To assess the features of panel members involved in the writing of the ACR-AC and identify alignment with research output and topic-specific research publications.A cross-sectional analysis was performed on the research output of panel members of 34 ACR-AC documents published in 2021. For each author, we searched Medline to record total number of papers (P), total number of ACR-AC papers (C) and total number of previously published papers that are relevant to the ACR-AC topic (R).p < 0.0001); however, proportion of relevant papers per topic (R/P) was higher for authors with < 5 ACR-AC papers (0.10) than authors with ≥ 5 ACR-AC papers (0.07).Three hundred eighty-three different panel members constituted 602 panel positions for creating 34 ACR-AC in 2021 with a median panel size of 17 members. Sixty-eight (17.5%) of experts had been part of ≥10 previously published ACR-AC papers and 154 (40%) were members in ≥ 5 published ACR-AC papers. The median number of previously published papers relevant to the ACR-AC topic was 1 (IQR: 0–5). 44% of the panel members had no previously published paper relevant to the ACR-AC topic. The proportion of ACR-AC papers (C/P) was higher for authors with ≥ 5 ACR-AC papers (0.21) than authors with < 5 ACR-AC papers  panel experts on ≥ 10 ACR-AC panels.Nearly 45% of the panel experts had zero median number of relevant papers.Fifteen panels (44%) had > 50% of members having zero relevant papers. The use of imaging studies in medicine has dramatically increased over the past two decades . As patiThe ACR-AC are developed and reviewed by “expert panels with leaders in radiology and other specialties” . The metGiven the importance of ACR-AC in regulatory compliance and clinical decision-making, the aim of this study was to assess the scholarly expertise of panel members involved in the formation of the ACR-AC by looking at their past research productivity and topic-specific publications. We focused on the ACR-AC published in 2021, during which a total of 18 new and 13 revised topics were added to the ACR-AC.We conducted a cross-sectional analysis of all the ACR-AC published in 2021. For each ACR-AC document, we extracted the name of the paper, author name(s), and their institutional affiliation(s). Once the information from the ACR-AC document was extracted, we searched the PubMed database to record the total number of papers (P), the total number of ACR-AC papers (C), the total number of first and non-first author papers relevant to the ACR-AC topic (R) for each listed author in the ACR-AC document. To search the previously published papers relevant to the ACR-AC topic, keywords were systematically selected by including the keywords from the ACR-AC document as well as from all the clinical variants described in the respective AC paper. The search was conducted in March 2022 by two independent authors  with 20% overlap (reviewed independently by both) and any conflicting information in the data was resolved after discussion with the senior author (A.M.). In May 2022, a third author (M.K.) independently extracted the data for 7 out of 34 panels for cross-verification. The data were collected in Microsoft Excel  and analyzed using STATA/ BE Version 17 .Summary statistics were used to analyze the results of this study. Categorical measures were summarized using rates and/or counts. Number of papers was analyzed using median and interquartile range because the data were not normally distributed as determined by the Shapiro–Wilk Normality test. The proportions were calculated by dividing the total number of ACR-AC papers and relevant papers to the total number of papers found on PubMed database for each author. Logistic regression was used to calculate the normal 95% confidence intervals for each demographic group. Differences between the two groups  were evaluated using the Student’s t test. Univariate followed by multivariate linear regression models were used to identify and confirm correlations and predictive factors for the total number of ACR-AC papers by each unique author.A total of 34 ACR-AC published in 2021 were included in the study with a total panel size of 602 members that included 383 discrete panel members. Of the 602 panel positions, 468 (77.7%) positions were filled by radiology and 134 (22.3%) by experts from other specialties. The panel size ranged from 12 to 22 members with a median size of 17 members per panel. 68 out of 383 (17.5%) panel members had ≥ 10 previously published ACR-AC papers and 154 (40%) had ≥ 5 published ACR-AC papers ; however, the proportion of relevant papers per topic (R/P) was higher for authors with < 5 ACR-AC papers  than authors with ≥ 5 ACR-AC papers , although this difference was not statistically significant (p = 0.08)  and the number of relevant papers (p = 0.011) with the total number of ACR-AC papers, on multivariate linear regression, only the total number of previously published papers had a significant and independent association (p = 0.0001).When compared to authors with < 5 ACR AC papers 229/374, 60%), authors with ≥ 5 ACR-AC papers  had a higher number of total PubMed publications , and higher number of relevant papers per assigned panel topic   had a median number of zero relevant papers per author and eight panels 23.5%) had a median of ≥ 5 relevant papers per author. Two panels (6%) had ≥ 10 median number of relevant papers per author  and used by the Agency for Healthcare Research and Quality (AHRQ) which emphasizes the maximal possible use of scientific evidence, while also recognizing that the expert panel consensus is needed in scenarios where sufficient literature is not available . The metp < 0.001) but a lower proportion of relevant papers  than their counterparts on less than five panels.Our study included 34 ACR-AC papers that were released in 2021. Six hundred two panel positions comprised 383 experts with a median panel strength of 17. The panel members had a median of 45 prior publications, however, the median number of relevant papers previously published by the panel members was only 1, with nearly 44% of the authors not having any prior publications on the topic of their assigned panel. Nearly half  of the panels had zero median number of relevant papers. 44% (15/34) of the panels had > 50% of members having zero relevant papers. Additionally, 17.5% of the experts were members of more than ten panels and 40% were members of more than five panels with the panel members being on a median number of four panels. These results show that a similar pool of experts is serving on multiple panels which may be a potential source of bias in the development of the AC. For experts serving on multiple panels, their work on the respective topics assigned to different panels was less compared to the experts who were on less than five panels. Additionally, the ACR-AC documents constitute 16% of the experts’ total publications while the relevant publications accounted for only 8% of their prior research output. These results are further potentially compromised with the experts on more than five panels having a higher proportion of ACR-AC publications  also issues guidelines relevant to the specialty . These gA review of 139 clinical practice guidelines in 2018 also found low percentages of guidelines reporting important aspects of authorship, panel selection process/criteria and characteristics of individual authors . ExternaThe methodology for the ACR Appropriateness criteria is the basis for the work of ESR Referral Guideline Subcommittee (RGSC) and is adhered to as far as possible. However, additional guidance principles have been established emphasizing that any changes or additional guidelines should be evidence-based to the extent possible and expert opinion should only function as a supplement when necessary.Our study has several limitations. While prior research output is one way to evaluate a member’s expertise, it is also important to consider that members with years of clinical experience without significant research output can add value and diversify the scope of the panels. Secondly, as per ACR-AC methodology, the expert panels may include research authors, who may be members in training including radiology residents and fellows as well as attending physicians. The stated goal is to provide them with mentoring and early exposure to the process of creating evidence-based guidelines. However, it is unclear how this is consistent with the RAND-UCLA Appropriateness method or with the stated objective of having “expert” panel members. Lastly, we did not classify the previous relevant papers into Scientific papers, review articles, case reports or commentary/opinions and included all types of PubMed publications based on the keywords.In conclusion, the ACR-AC are intended to be a helpful aid for physicians to optimize the use of imaging in different clinical scenarios. The importance of ACR-AC cannot be overstated given their national implementation under the medical appropriateness use criteria program. Our study provides the basis to reconsider staffing decisions when forming expert panels that will ultimately influence the healthcare delivery for millions of Americans every year.


