To verify the clinical efficacy of Zhang’s Xibi formula (ZSXBF) and explain the mechanism underlying its therapeutic effect.Preliminary elucidation of the clinical efficacy of ZSXBF in treating KOA in self-control studies, exploration of its mechanism of action with network pharmacology methods, and validation in animal experiments.In clinical studies, ZSXBF administration effectively improved patient quality of life and reduce pain. Network pharmacology was used to explore the possible mechanisms underlying its treatment effect, and after verification in clinical experience and animal experiments, it was found that ZSXBF regulated the expression of immune-related proteins such as IL-17, ERK1, and TP53 in mouse knee joints.ZSXBF, which is a traditional Chinese medicine compound that is used to clear heat and detoxify, can effectively improve the clinical symptoms of KOA patients, and its underlying mechanism includes the regulation of human immune-related proteins.The online version contains supplementary material available at 10.1186/s13018-023-04453-6. Osteoarthritis (OA) is a major cause of pain and disability worldwide , 2. OA iAt present, there is no definite and effective drug for the treatment of OA, and its clinical treatment mainly involves symptomatic treatment, for example, the use of nonsteroidal anti-inflammatory drugs (NSAIDs) to relieve pain; however, when NSAID treatment is ineffective, there are almost no drugs available for follow-up treatment . The lacZhang’s Xibi formula (ZSXBF) is the result of many years of experience in clinical practice by a famous teacher of traditional Chinese medicine in Zhejiang Province. This compound prescription, which is mainly composed of traditional Chinese medicine that is used to clear away heat and toxic materials, is considered to exert good effects in the treatment of KOA. It is generally believed that the heat-clearing and detoxification functions of this medicine may be related to the inhibition of immune responses that are caused by inflammation in the human body –9.However, the components of ZSXBF and the mechanism underlying its treatment effects against KOA have not yet been elucidated. Therefore, this study aimed to predict the key active ingredients and potential pharmacological mechanisms of ZSXBF in the treatment of OA through clinical experience, network pharmacology, and animal experimental methods.This study was approved by the Ethics Committee of Hangzhou Fuyang Traditional Chinese Medicine Bone Injury Hospital (No. 2023-LW-LC-003).Data from 146 KOA patients who were treated with ZSXBF at Fuyang Bone Injury Hospital in Hangzhou City between January 1, 2021, and December 31, 2021, were collected. To evaluate clinical outcomes, visual analog scale (VAS) and Leqiesne’s score examinations were performed one week before (baseline) and one month after treatment.ZSXBF consists of 6 Chinese herbs, including Dang-gui (DG), Chuan-niuxi (CNX), Ze-lan (ZL), Huang-bo (HB), Tu-fuling (TFL) and Mao-rensheng (MRS). The detailed data of ZSXBF are shown in Table The chemical components of the traditional Chinese medicine herbs in ZSXBF were searched in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) databases. Oral bioavailability (OB) and drug likeness (DL) were set as the screening criteria. In addition, eligible putative targets of these potential active compounds were identified in the TCMSP databases.Using knee osteoarthritis as the key word, associated target genes were identified by a joint search of the Genecards, Online Mendelian Inheritance in Man (OMIM), PharmGKB, Therapeutic Target Database (TTD), and DrugBank databases. All the collected target genes, which were in the form of abbreviations, were edited in the same Microsoft Excel table, and duplicates were removed.R was used to screen target genes that were shared by KOA and ZSXBF. Then, the STRING online tool was used to analyze the target genes to generate a protein‒protein interaction network (PPI). The enrich plot tool was used for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.https://pubchem.ncbi.nlm.nih.gov/) [http://www.rcsb.org/pdb/home/home.do). For docking analysis, all protein and molecular files were converted into PDBQT format with all water molecules excluded and polar hydrogen atoms were added. The grid box was centered to cover the domain of each protein and to accommodate free molecular movement. The grid box was set to 30 Å × 30 Å × 30 Å, and grid point distance was 0.05nm. Molecular docking studies were performed by Autodock Vina 1.2.2 (http://autodock.scripps.edu/).To analyze the binding affinities and modes of interaction between the drug candidate and their targets, AutodockVina 1.2.2, a silico protein–ligand docking software was employed . The molih.gov/) . The 3D All the botanical drugs in ZSXBF were provided by the First Affiliated Hospital of Zhejiang Chinese Medical University (Hangzhou China). After soaking in 10 volumes of distilled water for 1 h, DG, CNX, ZL, HB, TFL, and MRS were mixed  at a ratio of 6:15:15:9:25:30 for reflux extraction . Then, the solution was concentrated (3 g crude drug/mL).To determine the main chemical components of ZSXBF, high-performance liquid chromatography (HPLC) was used. We added 0, 1 ml, 2 ml, and 4 ml of ZSXBF soup to four round-bottomed flasks and added 20 ml of 70% methanol  solution. After weighing, the solution was ultrasonicated for 30 min, allowed to cool, and weighed again, and the extraction solvent was used to make up the lost weight. The mixture was shaken well and filtered, and the filtrate was taken to obtain the final product.An Ultimate ®UHPLC AQ-C18  chromatographic column was used. The mobile phase consisted of acetonitrile  and 0.2% acetic acid . The gradient elution procedure is shown in Table The botanical medicinal materials used in this study were sent to the Drug Testing Laboratory of Zhejiang University of Traditional Chinese Medicine Academy of Traditional Chinese Medicine for identification according to The Chinese Pharmacopoeia (2020 edition). The quality of ZSXBF was controlled, and the exact chemical composition was determined by ultra-performance liquid chromatography (UPLC). UPLC identified 18 chromatographic peaks representing 18 drug monomers  was performed on 10-week-old male C57BL/6 mice to establish the knee osteoarthritic model. Briefly, DMM surgery was performed on the right hind limbs of C57BL/6 mice. First, a 3 mm longitudinal incision was made on the medial part of the knee under anesthesia, and blunt dissection of the knee extensor muscles and patellar ligament (MMTL) was performed. Then, the MMTL was transected to destabilize the medial meniscus. Finally, the medial joint capsule was sutured, and the skin was closed. Sham surgery was also performed on C57BL/6 mice with a similar surgical approach but without manipulating the joint tissues.n = 6 in each group): the sham group, DMM group, and XBF group. In the XBF group, the drug was orally administered to the mice for 8 consecutive weeks  beginning on the day after DMM surgery.All the C57BL/6 mice were randomly divided into three groups . All the studies were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (20200622-07).3).All the mice were sacrificed at 8 weeks after surgery, and the right knee joints were collected. Then, microcomputed tomography (μ-CT) was used to analyze the knee joints. The area between the proximal tibia growth plate and the tibial plateau was chosen as the region of interest, and the parameters collected by μ-CT were percent bone volume , trabecular thickness , and bone mineral density  solution for 14 days, and embedded in paraffin. Then, 3 μm thick sections of the medial compartment of the joints were generated for Alcian Blue Haematoxylin/Orange G (ABH) staining to analyze gross cartilage structural changes. The thickness of the tibial cartilage was scored by three blinded observers.Immunohistochemistry (IHC) was performed to observe the expression of type II collagen , matrix metallopeptidase 13 , interleukin 17 , tumor protein p53 , mitogen-activated protein kinase 14 , and extracellular regulated protein kinase 1 .The baseline characteristics of the participants in the incidence and progression cohorts are shown in Table To evaluate the clinical efficacy of ZSXBF in the treatment of KOA, we scored the treatment outcomes of 146 patients. After treatment with ZSXBF, the VAS scores and Lequesne scores were significantly improved, indicating that after treatment with ZSXBF, pain levels improved, and walking and ability to perform activities of daily life improved (Table To prove the effect of ZSXBF treatment on cartilage and subchondral bone, which are the sites of injury in KOA, DMM surgery was performed on C57BL/6J mice, and ZSXBF was intragastrically administered for 8 weeks. After 8 weeks, knee joint tissue samples were taken for μ-CT scanning, and 3D reconstruction was performed to obtain representative images of the knee joints. The μ-CT scanning results showed that the DMM model group had significant subchondral bone sclerosis and cartilage loss and that the BMD, subchondral bone BV/TV, and Tb.Sp values were significantly different Fig. A, D, E. The Pharmacology Database and Analysis Platform database were used in this analysis. Six drugs in ZSXBF were identified in the TCMSP, and the TCMSP showed that CNX, DG, HB, TFL, ZL and MRS had 4, 2, 37, 15, 2 and 0 targets, respectively. After eliminating duplicate targets, 212 predicted target genes were identified. To determine the potential targets of OA, we searched 1817 OA-related target genes in the GeneCards, DrugBank, TTD, OMIM, and PharmGkb databases. To investigate the potential target genes of ZSXBF in the treatment of OA, Venn analysis was performed, and 114 genes were identified as overlapping genes between ZSXBF and OA targets (Fig. We used STRING to construct a PPI network to determine the key proteins in the treatment of OA with ZSXBF. The thresholds of betweenness, closeness, degree, eigenvector, information, LAC and subgraph were set in two steps based on the topology parameters calculated by Cytoscape. Finally, ten key proteins, including ESR1, AKT1, RELA, TP53, MAPK1, MAPK3, JUN, FOS, EGFR, and MAPK14, were identified (Fig. GO and KEGG pathway enrichment analyzes of 168 tandem targets were conducted with the database. Based on network pharmacological analysis, we found that the AGE-RAGE signaling pathway and PI3K-Akt signaling pathway may be the most relevant signaling pathways for the treatment of OA with ZSXBF Fig. A, B.Fig.To evaluate the affinity of the drugs in ZSXBF for their targets, we performed molecular docking analysis. The binding poses and interactions of the main drug candidate with the main protein were obtained with Autodock Vina v.1.2.2 (Additional file The network pharmacology results suggest the possible mechanism by which ZSXBF treats KOA. Combined with our understanding of ZSXBF TCM treatment, we aimed to verify whether ZSXBF regulates cellular immunity, and we performed IHC staining for IL-17A, TP53, MAPK14, and ERK1. The results showed that the expression levels of IL-17A, TP53, MAPK14 and ERK1 in the articular cartilage of DMM model mice were significantly higher than those in control mice, suggesting that the pathogenesis of knee osteoarthritis is closely related to the immune factor IL-17. The expression levels of IL-17A, TP53 and ERK1 in the treatment group were significantly lower than those in the DMM model group, and there was no significant difference in the expression level of MAPK14 (Fig. TCM is an ancient treatment method, and it has been widely accepted and applied in the treatment of knee osteoarthritis , 13. HowBefore verifying the mechanism, our study further elucidates the clinical efficacy of ZSXBF. Through a retrospective analysis of the clinical efficacy of ZSXBF, the agreement party, we found that ZSXBF can effectively relieve the pain of KOA patients and improve their quality of life according to Lequesne’s score. In this retrospective study, patients' own baseline levels were used as the control group.To compensate for the limitations of the self-control study, we further verified the therapeutic effect of ZSXBF in experimental animals in a random control study. The DMM model is a widely used and recognized mouse model of KOA , 15. We Then, the mechanism underlying the therapeutic effect of ZSXBF was analyzed through network pharmacology. In contrast to chemical drugs, TCM has complex components, and it has multiple effective compounds and targets. In recent years, a new method has been used to study the mechanism underlying drug treatment of complex diseases . FurtherThrough network pharmacology, we identified possible therapeutic targets of ZSXBF. These therapeutic targets were compiled and calculated from other researchers' studies on TCM components and KOA through a computer system. Some researchers studied CNX and found that it can protect against chondrocyte damage induced by IL-1β . ResearcChanges in the T-cell population have been observed in KOA, including an increase in the proportion of Th17 cells . A studyIn summary, in this article, we have preliminarily discussed the therapeutic effect and underlying mechanism of Zhang's prescription in the treatment of knee arthralgia. We believe that ZXBF has a clear therapeutic effect in clinical practice. After computer data analysis and experimental verification, we believe that the mechanism by which ZSXBF treats OA is related to IL-17, ERK1 and TP53. Combined with the published results of other researchers, we speculate that ZXBF can delay the progression of KOA by regulating the IL-17-mediated immune response.Additional file 1: Fig. S1. Binding mode of screened drugs to their targets by molecular docking. Binding mode of quercetin to TP53.


