Public open spaces (POS) are “publicly owned spaces such as parks, green areas, squares, marketplaces, streets and highways which are of public access”. Some attributes could increase or decrease participants’ attendance. Thus, reliable and valid audit tools are needed in order to measure these attributes. This study aimed to develop and validate a tool to assess POS features within the Mexico City context. The Mexican Public Open Spaces Tool (MexPOS) was developed based on (1) two validated POS audit tools, (2) several visits to the POS, (3) pilot testing, and (4) multiple work sessions with a group of specialists. The original tool included 181 items divided into nine sections. Trained personnel visited and evaluated 944 POS in Mexico City. An exploratory factor analysis was performed to examine the construct validity of the items and the relationship between the subscales. The final model resulted in seven factors: (1) Food and Wellness Environment (α = 0.15), (2) Maintenance (α = 0.81), (3) Amenities (α = 0.72), (4) Legibility (α = 0.59), (5) Security (α = 0.48), (6) Perceived Environment (α = 0.65), and (7) Urban Environment (α = 0.58). Our study highlights the relevance of using a validated tool to measure POS characteristics related to participants’ attendance to help assess infrastructure improvements and identify priority areas for changing socio-urban environments for physical activity. Public open spaces (POS) are “publicly owned spaces such as parks, green areas, squares, marketplaces, streets and highways which are of public access” . AccordiSome studies have evaluated POS globally. For instance, the project Public Spaces is an American organization, with an international impact that aims to identify the characteristics of successful POS. This project evaluated thousands of POS in the world and concluded that a “successful” space had four key qualities: (1) accessibility , 2) availability of a range of activities, (3) convenient and pleasant space, and (4) adequate space to meet and socialize . Similar availabiA systematic review of qualitative and quantitative studies on the characteristics of POS that influence adolescents’ physical activity found that the presence of trails, walking paths, playgrounds, and sports fields are associated with physical activity among this population . In the The Bedimo-Rung Assessment Tool–Direct Observation (BRAT-DO) is an extensive instrument, mainly used by researchers and health professionals, that evaluates the visible characteristics of the park, it has 181 items distributed into different sections such as generalities of the park, aesthetics, concession stands, drinking fountains, sports fields, among others . Likewisn = 629, aged 20–65) found that easy access and proximity to parks, aesthetics, and safety from crime were associated with moderate to vigorous physical activity; moreover, the perception of park safety was found to be a moderator of the association between physical activity and park use [Even though the tools mentioned above assess the physical attributes of POS, they fail to assess in depth some important aspects of POS in Mexico, such as the built environment around the POS  and perception of safety . A population-based study on adults in Mexico , nutrition (n = 6), urbanism (n = 2), architecture (n = 2), and public policy (n = 1) at the beginning of 2016. During this session, some characteristics of POS that could help foster active lifestyles were listed and discussed. Among these characteristics, they highlighted the presence of trees, pollution, safety, accessibility, location, lighting, presence of pets, advertising of foods that promote a sedentary lifestyle, sale of energy-dense foods, and hygiene.A first meeting was held at the National Institute of Public Health Mexico (INSP) with a group of specialists in physical activity  and south  of Mexico City were visited. This was a convenient selection of parks focused on spaces that have high participation of people who perform physical activity. These visits helped us to verify some of the characteristics listed at the first meeting and to include some others found inside  and outside  of the POS. After this visit, 16 items were created, including those related to the presence of pets, availability, quantity, quality, and cost of bathrooms, availability of garbage containers, smoke advertising, food, and beverage advertising, availability of drinking fountains, availability of energy-dense foods around the POS, availability of police stations, public transportation stops, availability of lighting, parking lots, outdoor gym and management of the POS. Later, this list was compared and complemented with the characteristics assessed by the Bedimo-Rung Assessment Tool–Direct Observation (BRAT-DO)  and the In the following meetings at the INSP, urbanists included some other questions related to mapping, quality of lighting, rules of procedure, name of the POS, areas of the park open to the public, memberships, accessibility, public transportation availability, streets around the POS, and security elements. Physical activity experts included items related to drinking fountains and their use. Nutritionists redesigned questions about food and beverage advertising and availability.Next, the new version of the audit tool was pilot tested by a group of urban planners in a metropolitan park in west-north Mexico City (Parque Bicentenario). During this visit, several adaptations were made to: (1) reduce the number of items, (2) eliminate items that measure similar characteristics or those that were not context-specific, (3) improve the wording of the items, and (4) include other POS attributes. Subsequently, two external volunteers from the research group applied the tool and provided feedback on the clarity of the tool’s instructions. The original version of the tool had 181 items and the following sections: (1) General park information, (2) General park features , (3) Accessibility, (4) Environment around the POS, (5) Roads/internal routes/tracks within the POS, (6) Security, safety and lighting, (7) Facilities and amenities , (8) Health, nutrition, and hygiene , and (9) Maintenance . Items were rated with three types of response scales: (1) Likert scales , (2) binary scales (yes/no), and (3) ordinal scales . The original version of the questionnaire in Spanish can be found in For this article, a POS was defined as green spaces , grey areas , or natural environments   were revAll POS that fitted our definition were included in the final sample. POS that were difficult to access , required an annual membership , were private  or did not serve a recreational purpose  were excluded from the final sample.2, (2) local park: 3000 to 10,000 m2, (3) neighborhood park: 400 to 3000 m2, (4) pocket park: 100 to 400 m2, (5) roundabout: has a higher proportion of sealed soil, vegetation or planters, and is planned to regulate vehicular traffic, (6) boulevard (Alameda): public space of up to 80,000 m2, with vegetation and bare or covered soil and paths or corridors for pedestrian traffic, (7) square (plaza): public space of up to 5000 m2, which has a greater proportion of sealed soil, has arboreal and/or shrubby vegetation. Planned for recreation, rest, or relaxation, (8) remnant: linear green space generally enabled along disused railway lines, rivers, streams, canals, and urban voids, and (9) garden: public space of up to 5000 m2, which has a higher proportion of sealed soil, has arboreal and/or shrubby vegetation, and is planned for recreation, rest, or relaxation [A total of 944 public open spaces (POS) distributed in all the municipalities of Mexico City were assessed between August 2017 to February 2018, with the original version of the tool by trained personnel. A list of POS was distributed among 3 fieldworkers at convenience. The most remote and insecure spaces (based on interviewers’ perceptions) were evaluated by the entire team (3 fieldworkers). On average, each fieldworker visited 15 POS each week. POS were categorized based on their typology into: (1) metropolitan park: ≥10,000 mlaxation .An exploratory factor analysis (EFA) was performed to examine the validity of the items and the relationship between the subscales. An EFA is a multivariate technique used to uncover the underlying structure of a set of observed variables  and the construct they measure  . In the Prior to the EFA, several assumptions  and pre-tests were checked to corroborate that this analysis could be performed. Regarding the assumptions, all data that were put into the model were either continuous or ordinal , and Yes/No variables (binary data) were treated as ordinal data assuming an underlying order “yes is higher than no” as has been done in previous research . The resp < 0.05) to reject the hypothesis [p = 0.001.Concerning the pre-tests, the Kaiser–Meyer–Olkin Measure of Sampling Adequacy (KMO) was performed to reflect the sum of partial correlations relative to the sum of correlations. KMO results vary between zero and one, where a value closer to one is better and a value lower than 0.5 indicates that an EFA might not be appropriate . The KMOpothesis . In the factor” command in STATA. Eigenvalues are indicators of the variance explained by a factor, they should be greater than one in order to account for at least as much variance as a single variable, hence the factors with an eigenvalue greater than one were extracted resulting in 13 factors. Thereon, commands “rotate” and “sortl” were used to rotate the factors for easier interpretability and to arrange the variables in descending order into the 13 factors each are loading. Alphas were calculated for each of the factors. All analyses were performed in STATA (Version 13), College Station, TX.Once the assumptions and pre-tests had been carried out the EFA was performed using principal factor analysis. To determine the number of factors, eigenvalues were calculated using the “A total of 944 POS were mapped and evaluated. Based on their typology, 26.7% were neighborhood, 24.5% locals, 20.9% metropolitans, 10% remnant, 7.3% squares, 4.5% pocket, 4.4% gardens, 1% roundabout, and 0.7% boulevards. Based on the municipality, the highest concentration of POS was located in four municipalities: Cuauhtémoc, Coyoacán, Iztapalapa, and Gustavo A. Madero (52.8%). In total, 14.4% of the POS was found in Cuauhtémoc municipality. Cuajimalpa, Magdalena Contreras, Milpa Alta, Xochimilco, and Iztacalco municipalities have least number of POS. Specifically, Milpa Alta, the second-largest municipality in Mexico City, possesses the lowest concentration of POS (0.3%) .From the first results of factor loadings (Model I) , a groupThe final model (Model IV) is presented in n = 6) were related to the land use around the park and the food and beverage advertising inside and outside the POS. All items were reverse items except for the land use around the POS, which was mainly institutional. In the Maintenance factor, items (n = 21) were related to the condition of the facilities  inside the park. Reverse items in this factor were the presence of garbage around the POS, hazardous waste , and animal feces.In the Food and Wellness Environment factor, items (n = 44) related to the courts , paths , outdoor gym equipment , and other POS features  in the POS. Moreover, in the Legibility factor, items (n = 15) were relevant