No significant differences were observed in myocardial work between spontaneous activation and LBBAP. Similarly, no significant differences in myocardial work were found between HBP and LBBAP. Conclusions: Both pacing modalities provide physiological ventricular activation without significant differences when compared to each other. Moreover, there were no significant differences in QRS duration between HBP and LBBAP. However, LBBAP demonstrated advantages in terms of feasibility, as it achieved better lead electrical parameters compared to HBP . Additionally, LBBAP required less fluoroscopy time  and procedural time  compared to HBP.Background: Physiological pacing has gained significant interest due to its potential to achieve optimal hemodynamic response. This study aimed to assess left ventricular performance in terms of electrical parameters, specifically QRS duration and mechanical performance, evaluated as myocardial work. We compared His Bundle Pacing (HBP) and Left Bundle Branch Area Pacing (LBBAP) to evaluate their effects. Methods: Twenty-four patients with class I or IIa indications for pacing were enrolled in this study, with twelve patients undergoing HBP implantation and another twelve undergoing LBBAP implantation. A comprehensive analysis of myocardial work was conducted. Results: Our findings indicate that there were no major differences in terms of spontaneous and HBP activation in myocardial work, except for global wasted work (217 mmHg% vs. 283 mmHg%;  What Is Known: His Bundle Pacing (HBP) and Left Bundle Branch Area Pacing (LBBAP) have been recognized as more physiological alternatives to traditional right ventricular pacing. LBBAP has shown greater feasibility compared to HBP, although direct comparison data between the myocardial work in HBP and LBBAP are limited. What the Study Adds: Our study contributes to the existing knowledge by demonstrating that both HBP and LBBAP provide physiological ventricular activation, with no significant differences observed between the two pacing modalities in terms of myocardial work and QRS duration. However, LBBAP showcased advantages, such as reduced need for fluoroscopy, shorter procedural time, and improved electrical parameters. These findings further support the potential of LBBAP as a favorable pacing option.Historically, dual-chamber pacing emerged as a superior alternative to single-chamber pacing due to its ability to maintain physiological atrioventricular synchrony whenever possible. However, the detrimental effects of mid- and long-term right ventricular (RV) pacing ,2,3,4 haThis study is derived from the EMPATHY study , which iThe inclusion criteria for this study were as follows: (1) class I or IIa indication for pacemaker implantation, based on the European guidelines ; (2) ageThe primary endpoint of this study was to compare LV myocardial work during spontaneous ventricular activation (SVA) with HBP or LBBAP. The secondary endpoint was to compare the change in LV myocardial work between stimulated activation with HBP and LBBAP and SVA.The index procedure involved the implantation of a pacemaker and simultaneous three-dimensional electroanatomical Mapping (3D-EAM). The right ventricle and the His bundle area were mapped non-fluoroscopically using a high-density mapping catheter, which was inserted via the femoral vein. The pacing leads were inserted via the left cephalic or axillary vein and positioned using a combination of 3D-EAM and fluoroscopy .For His bundle pacing, an active fixation lead  was placed using a non-deflectable sheath . The position of the His bundle lead was confirmed using unipolar and bipolar intracardiac electrograms, with standard criteria employed to determine selective and non-selective His capture ,17.Before the procedure, the thickness of the basal interventricular septum and the presence of a septal scar were assessed. The pacing leads were inserted via the left cephalic or axillary vein. The active fixation 3830 lumenless lead  and the non-deflectable C315His sheath, as well as the Biotronik Solia 60 stylet-driven lead  and Selectra 3D, were used as the pacing leads and delivery guiding catheters.The procedure began by advancing the guiding catheter into the right ventricle (RV) over a J wire. In the left anterior oblique (LAO) view (30–40°), contrast injection via the guiding catheter was conducted to delineate the septum. After counterclockwise rotation of the guiding catheter to achieve a perpendicular orientation to the septum, a pace map was conducted by exposing the tip of the pacing lead . The aimThe screwing technique varied depending on the type of lead used. For lumenless leads, 3–5 rapid rotations were applied based on the thickness of the ventricular septum while keeping the guiding catheter in position. For stylet-driven leads, the first screw was exposed, and after a pacing check, 2–3 rapid rotations similar to lumenless leads were performed. As the screw advanced into the interventricular septum, the paced QRS morphology exhibited a right bundle branch block (RBBB) pattern, with the notch at the nadir of the QRS in lead V1 shifting to the end of the QRS, indicating successful LBBAP . To summDuring the procedure, the pacing threshold was measured in the unipolar configuration at 0.4 ms, and the impedance was also determined in the unipolar configuration. The final R-wave amplitude was measured in the bipolar configuration. Successful LBBAP was considered when a deep septal lead position was achieved, and the paced QRS complex included a terminal R/r wave in lead V1, indicating a delay in right ventricle activation. In rare cases, a QS configuration  in V1 was accepted if a terminal R/r wave in lead V1 appeared during programmed stimulation or other features indicating LBBAP, as described below, were present.Global constructive work (GCW): the sum of work performed during myocardial shortening in systole and myocardial lengthening during isovolumetric diastole;Global wasted work (GWW): the sum of work performed by myocardial lengthening in systole and myocardial shortening during isovolumetric diastole;Global work index (GWI): the work performed throughout systole, specifically between mitral valve closure and opening;Global work efficiency (GWE): expressed as the percentage ratio of GCW to the sum of GCW and GWW.After the procedure, all patients underwent echocardiographic evaluation to assess various parameters. In patients with intrinsic rhythm, we performed the echocardiogram during spontaneous ventricular activation, setting the pacemaker to VVI mode at 30 bpm. For those without intrinsic rhythm, we analyzed previous echocardiographic exams conducted during intrinsic rhythm during the previous six months. In all cases, the echocardiogram was also performed during stimulated rhythm, with the pacemaker set to VVI or DDD mode, depending on whether it was a single or dual-chamber device and the presence of sinus rhythm or atrial fibrillation. The pacing rate was adjusted to achieve overdrive pacing. Basic information, such as LV volume (in mL), LV ejection fraction (LVEF) (in %), atrial volume (in mL), and the degree of valve diseases were collected. Global longitudinal strain (GLS) and myocardial work (MW) were analyzed as part of the evaluation. MW is a novel echocardiographic technique that utilizes speckle tracking analysis to estimate left ventricular performance by measuring the area under the pressure–strain loop curve derived from GLS and blood pressure ,21,22. AA twelve-lead ECG was performed and analyzed as well. Echocardiography and ECG evaluations were conducted in different conditions, including SVA and during HBP or LBBAP, respectively. The measurements for HBP and LBBAP were obtained during pacing at a fixed rate in DDD mode with an optimized AV delay or in VVI mode, depending on whether the patients were in sinus rhythm or atrial fibrillationContinuous variables were expressed as mean ± standard deviation when normally distributed, as estimated using the Shapiro–Wilk test, or as median and interquartile range. Not all variables were normally distributed. Categorical variables were expressed as numbers and percentages.LBBAP = (GCWLBBAP − GCWSVA)/GCWSVA] and multiplied for 100 to obtain percentage change.Differences between repeated measurements during SVA and HBP or LBBAP were assessed using the Wilcoxon rank sum test. Differences between independent measurements in HBP and LBBAP were assessed using the Mann–Whitney U test. In order to adjust for baseline parameters during SVA, the comparison of myocardial work during HBP and LBBAP was not made using relative values calculated as the difference between stimulated and spontaneous parameter divided by the spontaneous one [example: dGCWp values < 0.05 were considered statistically significant. The statistical analysis was performed using STATA, version 15.0 .A total of twenty-four patients were enrolled in this study, with four of them (two in each group) being female. The patients were matched for comorbidities and implant characteristics. The baseline clinical characteristics, procedural data, and electrical parameters are summarized in p = 0.68) or coronary artery disease (CAD) . Baseline LVEF was also comparable between the two groups .Furthermore, no significant differences were observed in the major cardiovascular comorbidities that could affect the implantation procedure. Specifically, there were no significant differences in the history of atrial fibrillation (AF)  and left atrial volume were similar between groups  for II- or III-degree atrioventricular block, two patients (17%) for brady–tachy syndrome, two patients (17%) for symptomatic brady–atrial fibrillation, and one patient (8%) for sick sinus syndrome. In the LBBAP group, the indications were: eight patients (67%) for II- or III-degree atrioventricular block, one patient (8%) for brady–tachy syndrome, two patients (17%) for symptomatic brady–atrial fibrillation and one patient (8%) for sick sinus syndrome.p 0.004). Similarly, intraprocedural fluoroscopy time was higher in the HBP group compared to the LBBAP group . Electrical lead parameters were assessed after implantation. Lead impedance was significantly lower in the HBP group compared to the LBBAP group . Lead bipolar sensing was significantly lower in the HBP group compared to the LBBAP group .Selective HBP was obtained in six patients (50%). LBBAP was feasible in all the patients. The procedural duration was significantly longer in the HBP group compared to the LBBAP group .The lead unipolar threshold was significantly higher in the HBP group compared to the LBBAP group . Similarly, the paced QRS duration did not exhibit a statistically significant difference between the HBP and LBBAP groups .The baseline data for electrocardiographic and myocardial work are presented in No significant differences were observed in any of the myocardial work indexes between the HBP and LBBAP groups.p 0.534) and GCW  between the two types of activation.In the first part of the analysis, a comparison was made between SVA and paced ventricular activation in the HBP group . No signp-value = 0.016), and GWE was significantly lower in HBP ventricular activation compared to SVA .However, GWW was significantly higher in HBP ventricular activation compared to SVA  mmHg% in SVA vs. 2320 (2073–2530) mmHg% in paced rhythm with p-value = 0.929; GWW was 137 (105–286) mmHg% in SVA vs. 264 (195–341) mmHg% in paced rhythm with p-value = 0.091; GWE was 93 (83–95)% in SVA vs. 87 (83–92)% in paced rhythm with p-value = 0.109 There were no statistically significant differences in terms of myocardial work index and global constructive work between spontaneous and paced activation in both LBBAP and HBP groups;(2)The relative difference in all myocardial work parameters between sinus and paced activation did not show statistically significant differences between the two groups.In our study, we assessed the impact of HBP and LBBAP on left ventricular performance, focusing on electrical and mechanical synchronization, in a subgroup of patients with a class I or IIa indication for pacemaker implantation. Our main findings are as follows:(3)The relative difference between spontaneous and paced QRS durations did not exhibit statistically significant differences between the HBP and LBBAP groups.Spontaneous and paced QRS durations were not statistically different in either group;Myocardial work (MW) is an emerging tool in studying myocardial mechanics . Unlike The duration of surface QRS has commonly been used as an approximate measure of electrical synchronization . In our p = 0.004; 13 vs. 6, p = 0.01, respectively), despite using 3D-EAM during HBP.HBP is a technically challenging procedure that requires time and the use of a backup right ventricular pacing lead . The sucAnother important aspect to consider is the type of capture achieved by HBP. It can be either selective, capturing only the His bundle tissue, or non-selective, involving fusion capture of the His bundle and adjacent myocardium . The typIn contrast, LBBAP is defined as capturing the subendocardial area on the left side of the interventricular septum, with or without simultaneous conduction system capture. It encompasses techniques such as Left Bundle Branch Pacing (LBBP), Left Fascicular Pacing (LFP), and Left Ventricular Septal Pacing (LVSP) . ConsequThe electrical performance of the implanted ventricular leads differed significantly between the two groups, with the HBP group exhibiting higher impedance, lower sensing, and higher unipolar threshold compared to the LBBAP group. These findings emphasize the clinical advantages of LBBAP over HBP, as LBBAP consistently exhibits superior electrical parameters.One of the limitations of our study is the relatively small sample size, with only 24 patients. However, given the scarcity of data comparing HBP and LBBAP in terms of myocardial performance, our findings provide valuable insights. Another limitation is that our study was conducted at a single center, which may introduce a potential limitation in terms of generalizability. However, this limitation also eliminates the potential bias associated with variations in expertise between different centers. Additionally, the assessment of mechanical synchrony was dependent on the quality of echocardiography images. Nevertheless, the use of speckle-tracking technology has been shown to reduce this limitation, as it has a feasibility rate of over 90% .Both His Bundle Pacing and Left Bundle Branch Area Pacing offer physiological ventricular activation with optimal electrical and mechanical synchrony. However, LBBAP emerges as a more favorable option due to its greater feasibility, reduced need for fluoroscopy, and shorter procedural time, effectively addressing the limitations associated with HBP.