Background: Childhood chronic non-infectious uveitis (cNIU) is a challenging disease that needs close monitoring. Slit lamp evaluation (SLE) is the cornerstone of ophthalmological evaluation for uveitis, but it is affected by interobserver variability and may be problematic in children. Laser flare photometry (LFP), a novel and objective technique, might be used in children with uveitis. Aim: The aim of this study was to attempt the use of LFP in cNIU clinical practice. Methods: Children, attending the Rheumatology Unit and who were scheduled to receive ophthalmological evaluation, were prospectively enrolled to concomitantly receive SLE and LFP. SLE was performed blind to LFP measure. Demographic, laboratory, clinical, and ophthalmology data were collected. Results: A total of 29 children (58 eyes) were enrolled, including 3 with juvenile idiopathic arthritis without uveitis (JIA-no-U), 15 with JIA-associated uveitis (JIA-U), and 11 with idiopathic chronic uveitis (ICU). We observed significantly higher LFP values in the eyes of children with uveitis compared to the others . Accordance between the SLE and LFP measures, at baseline  and during the follow-up , was detected. We evaluated significant correlation between LFP values and the presence of complications , especially with cataract formation . Conclusions: In this cohort, LFP measurements showed a good correlation with SLE. LFP values showed a positive correlation with the presence of complications. LFP might be considered as a reliable objective modality to monitor intraocular inflammation in cNIU. Chronic non-infectious uveitis is a challenging disease from a diagnostic and treatment perspective, due to the risk of severe complications, including blindness, if it is not promptly and correctly identified and treated ,2,3,4. UChildhood chronic non-infectious uveitis (cNIU) is frequently asymptomatic and eventually develops into severe ocular complications and visual loss. Persistent ocular inflammation and chronic use of corticosteroids can lead to the development of severe ocular complications including cataracts, glaucoma, posterior synechiae, and band keratopathy . In ordeHowever, this is a subjective technique that is burdened by interobserver variability. Additionally, it is not able to intercept subclinical changes in the blood-–aqueous barrier that can start in the earliest phase, and it may also easily miss a low number of cells, mirroring a persistent, sub-acute/chronic inflammation, thus eventually leading to complications. Additionally, in children, SLE might be challenging because of scarce collaboration.In the last few years, increasing progress has been achieved in quantifying intraocular inflammation in an objective way, and one of them is laser flare photometry (LFP). LFP is an objective, reliable, quantitative, and non-invasive technique that measures the light scattering of a helium–neon laser beam in the anterior chamber based on the concentration of proteins ,9,10. ThOur aim was to compare the LFP measurements with AC cell grading by SLE according to the SUN classification.This was a prospective observational study of JIA and cNIU children currently followed at the rheumatology and ophthalmology units of Meyer Children’s Hospital IRCCS. They underwent LFP assessment during a scheduled ophthalmology evaluation by SLE, between 15 February 2022 and 31 May 2023.All children who fulfilled the following inclusion criteria were included: (A) to be ≤16 years old when LFP was performed, (B) to undergo LFP measurement the same day of the SLE, (C) and to be diagnosed for JIA according to the ILAR definition  or cNIU Topical corticosteroid and cyclopentolate were permitted.Ethical approval was obtained from the Institutional Review Board of Meyer Children’s Hospital . The research was conducted in accordance with the tenets of the Declaration of Helsinki.Children enrolled in the study underwent a standard ophthalmology visit that included evaluation of visual acuity, intraocular pressure measurement, SLE, fundoscopy, complication evaluation, and LFP measurement. SLE was performed, independently, by an expert ophthalmologist (RP), while LFP was performed, blinded, in all the patients, by the same ophthalmologist (CdL) along with always the same technician (GD), as a part of the routine ophthalmological examination at each visit since the enrolment.Flare values were measured using a Kowa FM-700 laser flare photometer , which utilizes a diode laser to measure aqueous flare through a 0.3 × 0.5 mm sampling window. The obtained values are expressed as photon counts per millisecond (ph/ms). Each patient underwent six measurements, with the highest and lowest readings being excluded from the calculation. The average (denoted as AVG) and standard deviation (SD) were automatically calculated based on the remaining four flare measurements.-Demographic data including age, sex , ethnicity, presence of comorbidity, family history of autoimmune disease in a first-degree relative, underlying disease , age at onset (expressed in months), duration of disease (expressed in months).-Laboratory data including ANA, ANCA, HLA-B51, HLA-B27, and inflammatory parameters at onset if available (C-reactive protein (CRP) expressed in mg/dL , and erythrocyte sedimentation rate (ESR) expressed in mm/h ).-Characteristics of uveitis including laterality of uveitis , anatomical location according to SUN  , ocular hypotony (<5 mmHg), optic disc swelling, macular oedema defined as intraretinal cysts on optical coherence tomography scan, posterior synechiae, epiretinal membrane, band keratopathy, and choroidal neovascular membrane.-Visual acuity (VA) was recorded at onset and at each visit in LogMAR, and when this scale was not available, the appropriate conversion was performed , and VA -Inflammatory status of the eye was evaluated using SLE for the anterior chamber according to the SUN working group grading system defining anterior chamber cells and flare between 0.5+ and 4+ ,13,14,15-The treatments performed at each visit were recorded, including topical as well as systemic treatment.The following data were collected: p-value of <0.05 was considered to indicate statistical significance. Differences among categorical variables were assessed using the chi-square test or the Fisher exact test as appropriate. Specifically, the Fisher exact test was used for categorical variables with 2 categories, when 1 or more of the cell counts was less than 5. Continuous variables were compared with Student’s t-test or non-parametric tests such as the Kruskal–Wallis test as appropriate. Spearman’s rank correlations were performed to assess any relationship between baseline and follow-up LFP values and AC cell grades, and the correlation of these parameters with the ocular complications.Statistical analyses were performed using SPSS v28.0 for Microsoft. Continuous variables are described using mean and standard deviation or median and range depending on their distribution. Q–Q plots were used to test the assumption of normality. For dichotomous and categorical variables, proportions were used to assess the clinical characteristics of the population. A We collected the data of 29 children : 3 with Among the 26 patients with uveitis, 22 had a bilateral involvement (84.6%), 21 an anterior uveitis (80.7%), and 5 panuveitis (19.2%). Ocular symptoms were reported in 11 patients (42.3%), as pain in 4 (15.4%), redness in 11 (42.3%), photophobia in 6 (23.1%), and floaters/blurred visions in 4 (15.4%). The mean visual acuity at onset was 0.06 (SD ± 0.20) in the whole cohort, while in the JIA-U we observed a mean of 0.07 (SD ± 0.27), and in idiopathic of 0.056 (SD ± 0.13).At onset, nine patients (31%) showed ocular complications, with a median number of complications of 0 (IQR 0–2.5). The most frequent reported complications at onset were optic disc swelling in 12 eyes, posterior synechiae in 11 eyes, cataracts in 4 eyes, ocular hypertension in 2 eyes, band keratopathy in 1 eye, chorioretinal scar in 1 eye, and cystoid macular oedema in 1 eye.p = 0.025) and more frequently have anterior uveitis , while patients with idiopathic uveitis were less frequently ANA-positive , they more frequently had ocular symptoms at onset , and more complications compared to the other groups (p < 0.001)  .p = 0.007) (All the 29 patients underwent to at least one LFP measurement at a median age of 147 months (IQR 105–173), after a median duration of the disease of 51 months (IQR 16.5–101.25). The mean value of the first LFP was 14.73 ph/ms (±20.3) in the whole cohort, with significant differences among eyes with uveitis versus eyes without uveitis, according to SLE evaluation   Table 2p = 0.007p = 0.002 (p > 0.05).Analysis of LFP values in children with uveitis were then stratified according to uveitis activity and type of uveitis, based on SLE evaluations. At the first LFP assessment, the LFP median values of the eyes of subjects with active uveitis (15 eyes) were significantly higher when compared to the LFP median value of subjects with inactive uveitis (35 eyes), being 12.8 IQR 11.4–44.8 versus 8.7 IQR 5.8–12,  = 0.002 . We did 2 3.84, p = 0.05).At the time of the first LFP assessment, the mean AC of subjects with uveitis was 0.29 (±0.66), with 17 eyes with complications (29.3%), of whom 9 had cataracts (15.5%) and 14 posterior synechiae . Patientp < 0.001, LFP II ρ 0.460, p < 0.001, LFP III ρ 0.631, p < 0.001, LFP IV ρ 0.547, p = 0.006, LFP V ρ 0.767, p = 0.001) , the presence of cataracts , and the posterior synechiae .We observed significant correlations between the value of the LFP and the presence of complications at the different time points  (p < 0.001), at the third measurement , at the fourth , and at the last available follow-up too .Indeed, eyes which had complications showed higher LFP values compared to eyes which did not show complications at baseline  , at the This prospective study is one of the few studies that assesses the role of LFP in routine clinical practice to evaluate ocular inflammation in children with uveitis in comparison with SLE. We identified a consistent positive correlation between the LFP values and SLE performed by an expert ophthalmologist. We demonstrated the applicability of this novel technique in a cohort of children, where it was able to distinguish children with uveitis from children without uveitis, and we used this technique to monitor ocular inflammation in an objective way. Additionally, we highlighted a positive relation between higher LFP values and the presence of structural complications.Detection and monitoring of disease activity in uveitis is crucial for customizing the treatment of our children and preventing complications. The SUN grading system, which is the currently used referred system to evaluate ocular inflammation, shows several limitations. First of all, there is the subjective estimation of inflammation by an observer, even though there are clear rules to count the cells, and additionally the use of non-continuous variables does not allow estimation of minimal changes . Novel tIndeed, uveitis leads to an alteration of the blood–aqueous barrier that changes the protein composition, and the LFP technique also offers the possibility to detect minimal changes in the composition of humor aqueous in chronic uveitis, even though there are not evident changes in the number of cells in the anterior chamber.The first report of LFP use in this indication was published in 1972 