to the references or signs indicating characteristics of the POS , activities in the POS , and rules inside the POS . The items (n = 11) in the Security factor were features related to the protection against deliberate threats inside the POS  and to elements that might make a person feel unsafe . It should be mentioned that four out of eleven items (6.8 to 6.11) did not reverse load as expected.The Amenities factor comprised items (n = 14) relevant to the sensory aspects of the POS , as well as the number of trees and shade available. Reverse items were related to car traffic sounds, excessive noise, and people smoking inside the POS. Furthermore, in the Urban Environment factor, items (n = 17) were related to the type of streets around the POS , the presence of signaling devices such as pedestrian crossing lights and motorized vehicles stoplights, and ramps to guarantee access to the POS. Reverse items were having most streets around the POS primary, having sidewalks in a “regular” state, and having public transport stops and stations near the POS.The Perceived Environment factor comprised items . However, after conducting the factor analysis, the final model comprised seven factors or sections including food and wellness environment, maintenance, amenities, legibility, security, perceived environment, and urban environment. Internal consistency estimates for the latent variables showed strong alpha coefficients for Maintenance (α = 0.81), Amenities (α = 0.72) and Perceived Environment (α = 0.65), acceptable estimates for Legibility (α = 0.59), and Urban Environment (α = 0.58), and unacceptable values for Food and Wellness Environment (α = 0.15) and Security (α = 0.48) .At the time of this research, there was not a unique definition of POS, each public organization or government used a different definition, making it difficult to have a clear definition or understanding of these spaces. POS were also classified in accordance with the local urban norms defined by the federal, state, and local governments, where these spaces are defined mainly as green spaces and grey areas. However, a definition by the Authority of Public Spaces , which cAlthough there are many available instruments to measure POS worldwide ,20,21,22Second, there are some POS characteristics that could differ between countries and cities. This is the case of a big-sized and urbanized city such as Mexico City. This city possesses more than 900 POS divided into nine typologies. Despite considering a variety of characteristics within the instrument, some could be missed or not considered. For instance, the availability of restrooms could be a mandatory amenity within metropolitan parks, but not for the pocket, remnant, or plaza areas. This could generate an unfair estimation of the POS’ features, especially if the instrument was designed to generate an overall score  . In thisPark identification using national databases could be challenging in some countries. This is the case for Mexico City, where more than three public databases were searched and several physical scans were performed to generate a list of POS. In contrast, HICs have databases open to the public that describe park characteristics in detail ,36,37. TBased on validity values, it is complex to compare the obtained internal consistency estimates with other POS audit tools, as they do not report internal consistency measures. CPAT, EAPRS, PARA, and POST only report inter-rater reliability estimates (κ = 0.60–0.77) ,19,20,38The Cronbach alphas for Security and Food and Wellness Environment factors are low. As to Security, there were four out of eleven items that were expected to reverse load because of their nature. Items (6.8) Is there any evidence of threatening people or behavior? (6.9) Is there vandalism inside the park? (6.10) Is there poor street lighting in the area around the park? and (6.11) Have there been any security incidents so far this year? are measured as if the presence of it  adds up positively to the construct of Security. A possible reason for this is the complexity and “hour dependency” of measuring these items, for example, items (6.8) and (6.10) would depend on the time of the day of the fieldworker’s visit and their ability to record security incidents; (6.11) might depend on whether there is a park administrator to ask to or if incidents have been reported by the media.Food and Wellness Environment has also a low alpha factor (0.15). The measurement of the food environment is becoming relevant given its contribution to the health status of the population . SpecifiRegarding the Maintenance and Amenities factors, it could be argued that the high Cronbach alphas might be due to the high number of items in the analysis in both latent variables; nevertheless, these values are not so high (>0.95) as to indicate redundancy of the scale items . As for Among the strengths of the study is the use of a large dataset generated with complete information for 944 POS. To our acknowledge, this is the most comprehensive POS database created within Mexico City. The development of a Mexican context questionnaire that considered various elements of the context that contributes to healthy lifestyles  can be seen as a strength. Additionally, the corroboration of the assumptions and pre-tests contribute to a clear basis for using an EFA to assess construct validity. Moreover, the participation of a diverse group of field experts to define, based on the analysis results, the final EFA model adds to the strengths of the article. Finally, the MexPOS can be used in other cities of LMICs where the built environment can be similar to that of Mexico City.This study also has some limitations that should be addressed. First, the study is only representative of a single city in the country, and so far, has not been tested in other less urbanized areas. Second, the number of POS could be higher or lower within current years. For instance, pocket parks are no longer in operation. Third, some of the perception items  were difficult to capture, which could have altered the alphas.In conclusion, The MexPOS is a valid tool to measure the constructs of maintenance, amenities, legibility, perceived environment, and urban environment in POS. Although future research is needed in order to estimate the test–retest reliability, inter-rater reliability, and validity of this instrument, the MexPOS could help policymakers in evaluating the pre and post impacts of redesigning or renovating POS. In addition, this instrument could be used to improve and identify priority areas for changing socio-urban environments for physical activity based on typologies and/or municipalities, and to promote POS with ideal health conditions. This study could also contribute to making recommendations aimed to complement local and federal norms .


The new landscape of treatments for metastatic clear cell renal carcinoma (mRCC) is constantly expanding, but it is associated with the emergence of novel toxicities, adding to up to those observed in the tyrosine-kinase inhibitor (TKI) era. Indeed, the introduction of immune checkpoint inhibitors (ICIs) alone or in combination has been associated with the development of immune-related adverse events (irAEs) involving multiple-organ systems which, even if rarely, had led to fatal outcomes. Moreover, due to the relatively recent addition of ICIs to the previously available treatments, the potential additive adverse effects of these combinations are still unknown. A prompt recognition and management of these toxicities currently represents a fundamental issue in oncology, since it correlates with the outcome of cancer patients. Even if clinical guidelines provide indications for the management of irAEs, no specific protocol to evaluate the individual risk of developing an adverse event during therapy is currently available. A multidisciplinary approach addressing appropriate interventions aimed at reducing the risk of any insidious, severe, and/or dose-limiting toxicity might represent the most efficacious strategy to timely prevent and manage severe irAEs, allowing indirectly to improve both patients’ cancer-specific survival and quality of life. In this review, we reported a five-case series of toxicity events that occurred at our center during treatment for mRCC followed by the remarks of physicians from different specialties, pinpointing the relevant role of an integrated and extended multidisciplinary team in a modern model of mRCC patient management. Renal cell carcinoma (RCC) is an insidious neoplasm, accounting for approximately 2% of global cancer diagnoses and deaths, whose incidence will further increase worldwide. Cancers of the kidney and renal pelvis have rapidly become more common in the developed world over the past decades . AccordiIn KEYNOTE 426, the most common related grade 3 or higher adverse effects described are (≥10% patients in either group) as follows: hypertension , and diarrhea [46 (11%) vs. 23 (5%)]; deaths from adverse events (AEs) occurred in 19 (4%) of 429 patients in the pembrolizumab plus axitinib group   plus ipilimumab (1 mg per kilogram) intravenously every 3 weeks for four doses, followed by nivolumab (3 mg per kilogram) every 2 weeks. The second case involved a patient, treated before with pembrolizumab plus axitinib at a standard schedule  followed in second line with cabozantinib, who reported nephrological toxicities. The third and fourth cases entailed patients treated in the first-line treatment with standard pembrolizumab plus axitinib, during which they showed liver and cardiological toxicities, respectively. Finally, the fifth case was about a multidisciplinary management of dermatological toxicity due to cabozantinib. The severity of adverse events was graded according to CTCAE version 4.0. At the time of first oncological visit, all our patients signed informed consent in which the consent to the use of their data for research purposes is included.A 69-year-old man underwent right nephrectomy surgery in May 2019 for a renal carcinoma with sarcomatoid  and poorly differentiated clear renal cell components, pT3a pNx, stage III according to AJCC 2017. The postsurgery total-body contrast-enhanced computed tomography (CT) showed suspected pulmonary and mediastinal lymph node metastasis, confirmed by transbronchial needle aspiration (TBNA). According to the prognostic criteria of Motzer and Coll and Heng , 16, forIn December 2020, the patient had G2 asthenia, restriction of activities of daily living but not of personal care, dizziness, headache, non-alterations of vision, and G1 diarrhea. Laboratory tests showed hypoglycemia (72 mg/dl), hyponatremia, reduced levels of ACTH , and cortisol  at 8:00 a.m., TSH (0.18 µUI/ml), and FT4 (0.6 ng/dl). The ACTH stimulation test (1 µg) showed an insufficient adrenal response . Due to the headache and dizziness, a magnetic resonance imaging (MRI) of the brain was performed and highlighted the radiological signs of a meningeal irritation attributable to an hypophysitis Figure 2Then, in the presence of secondary adrenal insufficiency and secondary hypothyroidism, a diagnosis of immuno-related hypophysitis was placed, also supported by radiological imaging. The ICI was stopped, and an adrenal and thyroid replacement therapy  was administered. The immunotherapy was suspended for a month and resumed after normalization