SLC11A1 gene in the yak population, we conducted a comprehensive analysis of the regulatory region of the yak SLC11A1 gene by cloning gene sequences of different lengths. Through transient transfection experiments, we confirmed the promoter activity of four distinct-length sequences within the 5′ regulatory region of the yak SLC11A1 gene in the RAW264.7 cell line. Moreover, our analysis identified six novel SNPs in the 5′ regulatory region sequence of the SLC11A1 gene in yaks. Bioinformatic analysis emphasized the proximity of the −1057 G>T and −127 G>A loci to the transcription factor binding sites NF-1 and NF-1/L. This observation suggests their potential impact on NF-1 and NF-1/L transcription factor binding. In summary, this study serves as a foundational reference for future research on the connection between promoter region polymorphism of the yak SLC11A1 gene and disease resistance. It also lays the foundation for investigating the transcriptional regulation mechanisms of this gene, and opens the door to further exploration in this important field.Due to a lack of understanding of the 5′ regulatory region of the SLC11A1), also recognized as natural resistance-associated macrophage protein 1 (NRAMP1), represents a transmembrane protein encoded by the SLC11A1 gene. A variety of prior investigations have illuminated its involvement in conferring resistance or susceptibility to bacterial agents, positioning it as a promising candidate gene for breeding disease-resistant animals. Yaks (Bos grunniens), renowned inhabitants of the Qinghai-Tibet Plateau in China, stand as robust ruminants distinguished by their adaptability and formidable disease resistance. Notwithstanding these unique traits, there is scant literature on the SLC11A1 gene in the yak population. Our inquiry commences with the cloning of the 5′ regulatory region sequence of the Zhongdian yak SLC11A1 gene. We employ bioinformatics tools to identify transcription factor binding sites, delineating pivotal elements like enhancers and cis-acting elements. To ascertain the promoter activity of this region, we amplify four distinct promoter fragments within the 5′ regulatory region of the yak SLC11A1 gene. Subsequently, we design a luciferase reporter gene vector containing four site-specific deletion mutations and perform transient transfection experiments. Through these experiments, we measure and compare the activity of disparate gene fragments located within the 5′ regulatory region, revealing regions bearing promoter functionality and discerning key regulatory elements. Our findings validate the promoter functionality of the 5′ regulatory region, offering preliminary insights into the core and principal regulatory segments of this promoter. Notably, we identified single nucleotide polymorphisms (SNPs) that may be associated with important regulatory elements such as NF-1 and NF-1/L. This study provides a theoretical framework for in-depth research on the function and expression regulation mechanism of the yak SLC11A1 gene.The solute transport protein family 11 A1 ( SLC11A1 gene has emerged as a focal point of research, particularly as a pivotal candidate gene in investigations pertaining to disease resistance and susceptibility, including ailments such as bovine brucellosis and mastitis [SLC11A1 gene in comparison to their susceptible counterparts. A previous study identified single nucleotide polymorphisms (SNPs) in the bovine SLC11A1 gene that are associated with susceptibility to tuberculosis (TB) [SLC11A1 gene, which have been shown to cause changes to the lengths of two microsatellites (MS1 and MS2), have been identified in different breeds of cattle [SLC11A1 gene in 771 Chinese Holstein cattle, and found four unlinked SNPs, namely 6067 (A/G) and 6358 (C/T) in intron 9, 7155 (A/G) in exon 10, and 7809 (A/T) in intron 11. The correlation analysis of the different genotypes and the incidence rate of mastitis showed that 6358 (C/T) and 7155 (A/G) of the SLC11A1 gene had significant effects on the number of somatic cells and milk production (p < 0.05). There are a total of 16 random combinations of haplotypes in this population, and the CAAA haplotype combination has been preliminarily determined to be an advantageous haplotype. Individuals with this haplotype have low somatic cell numbers and high milk production, and can be used as candidate gene markers in disease resistance breeding screening for cow mastitis [SLC11A1 gene mRNA in peripheral blood mononuclear cells of mastitis-resistant and -susceptible cow herds. For the first time, they reported that the mRNA expression of the SLC11A1 gene in mastitis-resistant herds was higher than that in susceptible herds, and cows with high mastitis resistance can be screened based on this difference [SLC11A1 gene structure and the genotype in 135 Holstein cows, and found that there was a site mutation in exon 11 of the gene. The three genotypes formed by this point mutation were significantly related to the incidence rate of mastitis in Holstein cows, and this result shows that SLC11A1 is a candidate gene for mastitis resistance [SLC11A1 gene into fetal fibroblasts of cattle. Through somatic transfer, transgenic cattle with strong resistance to tuberculosis were ultimately obtained, further indicating that the SLC11A1 gene is an important candidate gene in disease resistance breeding research [The bovine mastitis ,3,4. An mastitis , who repsis (TB) , while isis (TB) . Additiof cattle ,9,10. Inmastitis . Nam et fference . Bagherisistance . Gao et SLC11A1 gene deviates from the conventional presence of TATA boxes or CAAT boxes [SLC11A1 gene in both humans and cattle, enabling transcription initiation even in the absence of TATA and CAAT boxes.The 5′ regulatory region of a gene plays a pivotal role in directly or indirectly regulating gene transcription. In addition to serving as the docking site for RNA polymerase, this regulatory region encompasses numerous transcription factor binding sites. Promoters serve as the controllers of transcription initiation and dictate the magnitude of gene expression. Earlier investigations have revealed that the promoter region of the bovine AT boxes . This deAT boxes . HoweverSLC11A1 gene has unveiled (GT)n microsatellite polymorphic sites, suggesting their potential to influence DNA conformation and, consequently, gene transcription and downstream expression [SLC11A1 gene promoter have revealed the presence of more than two transcription initiation start elements, which counteract the deletion of the TATA box. The expression of this gene is characterized by a high degree of cell specificity, with primary expression occurring in macrophages [SLC11A1 gene and transfected it into RAW264.7 cells [SLC11A1 gene. The bovine SLC11A1 gene stands as a pivotal player in regulating the susceptibility of cattle to infectious diseases. However, research in this domain remains somewhat limited, particularly concerning the promoter of the bovine SLC11A1 gene. A special report [SLC11A1 promoter gene sequence, which was subsequently recombinant and transfected into the RAW264.7 cell line. The results of this study indicated relatively weak promoter activity. Regrettably, research on the promoter of the yak SLC11A1 gene has remained conspicuously absent from the scientific literature.Recent research on the promoter region of the human pression . Studiesrophages . Holly e.7 cells . Their fl report  describePromoters, conventionally situated upstream of genes, hold a crucial role in identifying RNA polymerase and forming binding interactions with it, thereby promoting gene transcription processes. These promoters also encompass an array of binding sites typically located upstream of the 5′ terminus of the gene, which, in turn, control the participation of protein factors. Promoters are typically limited to a range of approximately 35 base pairs from the transcription initiation site, exerting the author’s control over gene transcription efficiency. The selection of appropriate promoters stands paramount, particularly in augmenting the expression of foreign genes . PromoteA total of 89 spleen samples were collected from Zhongdian yaks at a slaughterhouse located in Shangri La, within the Diqing Prefecture of Yunnan Province, China. Subsequently, these samples were immediately stored in a refrigerator at −80 °C to preserve them for subsequent experimental research. DNA was extracted from the spleen tissue of Zhongdian yaks using a tissue genomic DNA purification kit provided by Beijing TransGen Biotech Co., Ltd. .SLC11A1 gene, we assigned different names to the inserted fragments based on their positions in the gene. These names are as follows: SLC11A1-5′ (−1418~+342), SLC11A1-5′-1 (−1418~+58), SLC11A1-5′-2 (−797~+58), SLC11A1-5′-3 (−278~+58), and SLC11A1-5′-4 (−205~+58).In order to distinguish the various gene fragment sequences within the 5′ regulatory region of the yak SLC11A1-5′, SLC11A1-5′-1, SLC11A1-5′-2, SLC11A1-5′-3, and SLC11A1-5′-4, each featuring different lengths within the 5′ regulatory region. Additionally, the vector sequences from PGL3 were integrated into the primer design. These primers were synthesized by Shanghai Sangon Biotech Co., Ltd , and constructed to incorporate two critical restriction sites, namely KpnI and XhoI. The expected lengths of the amplified fragments were as follows: 1760 bp, 1476 bp, 855 bp, 336 bp, and 263 bp  (Gene ID: 282470), Bos indicus (zebu cattle) (109570553), bison (105003764), Bubalus bubalis  (102405015), Ovis aries (sheep) (443365), and Canis lupus familiaris (dog) (478909). Similarity analysis was conducted employing the BLAST program within the NCBI database, aligning the 5′ regulatory region sequence of the yak http://bioinfo.itb.cnr.it/~Webgene/wwwHC_tata.html (accessed on 10 October 2023). Foreseeing CpG Islands was accomplished utilizing the online analysis software accessible at http://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot (accessed on 13 October 2022). Further investigations involved the utilization of the amplified promoter sequence from the yak SLC11A1 gene, which served as a reference. Online tools available at http://www.fruitfly.org/seq_tools/promoter.html (accessed on 19 October 2022) and http://bioinfo.itb.cnr.it/~Webgene/wwwHC_polya.html (accessed on 20 October 2022) were then deployed to prognosticate promoter regions, ascertain transcription start sites, delineate termination sites, and identify transcription factor binding sites. In parallel, predictions regarding duplicate components within the sequence were made, utilizing the RepeatMask program accessible at http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker (accessed on 30 October 2022) and https://www.itb.cnr.it/sun/webgene/ (accessed on 1 November 2022).The predictive TATA box was identified via the online tool available at SLC11A1 gene in yaks were