Lymph node staging is very important for the prognosis of patients with gastric cancer. Currently, the internationally accepted lymph node staging method is the 8th AJCC staging, which divides lymph nodes into different stages according to the number of positive lymph nodes. This staging method is simple and convenient, but ignores the laterality of lymph nodes. In this paper, we for the first time combined the location and number information of positive lymph nodes to create a novel lymph node staging system for gastric cancer. After training cohort and validation cohort tests, this staging is more accurate in predicting the prognosis of patients than the 8th AJCC staging.Background: Lymph node metastasis location and number significantly affects the prognosis of patients with gastric cancer (GC). This study was designed to examine a new lymph node hybrid staging (hN) system to increase the predictive ability for patients with GC. Methods: This study analyzed the gastrointestinal treatment of GC at the Harbin Medical University Cancer Hospital from January 2011 to December 2016, and selected 2598 patients from 2011 to 2015 as the training cohort (hN) and 756 patients from 2016 as the validation cohort (2016-hN). The study utilized the receiver operating characteristic curve (ROC), c-index, and decision curve analysis (DCA) to compare the prognostic performance of the hN with the 8th edition of AJCC pathological lymph node (pN) staging for GC patients. Results: The ROC verification of the training cohort and validation cohort based on each hN staging and pN staging showed that for each N staging, the hN staging had a training cohort with an AUC of 0.752  and a validation cohort with an AUC of 0.812 . In the pN staging, the training cohort had an AUC of 0.728 , and the validation cohort had an AUC of 0.784 . c-Index and DCA also showed that hN staging had a higher prognostic ability than pN staging, which was confirmed in the training cohort and the verification cohort, respectively. Conclusion: Lymph node location-number hybrid staging can significantly improve the prognosis of patients with GC. According to the World Health Organization (WHO), gastric cancer (GC) was the fifth common global tumor incidence in 2020, and its mortality rate was ranked fourth, as it is responsible for approximately 770,000 deaths per year . Due to Although the number of positive lymph nodes can predict the prognosis of patients, Daniele et al.  found thThis study proposed a new hybrid lymph node staging through retrospective analysis and combined it with the advantages of location and number staging. In addition, the prediction ability and accuracy of the two systems were compared to propose a supplementary standard that can be used as the existing pN staging.The diagnosis of GC was based on the tissue samples obtained using preoperative gastroscopy and further confirmed via histopathological examination by a professional pathologist. In addition, the patient underwent routine preoperative examinations, including abdominal CT, chest CT, echocardiography, ultrasound of bilateral supraclavicular lymph nodes, electrocardiogram, routine hematology, and tumor marker examination. Every patient had more than 16 lymph node cleanings. Following lymph node removal, each patient was photographed and sent to the pathology lab separately. At least two pathology experts reviewed all pathological results. All staging was conducted in accordance with the 8th edition AJCC.http://www.sgihmu.com, accessed on 10 August 2022). Gender, age, tumor diameter, tumor location, pTNM stage, venous invasion, nerve invasion, and postoperative chemotherapy were included. Patients with negative surgical margins (R0) and standard D2/D2+ surgery were enrolled. The inclusion criteria for our study were as follows: ① patients who underwent radical gastrectomy in the Affiliated Cancer Hospital of Harbin Medical University between 1 January 2011, to 31 December 2016; and ② had regular follow-up for at least 5 years. Exclusion criteria included: ① history of the other malignancies; ② patients with preoperative neoadjuvant chemotherapy; ③ the postoperative pathological report was non-tumor; ④ patients with unclear lymph node metastasis or invasion depth reported by postoperative pathology; and ⑤ patients with a history of gastric surgery. A total of 3354 patients were included after the exclusion. Finally, 2598 patients were chosen as the training cohort (hN) from January 2011 to December 2015, and 756 patients were chosen as the validation cohort (2016-hN) from February 2016 to November 2016.A total of 4712 surgical patient in the Department of Gastrointestinal Surgery of Harbin Medical University Cancer Hospital from 1 January 2011, to 31 December 2016, were selected for this study. Patients’ clinicopathological data were saved in the GC information management system v1.2 of Harbin Medical University Cancer Hospital  that appeared when using univariate analysis were then entered into multivariate analysis. The optimal cut-off value of the number of positive lymph nodes was calculated using X-tile. The receiver operating characteristic curve (ROC) was used to calculate the accuracy of the recurrence scores in predicting recurrence and the area under the curve (AUC). Harrell Consistency Index (C-index) analysis and DCA test were performed using the survival package in R.Statistical analyses were performed using IBM, SPSS, and R. Chi-square or Fisher’s exact test was used for categorical variables. A Chi-square test was performed to analyze the relationship between recurrence and the clinicopathological characteristics of the patients. Factors with significant differences . Most tumors were located in the lower part , 403 (15.51%) followed by the middle part, 269 (10.35%), then the upper part, and 36 (1.39%) in the whole stomach. From hN0–hN3b, patients with tumors in the lower