of pituitary function. The patient still maintains a complete radiological response with an OS of 29 months.The incidence of thyroid disorders in course of immunotherapy is rarely higher than G2, due to the frequent monitoring of thyroid function that allows to an early detection. In particular, a meta-analysis of 28 studies, which included more than 7,500 patients, showed an incidence of hyperthyroidism and hypothyroidism, under combined anti-CTLA4/anti-PD1 treatment, of 8% and 13.2% versus 3.2% and 3.9% in the course of an anti-PD1 treatment, respectively . NowadayPituitary gland disorders are more frequent with anti CTLA-4 than with anti-PD1/PD-L1. The incidence of hypophysitis depends on the dose and drug administered: with ipilimumab, 3 mg/kg is 1%; with ipilimumab, 10 mg/kg is 16%; with nivolumab, 240 mg is 1.1%; and with ipilimumab, 3 mg/kg + nivolumab 240 mg reaches 8% . The pitIn conclusion, in the present case report, the front-line treatment, still in progress, has been allowed to reach a survival of more than 2 years. An adequate laboratory monitoring is mandatory to manage endocrine toxicities in advance. Of course, a more appropriate diagnostic classification of endocrinological toxicity, together with a more detailed of toxicity degree, is required. In case of G≥2 immuno-related endocrinological disorders, suspension of treatment is not mandatory. A multidisciplinary approach in the management of toxicity is essential to ensuring a correct cost/benefit balance for the patient, favoring therefore greater adherence to treatment while respecting an adequate quality of life. In summary, our case concluded that adequate laboratory monitoring is essential for early intervention in the management of endocrine toxicity. In the presence of endocrinopathy, an accurate diagnosis and a correct definition of the degree of toxicity are needed; if hypothyroidism and adrenal insufficiency occur during treatment with ICIs, it is extremely important to start replacement therapy but discontinuation of immunotherapy is almost never indicated. A multidisciplinary approach to the management of toxicities is essential to ensuring correct continuation of therapy for the patient and also greater adherence to treatment in accordance with an adequate quality of life.In June 2008, a 70-year-old man with a history of cerebral ischemia, atrial fibrillation, and hypertension experienced a persistent abdominal pain and weight loss (5 kg over a year). Imaging revealed a renal lesion of 58 × 62 × 55 mm in the upper pole and pars intermedia of the right kidney, suspected for neoplastic mass, with no other tumor lesions. Therefore, the patient underwent right nephrectomy, and the histological examination revealed a clear-cell type cancer . The follow-up was negative until a total body CT scan showed a relapse of disease in the lung, pancreas, and subcutaneous tissue . In January 2021, a first-line therapy for good risk with standard pembrolizumab plus axitinib was administered. Laboratory tests documented a baseline serum creatinine at 1.4 mg/dl. After 5 cycles, in May 2021, for the first time the renal function worsened (serum creatinine 2.1 mg/dl) with a negative urine test. Renal ultrasound did not show any sign of kidney obstruction . The patient carried out a nephrological evaluation, and it was decided to replace sartan-based antihypertensive therapy with a calcium antagonist in order to avoid concomitant renal medication damage. Hydration was preserved, and cancer treatment continued. After 2 weeks, creatinine was about 1.5 mg/dl. Oncologic therapy was not stopped. After 2 more cycles of pembrolizumab plus axitinib, acute renal dysfunction was observed again (serum creatinine 2.4 mg/dl). Therefore, according to the ESMO clinical guidelines about the management of immunotherapy-related nephritis .In September 2021, after nine cycles of therapy, a total body CT showed disease progression: the pancreatic nodule increased in size (from 1.5 × 1 cm to 3.5 × 5.5 cm) and a new lesion appeared in the second liver segment. Thus, we decided for a second-line treatment with cabozantinib 60 mg per day. After 3 cycles, in November, hypertension had worsened and there was a gradual, progressive deterioration of renal function: creatinine was about 1.95 mg/dl and urinalysis revealed proteinuria = 50 mg/dl and microhematuria. The daily urine protein loss was found to be about 1,200 mg. Hydration was started and cabozantinib 60 mg per day continued. After 2 weeks, serum creatinine was still about 1.85 mg/dl and daily urine protein loss was 1,000 mg. Then, in agreement between oncology and nephrology specialists, cabozantinib was reduced to 40 mg per day. After 2 weeks, serum creatinine was 1.7 mg/dl, daily urine protein loss was 600 mg, and after 2 more weeks creatinine was 1.6 mg/dl and daily 24-h urine protein loss was 250 mg.Then, the patient continued cabozantinib 40 mg per day, no more renal toxicity was observed, and the treatment was well-tolerated. In March 2022, total body CT showed stable disease and treatment with cabozantinib 40 mg per day is still ongoing Figure 3According to the advent of new oncological treatments , drug toxicity and especially renal toxicity are more frequent than before. As reported by ESMO clinical guidelines , for exaIn order to manage different types of toxicity, a timely treatment of renal injury is crucial and in this setting the nephrologist’s role is of primary importance inside the MDT.Furthermore, a new evolving field, namely, onco-nephrology, has emerged during the last few years. It includes the vast spectrum of renal disorders that can arise in patients with cancer. A differential diagnosis between progression of underlying renal disorders and secondary disorders due to oncological treatments or to the malignancy itself is essential in order to allow the oncologist to continue the antineoplastic therapy. Cancer therapy is increasingly prescribed in elderly patients, a population often already affected by multiple morbidities and preexistent CKD (chronic kidney disease). Therefore, it is important to consider how the presence of CKD, AKI (acute kidney injury), and other renal disorders may affect treatment options and outcome and how certain therapies may increase the risk of kidney toxicity .Regarding our case report, it is well known that VEGFR2 inhibitors lead to some adverse events such as proteinuria, hypertension, hand−foot syndrome, and kidney dysfunction whereas ICIs lead to other adverse events as autoimmune disorders, such as thyroiditis, colitis, skin disease, and different forms of nephritides.Clinically, renal adverse effects of anti-VEGF therapies are arterial hypertension, proteinuria, rarely nephrotic syndrome, AKI, or CKD. Various parts of the nephron can be injured; 42% of the total number of renal adverse effects is represented by renal impairment, 47% by metabolic disturbances, and hypertension in 11% .Podocytes and endothelial cells are involved, resulting in severe alteration of the architecture and function of the GBM .Proteinuria is described as one of the most common renal side effects of other anti-VEGF drugs and frequently occurs with hypertension . It is tProteinuria, hypertension, and kidney injury are closely related to the destruction of the integrity of the glomerular filtration barrier, composed of podocytes, a glomerular basement membrane, and endothelial cells. TKI-induced endothelial cell damage leads to the compensatory expression of pro-angiogenic factors and the formation of an abnormal endothelial−podocyte cross talk and podocyte injury .On renal biopsy from patients receiving TKIs, the most common pathological findings are thrombotic microangiopathy (TMA), MCD , and FSGS , as the result of direct cellular toxicity on endothelial cells and/or podocytes –42. In aDespite TKIs, ICIs can cause different autoimmune diseases known as immune-related adverse events (irAEs) . UsuallyA recent multicenter study identified three independent risk factors for development of ICI-AKI: 1) concomitant use of PPIs; 2) combination treatment with anti–CTLA-4 and anti–PD-1/PD-L1 agents; and 3) lower baseline eGFR .According to the results of this study, patients receiving PPIs, those receiving combination ICPI therapy, and those with a lower baseline eGFR may receive closer renal surveillance.In this setting, the figure of the onco-nephrologist is very important for the consultation and consideration of kidney biopsy, especially for patients with persistent stage 1 AKI, and those who develop stage 2 or 3 AKI.Indeed, in a recent review , patientKidney dysfunction under TKIs usually resolves with dose reduction or drug discontinuation, and it depends, in part, on the patient’s baseline serum creatinine. However, these patients also present many risk factors for CKD such as diabetes, old age, hypertension, and nephrectomy which can lead to chronic kidney failure. In the KDIGO Controversies Conference on onco-nephrology, for patients with CKD, TKIs may be used at a lower-than-standard dose and then increased according to individual tolerability . AccordiRecommendations for management of ICI-associated adverse renal effect have been recently summarized in a complete review by Hermann and Perazella .International guidelines suggest the following management of immune-related adverse events , 50, 51:ASCO guidelines also suggest the use of other immunosuppressive agents like mycophenolate mofetil, azathioprine, cyclophosphamide, and infliximab if corticosteroid therapy is not enough .Nevertheless, in Italy these immunosuppressive agents are not recommended in immune-related adverse events and so we cannot express an opinion on this subject.However, according to these findings, baseline renal function, urine analysis, and electrolytes are three of the most important things to monitor during cancer treatment with both TKIs and ICIs, especially in patients with comorbidities  that can cause one of the most difficult problems for making the differential diagnosis between collateral effects of antineoplastic drugs or preexistent diseases. Therefore, a complete evaluation of kidney function prior to oncological therapies is mandatory for prolonging the survival of our patients  , who experienced a progressive weight loss and dyspepsia, underwent abdominal ultrasound and CT scan which showed a huge expansive mass (10.5 × 7.8 cm) at the level of the middle and lower portions of the left kidney. She thus underwent left nephrectomy. Histopathological report diagnosed a clear cell-type RCC, grade 2 Fuhrman nuclear grading, pT2b pN0 according the AJCC TNM system. Follow-up was negative until June 2020, when a chest CT scan showed at the level of the left lung the increase of both a parascissural nodule, measuring 10 × 9 mm (previously 2.5 mm), and of a nodule in the posterobasal segment (measured 4.5 vs. 3.5 mm). She underwent atypical pulmonary resection, and the histological examination described a pulmonary localization of RCC. After 2 months, due to acute dyspnea, she underwent a further CT scan which showed left lung pleural effusion and necrotic solid tissue localized at the apex of the left lung (10 × 3.3 cm), which was infiltrating the pleura, pericardium, and fifth rib; additional pleural implants; carcinomatous lymph nodes; and mediastinal lymphadenopathies. The patient then had blood tests