submitted to the Guangzhou Vipotion Biotechnology Co., Ltd. for DNA bidirectional sequencing. Subsequently, the SeqMan program within the DNAStar software package (version 7.1) was employed to compare and analyze the sequencing results, with a focus on the identification of SNP mutations.The amplified products of the 5′ regulatory region of the SLC11A1 gene was subjected to comprehensive analysis. For any novel SNPs uncovered, a preliminary assessment of their biological significance was conducted based on the outcomes of sequence prediction analyses. Particular attention was given to assessing the precise location of these SNPs, determining whether they reside within or near crucial expression regulatory sites or within repetitive sequences. SNPs deemed to play a pivotal role in the regulatory landscape were singled out for further investigation.Utilizing online tools such as MatInspector professional 7.2.2, the sequence of the 5′ regulatory region of the yak 2-saturated humidity.In a sterile environment, the cell culture medium was removed from the culture flask and one to two gentle washes with PBS were performed. Subsequently, the cell flask’s base was rinsed with trypsin solution, followed by the removal of trypsin solution. The flask was then placed within a 37 °C incubator for a duration of 2–3 min. Subsequently, an appropriate volume of fresh culture medium enriched with serum was introduced to terminate the trypsin action. To ensure uniform mixing, a pipette was utilized to disintegrate cell clumps, followed by the transfer of the cell suspension into a new culture flask in accordance with the designated dilution ratio. The cells were then incubated within a 37 °C constant-temperature incubator under 5% COSLC11A1 gene was as follows: (1) empty vector group, (2) PGL3/SLC11A1-5′-1 (1476 bp), (3) PGL3/SLC11A1-5′-2 (855 bp), (4) PGL3/SLC11A1-5′-3 (336 bp), (5) PGL3/SLC11A1-5′-4 (263 bp), (6) PGL3/SLC11A1-5′-1 (1476 bp) + LPS/IFN-γ, (7) PGL3/SLC11A1-5′-2 (855 bp) + LPS/IFN-γ, (8) PGL3/SLC11A1-5′-3 (336 bp) + LPS/IFN-γ, and (9) PGL3/SLC11A1-5′-4 (263 bp) + LPS/IFN-γ. The experimental procedure was as follows: (1) Cells from the above-mentioned groups were harvested to preparate single-cell suspensions. (2) The number of cells was counted, and the cell density was adjusted to 8 × 105 cells/mL. (3) A 24-well plate was inoculated with 100 µL of the cell suspension (density: 8 × 105 cells/mL) and cultured at 37 °C with 5% CO2. (4) In a 125 µL DMEM (Dulbecco’s modified Eagle medium) serum-free medium, 0.8 µg of recombinant plasmid and 0.8 µg of pGL3 basic plasmid were added (a serum-free culture medium containing 5 μg/mL LPS (lipopolysaccharid) and 10 ng/mL IFN-γ in DMEM was used in groups 6, 7, 8, and 9). (5) In a 125 µL DMEM serum-free medium, 2 µL of Lipofectamine™ 2000  was added (a mixture containing 5 μg/mL LPS and 10 ng/mL IFN-γ (interferon-γ) in DMEM serum-free medium was used in groups 6, 7, 8, and 9). (6) Following steps 5 and 6, the plasmid was diluted and Lipofectamine™ 2000 was added, and left at room temperature for 20 min to form a complex (plasmid/Lipofectamine™ 2000). (7) Next, 250 µL of the complex (plasmid/Lipofectamine™ 2000) was added to the well containing cells and the culture medium. (8) The complex was incubated for 5 h, then fresh complete culture medium was introduced for transfection. (9) The plate was washed twice with PBS, and 100 µL of passive lysis buffer (PLB) was added to each cell well, and cell lysate was collected. (10) For each sample, 20 µL of cell lysate was added to the luminescence plate and the background value was recorded for 2 s using a bioluminescence detector (GloMax) . (11) Then, 100 µL of LAR II (Luciferase Assay Reagent II) solution was processed into each sample, and recorded for 2 s. (12) After recording, 100 µL of Stop and Glo® Reagent was introduced into each sample, and placed within a luminescence detector for recording.The experimental grouping for recombinant and empty vectors featuring various-length fragments of the 5′ regulatory region of the http://analysis.bio-x.cn/myAnalysis.php) (accessed on 9 March 2023). To delve into the intricate landscape of polymorphisms within our dataset, we harnessed a specialized program designed for the calculation of polymorphism information content (PIC) values. Moreover, the fundamental assessment of the Hardy–Weinberg equilibrium (H–W) was conducted employing the PopGen32 software.Utilizing SPSS 22.0 software, we computed both the mean and standard deviation of the experimental data within each group. Subsequently, one-way ANOVA was employed to scrutinize the data for any discernible significant distinctions across the experimental groups. The presentation of our findings was accomplished using GraphPad Prism 5. For sequence alignment, the Clusterx program (version 1.83) was enlisted to ensure precision in our analysis. The determination of genotype, allele, and haplotype frequencies was executed via the utilization of the online software (SLC11A1 gene. These sequences include SLC11A1-5′ (1760 bp), SLC11A1-5′-1 (1476 bp), SLC11A1-5′-2 (855 bp), SLC11A1-5′-3 (336 bp), and SLC11A1-5′-4 (263 bp).The electrophoresis results of PCR products are illustrated in SLC11A1-5′-1, PGL3/SLC11A1-5′-2, PGL3/SLC11A1-5′-3, and PGL3/SLC11A1-5′-4, were identified using restriction endonucleases KpnI and XhoI, respectively. The electrophoresis results showed consistency with the expected fragments, as shown in Four recombinant plasmids, PGL3/SLC11A1-5′ PCR products and the four recombinant plasmids, namely PGL3/SLC11A1-5′-1, PGL3/SLC11A1-5′-2, PGL3/SLC11A1-5′-3, and PGL3/SLC11A1-5′-4) to the corresponding fragment of the published SLC11A1 gene sequence surpasses the threshold of 99%. This result affirmed the successful cloning of the yak SLC11A1 gene 5′ regulatory region sequence in this endeavor and the construction of recombinant plasmids PGL3/SLC11A1-5′-1 and PGL3/SLC11A1-5′-2. These plasmids were instrumental for subsequent bioinformatics analyses of the 5′ regulatory region sequence of the bovine SLC11A1 gene and for facilitating the expression of the promoter EGFP reporter gene in PGL3/SLC11A1-5′-3 and PGL3/SLC11A1-5′-4.The results of BLAST analysis showed that the sequence similarity of our target fragment , as well as within other regulatory regions, such as TCTACCCTG (−1255 bp~−1246 bp), GGGATAAGG (−947 bp~−938 bp), TTTATTACT (−671 bp~−662 bp), CCTATTACTT (−428 bp~−419 bp), ATTACTTCCC (−425 bp~−416 bp), and TGAATCTTCA (−157 bp~−148 bp). Moreover, the Methprimer prediction results showed that there were no CpG islands in the region.In the 5′ regulatory region of the yak Our analysis pinpointed a promoter region within this region, consisting of 50 bases (GACCCAAGATTAAAGGGAGAGACCTGACTGCTTACAGGGTGAGGG), with the bold red letter ‘A’ signifying the transcription start site . SubsequIntriguingly, these binding sites were conserved at corresponding positions in the gene sequences of cattle, zebu, bison, water buffalo, sheep, and dogs, indicating a common regulatory framework among these species . The prediction results of the repetitive elements revealed the existence of three short and dispersed duplicate elements: BTALΜL1 (−642 bp~−450 bp), BOVTA (−625 bp~−443 bp), and BTCS (−565 bp~−432 bp). In addition, we also identified the duplicate DNA element, namely Charlie 8/hAT Charlie (−1154 bp~−1077 bp).SLC11A1 gene. These SNPs are accurately located at positions −1264 A>C, −1237 A>G, −1057 G>T, −127 G>A, −96 A>G, and −50 G>A  were in linkage equilibrium, while the remaining half exhibited linkage disequilibrium .The results obtained from the RepeatMask program analysis showed that none of the six SNPs were consistent with the repeat sequence. Notably, the −1057 bp G>T locus was positioned a mere five bases away from the transcription factor binding site NF-1 (−1062), while the −127 bp G>A mutation was at a distance of six bases from NF-1/L (−133).SLC11A1-5′-1, PGL3/SLC11A1-5′-2, PGL3/SLC11A1-5′-3, and PGL3/SLC11A1-5′-4 in RAW264.7 cells post-transfection. This observation indicated the promoter functionality of different-length gene sequences within the regulatory region of the yak SLC11A1 gene in the RAW264.7 cell line. However, changes in fluorescence intensities suggested differing relative fluorescence activities among these gene sequences. Using sea kidney luciferase as an internal control, the activity of each promoter was standardized (The results of cell transfection experiments demonstrated the expression of the recombinant plasmids PGL3/dardized .p < 0.05). Furthermore, the activity of the reporter gene containing a 1476 bp vector surpasses that containing a 263 bp vector (p < 0.05). This phenomenon highlights the variability of promoter activity detected in different missing fragments, indicating the presence of key regulatory elements in specific regions. Upon induction with 5 μg/mL LPS/10ng/mL IFN-γ, the fluorescence activity of the recombinant vector containing luciferase was significantly increased (p < 0.05). These results emphasized the basic promoter activity of the -205~58 gene sequence fragments.The results showed that the reporter genes constructed with different amplified 5′ regulatory region lengths exhibited promoter activity significantly higher than the empty vector , BOVTA (−625~−443 bp), and BTCS (−565~−432 bp), in addition to duplicate DNA elements . It is worth noting that the presence of repetitive elements in gene sequences may be due to the insertion of reverse transcripts. Such repetitive elements can serve as a protective mechanism that has evolved over time to safeguard the promoter, exon, and other regulatory elements from gene mutations that could potentially compromise their functionality. However, the specific roles and mechanisms associated with these repetitive elements remain areas of ongoing investigation and exploration.Interestingly, the distal promoter region of the SLC11A1 gene, it is imperative to identify the key regulatory elements and other important sequences within its 5′ regulatory region. In this experiment, the EGFP reporter gene was selected as a vehicle to examine the promoter functionality inherent within the 5′ regulatory region of the yak SLC11A1 gene. The EGFP reporter gene boasts several advantages, including minimal background interference in animal cells, the ability to observe directly under long wavelength ultraviolet or visible light, and ease of detection and high sensitivity. Previous studies have indicated that the target tissues for SLC11A1 gene expression are primarily the spleen, lungs, and related organs. The target cells include macrophages and reticuloendothelial cells derived from these organs, as well as peripheral white blood cells [In order to delve deeper into the molecular complexity of controlling the transcriptional regulation of the yak od cells ,27. Consp < 