resource Data).The lncRNA A heatmap of FPKM data for selected lncRNA genes was generated to show the expression for the two different cell-stages  in isolates - and the correlation between cell-specific (oocyte versus granulosa cell) isolates Figure .Extensive efforts to gain deeper understanding of RNA biology have yielded evidence of the diverse structural and regulatory roles in protecting chromosome integrity, maintaining genomic architecture, X chromosome inactivation, imprinting, transcription, translation and epigenetic regulation  and cumulus cells .In a few well-studied cases, such as NEAT1 and MALAT1 (NEAT2) indicates that paraspeckles are actively formed and present during human follicle development. Paraspeckle formation is initiated by transcription of the NEAT1 chromosomal locus and proceeds in conjunction with NEAT1 lncRNA biogenesis and a subsequent assembly step involving >39 paraspeckle proteins (PSPs). Interestingly, a study has shown that subunits of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodeling complexes were identified as paraspeckle components that interact with PSPs and NEAT1 lncRNA  and males (XY), one X chromosome randomly undergoes X chromosome inactivation (XCI) in female cells , expressed particularly in the primordial stage, as well as in primary follicles and in mural granulosa cell layers and the theca cell layers. Part of the GAS5 RNA structure mimics the glucocorticoid response element, enabling it to bind the DNA binding domain of the glucocorticoid receptor, thus inhibiting glucocorticoid induced transcription. GAS5 is further thought to regulate transcriptional activity of the androgen receptor. In line with this, GAS5 lncRNA has been found to repress the AR/androgen complex from binding to target through sequestering, thus repressing transcription  by the lncRNA HOTAIR, accelerating their degradation  repair foci. From this it can be concluded that NONO or related proteins are critical for DSB repair (Li et al., Oct4 promoter and positively regulates Oct4 gene expression in ES cells (Park et al., The upregulated expression profile of Several lines of evidence suggest paraspeckle proteins to be essential in cell fate determination, which is highly relevant for early developmental processes (Yamazaki and Hirose, FUS, EWS, and TAF15 are structurally similar multifunctional proteins that were initially discovered in the process of characterization of fusion oncogenes in human sarcomas and leukemias. As they are implicated in numerous central cellular processes such as gene regulation, genomic integrity maintenance and mRNA/microRNA processing, it is therefore not surprising to find them in many cellular contexts and in different cell types and tissues. The expression profile of the FET proteins were characterized in both the human (Andersson et al., The FET proteins also frequently exhibit gene translocation in human cancers (Paronetto, NEAT1 lncRNA, reducing the expression of FUS, and subsequently causing cell apoptosis. In combination with miR-548ar-3p, this regulates breast cancer cell apoptosis (Ke et al., It has been shown that FUS interact directly with We identified the presence of lncRNAs as well as the genes encoding the paraspeckle proteins, offering insights into how their transcripts are expressed during human follicle development. The study is descriptive in nature. As a proof of concept, we probed for the intracellular presence and localization of three selected paraspeckle proteins. It remains to be determined for several other proteins encoding by genes noted, as well as lncRNAs. In particular, our study indicates that they may be involved in cellular processes such as cell differentiation and cell integrity. This could be accomplished by their ability to control gene expression, epigenetics and mRNA turnover during follicle development.EE and KL-H conceived the study. PV performed bioinformatics analysis. EE, JN, and MI performed IMF. EE and KL-H analyzed RNA sequencing data and wrote the manuscript. All authors approved the final 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.