which showed levels within a normal range. According to the IMDC, for Karnofsky Performance Status below 80%, she was categorized prognostically as at an intermediate risk. Consequently, from January to May 2021, she underwent administration of six cycles of pembrolizumab + axitinib with partial radiological response, and her global clinical conditions improved. In May 2021, after the sixth cycle, routine biochemical tests showed an increase in serum transaminases, with a normal bilirubin value: the glutamic-oxaloacetic transaminase (GOT) value was 83 U/l  whereas the glutamic-pyruvate transaminase (GPT) was 142 U/l . According to the classification NCI-CTCAE (v.5.0) , the patDuring ICI monotherapies , liver enzymes’ increases in various orders of magnitude have been reported to cardiological toxicityoccur in around 2%–10% of patients , 55, 56.Liver-related adverse events (LRAEs) occurring during ICI treatment are usually reported and scored according to the CTCAE . It has At present, liver biopsy should be considered for patients with more severe liver toxicity (grade >3) or in case of uncertain diagnoses. Patterns of liver toxicity during ICI treatment are still currently scarcely characterized from a histological standpoint , and bioInterestingly, ICI-induced liver toxicities do not display the histological  and serological (absence of autoantibodies) features of an autoimmune hepatitis (AIH), but this notwithstanding, the current pharmacological management is mainly based on protocols derived from those used in the treatment of this liver disease . As receA 74-year-old woman, due to persistent cough and abdominal pain on the left side, underwent an abdominal ultrasound and a total body CT scan that showed a mass of 2 cm in the left kidney, suspected for primary tumor, and multiple nodular lung lesions. After 1 month, in December 2020, the patient underwent a left radical nephrectomy, whose histological examination revealed a clear-cell type RCC . After 2 months, the patient was referred to our center for the first oncological evaluation. The past medical history comprised systemic hypertension treated by angiotensin II receptor blockers and paroxysmal atrial fibrillation on direct oral anticoagulants (DOACs), allergic asthma on foster therapy, and type 2 diabetes mellitus treated by metformin. An echocardiogram showed a normal bi-ventricular dimension, wall thickness, and systolic function. The patient was in good general condition, the blood pressure was within the normal limit, the blood test showed normal renal function, and electrolytes were within limits, without proteinuria. According to the IMDC intermediate risk, a combination therapy with pembrolizumab and axitinib was started. The patient was instructed to monitor the blood pressure at home and to contact the clinic in case of hypertension or any new symptoms. After the third cycle of therapy, the patient reported asthenia and headache. The blood pressure was increased (180/90 mmHg). The patient was referred to a cardiologist. The electrocardiogram showed a sinus rhythm with a heart rate 90 bpm without repolarization changes. The blood pressure was persistently increased. Troponin showed a negative result. Echocardiogram showed a normal bi-ventricular systolic function with FEVS 61%. Antihypertensive therapy was implemented with the addition of amlodipine with good response to therapy. Furthermore, due to the drug interference between amlodipine and metformin by pharmacodynamic antagonism, the patient was closely observed for the risk of hypoglycemia.After 4 months, a total body CT scan was performed, showing stability of the disease. Blood test showed kidney function, electrolytes, and glucose levels within the normal limits, and no proteinuria. Cardiovascular evaluation showed normal ECG and normal blood pressure (140/80 mmHg). The patient was asymptomatic. Therefore, considering the stability of the disease, the results of the laboratory tests, the cardiovascular evaluation, and the improvement in symptoms, the patient continued the scheduled therapy, which is still ongoing with good tolerability Figure 5In this case, a combination of two different classes of agent were administered to the patient. Each agent holds the potential to determine different cardiovascular toxicities. One of the most common adverse reactions of axitinib is systemic hypertension. A meta-analysis including 77 studies showed that arterial thromboembolism, cardiac ischemia and cardiac dysfunction rate among cardiotoxic effect, and hypertension were the most common and clinically recognized adverse events with OR 5.28 [4.53–6.15] . AnotherGenerally, hypertension is an established risk factor for chemotherapy-induced cardiotoxicity, and poorly controlled blood pressure can influence outcomes for cancer patients. Therefore, continuous monitoring and medical treatment with antihypertensive agents are recommended for axitinib-associated hypertension. There is no general consensus on the best modality for blood pressure monitoring. The AIOM  guidelines recommend a weekly based monitoring in the first 8 weeks and specifically a blood pressure measurement before every cycle. In addition, guidelines recommend to obtain a good blood pressure before starting treatment . The ESMThe second agent administered to the patient was the ICI pembrolizumab. This agent can be associated with a spectrum of adverse effects mainly related to irAEs and can affect multiple organs including the cardiovascular system. Although rare, cardiovascular IrAEs can be fulminant . A recenAnticancer therapies utilized in GU cancer can have cardiac-related toxicities, and the collaboration between oncologist and cardiologist is crucial. One of the priorities of the cardio-oncology field is the possibility to improve the cardiovascular screening to mitigate risk factors for cardiotoxicity prior to the beginning of treatment and to identify high-risk patients requiring a closer follow-up Table 1.A 60-year-old male patient with mRCC treated with cabozantinib was referred to our department. His personal history showed type 2 diabetes mellitus, hypertension, and atrial fibrillation.In July 2012, he underwent surgery of left nephrectomy and histological examination showed renal clear cell carcinoma, pT2a. Therefore, the patient started clinical and radiologic follow-up.In July 2016, the total body CT scan showed a local relapse of disease and distant metastases, located in the paravertebral muscles, right gluteus muscle, bones, and lungs. In August 2016, the patient underwent a biopsy of the gluteus muscle, which confirmed the diagnosis of metastases from RCC. Therefore, he started first-line therapy with sunitinib 50 mg per day 4 weeks on/2 weeks off, from September 2016 to June 2017. Then, the total body CT scan showed a disease progression to the lungs and muscles. Considering the previous treatment, the patient started therapy with nivolumab and in July 2017 he underwent radiotherapy for muscular metastases  and stereotactic radiotherapy on a lung metastasis in July 2020. Nivolumab was administered until February 2021, when the total body CT scan showed a disease progression on the liver and pancreas.At this point, in April 2021, the patient started a third-line treatment with cabozantinib 60 mg daily. After 28 days, at the beginning of the second cycle of therapy, the patient reported erythema on the dorsal hands, not associated with pruritus. However, we decided to continue the therapy. One month later, at the beginning of the third cycle, we found a worsening of cutaneous toxicity, with lesions resembling cigarette burns Figure 6At this point, we asked a dermatologic consultant, in order to evaluate and treat these lesions. Since lesions were limited to the upper arms, with less than 30% of skin involved, and Nikolsky sign showed a negative result, we considered this skin eruption as prurigo-like and our dermatologist prescribed azithromycin 1 cp/day for 3 days, fluorescein lotion, silver nitrate gel, cicatrizin gel, zinc oxide, delicate hand cleanser, and nitrile gloves. The patient started this treatment without discontinuation of cabozantinib. At the subsequent visits, the patient showed a reduction in the skin toxicity Figure 6However, in August 2021, due to grade 3 gastrointestinal toxicity and an episode of syncope with hypotension, we discontinued treatment with cabozantinib, and a few months later, in October 2021, we decided to resume the cabozantinib at a lower dosage (40 mg).The total body CT scan, performed in November 2021, showed a partial response of the disease. The patient is still under treatment, with no other severe toxicity.Cutaneous adverse events may occur frequently with the use of cabozantinib . CabozanThe hand–foot syndrome  is a potentially painful dermatological condition, reported by 43% of the patient in the METEOR trial. The mechanism by which HFS develops is not fully understood; it is possible that the drug interferes with pericyte‐mediated endothelial survival mechanisms, leading to damage to the capillary endothelium in the hands and feet  or the iToday, most patients with mRCC receive systemic therapy that is ICI- or target-based, alone or in combination with each other, and may develop drug-related symptoms of different grades of severity. With the introduction of novel combinations, there was a dramatic improvement in the outcome of mRCC patients but also the occurrence of adverse events more difficult to manage, as compared with those observed with the previously used TKIs. Furthermore, due to the relatively recent introduction of these combinations in clinical practice, their cumulative dose adverse effects are still unknown. Furthermore, as the immunotherapy may affect any organs, related toxicities are often misunderstood, before becoming from moderate to severe. A prompt recognition and management of these toxicities represents a fundamental issue in oncological clinical practice, since it correlates with the outcome of cancer patients. Although both European and Italian guidelines give well-established protocol to treat immune-related toxicities according to different grades of severity , 67, a sIn this context, MDT evaluations should be provided in any cancer center, especially for those patients, not only the most elderly and fragile, who should be investigated for preexisting unhealthy conditions, which may require a prompt support to finalize their treatment’ program.In the present paper, we reported a case series of critical toxicities that occurred in our center during treatment for mRCC and a literature review, with the aim of supporting the MDT’s role in genitourinary cancer care. Indeed, the different specialized disciplines integrated in the genitourinary MDT have demonstrated to help oncologists by providing a better care to mRCC patients, mainly during treatment and follow up. Joining the efforts from different healthcare professionals improves patient management, by an early recognition of treatment side effects and relief of severe symptoms that may occur during treatment with both immune- or target-based therapy. In this way, by preventing and reducing drug-related adverse events, patients’ quality of life as well as adherence and compliance to therapies became better . AccordiMR and MP write the manuscript. All the authors revised and approved the manuscript.