0.05). This enhancement indicated the presence of positive regulatory elements in the differential regions between these plasmids. Such a phenomenon may be attributed to the positive regulatory role played by binding sites like alpha-CBF, alpha-IRP, CBF-B, alpha-CP1, AP-1, NF-Y, c-Myc, and NF-1 present in this region, all bolstering the promoter activity of the yak SLC11A1 gene. However, the exact factors responsible for this positive regulatory effect, as well as whether other factors are involved, still need to be further explored and validated through more targeted mutation analysis.Through transient transfection, a dual luciferase reporter gene EGFP system was constructed, followed by a luciferase activity assessment. These findings elucidated the promoter activity of these sequences with different lengths in the RAW264.7 cell line, confirming that plasmids spanning from −205 bp to +58 bp have basic promoter activity. Notably, the plasmid spanning from −1418 bp to +58 bp exhibited heightened promoter activity in RAW264.7 cells compared to the −205 bp to +58 bp plasmid . In a previous study [SLC11A1 promoter in RAW264.7 cells after LPS induction, with the highest induction effect observed at an LPS concentration of 1000ng/mL, significantly surpassing the blank control group (p < 0.05). This observation aligns with our research results. Notably, LPS induction did not significantly affect the promoter activity in the 293T cell line (p > 0.05). Although the promoter activity of each recombinant plasmid increased and decreased after LPS induction compared to those without LPS, the increase was statistically significant (p < 0.05), while the decrease was not (p > 0.05). In our experiment, we employed a 5 μg/mL concentration of LPS for induction, where a significant difference (p < 0.05) compared to the blank control was observed. The promoter activity of each recombinant plasmid induced by LPS exhibited a substantial increase compared to those without LPS (p < 0.05).To delve into the impact of LPS stimulation on the us study , the effSLC11A1 gene exhibiting basal promoter activity extended from the 5′ regulatory region of -89 bp to +58 bp [SLC11A1 gene depends on the SPL binding site [SLC11A1 gene in yaks, GR binding sites were located at −371 bp, −194 bp, −150 bp, −134 bp, and 227 bp, NF1 binding sites were located at −228 bp, −169 bp, −142 bp, −56 bp, and 160 bp, alongside transcription factors such as SP1 at −18 bp, −88 bp, 137 bp, and 261 bp, and TGT3 at −20 bp, which were conserved at the corresponding positions in the gene sequences of yellow cattle, zebu, bison, water buffalo, sheep, and dogs. These findings strongly suggest the pivotal role played by SP1 in the basal expression of the yak SLC11A1 gene.The transfection of RAW264.3 and 293T cell lines indicated that the region of the bovine o +58 bp . Consisto +58 bp , SP1 emeing site . SimilarSLC11A1 gene can be found in various databases, including NCBI and Ensembl. For instance, Bellamy et al. [SLC11A1 gene’s SNPs remain less well documented. Martinez et al. [SLC11A1 gene, focusing on Romance cattle and zebu cattle. Nevertheless, these studies, limited to specific cattle herds in specific regions, lack comprehensive representativeness. Therefore, this study employed direct sequencing to analyze the 5′ regulatory region (−1418 bp~+342 bp) of the SLC11A1 gene in yaks. The polymorphism analysis of the 5′ regulatory region in the yak SLC11A1 gene unveiled six novel SNP loci. Although these loci may not directly alter protein composition, they may exert regulatory roles in gene transcription and protein synthesis due to the presence of core promoters, distant regulatory elements, and remote regulatory promoter regions located upstream, downstream, proximal, and distal to the transcription starting points. These findings lay the foundation for further exploring the connection between SLC11A1 gene polymorphism and function of yaks [SLC11A1 gene is subject to regulation by transcription factors such as SP1, PU.1, NF-1, and NF-1/L [Comprehensive information on SNPs of the y et al.  reportedz et al.  did repo of yaks . Sequencd NF-1/L ,20. TherSLC11A1 gene by cloning gene sequences of different lengths, and confirmed the promoter activity of four distinct-length sequences within the 5′ regulatory region of the yak SLC11A1 gene in the RAW264.7 cell line. Moreover, our analysis identified six novel SNPs in the 5′ regulatory region sequence of the SLC11A1 gene in yaks. Bioinformatic analysis emphasized the proximity of the −1057 G>T and −127 G>A loci to the transcription factor binding sites NF-1 and NF-1/L. This observation suggested their potential effect on the binding of NF-1 and NF-1/L transcription factors. Collectively, this study serves as a foundational reference for future research concerning the connection between promoter region polymorphism of the SLC11A1 gene in yaks and disease resistance. In this study, we conducted a comprehensive analysis of the 5′ regulatory region of the yak


Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome associated with a high morbidity and mortality rate. Leucine supplementation has been demonstrated to attenuate cardiac dysfunction in animal models of cachexia and heart failure with reduced ejection fraction (HFrEF). So far, no data exist on leucine supplementation on cardiac function in HFpEF. Thus, the current study aimed to investigate the effect of leucine supplementation on myocardial function and key signaling pathways in an established HFpEF rat model. Female ZSF1 rats were randomized into three groups: Control (untreated lean rats), HFpEF (untreated obese rats), and HFpEF_Leu (obese rats receiving standard chow enriched with 3% leucine). Leucine supplementation started at 20 weeks of age after an established HFpEF was confirmed in obese rats. In all animals, cardiac function was assessed by echocardiography at baseline and throughout the experiment. At the age of 32 weeks, hemodynamics were measured invasively, and myocardial tissue was collected for assessment of mitochondrial function and for histological and molecular analyses. Leucine had already improved diastolic function after 4 weeks of treatment. This was accompanied by improved hemodynamics and reduced stiffness, as well as by reduced left ventricular fibrosis and hypertrophy. Cardiac mitochondrial respiratory function was improved by leucine without alteration of the cardiac mitochondrial content. Lastly, leucine supplementation suppressed the expression and nuclear localization of HDAC4 and was associated with Protein kinase A activation. Our data show that leucine supplementation improves diastolic function and decreases remodeling processes in a rat model of HFpEF. Beneficial effects were associated with HDAC4/TGF-β1/Collagenase downregulation and indicate a potential use in the treatment of HFpEF. Heart failure (HF) is a worldwide burden that affects around 26 million individuals. Approximately half of all HF patients present with a preserved left ventricular ejection fraction , and the prevalence is increasing (around 10% per decade) due to an aging population and an increase in other risk factors such as diabetes mellitus, arterial hypertension, and sedentary lifestyle [The HFpEF phenotype is characterized by diastolic dysfunction due to impaired left ventricular (LV) relaxation and/or increased LV stiffness; in other words, the heart loses its ability to appropriately store blood during diastole . The conThus far, most efforts to reduce morbidity and mortality in HFpEF have failed. Only the recently published EMPEROR-trail, which used Empagliflozin , a SodiuAnother promising therapy for HF includes nutritional intervention by leucine supplementation. Amino acids provide approximately 30% of the energy that sustains the contractile function and are used for the maintenance of ionic homeostasis in the heart ,8. This HDACs are an enzyme family that removes N-acetyl lysine from histone and non-histone proteins. Class II HDACs  are highly expressed in the heart ,15. ReceFurthermore, we recently demonstrated for the first time that leucine supplementation significantly reduces HDAC4 expression and positive nuclear localization induced by hindlimb immobilization . These fDespite these encouraging results, the mechanisms by which leucine supplementation protects the myocardium remain unclear. Thus, the purpose of this study was to investigate the impact of leucine supplementation on myocardial function in an established HFpEF rat model. Furthermore, we assessed leucine’s effect on important key signaling pathways that control myocardial function, examined mitochondrial respiratory capacity, stress, anabolic, catabolic, and fibrotic markers, as well as HDAC4 protein expression and tissue localization.n = 10, untreated lean rats receiving standard chow throughout the experiment); HFpEF , and HFpEF_Leu   were used in this study, taking into account that HFpEF is predominantly reported in the female phenotype. Obese rats carrying two leptin receptor mutations (fa: facp) develop HFpEF. Single or no mutation leads to a healthy, lean phenotype . The anileucine) . All aniLeucine supplementation started at 20 weeks of age, after HFpEF was established in obese animals ,20,21,22This study was approved by the local animal research council, TU Dresden, and the Landesbehörde Sachsen (TVV 26/2022).Blood serum was separated by centrifugation, and NT-proBNP levels were measured using RatNT-proBNP ELISA  according to the manufacturer’s protocol.3 − LVID;d3) and corrected by the factor 0.8.Rats were anesthetized with isoflurane (1.5–2%) and placed on a controlled warming pad with electrodes that continuously recorded breathing frequency, heart rate, and body temperature. Transthoracic echocardiography was performed using a Vevo 3100 system and a 21-MHz transducer  to assess cardiac function as recently described ,20,21,22Diastolic function was assessed in the apical 4-chamber view using pulse wave Doppler (for measurement of early (E) and atrial (A) waves of the mitral valve velocity) and tissue Doppler ) at the level of the basal septal segment in the septal wall of the left ventricle.Functional parameters  and stroke volume (SV)) and ratios of E/é and E/A) were computed by the Vevo LAB 3.1.1 software.Prior to organ harvest, invasive hemodynamic measurements were performed as recently described . In anesThe respiratory parameters of the total mitochondrial population were studied in saponin-skinned fibers of myocardial tissue. Respiratory rates were determined by using a Clark electrode  in an oxygraphic cell at 25 °C with continuous stirring. To avoid oxygen diffusion limitation, the oxygen concentration was increased to ~400 µmol/L by adding pure oxygen and was kept above 270 µmol/L throughout the experiment. Left ventricular muscle fibers were isolated in permeabilization solution (SolP) containing, in mmol/L: 2.77 CaK2EGTA, 7.23 K2EGTA, 6.56 MgCl2, 5.7 Na2ATP, 15 phosphocreatin (PCr), 20 taurine, 0.5 DTT, 50 K methane sulfonate, and 20 imidazole (pH 7.1) and incubated for 30 min in SolP with 50 µg/mL saponin. Permeabilized fibers were transferred to respiration solution (SolR) ) for 10 min to wash out adenine nucleotides and PCr. All steps were carried out at 4 °C under continuous stirring. Respiration rates of 1–5 mg of skinned fibers were measured at 25 °C in 1 mL of SolR containing 1 mg/mL bovine serum albumin. The following substrates were added sequentially and oxygen consumption was monitored: (I) glutamate (10 mmol/L), malate (2.0 mmol/L), (complex I state 2 respiration); (II) adenosine diphosphate ; (III) octanoylcarnitine ; (IV) cytochrome C ; (V) succinate ; (VI) rotenone ; (VII) FCCP ; (VIII) antimycin A . After the experiment, fiber bundles were blotted dry and weighed. Rates of respiration are given in nanomoles O2 per second per mg wet weight.Left ventricular tissue was homogenized in Relax buffer  and aliquots were used for enzyme activity measurements. Enzyme activities for citrate synthase (EC 2.3.3.1), pyruvate kinase (EC 2.7.1.40), and lactate dehydrogenase EC 1.1.1.27) were measured spectrophotometrically as recently described ,25,26,27 were meaSnap-frozen left ventricular tissue was homogenized in Relax buffer containing a protease inhibitor mix  and sonicated. Protein concentration was determined , and aliquots (10–40 µg) were separated by SDS-polyacrylamide gel electrophoresis.Proteins were transferred to a polyvinylidene difluoride membrane. To verify a homogeneous loading, membranes were stained with Ponceau S. Next, membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween  for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibody. Primary antibodies were as follows: rabbit anti-MAFbx , mouse anti-total OXPHOS , rabbit anti-AKT , rabbit anti-AMPKα , rabbit anti-pAMPKα , rabbit anti-PKA , rabbit anti-pPKA , mouse anti-MuRF1 , mouse anti-myogenin , mouse anti-eIF2Bδ , rabbit anti-myostatin , rabbit anti-HDAC4  and rabbit anti-GAPDH . After washing , the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody, and specific bands were visualized using enzymatic chemiluminescence . Densitometry analyses were performed using a 1D scan software package , and the GAPDH signal was used to normalize variations in loading.Total RNA was isolated from LV tissue using Qiazol reagent and the miRNeasy Mini Kit  following the standard protocols. cDNA was synthesized with the Revert AID™ H Minus First Strand Synthesis Kit  using oligo-dT primers. Real-time PCR was performed using the CFX384TM Real-Time PCR System  and Maxima SYBR Green qPCR Kit . The PCR program for all primer sets was as follows: 95 °C for 8 min prior to 40 amplification cycles, each consisting of 95 °C for 10 s, 58 °C for 15 s, and 72 °C for 30 s, with a final extension step at 72 °C for 2 min. Melting point analysis was performed to prove the identity of the PCR products. Relative quantification of gene expression was calculated using the ∆∆CT method with Polr2a and Rpl-32 as housekeeping genes using BioRad CFX Maestro 1.1 version 4.1.2433.1219 . Specific primer sequences are listed in Heart cryo-sections (10 µm) were fixed at room temperature in 4% PFA  for 10 min, washed (3 times for 5 min) with phosphate-buffered saline (PBS), and then permeabilized with PBS-T (PBS with 0.1% Triton X-100) for 30 min. Subsequently, the sections were incubated with blocking solution  for 1 h at room temperature and incubated at 4 °C with primary antibody rabbit anti-HDAC4  in diluent solution  overnight. Thereafter, slides were washed with PBS (3 times for 5 min), incubated with the secondary antibody  in diluent solution for 1 h and subsequently washed (3 times for 5 min) in PBS. The slides were incubated with DAPI  for 5 min and mounted with coverslips using Mount Fluor . Digital acquisitions and the nuclear colocalization analysis were performed using microscopy equipment (Echo RVL-100-M Revolve Fluorescence Microscopy). For cross-sectional area measurements (CSA), the myocardial heart sections were stained with Collagen IV , and ImageJ  was used to measure the heart fibers. Approximately 1000 fibers per group were analyzed.For fibrosis measurements, paraffin-embedded heart sections (3 µm) were stained with picrosirius red, and perivascular fibrosis around arteries, expressed as perivascular fibrosis ratio (PFR), was quantified as described by Dai and colleagues . PFR wasp < 0.05 was considered significant.Data analyses were performed using Prism Software (GraphPad Prism 7.0). Multiple comparisons were performed using either one-way ANOVA followed by Tukey’s posthoc test (for parametric data) or the Kruskal–Wallis test of one-way ANOVA followed by Dunn’s post hoc test (for non-parametric data). Data are presented as mean ± SEM, and Indicators of myocardial hypertrophy LV mass and septum thickness were decreased in HFpEF_Leu compared to HFpEF . Left veSystolic function was preserved in all groups at all times, as indicated by the similar ejection fraction . SystoliDiastolic function was significantly improved after four weeks of leucine supplementation (E/é: 11% reduction vs. HFpEF) and normalized after 12 weeks of treatment (E/é: 20% reduction vs. HFpEF) . LV stifGiven that the hemodynamics assay revealed a decrease in LV stiffness in HFpEF_Leu compared to HFpEF, we proceeded to histology analysis. Perivascular fibrosis was comparable between all groups, as shown in In addition, we assessed the LV fiber cross-sectional area and found a significant increase in HFpEF when compared to the control, which was reduced by leucine supplementation C, confirp = 0.098, Compared to the control, the expression of ANP mRNA was upregulated in HFpEF and tendentially reduced in HFpEF_Leu (p = 0.077) but back to the control in the leucine-treated group (Transforming Growth Factor Beta 1 (TGF-β1) expression levels were tendentially increased in HFpEF (ed group D. Expresed group E and Coled group F were inp = 0.061) , a key mediator of collagen maturation, was tendentially decreased in HFpEF_Leu compared to untreated HFpEF (= 0.061) G.The sequential addition of complex I and II substrates/inhibitors enabled complex- and substrate-specific monitoring of oxygen consumption .p < 0.005 vs. Control, p < 0.05 vs. Control, Basal respiration  did not differ between all three groups  was increased in HFpEF_Leu compared to HFpEF  ,29,30. TRegarding the catabolic pathway, myogenin, a transcription factor of MAFbx and MuRF1, was downregulated in both HFpEF groups A. The exConcerning the anabolic pathway, we analyzed the expression of one upstream marker AKT, E and oneLooking for the underlying mechanisms of the beneficial effect on diastolic function mediated by leucine supplementation, HDAC4, a protein highly expressed in the heart and involved in cardiac dysfunction ,17,31, wHDAC4 is inactivated by phosphorylation, and two important kinases mediating the inactivation of HDAC4 are AMPKα and PKA B. HDAC4 HDAC4 inactivation is accompanied by nuclear exportation, which reduces its action on target genes. Histological analysis of HDAC4 distribution in left ventricular tissue revealed a significantly increased number of HDAC4-positive nuclei in HFpEF that were reduced by leucine supplementation B, strengThe present study demonstrates that leucine supplementation improves diastolic function in a rat model of HFpEF. This was accompanied by reduced left ventricular stiffness, fibrosis, and hypertrophy . As poteTreatment of HFpEF remains challenging. Apart from SGLT2i  and exerIt is important to highlight that leucine supplementation improved diastolic function and was accompanied by reduced cardiac remodeling, indicating that an established HFpEF responds to the supplementary treatment. Since most patients are diagnosed after the disease has already developed, these findings encourage the use of leucine as a potential therapeutic treatment for HFpEF.In the literature, several benefits of leucine supplementation in HF conditions are documented, including atherosclerosis development prevention, decreased damage induced by myocardial ischemia/reperfusion (I/R) injury, and inhibited doxorubicin-induced pathological cardiac remodeling ,11,12,13Tissue remodeling and the presence of fibrosis disrupt the architecture of the myocardium and increase the development of cardiac dysfunction, affecting patients’ clinical outcomes ,34,35. TThe mechanism underlying leucine’s effect on fibrosis and hypertrophy might likewise be linked to HDAC4 suppression . TGF-β bMorio and collaborators  recentlyWe also measured specific enzyme activities that are modulated by HFpEF. CS is a marker for mitochondria content, and no significant differences were observed between groups, correlating with no changes in mitochondrial respiratory complex protein expression as determined by Western blot . ConcernThe main mechanism of action postulated for leucine is a stimulation of the mammalian target of rapamycin (mTOR), which is also known as a potent activator of muscle protein synthesis . In HF pMyogenin protein levels were reduced in both HFpEF groups compared to the control. The mechanisms involved in myogenin reduction during heart failure are not completely understood. However, our findings support previous work performed in cell culture, where doxorubicin-induced cardiotoxicity was associated with suppressed myogenin expression . AlthougSince HFpEF is a highly clinically relevant disease, it is important to point out how leucine supplementation has been evaluated as a translational treatment. Leucine supplementation has long been used as a therapeutic option, mostly for skeletal muscle ,58,59,60To put a comparison involving rodents and humans into context, high dosages of leucine (2.5 g/kg) have traditionally been employed in rodent studies, either by gavage or by adding to the food ,12,64,65Takata et al. proposed a protocol for testing the efficacy and safety of a BCAA (branched-chain amino acids) preparation used in combination with cardiac rehabilitation for patients with chronic heart failure . The autSeveral limitations of this study have to be mentioned. First, the limited number of experimental animals led to a high distribution regarding several parameters. This partly resulted in statistical insignificance or only tendencies, although a direction was apparent. Second, we have not addressed all of the components involved in the various pathways discussed, such as Smad3 in the panel of fibrotic markers. Further analyses  will be necessary to deepen our understanding of underlying pathways regarding the effects of leucine.Our data demonstrate that leucine improves diastolic function in HFpEF. This was accompanied by reduced remodeling and involved HDAC4/TGF-β1/Collagenases downregulation.