by Herbort et al. , where tThus, LFP offers an extremely objective instrument also able to detect minimal changes in the aqueous composition, with minimal collaboration from young patients compared to common SLE that, in in ambiguous situations, requires prolonged collaboration from young patients.The use of this novel technique in routine clinical practice has already been reported in limited pediatric studies ,15,21,22In accordance with recent studies, our study showed significantly increased LFP values in patients who showed structural complications such as cataracts and posterior synechiae, in a consistent way, regardless of inflammatory status ,21,23,24However, our data seem to be in contrast with Tappeiner et al., who described an additional association between ocular hypertension and the epiretinal membrane, but a possible explanation might be that none of our patients showed these complications ,20.Additionally, we did not evaluate any correlation between the LFP values and visual acuity as evaluated by other authors such as Gonzales et al. .However, caution should be posed when we compare our data with others, because we need to consider that our cohort of children showed a lower prevalence of complications and better visual outcomes compared to other cohorts ,21. AddiBefore drawing our conclusions, we need to point out several limitations of our study. First of all, we need to consider the small sample size of patients included, which did not allow us to draw firm conclusions, but we need to consider the rarity of the disease and the use of the novel technique. Additionally, the patients included in the study showed different anatomical subtypes. In particular, we included six patients with panuveitis at onset who, however, during follow-up mainly manifested an anterior involvement. Additionally, the aim of this study was to test the hypothesis that LFP is comparable to the standard SLE evaluation in a pediatric population. Thus, we were not able to consider a specific interval of evaluation. Most of the patients with uveitis underwent LFP during scheduled visits. Unfortunately, because of the exiguous number of patients included in this study and the different time points at which the patients were in their disease history, we were not able to determine a relationship between LFP and relapse and/or treatment.Furthermore, we have variable follow-up for the different patients.Laser flare photometry represents an optimal non-invasive, objective, and quantitative method to assess intraocular inflammation, with an acceptable profile for children. It showed a significant ability to distinguish patients with and without uveitis, with active and inactive uveitis, and with complications, with a significant correlation with SLE performed by an expert ophthalmologist. However, caution should be posed in the interpretation of these data, considering the sample size included in our study and short duration of follow-up. The increasing evidence about the use of this novel technique in childhood suggests enhancement of the use of LFP in the routine clinical ophthalmology visit, at least in tertiary centers, in combination with standard visits.


BackgroundThis study aimed to clarify the need for disease-modifying drug (DMD) treatment in elderly patients with multiple sclerosis (MS) aged 50 years or older. MS is an autoimmune, demyelinating disease of the central nervous system that predominantly affects young women. Various DMDs are effective in preventing relapses and slowing the progression of disability in patients with MS. Although disease activity in MS is believed to decrease with aging, a consensus on the appropriate DMD treatment for elderly patients with MS is lacking.MethodologyThis study included elderly patients with MS (>50 years old). We compared the occurrence of relapses, worsening of disability, and conversion to secondary progressive MS (SPMS) between patients with DMD treatment and those without. Logistic regression analysis was performed to determine the predictors of these outcomes. Confounding factors were adjusted using propensity scores.ResultsFrom January 1991 to October 2022, 76 elderly patients with MS were included. The mean age at the last visit was 57.4 ± 6.3 years, with 51 patients being female. The mean age of onset of MS was 37.1 ± 10.1 years. Fifty-four patients were included in the DMD treatment group. The overall relapse rate was 38% . No significant differences in relapse rates (p = 0.72) or in the Expanded Disability Status Scale (EDSS) scores were identified between the two groups. Kaplan-Meier curves showed no differences in the time to first relapse within five years between the two groups. Additionally, no significant predictors of relapse were identified. Among 61 patients with relapsing-remitting MS, 25% converted to SPMS during the observation period. Logistic regression analysis showed that older age at the final visit and the presence of brainstem lesions at the age of 50 years were associated with a higher rate of transition to SPMS.ConclusionsIn the present study, no significant difference was found in the rate of relapse, disability progression, and conversion to SPMS between the DMD treatment and untreated groups in elderly patients with MS. Therefore, in patients without long-term relapse, no poor prognostic functional factors or predictors of conversion to SPMS, discontinuation of DMDs may be considered. In addition, the presence of brainstem lesions at 50 years of age may predict the conversion to SPMS. Thus, the continuation of DMD or conversion to an appropriate DMD should be considered in patients with brainstem lesions at 50 years of age. Multiple sclerosis (MS) is an autoimmune, inflammatory, demyelinating disease of the central nervous system that is particularly prevalent in young women. It is characterized by multiple lesions occurring in the central nervous system that disseminate in time and space. The associated neurological symptoms are various. Approximately half of the affected patients convert from relapsing-remitting MS (RRMS) to secondary progressive MS (SPMS), in which disability progresses regardless of relapse within 15-20 years of disease onset . ConcernWe conducted a retrospective study among patients with MS who had a medical history of outpatient visits to our clinic between January 1, 1991, and October 31, 2022. The patients included in this study met the 2017 McDonald diagnostic criteria for MS . PatientStatistical analysisCategorical and continuous data were compared, respectively, using the chi-square test and Wilcoxon rank-sum test for non-parametric data. Statistical significance was defined as p-values <0.05. All statistical analyses were performed using JMP Pro 16.0.0 (512257SAS Institute Inc.).Logistic regression models were developed to identify the DMD treatment-associated factors. We evaluated the relationship between DMD treatment and relapse, worsening of EDSS scores, and conversion to SPMS. Additionally, time-to-event analysis using the Kaplan-Meier method was performed to determine the onset of relapse.Propensity score analysisTo account for the selection bias inherent to a retrospective study, a propensity score was calculated for each patient, which assessed DMD treatment for MS patients aged over 50 on observed covariates. The covariates used to build the propensity score were age at final visit, sex, age at disease onset, observation period, and disease subtype. All patients were stratified into quintiles based on their propensity scores and were included in the analyses. We assessed the adequacy of the propensity score specification by comparing the standardized differences in baseline covariates after stratification between the DMD and untreated groups. A standardized mean difference of <0.1 or p-values >0.05 was used to indicate no significant difference between the baseline covariates.We examined 76 elderly patients with MS (age >50 years). Among them, 51 were in the DMD treatment group. The mean age at the last follow-up was 57.4 ± 6.3 years (mean ± SD), and 55 patients were females . Additionally, we performed logistic regression analysis using propensity scores based on age at the last follow-up, sex, disease subtype, and observation period; no significant difference was found (p = 0.89)  at 50 years of age between patients with and without relapse, no statistically significant difference was identified between the groups. In the patients who presented relapse, the DMD treatment consisted of IFN-β in nine cases, GA in one, DMF in four, FTY in two, and natalizumab in one. In contrast, among patients without relapse, 13 were treated with IFN-β, nine with DMF, eight with FTY, two with siponimod, one with NTZ, and one with OMA.We investigated the conversion to SPMS in patients with RRMS aged 50 years in the DMD treatment group and untreated groups Table . The DMDThis study aimed to clarify the need for DMD treatment in elderly patients with MS aged 50 years or older. Therefore, our study compared disease activity and conversion to SPMS in elderly patients with MS between the DMD treatment and untreated groups. Generally, disease activity, such as relapse and the presence of new or Gd-enhanced lesions on MRI, decreases in elderly patients with MS. According to a previous study, the ARR of MS decreases with age; for individuals in their 20s and 30s, the ARR is 0.26 in females and 0.32 in males, whereas for those in their 50s, it decreases to 0.15 in females and 0.14 in males . A previAlthough the disease activity seemed to decrease after 50 years of age, some patients present disability progression. Recently, SPMS, which is characterized by disability progression, regardless of relapse, has gathered attention. In MS, approximately half of the patients convert to SPMS within 15-20 years of disease onset . AdditioIn our study, patients in the DMD treatment group had a higher number of poor prognostic factors, such as the presence of spinal cord lesions and increased IgG index, than those in the untreated group Table  12]. Ov. Ov12]. The pathogenesis of SPMS is unclear, and no effective DMD can suppress the progression of disability until now. However, many recent studies have been conducted on SPMS, for example, patients with SPMS who have meningeal B-cell aggregates show several distinct clinical characteristics, including early disease onset, early age at wheelchair dependence, and more pronounced cortical demyelination . These rThe limitations of this study include that patients in the DMD treatment group tended to possess poorer prognostic factors, such as the presence of spinal cord lesions and increased IgG index, compared with the untreated group . AlthougThe present study identified no significant differences in relapse, EDSS progression, and conversion to SPMS between the DMD treatment and untreated groups in elderly patients with MS. Although some patients aged over 50 years exhibited relapse or deterioration, some patients remained without relapse or disability progression, suggesting that the discontinuation of DMD treatment may be considered in some elderly patients without poor prognostic factors. Patients aged 50 years with brainstem lesions have a higher risk of conversion to SPMS compared to patients without such lesions. Therefore, appropriate administration and/or continuation of DMDs that are effective in SPMS, such as siponimod and OMA, should be considered for 50-year-old patients with brainstem lesions.