The asymmetrically localized PINs usually determine the directionality of intercellular auxin flow. Different internal cues and external stimuli modulate PIN polar distribution and activity at multiple levels, including transcription, protein stability, subcellular trafficking, and post-translational modification, and thereby regulate auxin-distribution-dependent development. Thus, the different regulation levels of PIN polarity constitute a complex network. For example, the post-translational modification of PINs can affect the subcellular trafficking of PINs. In this review, we focus on subcellular trafficking and post-translational modification of PINs to summarize recent progress in understanding PIN polarity.Auxin regulates plant growth and tropism responses. As a phytohormone, auxin is transported between its synthesis sites and action sites. Most natural auxin moves between cells  During clc2 clc3 double mutants show that disruption of clathrin formation influences hypocotyl growth by changing PIN3 lateral localization and causes PIN3 relocalization after exposure to blue light , display defective PIN localization P2, which influences the formation of clathrin vesicles, is required for the establishment of PIN polarity  and Golgi BFA resistance factor (GBF) initially  are recruited to change the Arf type from GDP to GTP . Brefeldnitially . Among Gnitially . GNOM isnitially , 2014. Gnitially . Inhibitnitially . Localiznitially . GNL1 plnitially . In contnitially . There initially . In addinitially . Some renitially .RGTB1, which catalyzes the Rab prenylation to assist Rab to bind more stably to the membrane, impairs the recycling of PIN1 and PIN3 , FORKED1-LIKE (FL), and SCARFACE (SFC) were detected in the TGN and were proposed to influence the secretory pathway that transports PIN1 to the apical PM during leaf vein development . CHOLINEsec6, sec8, and exo70 mutants, show that the exocyst directly influences polar exocytosis of PINs . In addition, certain phosphatases dephosphorylate PINs  and WAG2; pid mutants are phenotypically similar to pin mutants, the relationship between these two genes was studied , a member of the AGC family, was first found to be related to PINs. In  studied . PID wasignaling . Benjamiignaling . Overexp mutants . Given t mutants . In addi mutants . WAG1 an mutants . These t mutants . More im mutants . PID2 ha kinases . Despite kinases . Some stactivity . The chaactivity . Thus, aTwo important factors upstream and downstream of PID that regulate the PIN polarity should be mentioned. The first protein is associated with lipids. PHOSPHOLIPASE D (PLD) is responsive to many environmental signals. Under salt stress, PLD ζ 2 is involved in the endocytosis of PIN2 . Also, sd6pk mutants show some similar morphological phenotypes to pid mutants, some differences between D6PK and PID have been noted. Loss-of-function and overexpression of D6PK do not change PIN polarity (The D6 PROTEIN KINASE (D6PK) belongs to the AGC family. It is generally accepted that D6PK as well as D6PKL1, D6PKL2, and D6PKL3 phosphorylate S1, S2, S3, S4, and S5 of PINs  and CANALIZATION-RELATED RECEPTOR-LIKE KINASE (CANAR) are kinases that influence PIN1 polarity and respond to auxin . The kinpp2a subunits, whereas knocking out PID rescues pp2a A subunit mutants  is a heterotrimeric protein consisting of two regulatory subunits, A and B. It is involved in many processes that counteract PID. The PP2A A subunit is able to dephosphorylate PIN1 to change the polarity. Overexpression of PID further impairs mutants of  mutants . In addipression . Interespression . An addipression . Howeverpid mutants is basal and in PID-overexpression mutants is apical; in crk5 mutants is apical), but the phosphorylated PINs are observed on each side of the PM , the addition of nitric oxide (NO), is controlled by NO concentrations and denitrosylation catalyzed by thioredoxin and S-NITROSOGLUTATHIONE REDUCTASE GSNOR; . The lacThe PIN-mediated short-distance transport of auxin is important to regulate plants growth and for tropic responses. A model to explain the distribution of PINs has been formulated. PINs are secreted, recycled or degraded through intracellular trafficking, in which GNOM plays a role to recycle the basal PINs, and PINs can be phosphorylated to change the polarity. However, some gaps in this model remain. Only GNOM or GNOM-like ARF-GEFs are known to be responsible for the recycling, but additional factors, such as the myosin and other Arf and Rab proteins are still poorly studied . Given tSC wrote the original manuscript and made the figures. SC and YW revised and completed the writing of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Hainan Yazhou Bay Seed Laboratory (B21HJ0220), National Natural Science Foundation of China (31871537 and U2003115) and Zhejiang Provincial Natural Science Foundation of China (LR21C020001).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Exostoma is a group of stenotopic and rheophilic glyptosternine catfishes distributed in South and Southeast Asia. So far, comprehensive studies on mitogenomics referring to this genus are very scarce. In this study, we first sequenced and annotated the complete mitochondrial genomes of Exostoma tibetanum and Exostoma tenuicaudatum—two sympatric congeners from the lower Yarlung Tsangpo River, Tibet, China. The mitogenomes of both species contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, one light-strand origin of replication, and one control region, with lengths of 16,528 bp and 16,533 bp, respectively. The mitogenome architecture, nucleotide composition, and codon usage of protein-coding genes were almost identical between the two Exostoma species, although some estimated parameters varied. Phylogenetic analysis strongly supported the monophyly of Exostoma in the subfamily Glyptosternae, and Exostoma tibetanum had the closest relationship to Exostoma tenuicaudatum. The divergence time estimation demonstrated that these two species diverged approximately 1.51 Ma during the early Pleistocene, which was speculated to be triggered by the river system changes caused by the uplift of the southeastern Tibetan Plateau. Selection pressure analyses indicated that all protein-coding genes of Exostoma species underwent a strong purifying selection, while minority positive sites from NADH dehydrogenase complex genes were detected. These findings are expected to promote our understanding of the molecular phylogeny of the genus Exostoma and provide valuable mitogenomic resources for the subfamily Glyptosternae.The genus  PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), one light-strand origin of replication (LO), and one control region (D–loop) [The mitochondria are important semi-autonomous organelles in eukaryotic cells as their main venues of aerobic respiration, and generally the main energy production center of ATP for eukaryotes. The typical mitochondrial genome (mitogenome) is a closed-ring and double-stranded compact DNA molecule. For vertebrates, the length of the mitogenome ranges from approximately 15 to 20 kb, and comprises 13 protein-coding genes ((D–loop) .Exostoma Blyth 1860—a clade of glyptosternine catfishes—can be distinguished by their having a continuous post-labial groove, flattened ventral surface of the body with horizontally patulous paired fins specified for adhesion, and distally flattened oar-shaped homodont dentition in two separated patches on both the upper and lower jaws [Exostoma have been described and validated [Exostoma have been recorded, namely, Exostoma tibetanum and Exostoma tenuicaudatum [The subfamily Glyptosternae is a specialized group of catfishes belonging to the order Siluriformes and family Sisoridae, and is found primarily in the Tibetan Plateau and adjacent drainage basins. The genus wer jaws ,6. Fishewer jaws . So far,caudatum .Exostoma species have been published [Exostoma are required to improve the accuracy of mitogenome features of this genus, as well as to promote the analysis of their phylogenetic positioning and evolutionary history. Keeping this background in mind, herein, we first sequenced, annotated, and characterized the complete mitogenome sequences of two sympatric Exostoma species distributed in the lower Yarlung Tsangpo River drainage, and then analyzed the mitogenome features of both species with respect to genome architecture, nucleotide composition, and the codon usage of PCGs. Then, we performed the phylogenetic analysis to determine the relationship of the genus Exostoma within the subfamily Glyptosternae based on the concatenated PCG sequences, as well as to estimate the divergence time and the gene selection pressure of this genus.To date, the complete mitogenome sequences have been determined for dozens of glyptosternine catfishes. However, very few documents of the mitogenomics of ublished . TherefoExostoma tibetanum and Exostoma tenuicaudatum were collected from the Xigong River—a tributary of the lower Yarlung Tsangpo River in Motuo County, southeast Tibet, China . After collection, the muscle tissues were taken and preserved in dehydrated alcohol and stored at −20 °C. Total genomic DNA was extracted from muscle tissue using the TSP201 Animal Tissue DNA Extraction Kit  according to the manufacturer’s instructions. The concentration and quality of DNA were assessed by agarose gel electrophoresis.The samples of Exostoma tibetanum and 4126 Mb for Exostoma tenuicaudatum were generated. The raw sequencing reads were trimmed and filtered using SOAPnuke to remove adapter contamination and low-quality reads, with a cutoff of Phred quality scores of Q20. Then, about 3999 and 4104 Mb of clean data were obtained for Exostoma tibetanum and Exostoma tenuicaudatum, respectively.Extracted genomic DNA was sent to Novogene  and prepared for sequencing on the Illumina HiSeq platform . High-quality DNA samples were randomly broken into fragments for paired-end sequencing (2 Â 150 bp), and DNA libraries were constructed according to the procedure of Illumina DNA library construction. Approximately 4013 Mb of raw data for Exostoma gaoligongense [Assemblies of the clean reads obtained de novo were conducted using SPAdes 3.9  to obtaigongense . Composigongense . The nucgongense . The RSCgongense .Bagarius yarrelli, Glyptothorax annandalei, and Pseudecheneis sulcatus were selected as the outgroup. Their distribution information, GenBank accession numbers, and mitogenome size are given in PCG and the three codon positions were conducted in DAMBE v6 by comparing the index of substitution saturation (Iss) against the critical Iss (referred as Iss.c) at which the sequences began to fail to recover the true tree [PCGs were concatenated to a combination of sequences set without a termination codon to detect the phylogenetic relationships between the two Exostoma species and their congeners. Moreover, we also compared the topologies of each PCG and the concatenated PCGs using the Shimodaira–Hasegawa test from the “phangorn” package in R software to examine the statistical significance of alternative topologies [The mitogenomes of 18 glyptosternine catfishes, covering 9 genera obtained from the NCBI GenBank—including the two new mitogenomes determined in the present study—were used to perform the phylogenetic analysis. rue tree . Then, tpologies ,18.PCGs were determined using Bayesian inference (BI) and maximum likelihood (ML) methods. The best-fit nucleotide substitution model was identified utilizing the corrected Akaike information criterion with PartitionFinder v2.0 on the PhyloSuite platform [Phylogenetic trees inferred from concatenated platform . The BI platform , with thplatform  using thExostoma species was estimated based on a dataset of 13 PCG sequences with a relaxed molecular clock method in the program BEAST v.1.10 [Bagarius yarrelli (5.3~1.8 Ma) was set as the calibration point [The divergence time of T v.1.10 . The foson point . A Yule on point . The conon point , and wason point .PCGs is often characterized by the nonsynonymous-to-synonymous substitution ratio , which is an effective indicator applied for detecting the direction and