Anti-tumour necrosis factor (TNF)-α agents have been increasingly used to treat patients affected by inflammatory bowel disease and dermatological and rheumatologic inflammatory disorders. However, the widening use of biologics is related to a new class of adverse events called paradoxical reactions. Its pathogenesis remains unclear, but it is suggested that cytokine remodulation in predisposed individuals can lead to the inflammatory process. Here, we dissect the clinical aspects and overall outcomes of autoimmune diseases caused by anti-TNF-α therapies. Anti-tumour necrosis factor (TNF)-α agents are increasingly being used for the treatment of patients affected by inflammatory bowel disease (IBD) such as ulcerative colitis (UC) and Crohn’s disease (CD), as well as rheumatologic and dermatological disorders such as rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), psoriatic arthritis (PsA), and psoriasis. Notably, the majority of these drugs can neutralise both forms of TNF-α, either by binding the trans-membrane form (m-TNF-α) or by blocking the soluble form (s-TNF-α) . Only inWith the widespread use of anti-TNF-α agents, concerns about any unexpected effects have been raised. Indeed, over the past 20 years, the widening use of biologics in several autoimmune diseases has been related to a new class of adverse events called paradoxical reactions. These immune-mediated processes appear paradoxically during treatment and can virtually affect several organs, including the skin, liver, lungs, kidneys, peripheral and central nervous system, vascular system, and bowels . DespiteAnti-TNF-α therapy can cause a wide range of dermatological conditions, including local skin irritation or reaction, increased skin infection rates, psoriasis, eczema, anti-TNF-induced cutaneous lupus erythematosus (ATIL), and alopecia areata (AA). Other reactions rarely occur, such as granuloma annulare, lichen planus, vitiligo, and cutaneous vasculitis. In patients with IBD, some of the above complications, such as erythema nodosum and pyoderma gangrenosum, could be extraintestinal manifestations related or not to clinical exacerbations. Thus, temporal associations with biological therapy could differentiate between disease- and drug-related complications. These reactions are managed by stopping anti-TNF-α treatment, switching to a different TNF-α inhibitor or ustekinumab, switching to another systemic immunosuppressant for treating the underlying dermatologic condition, or continuing anti-TNF-α therapy with the addition of topical or systemic skin-directed treatments, depending on the severity and extension of the cutaneous lesions.Psoriasis is a chronic autoimmune disease characterised by raised red, itchy, scaly patches, most commonly on the knees, elbows, trunk, and scalp. TNF-α antagonist-induced psoriasis, which can emerge de novo or exacerbate a pre-existing form, is the most common dermatological adverse reaction linked to anti-TNF therapy ,5. In mon = 17) and rheumatoid arthritis (n = 2) patients with new-onset inflammatory skin lesions during anti-TNF-α therapy [In contrast, PP is caused by the absence of TNF-α and represents type I interferon (IFN)-driven innate inflammation . Stoffel therapy , evidenc therapy . This si therapy . Among t therapy .IFX and ADA are the most frequently associated with psoriasis among anti-TNF drugs. Collamer et al. reviewed 207 cases of new-onset psoriasis, of which 43% of patients had RA, 26% seronegative SpA, and 20% IBD . The mosBesides the adult setting, little is known about paediatric IBD patients. PP appears to be the most frequent side effect in paediatric CD treated with IFX. Perianal lesions and young age at anti-TNF initiation represent the most significant risk factors .Following the introduction of anti-TNF, ATIL has become a new and increasingly recognised clinical entity . All antOverall, the first case of drug-induced lupus (DILE) was reported in 1945  as a comA large Australian cohort study including 454 patients treated with anti-TNF-α therapy (300 with IFX and 154 with ADA) reported an incidence of ATIL of 5.7% for IFX and 0.6% for ADA . These rAlopecia areata (AA) can be described as a non-scarring inflammatory pathology with a sudden onset and rapid hair loss in some regions of the scalp and body. It can be divided into alopecia focalis (monolocularis or multilocularis), which can occur in one or multiple areas of the scalp; alopecia totalis, which happens all over the scalp; alopecia universalis, which is a complete loss of scalp and body hair. It is linked to a multifactorial process involving an immuno-mediated response and the overexpression of proinflammatory cytokines such as TNF-α, interleukin-1-α, and IFN-γ. According to this evidence, a role for anti-TNF in the pathogenesis of AA seems unlikely. Nevertheless, few reports have shown this connection , such asAmong dermatological adverse effects, reports have associated TNF-α inhibitors with the onset of lichenoid eruption (LE). Lichen planus is a recurrent, itchy inflammatory rash with single, small, polygonal papules that can merge to form rough, scaly plaques, often accompanied by injury to the oral cavity or genitals. Diagnosis is usually clinical and can be supported by a skin biopsy. Treatment generally requires topical or intralesional corticosteroids. A case report described three IBD patients who developed oral lichen planus after starting anti-TNF therapy . AnotherVitiligo is a long-term skin condition characterised by patches of the skin losing its pigment. Hypotheses for its pathogenesis include immune-mediated destruction of epidermal melanocytes . VitiligConversely, new-onset cases following biological treatment have emerged . In thisIn addition, several cases of anti-TNF-mediated dermatomyositis/polymyositis have been described . Here, tThe primary concerns about anti-TNF-α therapy-related liver injury refer to the increased susceptibility towards chronic infections, especially towards the reactivation of the hepatitis B virus (HBV) . AutoimmClinical manifestations range from asymptomatic abnormalities in liver function tests, self-limiting after drug discontinuation, to more severe damage, requiring corticosteroid therapy. Tobon et al. described two cases of severe liver disease: a 39-year-old woman who was treated with IFX for eight months and developed cholestatic liver disease and hepatic failure with hepatic encephalopathy and ascites and finally underwent liver transplantation, and a 54-year-old woman who was diagnosed with acute hepatitis with positive ANAs after 17 months of IFX therapy and was treated with prednisolone and azathioprine . Cases oAnti-TNF-related lung autoimmune disorders are mainly represented by interstitial lung diseases (ILDs) and pulmonary fibrosis, the most severe manifestation. However, the association between these drugs and developing or exacerbating ILDs is still controversial . ExperimIn contrast, case reports have described ILDs and pulmonary fibrosis as paradoxical side effects of anti-TNF-α agents. These drugs may both induce new lung injury and worsen pre-existing ILDs. Most available data refer to patients affected by rheumatoid arthritis ,53. The Similarly, even if less frequently, developing or exacerbating ILDs under biological therapy has also been reported in diseases other than rheumatoid arthritis, such as psoriatic arthritis/psoriasis, ankylosing spondylitis, systemic sclerosis, and IBD ,58,59. TConversely, it has been suggested that anti-TNF-α might exacerbate pre-existing ILDs compared to non-anti-TNF-α biological therapy . In a coCompared to other anti-TNF-mediated adverse events, autoimmune renal disorders are uncommon. They are most often described in patients affected by rheumatic diseases, especially rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis, but also in other conditions, such as psoriasis and IBD. ETA is the drug most frequently implicated, followed by ADA and IFX . Renal aAcute non-granulomatous tubulointerstitial nephritis has also been reported in an HLA-B27-positive patient with axial spondylarthritis and CD treated with ADA . SporadiAn increasing number of therapeutic agents have been associated with vasculitic syndromes. Among these, TNF-α inhibitors play a central role. Cutaneous manifestations with palpable purpura and leukocytoclastic vasculitis (LCV) are usually the most common upon histological examination, while systemic vasculitis mainly affects the peripheral nervous system and the kidneys ,77,78. ODemyelination has been described as a consequence of anti-TNF-α therapy . A revieSarcoidosis is a systemic inflammatory disorder characterised by non-caseating granuloma, which commonly affects the lungs, lymph nodes, and salivary glands, but rarely other organs . In recent years, TNF-α inhibitors have been used for its treatment. However, there have been isolated cases of sarcoidosis occurrence during anti-TNF therapy. Daïen et al. estimated a frequency of 1:2800 . In mostPropionibacterium acnes was detected in the granulomas, suggesting that P. acnes infection may show aberrant responses to drugs such as TNF-α inhibitors and immune checkpoint inhibitors, consequently leading to drug-induced sarcoidosis [Interestingly, Isshiki et al. described the case of an RA patient who developed sarcoidosis six months after the introduction of ETA and improved without therapeutic intervention after its cessation . In thiscoidosis . Furthercoidosis . This phcoidosis ,91.Furthermore, several studies have suggested that monoclonal antibodies, in contrast to ETA, inhibit the expression of the IL-17 and IFN-γ cytokines intensely involved in granuloma formation. In contrast, some reports have shown sarcoid-like reactions in patients treated with IFX and ADA ,93. In tBoth paradoxical worsening of IBD and new-onset cases induced by anti-TNF-α agents have been reported in patients with different inflammatory diseases, especially AS, JIA, and psoriasis. CD has been observed more frequently than UC, with most cases attributed to ETA ,96. In cAnti-TNF-α agents has significantly improved the outcomes of IBD and dermatological and rheumatological conditions. Paradoxical events due to these therapies are not commonly reported in the literature, especially compared to other side effects. In recent years, a growing number of cases and a more extensive range of clinical manifestations have been reported because of their increased use. The pathogenesis of the paradoxical reactions is not completely clarified yet. However, several mechanisms involved have been hypothesised and are summarised in