Cell death is central to development, organismal homeostasis, and immune responses. The cell death field has experienced tremendous progress by delineating the molecular programs specific to each of the apoptotic and inflammatory cell death pathways. Moreover, the discovery of the inflammasomes and pyroptosis and necroptosis pathway regulators have provided the genetic basis for the programmed inflammatory cell death pathways. Earlier research highlighted the unique regulation of each of these pathways, but emerging studies discovered co-regulation and crosstalk between these seemingly different cell death complexes. The progress in this area has led to an idea that master regulators play central roles in orchestrating multiple cell death pathways. Here, we provide a brief review of the master regulators, the innate immune sensor ZBP1 and the essential cell survival kinase TAK1, that play vital roles in the regulation of RIPK1/RIPK3–FADD–caspase-8 cell death complex assembly and its versatility in executing Pyroptosis, Apoptosis, and Necroptosis, which we dubbed here as PAN-optosis. Furthermore, we discuss the implications and therapeutic potential of targeting these master regulators in health and disease.ZBP1 and TAK1 regulate PAN-optosis. Cell death is fundamentally important for organismal development, homeostasis, and host response to infection. Several cell death pathways have been identified with specific genetically encoded requirements; these have collectively been named programmed cell death pathways  is a single-stranded RNA virus which infects millions worldwide and poses a major threat to public health. Early reports provided the first evidence of a crucial role for nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) in sensing IAV infection and dsRNAs to assemble the inflammasome complex via the adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and drive caspase-1–mediated inflammation -containing proteins and is known to activate necroptosis via RIPK3, another RHIM-containing protein  and their crosstalk with FADD–caspase-8–mediated pathways improved our understanding of the apoptotic and necroptotic pathways and their intricate regulatory mechanisms  gene that results in debilitating inflammatory arthritis similar to that of patients with chronic multifocal osteomyelitis (cmo), is rescued when caspase-8 is inactivated in combination with either caspase-1 or the NLRP3 inflammasome (Ferguson et al., Pstpip2cmo mice. Together, these findings support the convergent evolution and shared mechanism of PAN-optosis with potential implications in the development of inflammatory disease.Similarly, genetic deficiency of the NF-κB and MAPK components suggests a role for the necrosome components RIPK1 and RIPK3 in promoting NLRP3 inflammasome activation, triggering multiple forms of cell death and the development of inflammatory diseases (Ikeda et al., Recent work has resulted in tremendous progress in our understanding of cell death pathways with the discovery of novel regulators and the crosstalk between pyroptosis, apoptosis, and necroptosis. These studies also revealed the existence of master regulators such as ZBP1 and TAK1 that act as key signaling nodes for cell death. Recent progress in this area has also revealed an intimate connection between the RIPK1/RIPK3–FADD–caspase-8 signaling complex and the execution of PAN-optosis. It is clear that the PAN-optotic death complexes activated by the master regulators are multifaceted in nature with the potential to execute diverse forms of caspase-mediated cell death and promote necroptosis when caspases are inhibited. Together, these findings lay the foundation for the concept of master regulators controlling multifaceted cell death, PAN-optosis. Future studies should reveal additional mechanisms of PAN-optosis to better understand its evolutionary relevance and significance in health and disease. Further understanding of the master regulators of PAN-optosis may also allow us to develop superior therapeutic approaches to target cancer, infection, and inflammatory diseases.RM, SK, and T–DK drafted and edited the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rosette-forming glioneuronal tumor (RGNT) is a rare tumor which has been first reported as the fourth ventricle tumor by Komori et al and is classified as a distinct clinicopathological entity by the WHO Classification of Tumors of the Central Nervous System as in 2007. Although RGNTs were reported to occur in both supratentorial and inflatentorial sites, only 4 case reports of spinal RGNT have been demonstrated.A 37-year-old female presenting with slowly progressing right-sided clumsiness. Cervical magnetic resonance imaging revealed a spinal intramedullary tumor between the C2 and C5 levels.Pathological analysis showed unique biphasic cellular architecture consisting of perivascular pseudorosettes dominantly with few neurocytic rosettes and diffuse astrocytoma component. The tumor cells composed of perivascular pseudorosettes showed positivity for both synaptophysin and glial markers such as GFAP and Olig2. Therefore, the diagnosis of RGNT was made.Gross total resection of the tumor was achieved. No adjuvant chemotherapy nor radiotherapy was conducted after operation.At 2 years after the operation, no recurrence was observed.Although RGNT arising from the spinal cord is extremely rare, we need to consider the tumor as a differential diagnosis for intramedullary spinal cord tumors. It is now recognized as a new entity and was later classified as grade I tumor by World Health Organization. RGNT has 2 distinctive features: a glial component and a neurocytic component forming neurocytic rosettes and/or perivascular pseudorosettes. In addition, positivity of synaptophysin in the neurocytic component is important feature in the diagnosis of RGNT. Despite of these pathological characteristics, the diagnosis of RGNT is difficult mainly because of its rare incidence. RGNT most commonly arises from the forth ventricle but was reported to arise in the cerebellum, brain stem, pineal region, third ventricle, lateral ventricle, hypothalamus, and optic chiasm later. RGNT of the spinal cord origin is rare and to date, there have been only 4 case reports of spinal RGNT in the English literature.