The COVID-19 pandemic may have had an impact on healthcare-associated infection (HAI) rates. In this study, we analyzed the occurrence of HAIs in a neonatal intensive care unit (NICU) of the Umberto I teaching hospital in Rome before and during the pandemic. All infants admitted from 1 March 2018 to 28 February 2022 were included and were divided into four groups according to their admission date: two groups before the pandemic (periods I and II) and two during the pandemic (periods III and IV). The association between risk factors and time-to-first event was analyzed using a multivariable Cox regression model. Over the four-year period, a total of 503 infants were included, and 36 infections were recorded. After adjusting for mechanical ventilation, birth weight, sex, type of delivery, respiratory distress syndrome, and previous use of netilmicin and fluconazole, the multivariable analysis confirmed that being hospitalized during the pandemic periods (III and IV) was the main risk factor for HAI acquisition. Furthermore, a change in the etiology of these infections was observed across the study periods. Together, these findings suggest that patient management during the pandemic was suboptimal and that HAI surveillance protocols should be implemented in the NICU setting promptly. Staphylococcus aureus, coagulase-negative Staphylococci, and Enterococci. In addition, recent years have recorded a considerable increase in HAIs sustained by Gram-negative bacteria and fungi, especially Candida spp., which are mainly responsible for ventilator-associated pneumonia (VAP) and urinary tract infections, but also for peritonitis and meningitis [Healthcare-associated infections (HAIs) are among the most serious preventable complications in neonatal intensive care units (NICUs) . Pretermningitis ,4.Pseudomonas aeruginosa bacteremia [According to a study conducted in 2016/2017  using thcteremia . During cteremia ,10, inclcteremia .While the impact of the pandemic on incidence rates of nosocomial infections in adult ICUs has been investigated ,11,12,13Staphylococci were considered as susceptible or resistant to oxacillin and/or glycopeptides [In this cohort study, we retrospectively analyzed patients hospitalized in the NICU of Umberto I teaching hospital of Rome from 1 March 2018 to 28 February 2022. Patients were followed until discharge or 23 March 2022. The NICU has a total of six beds in which healthcare providers take care of critically ill babies born in this hospital or coming from other hospitals in Rome and the Lazio region via the Neonatal Emergency Transport Service. We followed the STROBE guidelines to report our findings. Microorganisms’ antimicrobial susceptibility profiles were defined according to the classification proposed by Magiorakos et al.  , type of delivery, admission diagnosis , exposure to invasive devices , use of antimicrobial agents (days), site of infection, date of infection onset, and microorganism isolated are routinely collected. An infection is considered to be healthcare-associated if it occurs 48 h after birth or admission. The surveillance system records central/umbilical line-associated bloodstream infections (CLABSIs) and ventilator-associated pneumonia (VAP) and any other type of infection that occurs during hospitalization, the diagnosis of which is determined by an infectious disease specialist. All infections are defined according to the standard diagnostic criteria published by the Center for Disease Control and Prevention (CDC), adapted to neonatal pathology .Patients were divided into four groups according to their admission date: period I, from 1 March 2018 to 28 February 2019; period II, from 1 March 2019 to 29 February 2020; period III, from 1 March 2020 to 28 February 2021; and period IV, from 1 March 2021 to 28 February 2022. The date 1 March 2020 was set as the cut-off date between prepandemic and pandemic years. Then, the two preceding and following calendar years were identified for the analysis in order to investigate four equally long time intervals. For each period, descriptive statistics were obtained using means and standard deviations for continuous variables and proportions for dichotomous and categorical variables. For the univariable analysis, the Kruskal–Wallis test was used to compare continuous variables across study periods, whereas Pearson’s chi-squared test was used for dichotomous and categorical variables. BW was categorized accord according to the Center for Disease Control/National Healthcare Safety Network (CDC/NHSN) classification [p-value < 0.05 as cut-off. The proportionality assumption was checked by testing the statistical significance of interaction terms involving failure time, each one at a time. Multicollinearity was checked using as threshold a variance inflation factor of 5. All analyses were performed using STATA , version 17.0. A two-sided p-value < 0.05 was considered statistically significant.Time-to-first HAI  was estimated by survival analysis. Firstly, we estimated the Kaplan–Meier survival function for each period of hospitalization, and survival curves were compared with the log-rank test. Then, the association between period of hospitalization and time-to-first event was assessed through a multivariable Cox regression model for proportional hazard, which provided estimates of the adjusted hazard ratio (aHR) and its associated 95% CI. The main exposure of interest, period of hospitalization, was adjusted for the potential confounders of the association based on expert knowledge . As a reFrom 1 March 2018 to 28 February 2022, 564 neonates were hospitalized in the Umberto I teaching hospital of Rome, of which 503 were included in the HAI surveillance system . The higAs for the use of invasive devices, slightly more patients had a central line in periods I and II  compared to periods III and IV , but the average cumulative use in those who had a central line was highest in period III (19.6 days). Similarly, a lower number of patients underwent mechanical ventilation during the pandemic, especially in period IV (9.8%), but the highest average cumulative exposure was observed in period III (14.7 days). Antibacterial consumption was quite high; approximately three out of four patients were administered ampicillin, whereas two out of three were prescribed netilmicin in each period. However, while the cumulative average use of ampicillin was similar throughout the study, the average exposure to netilmicin was reduced in period IV. Fluconazole, on the other hand, was used in a lower proportion of patients (from 6.8% to 16.7%) with the highest average exposure in periods II and III. Lastly, a greater number of deaths were observed in periods I and II, accounting for slightly higher mortality rates (2.5 deaths per 1000 patient days (95% CI: 1.0–5.9) in period I and 3.5 per 1000 patient days (95% CI: 1.7–7.3) in period II).The prevalence of patients with at least one HAI was greater during the pandemic than prepandemic , with the highest proportion of patients with at least one CLABSI or VAP in period III . A totalDifferentiating by type of HAI, the CLABSI and VAP incidence rates showed the same trend, with the highest values from March 2020 to February 2021 . HoweverSerratia marcescens was the pathogen responsible for half the HAIs diagnosed in period I (5.0%), whereas Klebsiella pneumoniae was primarily detected in period II (5.0%). By contrast, Staphylococcus aureus (22.5%) and coagulase-negative Staphylococci (12.5%) were most frequently isolated in period III, as well as in period IV, where infections mainly involved coagulase-negative Staphylococci (27.5%), followed by Escherichia coli (5.0%). Other microorganisms, such as Haemophilus influenzae, were less frequently detected in periods II and III. As for microorganisms’ antimicrobial susceptibility patterns, out of 21 pathogens that could be classified according to the Magiorakos criteria [Staphylococci, the number of such microorganisms resistant to oxacillin increased from 4 in period III (25.0%) to 10 in period IV (62.5%), while only 1 isolate was found to be resistant to both oxacillin and glycopeptides in period IV (6.3%).The etiology of HAIs across the four study periods varied . Serraticriteria , no multp = 0.042)  . SurvivaA multivariable analysis  showed tAmong the indirect consequences of the COVID-19 pandemic, an increase in the incidence of HAIs has been frequently reported, especially in some wards, such as ICUs ,22. HoweS. aureus outbreak in our NICU that accounted for most VAP recorded between May and July 2020. Gram-negative bacteria, particularly S. marcescens and K. pneumoniae, were the pathogens most frequently responsible for HAIs before the pandemic, in line with data from the literature in which they were often involved in NICU outbreaks [S. aureus and oxacillin-resistant coagulase-negative Staphylococci, usually the main causes of HAIs in infants [As for the HAI type, both CLABSI and VAP rates increased during the pandemic, even though the CLABSI increment was not significant in the univariate analysis, probably due to reduced statistical power. Indeed, despite the lower number of neonates using central lines in these years, the higher average exposure, especially in period III, may at least partially explain the peak in CLABSI incidence recorded between March 2020 and February 2021 ,28,29. Iutbreaks ,31. In c infants ,33. This infants ,35,36,37 infants .However, our study confirmed that a major risk factor for the occurrence of HAIs in NICUs was low BW ,40. ThisThis study has several strengths and limitations. The main strength is the ability to compare data over time. Because data were collected as part of a four-year continuous surveillance system routinely carried out by the Department of Public Health and Infectious Diseases, a potential bias in the results due to overworked NICU staff is unlikely. In addition, to the best of our knowledge, this is one of the few studies that describes and identifies risk factors related to HAI occurrence in a NICU during an emergency period, such as the COVID-19 pandemic. In this regard, we plan to conduct new studies in the near future on the occurrence of HAIs in NICU and ICU settings as the pandemic progresses. In contrast, the first limitation is represented by the fact that we do not have data on the SARS-CoV-2 positivity or negativity status of the infant mothers, even though all hospitalized infants were tested and were SARS-CoV-2 negative. Second, patients discharged from the NICU were no longer under surveillance, although only the most stable patients were chosen for transfer. Third, even though we adjusted for the main risk factors, namely demographic characteristics and use of invasive device and antibiotics, we may have not fully accounted for clinical severity, meaning that some residual confounders may be still present. However, this bias is likely to be constant across time periods. Lastly, we did not study the impact of HAIs on patient mortality, although this was not a goal of our research. Further studies should be conducted to address this issue, together with research that analyzes hand hygiene compliance, which could be of interest to better understand the mechanisms behind any increase or decrease in the incidence rates.We found higher rates of HAIs in our NICU during the COVID-19 pandemic. This, coupled with the fact that the microorganisms involved were different across the study period, suggests a crucial role for patient management and underlines the importance of implementing effective HAI prevention and control strategies ,51. Beca