magnitude of selective pressure on amino acid substitution. The ω values greater than 1, equal to 1, and less than 1 indicate purifying selection, neutral evolution, and positive selection, respectively [Exostoma species, the codeml program with the branch model (free-ratio model parameters) was performed for each PCG and the concatenated PCGs in EasyCodeML [ω values for each clade on the tree. Furthermore, in order to detect the positive selection on certain codons instead of entire genes, we applied the branch-site model with a likelihood ratio test under the null hypothesis of the substitution rate ω fixing to 1. To be specific, we respectively set Exostoma tibetanum, Exostoma tenuicaudatum, and Exostoma gaoligongense to the foreground, which computed the ω value for each codon in 13 PCGs of 3 species.The natural selection acting on ectively ,28. To csyCodeML , which cExostoma tibetanum (GenBank accession no. ON641840) and Exostoma tenuicaudatum (GenBank accession no. ON641841) was 16,528 and 16,533 bp in length, respectively. The mitogenome content, gene arrangement, and gene-coding strands of both species were in accordance with the vertebrate consensus [PCGs, two rRNAs, 22 tRNAs, one D–loop, and one LO. The total length of the PCGs was 11,411 bp, accounting for 69.0% of the entire mitogenome for both species. Of all of the components, one PCG (ND6), eight tRNAs , and LO were located on the light (−) strand, while the remaining genes were encoded on the heavy (+) strand  strand . There wExostoma tibetanum and 55.2% in Exostoma tenuicaudatum. The content of A + T in different regions for Exostoma tibetanum was 54.4% (PCGs), 53.8% (rRNAs), 55.6% (tRNAs), and 62.4% (D–loop); while it was 54.8% (PCGs), 54.4% (rRNAs), 56.4% (tRNAs), and 59.6% (D–loop) for Exostoma tenuicaudatum. For PCGs, to be specific, the second and third codons were also A/T-biased in nucleotide composition, except for the first codon (48.1% for both species). This base pattern is consistent with most previously reported mitogenomes of Siluriformes catfishes [Exostoma tibetanum and 0.013 to 0.523 for Exostoma tenuicaudatum, except for the second codon position of the PCGs, which implied that A had more content than T (−0.371 for Exostoma tibetanum and −0.367 for Exostoma tenuicaudatum). In contrast, except for the CG skewness for the tRNA regions close to zero (0.0009 for Exostoma tibetanum and −0.002 for Exostoma tenuicaudatum), all of the other CG skewness values were negative—from −0.631 to −0.042 for Exostoma tibetanum, and from −0.635 to −0.050 for Exostomatenuicaudatum—indicating a higher content of C than G  as the stop codon, whereas ND6 of Exostoma tibetanum uses AGA as the stop codon, and four genes  exhibit incomplete stop codons (T or TA)  . Despitetebrates ,36.PCGs codons, in spite of a few divergences (Exostoma tibetanum were CUN (2Leu) with 523 times, GCN (Ala) with 337 times, and UAY (Tyr) with 333 times, whereas the most frequently used codons in Exostoma tenuicaudatum were CUN (2Leu) with 529 times, GCN (Ala) with 325 times, and AUY (Ile) with 291 times. UGY (Cys) was the least used PCG codon, with 30 times in Exostoma tibetanum and 31 times in Exostoma tenuicaudatum. Moreover, the RSCU values of the 13 PCGs for both mitogenomes were calculated, and are visualized in Both mitogenomes showed coincident distribution patterns for the concatenated ergences . The mosPCG and the concatenated PCGs, the test of substitution saturation indicated that the values of Iss were all significantly smaller than Iss.c (p < 0.01), suggesting no saturation in the PCGs dataset, rendering it suitable for phylogenetic analysis. First, phylogenetic trees were generated based on the 13 single PCGs and the concatenated dataset. The Shimodaira–Hasegawa test suggested that the 13 topologies inferred from single PCGs showed a significant reduction (p < 0.01) in log-likelihood (ln L), and were statistically different from the tree topology inferred from concatenated PCGs  monophyletic group in the subfamily Glyptosternae. Specifically, E tenuicaudatum is a sister clade to its sympatric congener Exostoma tibetanum, with a Kimura two-parameter distance of 9.2%. Then, both species were clustered with Exostoma gaoligongense, and next grouped with species of other glyptosternine genera.For each ted PCGs . Meanwhias given . As the e et al.  inferrede et al. . In addiExostoma tibetanum and Exostoma tenuicaudatum arose 1.51 Ma (95%HPD: 0.25–3.12 Ma) during the early Pleistocene. The divergence time of Exostoma gaoligongense and the two aforementioned species was computed at 2.27 Ma, with a 95%HPD of 0.47–4.49 Ma (Exostoma species to 5.52 Ma during the late Miocene (5.33–23.03 Ma). This discrepancy may account for the inclusion of more Exostoma species in the present study so as to determine the accurate topology of the evolutionary tree. Peng et al. [Exostoma—branched off from their ancestors, facilitating the unique glyptosternine fish diversity patterns in this region.The divergence time with 95% highest posterior density interval (95%HPD) estimated for the most recent common ancestor of –4.49 Ma . These rg et al.  concludeg et al.  indicateg et al. ,44. It iω values for the three Exostoma species yielded from the branch model in the codeml program showed that the ω values of the 13 PCGs, as well as the concatenated genes, were all less than 1, ranging from 0.0001 (COX2 of Exostoma tibetanum and Exostoma tenuicaudatum) to 0.5699 (ATP8 of Exostoma gaoligongense)  are potential barcoding markers for the delimitation of Exostoma species. Ma [ω values of basal glyptosternine fishes are lower than those of specialized species, implying that this group of species has undergone rapid evolution of the subfamily Glyptosternae through the dispersal process. The estimation results of this study reveal that Exostoma is a relatively old fish group in the subfamily Glyptosternae, which is consistent with Ma’s conclusions.The separate teleosts ,46,47. Trganisms ,49,50. Tcies. Ma  has indiExostoma tibetanum as the foreground, the model detected moderately significant output of the positive site at ND1, with residue 103 as a positively selective site at a 90% confidence level. When Exostoma gaoligongense was selected as the foreground, the model determined significant evidence at ND6, with residue 46 deemed to be a positively selected site at a 95% confidence level  for the first time. The circular mitogenomes displayed lengths of 16,528 bp and 16,533 bp, respectively. Both mitogenomes encoded a typical set of 37 genes with conservative gene arrangement, which contained 13 PCGs, 2 rRNAs, and 22 tRNAs, together with one LO and one D–loop. Additionally, the nucleotide composition, skew values, codon usage, and RSCU of the 13 PCGs, were comparatively analyzed. Phylogenetic analysis inferred from the PCG sequences demonstrated that three included Exostoma species are clustered into monophyletic clades nested within the subfamily Glyptosternae, of which Exostoma tibetanum is a sister clade to Exostoma tenuicaudatum. Estimation of divergence time revealed that the speciation of both sympatric species might have been triggered by river system changes in the lower Yarlung Tsangpo River, accompanied by the uplift movement in the southeastern Tibetan Plateau during the early Pleistocene. The selective pressure analysis for Exostoma species demonstrated homogeneous purification selection for each gene, with several positively selective sites detected in NADH dehydrogenase complex genes, which may be associated with the adaptation to their cold and hypoxic habitats. The molecular data obtained could provide insights into the mitogenome architecture and molecular phylogeny of Exostoma fishes. In the future, more species of Exostoma should be included in the mitogenomics analysis to promote a better understanding of the molecular evolutionary processes of this genus.In this study, we sequenced and annotated the mitogenomes of two sympatric catfishes of


The discovery of a large number of small pulmonary nodules and early diagnosis of lung cancer in the diabetic patients prompt us to re-examine the relationship between diabetes and the occurrence and development of lung cancer. The aim of this study was to explore the underlying metabolites changes in diabetes with NSCLC or benign nodule patients, and further to investigate the association of serum IGF-1 level and differentially expressed metabolites (DEMs). An untargeted metabolomics method was used to detect the changes of metabolism in diabetic patients with NSCLC on the platform of HR-MS. Serum level of IGF-1 was measured by ELISA. The patients were divided to three groups, DM, DLB (nodule), and DLC (cancer). we have identified numerous DEMs, which include amino acid, choline, and fatty acid derivatives. Further analysis of the involved metabolic pathways suggested that linoleate metabolism, tryptophan metabolism, histidine metabolism, putative anti-Inflammatory metabolites formation from EPA, and arachidonic acid metabolism were considered to be the most significant metabolic pathways between groups. Networks analysis suggested that a series of metabolites were associated with serum IGF-1among the three groups, which can be divided into 6 categories. Nine metabolites have been identified as the main DEMs among the DLC, DLB, and DM groups. In conclusion, metabolomics is a powerful and promising tool for the cancer risk evaluation in diabetic patients. Our results suggest that decreased IGF-1 level is associated with restrained amino acid metabolism in NSCLC with diabetes mellitus. The IDF Diabetes Atlas estimates that the global prevalence of diabetes mellitus (DM) is over 10% in 2021 .According to the Global Cancer Statistics 2020 , lung caHowever, the underlying mechanism has been constantly explored. Many factors have been reported to be involved in the development and progression of cancer in DM, including hyperinsulinemia , elevateWith the continuous prevalence of COVID-19, chest CT has become an ordinarily screening program for diabetic patients, followed by the discovery of a large number of small pulmonary nodules and early diagnosis of lung cancer in the clinical practice. Is this just an accidental event, or an inevitable result of diabetes? This problem forced us to re-examine the relationship between diabetes and the occurrence and development of lung cancer. Metabolomics, as a semi-quantitative analysis method of metabolites, can achieve the purpose of quantitative measurement of most endogenous metabolites in a single blood sample . MetabolIn this Study, we conducted the serum IGF-1 analysis and the untargeted metabolomic strategy in the population of diabetes with lung cancer and nodules. This study aimed to highlight the mechanistic insights involved in the association between diabetes and lung cancer.All the participants enrolled in this study were from the department of Endocrinology and Thoracic Surgery in Xuzhou Central Hospital since April 2019 to July 2021, who were informed of the relevant benefits and risks in writing and voluntarily signed the informed consent. Study population include three groups  ≤ 30 kg/m^2; Diabetic patients with no abnormal pulmonary CT were taken as the DM group; Diabetic patients with small lung nodules but no malignant signs, were enrolled in the DLB group; For those with malignant signs, such like irregular edge, burr sign, halo sign, calcification, ground glass density, and infiltrative growth, who eventually undertaken surgical resection and confirmed as NSCLC by pathology, were taken as DLC group. Exclusion criteria: Patients with a past history of cancer, pneumonia and any other site’s acute inflammation, hypertension, acute heart infarction, severe hepatic lesion, renal dysfunction, severe anemia, and other serious situations.Anthropometric measurements were obtained from a clinical questionnaire, including age, gender, age, smoking status, and other basal information. Weight and height were measured in kg and cm, respectively. BMI was calculated as weight (kg)/height^2 (m^2). Blood pressure measurement was taken by a physician using a