–6 Although the molecular analysis of RGNT of the fourth ventricle revealed higher mutation in FGFR1 and PIK3AC, only 1 case of RGNT of spinal cord was previously analyzed about these mutations.,7 We here showed the spinal RGNT case with radiological, pathological, and molecular findings with review of the literature. The patient has provided informed consent for publication of the case.Rosette-forming glioneuronal tumor (RGNT) is a rare tumor usually arising from the forth ventricle. RGNT was first described in 1998 by Komori et al2h8 on the left side. Magnetic resonance imaging (MRI) showed the well-defined lesion in the cervical spinal cord (C2–5), which showed the low intensity with T1-weight imaging , hyperreflexia of the right upper and both lower extremities, and hyperalgesia below TBRAF genes and fluorescence in situ hybridization showed no 1p/19q-co-deletion nor KIAA1549-BRAF fusion.Pathologically, the tumor composed of cells with small, round nuclei showed perivascular pseudorosette where the tumor cells surrounded a small vessel with little nuerocytic rosette and diffuse astrocytoma, which was diagnosed as a WHO grade I RGNT featured by the biphasic histopathology consisting of glial and neurocytic components Fig. A. Both oPostoperatively, right hemiplegia was worsened to MMT grade 3. After 4 months of rehabilitation, the patient's neurological symptoms were alleviated, and independent walking was achieved, although clumsiness of right hand and hypalgesia below neck were persistent. No recurrence of the tumor has been observed at the 2-year follow up.3,8 As the present case was characterized by predominant perivascular pseudorosette formations with few neurocytic rosettes and glial component, we considered RGNT showed wide range of glial to neurocytic/perivascular pseudorosette component. Pathologically, the initial histological examination by H&E staining impressed ependymoma; however, the immunohistochemistry (IHC) is much different from ependymoma. Synaptophysin and glial markers such as GFAP and Olig2 were positive in the perivascular pseudorosettes but negative for EMA. Neurocytic components in RGNT have reported synaptophysin positivity in IHC but negative for GFAP. However, Chakraborti et al demonstrated both synaptophysin and GFAP in the neurocytic component of RGNT in immunohistochemistry and confocal analysis showed the positivity for stem cell marker, CD133. They suggested the RGNT originated from the progenitor cells of the subependymal plate which could differentiate biphenotypically. Duan et al reported 2 cases of spinal RGNT and documented that the tumors were found to originate from central canal of spinal cord. As our case also suggested the central canal origin tumor during operation, we suspected this tumor might originate from the progenitor cells of the subependymal cells in the central canal of spine, which can differentiate both glia and neuron.We have reported a case of spinal RGNT occurred in 35-year-old female with distinct clinicopathological features including unusual morphology and genetic results. RGNT is rare tumor which affects commonly fourth ventricles of young to middle-aged adults with female predominance and showed 2 distinctive morphologies: a glial component, whose morphology resembles pilocytic astrocytoma, and a neurocytic component forming neurocytic rosettes and/or perivascular pseudorosette.–6,8 and the current case is the fifth case report (Table RGNT commonly arises from the forth ventricle; however, 4 case reports of spinal RGNT have been described in the English literature,rt Table . The age In our case, as the intensity of Gd enhancement was very slight, ependymoma was not likely and possibility of low-grade astrocytoma was also unlikely because of well-defined boundaries. We thought that PA could not be denied because of its clear margin with weak enhancement on Gd contrast MRI; however, this patient's age was relatively older than the predilection age. As the spinal RGNT is very rare and shows relatively similar neuroimaging characteristics with other intramedullary tumors such as ependymoma and PA, the preoperative diagnosis is difficult.The preoperative differential diagnosis of RGNT in the spinal cord includes ependymoma, low-grade astrocytoma, and pilocytic astrocytoma. Kitamura et al have reported FGFR1 and PIK3CA mutations were found 46.2%and 44.4%, respectively and these mutation were detected in both glial and neurocytic components of RGNT. Among the 4 spinal RGNT cases, Bidinotto et al only examined hotspot mutations of FGFR1and PI3KCA; however the hotspot mutations of them were not detected. We also failed hot mutations of FGFR1, PIK3AC, BRAF, and IDH1/2 hotspot mutations. Although we do not have enough data of the molecular results of spinal RGNT, the genetic features of spinal RGNT might be different from the fourth ventricle origin.There are still limited number of reports about the genetic characteristics of RGNT. Gessi et al first reported FGFR1 mutations in RGNT of the fourth ventricle, hypothesizing the molecular similarities between RGNT and PA. All spinal RGNTs underwent complete resection without recurrence. As the rarity of spinal RGNT, it may be misdiagnosed as ependymoma or astrocytoma without immunohistochemistry; however, both clinical information including the patient's age, sex, and the tumor location and pathological findings such as biphasic pattern consists of neurocytic and glial components lead to narrow the differential diagnosis. It is important to accumulate spinal RGNT cases to reveal the true nature including molecular features and clinical course of the disease.Surgical resection is reported to be the first choice for the treatment of spinal RGNT and no adjuvant therapy can be recommended. In intracranial RGNT, recurrence-free survival rates have been reported to be 92.9% at a mean follow-up period of 22.5 months.Conceptualization: Mishie Tanino, Kazutoshi Hida.Data curation: Shuji Hamauchi, Toru Sasamori, Shunsuke Yano.Methodology: Mishie Tanino.Supervision: Shinya Tanaka.Writing – original draft: Shuji Hamauchi.Shuji Hamauchi orcid: 0000-0003-2760-5897.