Cocoa shell is a by-product of cocoa manufacturing. We obtained an aqueous extract (CSE) rich in polyphenols and methylxanthines with antioxidant and vasodilatory properties. We aimed to evaluate the effects of CSE supplementation in aged hypertensive rats on blood pressure and the mechanism implicated. Eighteen-month-old male and female rats exposed to undernutrition during the fetal period who developed hypertension, with a milder form in females, were used (MUN rats). Systolic blood pressure  and a blood sample were obtained before  and after CSE supplementation . Plasma SOD, catalase activity, GSH, carbonyls, and lipid peroxidation were assessed (spectrophotometry). In hearts and aortas from supplemented and non-supplemented age-matched rats, we evaluated the protein expression of SOD-2, catalase, HO-1, UCP-2, total and phosphorylated Nrf2 and e-NOS (Western blot), and aorta media thickness . MUN males had higher SBP compared with females, which was reduced via CSE supplementation with a significant difference for group, sex, and interaction effect. After supplementation with plasma, GSH, but not catalase or SOD, was elevated in males and females. Compared with non-supplemented rats, CSE-supplemented males and females exhibited increased aorta e-NOS and Nrf2 protein expression and cardiac phosphorylated-Nrf2, without changes in SOD-2, catalase, HO-1, or UCP-2 in cardiovascular tissues or aorta remodeling. In conclusion, CSE supplementation induces antihypertensive actions related to the upregulation of e-NOS and Nrf2 expression and GSH elevation and a possible direct antioxidant effect of CSE bioactive components. Two weeks of supplementation may be insufficient to increase antioxidant enzyme expression. Cardiovascular diseases (CVD) are collectively one of the leading causes of morbidity and mortality and are an important contributor to the burden for health systems worldwide. Hypertension is the main modifiable risk factor of CVD, which remains very prevalent, particularly in aged populations, despite pharmacological efforts to control it. To address the risks of high blood pressure to health, a multilevel approach, including pharmacological and nonpharmacological interventions, has been proposed . OxidatiTheobroma cacao L.) processing produces a significant amount of waste, with 90% of cocoa fruit’s total weight being discarded [Food waste is a rising environmental problem, and it is of interest to recycle its by-products. Cocoa . The rats and experimental procedures were approved by the Ethics Review Board of the Universidad Autónoma de Madrid and by the Regional Environment Committee of the Comunidad Autónoma de Madrid .The experimental model of hypertension was induced via exposure to undernutrition during fetal life through maternal undernutrition during gestation (MUN rat model). This intervention induces hypertension in males at the age of 6 months, while females exhibit a mild increase in blood pressure only during aging ,19. BrieRats from the same sex were housed with poplar bedding in type III or type IV cages according to their weight in groups of 3–5 per box; maintained under controlled conditions ; and fed with a standard diet  containing 55% carbohydrates, 22% protein, 4.4% fat, 4.1% fiber, and 5.4% mineral. Water was provided ad libitum. Rat health and welfare were regularly monitored by the staff at the Animal House Facility. 2 exposure followed by exsanguination, and thoracic aortas and hearts were dissected. From the aorta, a 1 cm length segment immediate to the aortic arch was fixed in 4% paraformaldehyde and stored. From the hearts, the atria were discarded. The remaining aorta and ventricles were snap-frozen and kept at −80 °C. In a group of age- and sex-matched MUN rats without supplementation, the same tissues were collected for comparison.Blood pressure was evaluated using tail-cuff plethysmography (see protocol below), and at the end of the third measurement, blood was collected from the tail in tubes containing 5% heparin. Thereafter, the rats were supplemented with CSE . The supplementation was based on voluntary ingestion using a gelatin cube containing the exact dose of CSE based on the weight of the rat (see protocol below). At the end of the supplementation period, blood pressure was measured again, and blood was collected. The rats were sacrificed with COSystolic blood pressure (SBP) was assessed via tail-cuff plethysmography coupled with a pressure acquisition system , as previously described . The ratThe measurements were performed over 3 consecutive days, always by the same qualified researcher. The data from the first day were not included in the statistical analysis since it was the day of adaptation to the procedure.3 size cubes. Both neutral and CSE-gelatins were left at 4 °C for solidification, and the individual cubes were extracted and frozen until used. CSE supplementation was performed using gelatin cubes and voluntary ingestion, as we previously described . The cubFor voluntary ingestion, the rat was first trained with neutral gelatin cubes until full acceptance by placing the animal in an empty box without bedding with the cube for 2 h. This procedure was repeated daily until the cube was fully ingested, indicating acceptance . Then, supplementation was initiated with the CSE-containing cubes at a dose of 250 mg/kg/day. To adjust the dose, the rats were weighed weekly. The CSE gelatin cubes were given for 2 weeks, 5 days/week (excluding weekends). We used this dose and regime of administration since we previously demonstrated, through a metabolomic study, the presence of bioactive ingredients from CSE in rat plasma with only 4 days of supplementation at 250 mg/kg/day and a further increase after a total of 8 days with a similar 2-day cessation of supplementation during the weekend .The collected blood samples were centrifuged for 10 min (900 g at 4 °C), and the plasma was aliquoted and stored at −80 °C until use.Protein content was assessed with a Bradford protein content assay , and absorbance was measured in a microplate reader  at 595 nm, using bovine serum albumin as standard.Reduced glutathione (GSH) was assessed as previously described . A fluorPlasma catalase activity was assessed using a kit  according to the manufacturer’s instructions and expressed as U catalase/mg protein in the sample. Absorbance was read at 540 nm in a microplate reader. Superoxide dismutase (SOD) activity was also assessed using a kit , according to the manufacturer’s instructions, as previously described, . AbsorbaPlasma carbonyl levels were evaluated as previously described  with a 2Plasma lipid peroxidation was assessed using a kit  that analyzes malondialdehyde (MDA) and 4-hydroxy-trans-2-nonenal (HNE) concentrations, as previously described . The exp4P2O7, 1 mM DTT, 20 mM HEPES, 20 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 2 mM phenylmethylsulfonyl fluoride (PMSF), 1 µL/mL leupeptin, 1 µL/mL aprotinin, and 0.5 µL/mL N-alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK-hydrochloride). Thereafter, the tissue was centrifuged , and the supernatant was collected and stored at −80 °C. Bradford protein assay dye  was used to assess protein content in the samples, measuring absorbance in the microplate reader at 595 nm. The levels of protein expression were assessed via Western blotting, as previously described for the heart  and aortPrimary antibodies were incubated overnight at 4 °C. After washing, the secondary antibodies  were incubated for 90 min. The blots were washed and incubated in commercially enhanced chemiluminescence reagents , and the resulting bands were detected using a ChemiDoc XRS + Imaging System . To ensure equal sample loading, all blots were re-incubated with glyceraldehyde 3-phosphate dehydrogenase (GADPH). Blots were quantified using the Image Lab 6.1 software , and the expression levels of each protein were normalized with GADPH.The antibodies we used, with their dilution, purchase company, molecular weight (MW), SDS-PAGE %, and protein loading quantity, are shown in All the gels used in the study are shown in TM image analysis software  was used to quantify media thickness.Two rings were cut from the PFA-fixed aortic segment and mounted directly on a slide provided with a small well filled with CITIFLUOR-AF mounting medium . The rings were visualized with a laser scanning confocal microscope  at excitation 488 nm/emission 500–560 nm, the wavelength at which elastic lamellae can be detected via autofluorescence, showing the limits of the arterial media. Single images were captured with a ×10 air objective, and the MetaMorphrio, dplyr, compareGroups, ggpubr, devtools, and ggplot2 packages. Given the small sample size, data were expressed as median and interquartile range [Q1; Q3. Statistical analysis was performed with 2-way ANOVA, analyzing the effects of sex and group , extracting the interaction effect between them. Significance level was established at a p-value < 0.05.Statistical analysis was performed with the R software  within the RStudio interface using the p-values of the group and group and sex interactions  but not between sexes  or the interaction effect , suggesting the effect of CSE on active cardiac Nrf2 in both sexes.In the hearts, there was no difference in total Nrf2 expression between MUN rats receiving CSE compared with non-supplemented rats, neither in males nor in females, as shown by the non-significant een them A. Howeven effect B. The rep-value for the group but not for sex or the interaction effect , and it was near statistical significance for sexes  and the interaction . These data suggest that, in aortas, CSE supplementation had a larger effect on active Nrf2 in male rats.In the aortas, the total Nrf2 expression was higher in rats receiving CSE, both in males and females, as shown by the significant n effect C. Howeven effect D. The rep-values for group, sex, and the interaction effect , as shown by the non-significant  effects . These dSimilarly, in the aortas, no statistical differences were detected in the expression of any of the evaluated antioxidant enzymes between rats receiving CSE and those without supplementation considering group, sex, or the interaction between them , indicatp-value for the group. The thickness was smaller in females, as shown by the significant p-value between the sexes  with previously demonstrated antioxidant and vasodilatory effects in vitro. Our main findings were that CSE supplementation was able to reduce blood pressure in male MUN rats, likely because of an upregulation in the expression of e-NOS and Nrf2 in cardiovascular tissues and the elevation of GSH, although we cannot discard a direct antioxidant effect due to CSE bioactive components. Despite the significant increase in Nrf2 in hearts and aortas, we did not detect a significant upregulation in antioxidant enzyme expression in these tissues or an improvement in vascular remodeling, probably because of the short duration or insufficient dose of CSE supplementation in counteracting the severity of the damage in the aged hypertensive rats.The present work aimed to evaluate the 2, (−)-epicatechin, (+)-catechin, and important amounts of methylxanthines (theobromine and caffeine) [The chocolate manufacturing industry generates considerable amounts of waste yearly with negative environmental consequences, making the re-utilization of its by-products highly desirable . Cocoa saffeine) . Furtheraffeine) . in vitro, in cell cultures, and in arteries [CVD continues to rise despite considerable progress in prevention and treatment  and is aarteries ,24, suggarteries ,31. We carteries . The MUNarteries , aorta harteries , and cararteries . We usedarteries . MUN femarteries , as obseSince the MUN rat model of hypertension has been fully characterized and compared with controls, in the present study, we did not use control rats and only evaluated the effect of CSE on the MUN rats, comparing males and females. Our data demonstrated that short CSE supplementation reduced blood pressure in MUN males but not in females. We suggest that sex differences in the response to CSE may be related to the higher degree of hypertension and oxidative damage in MUN males. This was evidenced in the present study by the larger plasma carbonyl levels, a biomarker of oxidative damage to proteins. These results are in accordance with our previous data showing larger oxidative stress in MUN males throughout their lifespan, from weaning to aging, due to both antioxidant deficiency and the increased expression of ROS-producing enzymes in cardiovascular tissues ,21,33. FThe blood-pressure-lowering effects of CSE are likely due to the presence of bioactive components in CSE, rich in phenolic compounds and methylxanthines . In humain vitro vasodilator action of CSE and the above-mentioned molecules through superoxide anion scavenging and NO protection from degradation [Regarding the mechanism implicated in blood pressure reduction by CSE supplementation in our experimental model, direct or indirect actions from bioactive components of CSE are possible. Regarding direct effects, we have evidence suggesting that CSE bioactive components (at least caffeine and theobromine) can reach plasma after 7 days of supplementation at 250/mg/kg/day, as can the radation . We alsoradation . Thereforadation .A second possibility is the indirect effect of CSE components stimulating the expression of enzymes relevant to cardiovascular function. According to our data, one of the possible effects is the stimulation of e-NOS expression in the aorta, which may counteract the endothelial dysfunction we previously observed in MUN rats . A recenBesides e-NOS expression and NO synthesis, CSE may also improve NO bioavailability through antioxidant action . We explWe evaluated those enzymes that have been reported in the literature to be stimulated by Nrf2, such as SOD, catalase, and HO-1 ,26, and Our CSE supplementation regime was not able to increase plasma SOD or catalase activities, but a significant elevation of GSH was observed. This increase may be related to Nrf2 upregulation, which is able to stimulate its synthesis through the activation of glutamate–cysteine ligase, the rate-limiting enzyme responsible for GSH synthesis . AnotherThe present study demonstrates the capacity of CSE supplementation to reduce blood pressure in aged male hypertensive rats through the stimulation of e-NOS and Nrf2 expression and GSH, together with the possible direct antioxidant effect of circulating bioactive components, improving NO availability and vasodilatation. We did not observe an elevation in antioxidant enzymes, probably because of the short duration of the treatment or low doses. There was a sex-dependent effect for CSE on blood pressure reduction, with a larger effect on males despite a similar degree of enzyme expression, which may be related to the higher oxidative damage in MUN males. However, the influence of sex on the CSE response is an aspect to be considered in future clinical trials with this extract. In summary, our data support the beneficial effects of cocoa shell by-products in the context of hypertension and aging and the possibility of conducting a clinical trial to develop food ingredients or nutraceuticals for human use, and they also contribute to the circular economy. The adjustment of doses and the length of supplementation are aspects that deserve to be explored in future studies.