mercury sphygmomanometer, which was after 30 minutes rest at least.Blood samples were collected after fasting for at least 10 hours and used for the detection of hepatic function, renal function, lipid profile, fasting blood glucose (FBG), and 2 hours postprandial blood glucose (2hPG). All of the measurements were taken at the clinical laboratory of Xuzhou Central Hospital. Serum samples for the metabolomics analysis were frozen at -80°C. An ELISA KIT  was used to assay the serum levels of IGF-1. The detection process refers to the instructions of ELISA kit. The standard sample shall be diluted. After adding the sample and enzyme, incubate for 10 minutes at 37°C. Finally, the OD value will be obtained at 450nm by using the microplate reader.Serum samples were pretreated before detection by HPLC-MS. In this process, 200ul serum was taken, 1200 μ L of precooled methanol and acetonitrile were used for the sample protein precipitation. After 10 minutes standing, the mixture was centrifuged at 12000g for 10 minutes. The supernatant was volatilized and dried by rotary evaporation to obtain dry powder. Finally, acetonitrile (50μ L) was used to dissolve the dry powder.LC separation was performed on the Waters E2695 Ultra HPLC  platform using the Fortis type C18 column  at a column temperature of 40°C. Water/acetic acid  is used as solvent A and methanol is used as solvent B in the mobile phase. During the injection, take 5 ul sample volume. Gradient is 0 min, 95% (A); 1 min, 75% (A); 3 min, 55% (A); 6 min, 5% (A); 12 min, 5% (A). The post-treatment time is 2 min with a flow rate of 0.3 ml/min. Positive and negative modes for performing MS analysis in full scan mode on the Thermo HFX mass spectrometer . The solution is injected into an ESI source and then at a rate of 0.3 ml/min, capillary of 4000 V, dry gas of 12 L/min, and dry gas temperature of 350°C. Optimization of the MS conditions was repeated for maximum detection sensitivity. Prior to sample analysis, in order to guarantee system stability, quality control (QC) samples were tested 6 times and mixed with all serum samples.The deconvolution of LC-MS spectrum was analyzed by Thermo Data Analysis software, including baseline correction, noise process, and peak alignment. Retention time (m/z) formed matrix analysis was performed by compound detection. R4.1.3 software was preformed to analyze the metabolites data from LC-MS. The principal component analysis (PCA) was used to show the difference among three groups , and theThe basic clinical characteristics of the study subjects were presented in To ensure the stability of our HPLC-MS system, we both conducted the positive ion mode and the negative ion mode. The QC samples results suggested to be clustered with a small relative standard deviation (RSD) of different peaks, which showed the stability of our HPLC-MS system. The raw MS data in the Thermo Data Analysis software was used to establish data matrix and then analyzed by OPLS-DA and PCA. As shown in the OPLS-DA was further conducted to investigate the DEMs between each two groups. Serum samples were clearly distinguished from groups either in the positive mode or in the negative mode. The Q2Y and R2Y values between the diabetes group and diabetes with lung cancer group were 0.978 and 0.990, 0.946 and 0.995, respectively (Based on the database KEGG and inhouse reference metabolites, Thermo Data Analysis software was used to annotate the peaks of the LC-MS chromatograms. To identify the DEMs among the three groups, further analysis of those metabolites with fold change >2 or <0.5 was performed. There were 3605 down-regulated and 328 up-regulated metabolites between diabetes and cancer groups (MetaboAnalyst web tool was used for the metabolic pathway enrichment analysis between two groups, DLC vs DM, DLB vs DM, and DLC vs DLB, separately. Metabolic pathways involved in linoleate metabolism, C21-steroid hormone biosynthesis and metabolism, histidine metabolism, 3-oxo-10R-octadecatrienoate beta-oxidation, tryptophan metabolism, D4&E4-neuroprostanes formation, putative anti-Inflammatory metabolites formation from EPA, and arachidonic acid metabolism were considered to be the most significant metabolic pathways between DLC and DM (As shown in Networks analysis suggested that a series of metabolites were associated with serum IGF-1 between DLC and DM group, of which the correlation coefficient is > 0.5 or < - 0.5 were shown in According to the general data of the study population, there was no significant difference in the age, sex, smoking status, BMI, course of diabetes, glycated hemoglobin among the group of DM, DLB and DLC. The consistency of baseline ensures the reliability of subsequent analysis.Since the relationship between serum IGF-1 and cancer was not always in consistency, we performed the detection of IGF-1 concentration in the serum of DM, DLB and DLC. The result comes to be decreased IGF-1 levels in DLC and DLB group compared with DM. Kentaro once reported that serum IGF-1 levels were significantly decreased in Japanese patients with uncontrolled T2DM . The parTo further explore the effect of diabetes on the development and progression of NSCLC, untargeted metabolomics analysis was used to examine the metabolite changes in serum of NSCLC and lung benign nodule in diabetes mellitus on the platform of HPLC-MS. In order to get a better distinction, we subjected the mass spectrometry data to OPLS-DA and PCA analyses. Numerous DEMs have been identified in the present study, especially those involved in amino acid metabolism, which is essential for lung cancer cell proliferation and regulation of immune cell function , 24. Ourin vivo, through which to participate in cancer immune regulation (According to the DEMs, we analyzed the involved metabolic pathways. The linoleate metabolism, tryptophan metabolism, histidine metabolism, putative anti-Inflammatory metabolites formation from EPA, and arachidonic acid metabolism were considered to be the most significant metabolic pathways between group DLC and group DM. Interestingly, we also found the alteration of tryptophan metabolism pathway between DLC and DLB, so does the comparison of DLB and DM, while the tryptophan metabolism pathway was considered as the potential pathway in the pathogenesis of several lines of cancer. Tryptophan can be enzymatically hydrolyzed into kynurenine gulation . Our resgulation  has repogulation . The dowIn conclusion, metabolomics is a powerful and promising tool for the cancer risk evaluation in diabetic patient. Our results suggest that decreased IGF-1 level is associated with restrained amino acid metabolism in NSCLC with diabetes mellitus.The original contributions presented in the study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by The Ethics Committee of Xuzhou Central Hospital. The patients/participants provided their written informed consent to participate in this study.HG, HH, and CL designed the experiments and analyzed the data. HG, HL, YL, CL, FZ, YM, JL, SS, YY and XL performed the experiments. HG and YL supervised the study. HG, HL, and FZ wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Science and Education Project for Young medical talents, Jiangsu Province, China, No. QNRC2016388; The social development project of the Xuzhou Municipal Science and Technology Bureau, No. KC21231.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


DMPK locus and neighbouring genes, in distinct DM1 tissues and derived cells, representing six DM1 subtypes, by bisulphite sequencing. In blood, we found no differences in CpGi 74, 43 and 36 in DNA methylation profile. In contrast, a CTCF1 DNA methylation gradient was found with 100% methylation in congenital cases, 50% in childhood cases and 13% in juvenile cases. CTCF1 methylation correlated to disease severity and CTG expansion size. Notably, 50% of CTCF1 methylated cases showed methylation in the CTCF2 regions. Additionally, methylation was associated with maternal transmission. Interestingly, the evaluation of seven families showed that unmethylated mothers passed on an expansion of the CTG repeat, whereas the methylated mothers transmitted a contraction. The analysis of patient-derived cells showed that DNA methylation profiles were highly preserved, validating their use as faithful DM1 cellular models. Importantly, the comparison of DNA methylation levels of distinct DM1 tissues revealed a novel muscle-specific epigenetic signature with methylation of the CTCF1 region accompanied by demethylation of CpGi 43, a region containing an alternative DMPK promoter, which may decrease the canonical promoter activity. Altogether, our results showed a distinct DNA methylation profile across DM1 tissues and uncovered a novel and dual epigenetic signature in DM1 muscle samples, providing novel insights into the epigenetic changes associated with DM1.Myotonic dystrophy type 1 (DM1) is a progressive, non-treatable, multi-systemic disorder. To investigate the contribution of epigenetics to the complexity of DM1, we compared DNA methylation profiles of four annotated CpG islands (CpGis) in the  Myotonic dystrophy type 1 (DM1) is an autosomal dominant inherited, multi-systemic disorder, with predominant muscle involvement and an estimated incidence of 1:8000 . These cEpigenetics is defined as heritable changes that do not affect the DNA sequence itself but influence gene expression and it includes DNA methylation, histone modifications, and non-coding RNAs . DNA metDMPK) gene (the gene carrying the disease causing CTG expansion in its 3′ untranslated region) and neighbouring genes (henceforth referred to as the DMPK locus) contain several CpG islands (CpGis). CpGi 374 has gained the most attention because this 3.5 kb island contains the expanded repeat. In addition, the CTG repeat is flanked by two CCTC-binding factor (CTCF) binding sites, named CTCF1 and CTCF2. Early studies suggested that the two CTCF binding sites together with the expanded repeat established an insulator element between the DMPK promoter and the six homeobox 5 (SIX5) enhancer, affecting chromatin dynamics [DMPK coding sequence [DMPK gene body , and raises an interest for the study of the epigenetic state of the other CpG islands located at the DMPK gene in DM1.The dystrophia myotonica protein kinase  gene, CpGi 74, and two in the DMPK gene, namely CpGi 43 and 36; however, nothing is known about the epigenetic state of these CpG islands in DM1. To date, only one publication has looked at the entire DMPK locus and this was solely done in control tissues and cell cultures [DMPK gene neighbourhood, in regions containing CpGi 43 and CpGi 74, respectively. Furthermore, CpGi 43 overlaps with a proposed alternative DMPK promoter, which could be tissue-specific and epigenetically regulated. Therefore, the main goal of this study was to elucidate the DNA methylation profiles across the four CpG islands residing in the DMPK locus in distinct DM1 tissues and tissue-derived cells across the different clinical phenotypes. Our results showed a CTCF1 DNA methylation gradient in blood of the developmental cases and CTCF1 methylation correlated to disease severity and CTG expansion size. Methylated cases showed a higher chance of maternal transmission and CTCF1 methylation in the parent was associated with a contraction of the CTG expansion upon generational transmission. Notably, DM1 patient-derived cells preserved the DNA methylation profiles observed in tissues. Finally, our results showed a DM1 muscle-specific epigenetic landscape, with a loss of methylation at CpGi 43, a region containing an alternative DMPK promoter, accompanied by a gain of methylation in the CTCF1 region in muscle and muscle-derived cells. Altogether, our results offer novel insights into the epigenetic changes in DM1.The cultures . InteresThis study was approved by the Ethics Committee of the University Hospital Germans Trias i Pujol and was performed in accordance with the Declaration of Helsinki for Human Research. Written informed consent was obtained for all participants. The study included 65 DM1 patients and 8 controls with no previous family history of