Tumor necrosis factor-alpha (TNF-α) is a pleiotropic immune cytokine that belongs to the TNF superfamily of receptor ligands. The cytokine exists as either a transmembrane or a soluble molecule, and targets two distinct receptors, TNF-α receptor 1 (TNFR1) and TNF-α receptor 2 (TNFR2), which activate different signaling cascades and downstream genes. TNF-α cellular responses depend on its molecular form, targeted receptor, and concentration levels. TNF-α plays a multifaceted role in normal physiology that is highly relevant to human health and disease. In the central nervous system (CNS), this cytokine regulates homeostatic functions, such as neurogenesis, myelination, blood-brain barrier permeability and synaptic plasticity. However, it can also potentiate neuronal excitotoxicity and CNS inflammation. The pleiotropism of TNF-α and its various roles in the CNS, whether homeostatic or deleterious, only emphasizes the functional complexity of this cytokine. Anti-TNF-α therapy has demonstrated effectiveness in treating various autoimmune inflammatory diseases and has emerged as a significant treatment option for CNS autoimmune diseases. Nevertheless, it is crucial to recognize that the effects of this therapeutic target are diverse and complex. Contrary to initial expectations, anti-TNF-α therapy has been found to have detrimental effects in multiple sclerosis. This article focuses on describing the various roles, both physiological and pathological, of TNF-α in the CNS. Additionally, it discusses the specific disease processes that are dependent or regulated by TNF-α and the rationale of its use as a therapeutic target. Tumor necrosis factor-alpha (TNF-α) is a pleiotropic immune cytokine belonging to the TNF superfamily of receptor ligands. It is involved in several homeostatic and inflammatory processes, and plays a major role in autoimmune and inflammatory diseases –3. AlthoTNF-α is one of the main cytokines regulating inflammation in the human body . It is pA number of studies have implicated TNF-α in autoimmune inflammatory diseases of the CNS . It playIn this review, we discuss the role of TNF-α in the pathogenesis of the main autoimmune disorders of the CNS and its significance as a therapeutic target.TNF-α exists as a transmembrane and a soluble molecule, and targets two natural receptors, TNFR1 and TNFR2 . TransmeActivation of TNFR1 by TNF-α triggers the formation of four distinctive molecular complexes , 22. TheL), which has a dual role in apoptosis and caspase activation . Complex IIb is similar to IIa, but also contains RIPK3. The formation of this complex is independently regulated by the long isoform of FLICE-like inhibitory protein . GABAAR mediate inhibitory neurotransmission and their downregulation leads to a reduction in the activity of the inhibitory synapses . By alteTNF-α exerts various effects on the CNS beyond its influence on receptor expression in the synaptic cleft. One notable effect is its ability to modulate the release of glutamate from astrocytes, altering the local concentration of this excitatory neurotransmitter . TNF-α sFurthermore, TNF-α plays a role in the regulation of oligodendrocyte proliferation and myelin formation, although the precise mechanisms are not fully understood , 35. TNFTNF-α plays a crucial role in the induction and augmentation of inflammation within the CNS . It is pOne of the key functions of TNF-α in CNS is its involvement in the regulation of excitotoxicity and neuroinflammation . These t1) TNF-α contributes to excitotoxic cell death in neurons by altering the balance between excitatory and inhibitory signals, leading to an increased influx of calcium into the cells  38). Th. Th38). 2) TNF-α also regulates the release and re-uptake of glutamate in astrocytes . Moreove3) Activation of microglial cells and their induction to the proinflammatory M1 phenotype are medicated by TNF-α , 40, 41.4) TNF-α plays a crucial role in the disruption of the blood-brain barrier and the infiltration of inflammatory cells and molecules into the CNS. This process, medicated by the TNFR1,is associated with increased expression of adhesion molecules (VCAM and ICAM) by endothelial cells, cell injury (necroptosis) and compromised barrier function , 43.Neuroinflammation is a common pathological characteristic of several CNS disorders. It is characterized by the activation of glial cells, demyelination and neurodegeneration . In autoSarcoidosis is a multisystemic inflammatory disease of unknown etiology characterized by granuloma formation in the affected organs. It can involve the CNS as a primary disorder (neurosarcoidosis) in 10-20% of all cases or as a secondary disorder in 5-20% of all systemic cases , 45. NeuThe unifying pathological findings in sarcoidosis are non-caseating granulomas. These granulomas consist of multinucleated giant cells and epithelioid macrophages surrounded by a rim of lymphocytes, mainly CD4 T cells and occasional B and CD8 T cells , 50. TheThe essential role of TNF-α in granuloma formation is further supported by the clinical efficacy of anti-TNF-α therapies. These therapies have demonstrated effectiveness in controlling neurosarcoidosis, decreasing lesion size and activity, and improving treatment outcomes . InflixiMultiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS . It is rCNS inflammation in MS is a complex and multifocal process involving the interactions between the innate and adaptive immune systems. Autoreactive T cells are thought to arise in the peripheral immune system when immune tolerance fails, leading to the presentation of myelin-mimicking protein epitopes , 65. SevSimilarly, TNF-α is upregulated in experimental autoimmune encephalomyelitis (EAE), the most commonly studied animal model of MS. Administration of TNF-α in these mice has shown to exacerbate the course of the disease , 71, 72.Additionally, anti-TNF-α therapy, commonly used in the treatment of rheumatological disorders, has been associated with unexpected demyelinating events , 76. TheBehçet’s disease is a systemic inflammatory vasculitis of unknown origin . The disThe etiology and pathogenesis of Behçet’s disease are not completely understood and are likely multifactorial . TraditiThe lack of a definitive etiology only emphasizes the complexity of this disease, a fact that also applies to its immunepathogenesis , 88. In In Behçet’s, TNF-α is produced by macrophages, NK cells, T helper (Th)1 cells, Th22 cells, endothelial cells, neutrophils, and monocytes. Its production is triggered by enhanced signaling of Toll-like receptors (TLRs) . TLR stiGiven its pivotal role, TNF-α has become one of the main therapeutic targets for Behçet’s disease , 85, 88.The most compelling evidence of the role of TNF-α in neuroinflammation and CNS diseases is provided by experimental and clinical studies on therapeutic blockade of this cytokine , 39, 82.The main pharmacological effect of anti-TNF-α therapies is the neutralization of sTNF-α. Another important mechanism of action is antibody-dependent cellular cytotoxicity (ADCC) , 100. WhThe unintended outcomes and the failed MS trials with anti-TNF-α therapies could be explained by the pleiotropic effects of TNF-α and the different downstream effects of TNFR1 and TNFR2 blockage. Understanding the positive impact of TNFR2 on oligodendrocyte homeostasis and remyelination has led to the development of selective TNFR blockage therapies , 81. OneTNF-α is one of the most widely studied cytokines. It activates different signaling pathways with diverse cellular effects, ranging from cell proliferation and synaptic plasticity to neuron cell death and neuroinflammation. The pleiotropism of TNF-α and its various roles in the CNS, whether homeostatic or deleterious, only emphasizes the functional complexity of this cytokine. Recognizing its master role in acute and chronic inflammation has spurred the development of a new class of biologic therapeutics. Anti-TNF-α therapy has proven to be effective in treating various autoimmune inflammatory diseases and has become a commonly used treatment approach for CNS autoimmune diseases. Additionally, newer and more specific therapeutics with selective blockage are under development, offering potential advancements in targeting specific aspects of TNF-α for therapeutic purposes. Future studies will be paramount in fully understanding the spectrum of TNF-α functions in the CNS and expanding the clinical applications of anti-TNF-α therapy.The author confirms being the sole contributor of this work and has approved it for publication.