Surgical intervention is necessary for resolving the symptoms of the spinal cord and nerve compression caused by symptomatic metastatic epidural spinal cord compression. However, surgeons are constantly seeking ways to improve surgical efficiency and safety. This study aims to evaluate the efficacy of 3D simulation/printing-assisted surgery for symptomatic metastatic epidural spinal cord compression of the posterior column.P < 0.05 was considered statistically significant.We retrospectively analyzed the clinical data of patients who underwent surgical treatment for symptomatic metastatic epidural spinal cord compression of the posterior column in our hospital from January 2015 to January 2020. The simulated group underwent a 3D digital simulation of the lesion area using imaging data before surgery. Twelve patients in the simulated group also received 3D printing, while the direct surgery group did not receive any 3D simulation or printing. All patients were followed up for at least 2 years. We collected clinical data, including operation time, intraoperative blood loss, pedicle screw adjustment rate, intraoperative fluoroscopy times, the incidence of dural injury and cerebrospinal fluid leakage, VAS score, postoperative neurological function improvement, and tumor recurrence. Statistical analysis was performed using SPSS23.0, and A total of 46 patients were included in this study, with 20 in the simulated group and 26 in the non-simulated group. The simulated group had better operation time, intraoperative blood loss, screw adjustment rate, fluoroscopy times, and incidence of dural injury/cerebrospinal fluid leakage compared to the non-simulated group. The VAS scores of the two groups improved significantly after the operation and at the last follow-up compared to before the operation. However, there was no statistically significant difference between the two groups. There was also no statistically significant difference in neurological function improvement between the two groups. In the simulated group, 25% of patients relapsed, while in the non-simulated group, 34.61% of patients relapsed. However, there was no statistical difference between the two groups.Preoperative 3D simulation/printing-assisted surgery is a practical and feasible approach for treating symptomatic metastatic epidural spinal cord compression of the posterior column. Common primary tumor types include lung cancer, breast cancer, prostate cancer, liver cancer, kidney cancer, and cervical cancer . SurgicaThe incidence of metastatic epidural spinal cord compression (MESCC) accounts for 5% to 10% of all malignancies . About o3D simulation/printing effectively uses CT imaging data to project human tissues and organs outside the body, creating visual three-dimensional digital models or physical models with equal proportions. These techniques display 3D information about the human body structure with higher resolution , providi2.2.1.We conducted a retrospective analysis of clinical data from patients with metastatic epidural spinal cord compression of the posterior column who underwent surgical treatment at our hospital between January 2015 and January 2020. The inclusion criteria were as follows: (1) confirmation of spinal metastases by CT-guided biopsy; (2) symptoms of epidural spinal cord compression, including adicular pain, ataxia, motor weakness, sensory disturbances, and/or bladder dysfunction; (3) lesions affecting only the posterior column of the spine, not the anterior and middle columns; (4) Involvement of no more than 2 segments without multiple spinal metastases; (5) the patients were older than 18 years. (6) all surgical procedures were performed by the same medical staff; and (7) patients still alive at the last follow-up. A total of 46 patients were included in this study, with 20 cases in the simulated group and 26 cases in the non-simulated group. In the simulated group, all patients underwent a 3D digital simulation of the lesion area using imaging data before surgery. Of these, 12 patients received further 3D printing, while those in the non-simulated group did not undergo any simulation or printing. All patients were followed up for at least 2 years. This retrospective study was supported by the ethics committee of our institution.2.2.To obtain three-dimensional data of the spine, spine three-dimensional reconstruction computed tomography was used, and the data was imported into reconstruction software (Mimics Innovation Suite 21.0) to construct a 3D model. Different colors were used to identify the location and extent of the intraspinal tumors. This model was used to clarify the adjacent and surrounding invasion of the intraspinal tumors. Although 3D printing based on 3D modeling was optional for the patient because it was an additional expense, FARSOON-SS402P  was used for those who chose to undergo the procedure.2.3.The patients were placed in a prone position under general endotracheal anesthesia. A posterior median incision was made at the lesion site to expose the ackes, lamina, facet joints, and transverse processes. The metastasis location and boundary were then exposed and defined. With the help of C-arm fluoroscopy, the pedicle screw was inserted into the diseased segment vertebra, and the connecting rod was fixed to retain sufficient space for tumor resection. Laminectomy and adnexectomy were performed to remove the tumor with margin negative resection. We carefully separated the contact interface between the tumor and the dura and performed detailed blunt separation where the dura was surrounded and adhered. Bone grafting was then performed, followed by complete hemostasis and placement of a drainage tube. Finally, the surgical incision was closed, and specimens were collected for pathological examination.2.4.Physicians who planned to participate in the operation carefully discussed and evaluated the 3D virtual model or 3D printed solid model one day before the operation. The evaluation included presetting reasonable screw entry points and screw entry directions, determining the negative resection margin of the tumor, identifying the tumor's adjacent relationship with important blood vessels and nerves, and predicting the risk of tumor resection and decompression based on the degree of tumor compression on the dura. Half an hour before the start of the operation, all physicians participating in the operation would re-evaluate the operation plan based on the 3D virtual model or 3D printed solid model. During the operation, it was particularly important to expose the key anatomical structures of the patient's surgical site. During the pedicle screw insertion, the surgeon compared the actual screw insertion point and direction with the 3D model. If there was an obvious deviation, it needed to be re-evaluated. During tumor resection, the surgeon first observed the tumor boundary with the naked eye, then compared it with the preset tumor-negative resection boundary in the 3D model, and made reasonable corrections to the resection boundary. During dura mater decompression, the more oppressed the dura mater was and the riskier the operation, the more careful the operation should be. Additionally, special attention should be given to the adhesion between the dura and the tumor.2.5.P < 0.05 was considered statistically significant. Continuous data with a normal distribution were analyzed using a t-test, categorical data were analyzed using the chi-square test, continuity-corrected chi-square test, or Fisher's exact probability method, and graded data were analyzed using the Mann–Whitney U-test.Clinical data, including baseline information such as gender, age, lesion site, lesion levels, pathological sources, Spine Instability Neoplastic Score(SINS)  and foll3.3.1.3.2.The simulated group had an operation time of 1.94 + 0.29 h, and intraoperative blood loss of 382.50 + 94.97 ml. In contrast, the non-simulated group had an operation time of 2.15 + 0.25 h and intraoperative blood loss of 478.85 + 125.83 ml. In the simulated group, a total of 146 pedicle screws were implanted, with a screw adjustment rate of 12.33% and an average of 1.30 + 0.47 intraoperative fluoroscopy times. In contrast, the non-simulation group had a total of 186 pedicle screws implanted, with a screw adjustment rate of 21.50% and an average of 1.73 + 0.67 intraoperative fluoroscopy times. Three patients in the simulated group experienced dural damage/cerebrospinal fluid leakage, while 11 patients in the non-simulated group did. None of the patients experienced hematoma, internal fixation failure, internal fixation loosening or fracture, wound infection, bone graft nonunion, and lower extremity venous thrombosis. The statistical analysis indicated that the operation time, intraoperative blood loss, screw adjustment rate, radiographic times, and incidence of dural damage/cerebrospinal fluid leakage were all better in the simulated than in the non-simulated group. During the two-year follow-up period, 25% of patients in the simulated group and 34.61% in the non-simulated group experienced a relapse, but there was no statistical difference between the two groups. The results are shown in 3.3.Both groups presented with varying degrees of low back pain symptoms before the operation. Fortunately, after surgical treatment, these symptoms were significantly relieved compared to preoperative levels. However, there was no significant difference in VAS pain scores before the operation and 1 week after the operation between the two groups. The patients in both groups had varying degrees of spinal cord hypofunction due to dura compression before the operation, but there was no neurological deterioration after the operation. Neurological function was assessed using the ASIA grading method, and the postoperative neurological function improvement was graded as follows: significant improvement (ASIA grading increased by 2 levels), slight improvement (ASIA grading increased by 1 level), or no improvement (ASIA grading maintained at the same level). The results showed that there was no significant difference in the improvement of neurological function between the two groups. 