neuromuscular disorders (recruited from the traumatology department in whom surgery was needed). DM1 diagnosis was confirmed or discarded with triplet primed-PCR in all the study participants. Clinical information of DM1 patients was obtained from the medical records and updated in the last visit by neurologists. Patients were subdivided into five different categories based on age of onset: congenital = first year of life, childhood = 1–10 years, juvenile = 11–20 years, adult = 21–40 years, late-onset > 40 years. Additionally, a group of asymptomatic patients was added. For three patients the exact year of age of onset could not be determined, but based on the clinical information all three were classified as adults. Clinical information included family history, and details on the last ophthalmological, cardiological and respiratory examination by the corresponding specialists, including the electrocardiograms, echocardiograms and spirometry tests performed in the last year. A full neurological work-up was performed by neurologists, including the presence of myotonia, ptosis, axial and facial weakness, muscle strength with the manual Medical Research Council (MRC) scale, and muscle impairment by the Muscular Impairment Rating scale (MIRS). In addition, the presence of cataracts, alopecia, intestinal problems, and sleep disturbances were catalogued and the functional status and degree of disability were evaluated using the DM1-Activ questionnaire and modified Rankin Scale (mRS), respectively.A total of three different samples from patients and controls were obtained: blood, muscle biopsy, and skin biopsy. Blood was obtained from all patients and lymphoblastoids were isolated when possible. From a subset of patients and controls, an additional muscle  and skin biopsy were taken, of which muscle and skin-derived cell cultures were obtained. Of note, to increase the number of biopsies, a subset of biopsies for which no blood was available were included. They included three extra patients and two controls. All samples were obtained at the same time and processed as described previously by Ballester-López and collaborators . Cell isGenomic DNA was isolated from peripheral blood by the use of the QIAamp DNA mini kit , the PureLink genomic DNA mini kit  or a simple salting procedure, as previously described by Miller and collaborators . Genomicwww.gelanalyzer.com, by Istvan Lazar Jr. and Istvan Lazar Sr., accessed on 16 February 2022). The CTG size of the progenitor (ePAL), which is the lowest range of band thought to originate from the transmitting parent, and the modal allele, which shows the densest collection of CTG sizes and thought to be the most representative size for the patient at that specific time.To estimate the length of the expanded progenitor allele, a specific long PCR with digested DNA, followed by a Southern Blot was carried out. First, 250 ng DNA was digested with EcoR I , according to the manufacturer’s protocol. Then, 750 pg of digested DNA was used in the subsequent PCR, with the Expand Long Template PCR System  and primers DM-C and DM-DR , accordiDMPK locus, divided into five individual areas, was studied by using bisulphite treatment and subsequent Sanger sequencing. For CpGi 74, CpGi 43 and CpGi 36, 19, 15 and 17 CpGs were studied respectively (The methylation status of four annotated CpG islands (CpGi) in the A total of 200–400 ng of DNA was bisulphite treated using the EZ DNA Methylation Gold kit , according to the manufacturer’s guidelines. Bisulphite-treated DNA was amplified using nested and hemi-nested PCR for the CTCF1 and CTCF2 region, located in the CpGi 374 surrounding the CTG expansion, previously described by Barbé and collaborators , with soAmplicons were purified using Illustra™ ExoProStar 1-Step , according to the manufacturer’s protocol. This was followed by sequencing using the BigDye Terminator v3.1 cycle sequencing kit , following the manufacturer’s guidelines. Afterwards, amplicons were run on an ABI Prism 3130 Genetic Analyzer  and analysed using Chromas software version 2.6.6 or FinchTV software version 1.5.0, as detailed in Carrió et al. 2016 . Sodium Dichotomous variable CTCF1 methylation status, the occurrence of abnormal methylation upstream of the repeat, was modelled as a dependent variable using a logistic regression model, against the independent variable ePAL and modal allele. The program used was SPSS 28.0.0.0 and the significance level was set at 0.05.n = 6), childhood (n = 6) and juvenile (n = 23) will also be referred to as the developmental cases, whereas adult (n = 22), late onset (n = 6) and asymptomatic (n = 2) will be referred to as the non-developmental cases. Age of onset ranged from first year of life until 67 years, with a mean of seven years for childhood, 15 years for juvenile, 31 years for adult and 52 years for late-onset. The presence of seven families was identified in this cohort. In 59 out of 65 patients, the CTG expansion could be sized, ranging from 115 to 1011 CTG repeats. Detailed information on the clinical phenotypes can be found in For this study, DM1 patients from six different subcategories were included. The first five categories are the different established clinical phenotypes: congenital, childhood, juvenile, adult and late-onset. The sixth category is a special subset of patients, which are known to carry the CTG expansion, but are as of yet asymptomatic. Congenital  and moderate facial weakness (one patient). Dysarthria was present in all patients, but to a higher degree in methylated patients, where it ranged from moderate to severe, compared to mild to moderate in non-methylated patients. Axial and limb weakness was also more pronounced in the methylated versus the non-methylated group. In the methylated cases, 2/3 had a severe limb weakness : one of the patients had a proximal and distal weakness, requiring a wheelchair and the other patient showed a proximal and distal weakness pattern needing just a cane for walking. The last one of the methylated childhood cases had a mild distal weakness . In the non-methylated group, all of the patients showed a mild weakness (MRC of 3–4), two of the patients with a distal pattern and one patient with proximal/distal weakness.Cardiac manifestations were more severe in the methylated group, as 2/3 patients had a pacemaker, whereas in the case of the non-methylated group only mild changes in electrocardiogram were seen, such as a mild first grade AV block in 2/3 cases. Two of the methylated patients and one of the non-methylated patients used ventilatory support. Cognitive manifestations were noticed in all methylated childhood DM1 patients: two of the patients had severe cognitive delay, while moderate learning difficulty was observed in the third patient. In the non-methylated group, just one patient had learning difficulties, while the others showed no mental disabilities. All methylated patients experienced hypersomnolence and none of the non-methylated patients suffered this symptom. Intestinal rhythm dysfunction was found exclusively in the methylated patients, while alopecia was found in the non-methylated patients only.At the time of revision, all patients had some degree of dependence in the mRS. In the methylated group, 2/3 needed help in the basic activities of daily life but did not require continuous supervision (mRS 4), while the other one required help for instrumental activities (mRS 3). The average score in the DM1-ACTIV scale was 12.33. In the non-methylated group, we found one patient with mRS 4, another one with mRS 3 and the last one had a milder dependence (mRS 2) and the average score in the DM1-ACTIV scale was 28.33, which means they were better at performing daily and social activities. Considering all this data as a whole, there seemed to be a more severe muscular, cardiac, and cognitive manifestation of the disease in the methylated childhood cases.n = 3) and non-methylated group (n = 20). Although, this might be due to the low number of cases, and analysis of a larger cohort of this DM1 subcategory would be needed to address the impact of CTCF1 methylation on this clinical subtype  and between the modal allele and the methylation status (p = 0.001), which suggests that the larger the ePAL/modal, the more likely methylation at the CTCF1 regions is going to occur.As mentioned before, methylation of the CTCF1 region is almost exclusively found in the developmental subtypes. These subtypes are associated with a higher disease severity and overall greater CTG expansion sizes. To see whether methylation status in DM1 patients is associated with the CTG expansion size, we performed a logistic regression on the entire cohort using the ePAL and modal allele. This revealed a positive association between ePAL and methylation status of the CTCF1 region in DM1 patients . From these biopsies, cells were isolated to assess whether cellular models accurately reflect the origin tissue in terms of DNA methylation status. In CpGi 74 and CpGi 36, high DNA methylation levels were found in skin and muscle of both DM1 patients and controls, similar to what was found in blood  were highly methylated, while CpGi 43  was unmethylated [Previously, it was reported that in leukocytes derived from control individuals, CpGi 36 and 74 , as well as cell type-dependent differences in promoter usage, according to epigenetic features [DMPK promoter (in CpGi 43) was suggested, by showing its hypomethylation together with hypermethylation of the canonical promoter, strong binding of CTCF and high levels of H3K4me3 in control leukocytes [DMPK expression levels in DM1 myogenic samples.Buckley and collaborators extensively studied the epigenetics in the features . The datfeatures . Conversukocytes . InteresDMPK and SIX5 expression. The loss of the insulator activity by DNA methylation would allow the interaction of the SIX5 enhancer with the DMPK promoter, increasing DMPK expression, while reducing SIX5 expression [DMPK and neighbouring genes, showed that CTCF regions and intragenic regions of DMWD and radial spoke head 6 homolog A genes (RSPH6A), located next to the DMPK gene, displays enhancer chromatin features in control muscle cells [DMPK lies in the middle of a chromosomal domain with three genes preferentially expressed in testis, indicating that its expression, mainly in skeletal muscle and heart, has to be tightly regulated. Additionally, Brouwer and collaborators showed an increase in the H3K9me3 chromatin repressive mark, together with gain of DNA methylation, in the CTCF1 region (and to a lesser extent in CTCF2) in DM1 mice hearts, which correlates with decreased DMPK expression [DMPK promoter and these potential myogenic enhancers, further experiments are needed.Additionally, this DM1 tissue-dependent demethylation was accompanied by a gain of methylation in the CTCF1, but not the CTCF2 region. Previous studies showed that CTCF, a transcription factor that can function as an insulator, binds strongly to CTCF1, but not CTCF2, in a methylation-dependent manner . Importapression . Notablyle cells . This ispression . To addrFinally, this study addressed for the first time the DNA methylation status of patient-derived DM1 cells. The availability of several DM1 tissues and their corresponding tissue-derived cells gave us the opportunity to determine that most cellular models maintained the DNA methylation state observed in the original tissue. However, in some cases, we observed a slight gain of methylation in cultured cells  versus the corresponding tissues. This can be explained by the observation that cellular models, especially immortalized cell lines or primary cell cultures that have been in culture for a substantial amount of time, can increase DNA methylation levels ,42,43 anDMPK gene body of DM1 muscle samples, which provided novel insights into the epigenetic changes occurring in DM1.In conclusion, our results showed a distinct DNA methylation profile across DM1 tissues and uncovered a novel, dual epigenetic signature involving a gain of DNA methylation in the flanking region of the CTG expansion, accompanied by specific DNA demethylation in the