4.The surgical methods for treating spinal metastases are rapidly developing, with minimally invasive techniques being a trend. Currently, minimally invasive techniques for spinal metastases treatment mainly include percutaneous instrumentation, mini-open approaches for decompression, and tumor removal with or without tubular/expandable retractors and thoracoscopy/endoscopy . Researcen bloc spinal resection should not be ignored, particularly in resection of more than two levels (Although minimally invasive, navigation, and robotics technologies have significantly improved the efficiency and safety of spinal metastases resection, traditional open surgery remains an indispensable option for surgical treatment of MESCC, especially for giant tumors, multi-segment tumors, and tumors with complex anatomy. Palliative resection is a relatively traditional approach aimed at relieving nerve compression, rebuilding spinal stability, and improving the patient's quality of life. For solitary or short-segment vertebral metastases, the en-bloc technique has become a classic and mature approach, and its safety and efficacy have been recognized by the industry . Howevero levels . In metao levels , multidio levels , 3D prino levels , and into levels , and theIn the treatment of MESCC of the posterior column, the open posterior approach provides the surgeon with a clear view of the surgical site, enabling a comparison with the preoperative surgical planning based on 3D modeling/printing physical model. The use of 3D modeling/printing can provide additional information to surgeons, such as determining the tumor boundary, identifying the negative resection range, and assessing the relationship between the tumor and the dura, blood vessels, and nerves. However, when dealing with MESCC of the anterior and middle columns, the surgical field of view is often limited, restricting the surgeon's ability to follow a physical model-based surgical plan, even with the assistance of 3D modeling/printing. Therefore, this study emphasizes the usefulness of 3D simulation/printing technology in the treatment of MESCC of the posterior column.Previous experience has shown that in the fields of neurosurgery and spine surgery, 3D printing can be used for anatomical training, surgical simulation, tumor biopsy, and resection, and for designing unique intraoperative implants and surgical equipment. In cases involving unfamiliar anatomical regions or complex neurovascular structures, virtual preoperative simulation can provide more valuable information than simple 2D images or limited CT-based 3D reconstructions . 3D prinOur study focused on 3D simulation/printing-assisted surgery for symptomatic metastatic epidural spinal cord compression of the posterior column for the first time, and our results were consistent with previous studies. 3D simulation/printing can provide surgeons with effective preoperative planning, allowing for the prediction of key points and potential risks, thereby increasing surgical proficiency and accuracy during the operation. Our research showed that 3D simulation/printing assisted surgical treatment of symptomatic metastatic epidural spinal cord compression of the posterior column can reduce operation time, intraoperative blood loss, screw adjustment rate, fluoroscopy times, and incidence of dural injury/cerebrospinal fluid leakage, making the operation more efficient and safer. These results illustrate that 3D simulation/printing-assisted surgery is a practical and feasible option for treating symptomatic metastatic epidural spinal cord compression.3D printing also has many disadvantages that cannot be ignored. For example, it is expensive and often not covered by national health insurance, making it less accessible for financially disadvantaged patients who may be reluctant to accept this additional expense. Furthermore, due to the limited availability of 3D printing technology in some hospitals, third-party cooperation may be necessary, leading to increased difficulty in communication between doctors and patients or potential patient mistrust. Consequently, the use of 3D printing is optional for patients at our institution. After receiving a sufficient explanation from the doctor, patients can weigh the pros and cons and make an informed choice. Therefore, the wider adoption of 3D printing technology still has a long way to go. Fortunately, 3D simulation can also meet clinical needs and achieve desirable outcomes.Additionally, there is no clear evidence that preoperative 3D simulation/printing has a positive effect on tumor recurrence or survival time after resection. The decision of whether further radiotherapy/chemotherapy is necessary after surgery depends on the pathology of the metastatic tumor and the treatment status of the primary tumor. Several studies have demonstrated that preoperative adjuvant chemotherapy and postoperative further chemotherapy and radiotherapy may improve the prognosis and survival of patients with various types of spinal metastases –35.5.The use of preoperative three-dimensional simulation/printing-assisted surgery is a practical and feasible approach for treating symptomatic metastatic epidural spinal cord compression. It can significantly reduce the operation time, intraoperative blood loss, screw adjustment rate, fluoroscopy times, and the incidence of dural injury and cerebrospinal fluid leakage.


Obesity is a common, serious, and costly disease. More than 1 billion people worldwide are obese—650 million adults, 340 million adolescents, and 39 million children. The WHO estimates that, by 2025, approximately 167 million people—adults and children—will become less healthy because they are overweight or obese. Obesity-related conditions include heart disease, stroke, type 2 diabetes, and certain types of cancer. These are among the leading causes of preventable, premature death. The estimated annual medical cost of obesity in the United States was nearly $173 billion in 2019 dollars. Obesity is considered the result of a complex interaction between genes and the environment. Both genes and the environment change in different populations. In fact, the prevalence changes as the result of eating habits, lifestyle, and expression of genes coding for factors involved in the regulation of body weight, food intake, and satiety. Expression of these genes involves different epigenetic processes, such as DNA methylation, histone modification, or non-coding micro-RNA synthesis, as well as variations in the gene sequence, which results in functional alterations. Evolutionary and non-evolutionary  factors have shaped the genetic predisposition or protection from obesity in modern human populations. Understanding and knowing the pathogenesis of obesity will lead to prevention and treatment strategies not only for obesity, but also for other related diseases. Obesity represents a burden on public health. Its prevalence is increasing at an alarming rate worldwide, and it ranks fifth among causes of death worldwide ,2,3,4.https://ibdofoundation.com/, accessed on 12 May 2023) [The incidence of obesity has tripled in the last few decades, such that more than two thirds 70.2%) of the United States adult population is overweight or obese, and almost half of adults 48.5%) live with prediabetes or diabetes, conditions strictly linked to obesity. The prevalence of being overweight and obesity in adults in Europe is 34.8% and 12.8%, respectively % live wi. The pre0.2% of tThere are several possible mechanisms leading to obesity. The traditional view is usually that the main cause is the significantly more excess energy stored than the energy the body used. The excess energy is stored in fat cells, thereby developing the characteristic obesity pathology . HoweverGenetics certainly play an important role in the genesis of the obesity phenotype. Just think that genetic variation between people accounts for 50 to 70% of the difference in BMI, but genetics are complex. The amount of body fat is affected by many different factors, including how efficiently the digestive system extracts nutrients from food, how easily nutrients are stored as fat or burned as fuel, and how hungry we feel. Each of these factors is influenced by hundreds of genes. The contribution of each of these genes is not defined. While there are some genetic variants that greatly increase the risk of obesity, these are rare. For most of us, each gene contributes only a small fraction of risk, but all together, they can mean the difference between being naturally thin and having to struggle to maintain a healthy weight. Even people with a similar risk of obesity may have different genetic reasons for that risk.Because of this, different people may need different approaches to weight management. The heritability of obesity and body weight in general is high, and a small number of confirmed monogenic forms of obesity have been identified ,15. GenoWe carried out an informal and non-systematic search by selecting analyses and genetic association studies to detect all possible clues suggesting the role of the genome in influencing the obesity phenotype and to find possible therapeutic strategies based on selective targets. We will try to analyze the contribution of the obesity susceptibility genes that we have been carrying around for millennia and how these only recently, in combination with the modern environment, make us obese.Monogenic forms of obesity are rare. These are caused by mutations in a single gene ,21,22,23MC4R) gene (OMIM*155541) [MC4R is a G protein-coupled receptor that is critical in the leptin–melanocortin pathway. MC4R is expressed in districts, such as the hypothalamus, as well as in adipocytes, but its deficiency in mice leads to obesity with a framework that includes hyperphagia and hyperinsulinemia [The most common monogenic form of obesity is caused by mutations in the melanocortin-4 receptor (*155541) ,24. MC4Rulinemia . In humaulinemia . MC4R-asulinemia .LEP, OMIM*164160; LEPR, OMIM*601007) have been described since the end of the 1990s. They are considered to have biological roles related to the proteins encoded by these genes [Mutations in leptin and leptin receptor genes  is an appetite inhibitory gene that produces peptides that regulates body weight, and, among them are α- and β-melanocyte stimulating hormone (MSH) [PCSK1 prohormone convertase 1 encodes for PC1 Proconvertase 1 (OMIM*162150), and it is involved in processing peptide hormones, such as POMC [PCSK1 gene [PCSK1 gene. Most of those patients have early-onset obesity and hyperphagia [Among the recognized genes, some encode for components of the melanocortin pathway, such us ne (MSH) ,34. Mutane (MSH) ,36. In pne (MSH) . In fact as POMC . StudiesSK1 gene ,40. In perphagia ,42.NPY gene, which encodes neuropeptide Y (OMIM*162640), a potent hypothalamic orexigenic peptide [the NPY gene and obesity. In fact, administration of neuropeptide NPY causes increased food intake and obesity. Neuropeptide Y neurons inhibit POMC neurons that decrease appetite, and they activate orexin- and melanin-concentrating hormones, which are appetite-inducing peptides [Another gene of interest is the  peptide . There ipeptides .FTO gene (OMIM*610966), known as “the fat mass and obesity associated gene”, which is an obesity susceptibility gene [FTO and obesity is documented in different populations [FTO inactivation in mice leads to postnatal growth retardation and a decrease in adipose tissue. Those lean mice have an increase in energy expenditure and systemic sympathetic activation and a decrease in hyperphagia, which is interrelated with spontaneous locomotor activity [FTO gene, called rs9939609 A, correlates with a lower feeling of satiety following meals. AA individuals, homozygous for this form of the gene, in fact have altered circulating levels of the hormone ghrelin, a central element of the neuroendocrine control of energy homeostasis [IRX3 and IRX5 that are one Mb apart [FTO maps, and they elegantly demonstrated that coding variants of FTO have pleiotropic effects across multiple tissues, whereas non-coding variants mediate their phenotypic effects by specific tissue and temporal regulation of gene expression. These results demonstrate that the genetic architecture of associated loci in this region associated with obesity may result in broad pleiotropy, allelic heterogeneity, shared allelic effects between tissues, and temporally limited effects.An Important recognized gene is the ity gene . Howeverulations ,47,48. Factivity . Intereseostasis . HoweverMb apart ,52, suggMb apart  examinedGenes with high penetrance towards the obesity phenotype are also found in syndromic forms of obesity manifesting with additional phenotypes and often presenting as part of a distinct genetic syndrome  Table 1Table 1. SIM1 OMIM*603128) gene, which encodes a transcription factor active during neurogenesis, proposed as a candidate for involvement in certain dysmorphic features , brain development abnormalities, and/or disability cognition of Down syndrome [Some of the most serious complications of this syndrome are related precisely to obesity and hyperphagia ,56. It isyndrome . For thisyndrome .FMR1 gene (OMIM*309550). It is associated with retardation and phenotypic features, such as prominent ears and elongated face. In more than 30% of those affected, obesity problems are found [Studies also delve into fragile X syndrome due to the repetition of CGG triplets (over then 200), which results in the silencing of the re found . In somere found ,61.BDNF gene (brain-derived neurotrophic factor) (OMIM*113505), suggesting that obesity phenotype is due to the alteration in this gene [Another less prevalent syndrome is WAGR syndrome, caused by 11p13 deletions, and about 30% of those affected have obesity. Most of them have a deletion in the his gene ,63.As with all complex and multifactorial traits, many susceptibility genes contribute to the obesity phenotype. Polygenic obesity is certainly the most common form of obesity, which inevitably results from the interaction between obesogenic environmental factors and variants of different risk alleles. Unlike monogenic and syndromic obesity, it is more difficult to study this variety of obesity because, hypothetically, every individual has a different number of variants, which can contribute to the phenotype with a different “weight” and with complex allelic interactions that are difficult to evaluate . NumerouOne of the first obesity genes identified is FTO was described in monogenic form. Some genetic variants increase the risk of obesity, while others reduce it. Even the high-risk forms of FTO have little effect on body fat among people who exercise a lot or eat low-fat diets. FTO gene encodes a nuclear protein of the non-heme iron superfamily related to AlkB and 2-oxoglutarate-dependent oxygenase. Recently, FTO was shown to mediate m6A demethylation of long-spliced element 1 (LINE1) RNA in mouse embryonic stem cells (mESCs), by regulating LINE1 RNA abundance and local chromatin status, which, in turn, modulates the transcription of LINE1-containing genes. These results suggest broad effects of LINE1 RNA m6A demethylation by FTO in mammals .Numerous SNPs identified as susceptibility genomic biomarkers for obesity are indeed found in more or less extensive phenotypes of obesity as BMI, adiposity traits, adipogenesis, lipid metabolism, insulin secretion, and genes involved in neuro-circuits of appetite and satiety, such as BDNF, NEGR, IRS1, and INSIG2 ,69,70,71Some considerations, however, can be made based on the GWAS results. In fact, looking at the data obtained from a very broad meta-analysis both in terms of subjects studied almost 340,000) and of number of polymorphisms (SNPs), it needs to be underscored the fact that all of the 97 loci associated with BMI affect genes expressed at the level of the central nervous system [0,000 andABCC5, ARID1B, CD247, CHD3, CNTN1, CPNE6, EEFSEC, FAM53B, GABBR1, IGFBP6, KCNQ1, MAD1L1, RPS6KA2, SNO2, SH2B2, SLC43A1, SLCO3A1, STK40, and SYNJ2 are characterized by changes in DNA methylation [IGF2 gene (Data obtained by comparing unexposed siblings) [INSIGF2, GNASAS1, MEG3, IL10, and LEP [MMP7, KCNK4, TRPM5, and NFKB1) in cord blood DNA [Genes are not “naked” in our genome but are subject to chemical modification regulating gene activities ,76. Thishylation ,84. Intehylation . Individhylation ,87. Peopiblings) . Mothers and LEP . Other slood DNA . Offsprilood DNA .Micro-RNA (miRNA) dysregulation has also been observed in obesity ; miRNAs The study of the epigenetics of obesity is of particular interest for developing and activating prevention programs at the population level. Indeed, as stated by Danielle Reed, “Epigenetics is sort of like writing in pencil, whereas genetics is really writing in pen” . This mePLIN1 14995 A > T carriers [IRS1 rs2943641 CC genotype [PPM1K rs1440581 have greater weight loss on a low-carbohydrate diet [FGF21 region improves the risk of developing obesity, determining the preference for carbohydrate intake [It is conceivable to foresee that, in the coming years, genetic analysis technologies will identify obesity susceptibility genes, and PRS algorithms will be available on a large scale to identify individuals at high risk of developing obesity and related diseases from birth or before. A child with a predilection toward a genetic form of obesity may be treated with a particular diet, or they may be metabolically “reprogrammed” with the administration of drugs, hormones, and, perhaps, in some rare cases of monogenic obesity, with gene therapy. All this presupposes an accurate medical history and adequate genetic counseling before and after the test. A detailed family history, as well as psychosocial history, diet assessment, and physical activity/exercise are key elements in the diagnostic and therapeutic process of obesity. In fact, only after excluding endocrine causes of obesity and syndromic forms will it be possible to carry out genetic or epigenetic tests to identify adequate and actionable therapeutic targets. Identifying the genetic variants associated with diet-related diseases to study the variability of an individual’s response to diet and nutrition is now considered an interesting and innovative line of research . Studiescarriers . Anotherate diet . Additioe intake . These se intake .It is indisputable that food has a huge impact on mental and physical health. Genetic and biochemical knowledge today make the time ripe for clinical trials of specific approaches to the prevention or treatment of diseases, such as obesity, using food as medicine .POMC, PCSK1, or LEPR genes and Bardet Biedl syndrome. The advantage of this drug is that it acts directly on the MC4R receptor, bypassing multiple targets, which may be mutated in the leptin pathway.The Food and Drug Administration  has approved two drugs intended for patients with genetic causes of obesity: metreleptin and setmelanotide. The other drugs, such as semaglutide, liraglutide, phentermine–topiramate, and naltrexone–bupropion is approved for weight loss in the general population and can be used to treat patients with genetic obesity. Metreleptin is a leptin analog used to treat patients with congenital generalized lipodystrophy in leptin-deficient patients with mutations in the leptin gene. However, metreleptin cannot be used in patients with leptin receptor mutations or mutations downstream of the leptin signaling pathway. The use of this drug is monitored by the FDA’s Risk Evaluation and Mitigation Strategies (REMS) Panel. Setmelanotide is a MC4R agonist used in obese patients with genetic mutations in the HOXB1, PRKCZ, SLC38A10, and SECTM1 genes [From what has been described above, it is, therefore, possible to state that distinct interventions of a nutritional or pharmacological type can modify the epigenome of the body for the benefit of patients with obesity. However, in some cases, it is a necessary recourse to resort to bariatric surgery. This, too, can determine epigenomic changes, such as the pattern of exosomal micro-RNAs of adipocytes, and it can cause epigenetic changes in differential methylated regions in M1 genes .POMC and PCSK1, with results not significantly different from noncarrier patients of these mutations [LEP, LEPR, SIM1 and PCSK1 genes demonstrated significantly less weight loss at six years than patients without the variant [MC4R variants, studies show how there is an identical weight loss at one year for patients with the MC4R variants and the control group [MC4R patients have insufficient weight loss [Regarding surgical treatment, according to some studies, outcomes are worse in patients with Prader-Willi syndrome than in patients with common obesity . Accordiutations . An addi variant . Concernol group  or withool group . Cooimanght loss .RUNX1, NDUFC2, THADA, MEF2A, and PRKAA2 genes. Indeed, it has been shown that, in patients who lose weight, their methylation profiles of the RYR1, TUBA3C, and BDNF genes resemble those of lean individuals [Even regular exercise can be considered an “environmental therapy”, as it can cause widespread changes in DNA methylation in the ividuals ,85,100.CREBRF gene [INSIG2 gene, present in 10% of individuals of different ethnic origins, suggesting that it is ancient and is consistent with the hypothesis that such alleles only became deleterious in modern times [FTO and TCF7L2 genes [GPR75 LOF variants against weight gain and the related disruption of glucose and insulin metabolism. This could suggest the development of inhibitors of GPR75 cellular functions as potential therapeutic drugs for obesity. Interestingly, functional SNPs within GPR75 were found recently within the evolutionary constraint DNA regions, which represent 10.7% of the human genome, which is conserved in more than 98% of the 240 placental mammals analyzed [A century ago, obesity was rare. Now, people around the world are gaining weight, with 70.2% of adults in the US currently being overweight or obese. Obesity is linked to increased rates of health problems, such as cardiovascular disease and diabetes. Why the sudden change? Is obesity in our genes or has something in our environment made us fat in recent decades? The answer is both. People vary in how easily they gain weight, and much of this variation is encoded in genes and passed down through families. However, the rise in obesity has occurred too rapidly to be caused by genetic changes. Our genes are the same as they were 10,000 years ago, and it is known that it takes at least 184–1840 generations of natural selection  for a human population to undergo significant genetic change. As obesity rates have nearly tripled in just a few generations, the increase likely has more to do with changes to our environment and lifestyle than our genes . GeneticBRF gene ,111,112.BRF gene , but theL2 genes . These rL2 genes , using wanalyzed .Large-scale human exome sequencing, coupled with the analysis of methylation episignatures across the genome, is likely to become an increasingly important entry point for uncovering mechanistic insights into the biology of obesity in mammals such as humans.In conclusion, obesity is a complex health problem influenced by various factors, including behavior, genetics, and the environment. Understanding the genetic effects on appetite regulation is crucial for effective treatment and prevention of obesity. Genetics play a significant role in the regulation of appetite and the development of obesity. Exploration of genetic factors and metabolic pathways is critical for the precise treatment and prevention of obesity.