part decreased gradually, while patients with tumors in the middle, upper part, and whole stomach increased gradually. The proportion of hN0–hN3b decreased gradually from T1 to T2 and increased gradually from T3 to T4. BMI, CEA, CA-199, RLNs, MLNs, and RLNs showed significant differences under different hN stages , hN1 483 (18.59%), hN2 443 (17.05%), hN3a 223 (8.58%), and hN3b 158 (6.08%). The average age was 57.97 ± 10.05. There were 1909 males (73.48%) and 689 females (26.52%) (N stages .p = 0.859). With the progress of the hN stage, the proportion of tumors located in the lower part of the stomach gradually decreased, while the proportion of tumors located in the upper part and the whole stomach gradually increased. BMI, CEA, CA-199, RLNs, MLNs, and RLNs showed significant differences under different hN stages , 104 (13.76%), 145 (19.18%), 57 (7.54%), and 70 (9.26%), respectively. Furthermore, 405 (53.57%) patients were less than 60 years, and 351 (46.43%) patients were more than 60 years old  and 0.728 (95%CI: 0.708–0.749), respectively. Furthermore, the AUC of 2016-hN and 2016-pN for the validation cohort were 0.812 (95%CI: 0.780–0.845) and 0.784 (95%CI: 0.754–0.824), respectively a,b.For hN and pN, C-index was calculated under the conditions of differentiation degree a, T stagDCA results showed that in the validation cohort, hN staging and hTNM staging were better than pN and pTNM in the 3rd and 5th years a–d, withp < 0.01 among each stratum. The hN stage had significant statistical differences under each N stratum, indicating that the hN stage had a good stratification  rate of each subgroup under the condition of hN in the training cohort and the validation cohort are shown in fication In order to determine the independent risk factors affecting the prognosis of patients under the hN stage, Cox risk regression model analysis was performed. Univariate analysis showed that BMI, CEA, CA-199, MLNs, RLNs, LNRs, age, tumor location, hN stage, pT stage, hTNM stage, pathological tumor type, degree of differentiation, and tumor size were statistically significant. Multivariate analysis showed that BMI, CEA, CA-199, RLNs, age, tumor location, hN stage, pT stage, hTNM stage, and pathological type were independent risk factors related to the prognosis of patients . Based oLymph node metastasis is one of the most critical factors affecting the survival of patients with GC, as it can lead to the recurrence of GC ,18,19. TMine et al.  found thp = 0.036. It was found that there was a significant difference in the number of metastatic lymph nodes in different sites. The number of metastatic lymph nodes in the greater curvature was significantly higher than in the lesser curvature and peripheral lymph nodes. Previous studies have shown that the metastatic ability of lymph nodes is related to the distance of the lymph nodes from the tumor. According to the distance from the tumor location to the lymph nodes, the tumor in the greater curvature is more likely to metastasize to the 6 group lymph nodes outside the greater curvature [In order to solve the above problems, Choi et al.  created urvature , which iurvature ,25. Moreurvature  found thThe latest 8th AJCC staging has improved the predictive performance of pTNM staging for the prognosis of different patients . HoweverIt was found in this study that the C-index of hN was higher than that of pN except for TNM I patients. In stage I patients, the C-index of hN and pN was the same, which was 0.520, and the C-index was between 0.5–0.6, showing poor predictive ability. The possible reason is that according to the 8th edition AJCC staging criteria , pTNM I In this study, we found that with the gradual progress of the hN stage, the proportion of tumors located in the lower part gradually decreased, while the proportion of tumors located in the gastric body, cardio, and whole stomach gradually increased . TherefoVerlato et al.  found thThis staging system also has some limitations. Firstly, this is a retrospective study, and the sample recording process may be biased. In addition, this is a single-center study. More patients and testing centers could make this study more accurate and reliable. Secondly, the limitations of the hybrid staging system arise from its complexity. The system requires more preliminary preparation and staging calculation than the 8th AJCC staging system. This can increase the workload for surgeons and pathologists, who need to record the lymph node clearance. The hybrid staging system is also more complex than AJCC N staging, which utilizes only the number of positive lymph nodes. Physicians may require some study and practice to become proficient in its application.Both the number and location of positive lymph nodes significantly impact the prognosis of patients with GC. The lymph nodes location-number hybrid staging system can improve the prognostic value of GC patients and enhance the correlation between the mixed staging and the tumor’s location and biological characteristics to improve the prediction efficiency. It can be used as a new installment instead of the 8th AJCC staging.


Rheumatoid arthritis (RA) is a debilitating autoimmune disorder with an inflammatory condition targeting the joints that affects millions of patients worldwide. Several unmet needs still need to be addressed despite recent improvements in the management of RA. Although current RA therapies can diminish inflammation and alleviate symptoms, many patients remain unresponsive or experience flare-ups of their ailment. The present study aims to address these unmet needs through in silico research, with a focus on the identification of novel, potentially active molecules. Therefore, a molecular docking analysis has been conducted using AutoDockTools 1.5.7 on Janus kinase (JAK) inhibitors that are either approved for RA or in advanced phases of research. The binding affinities of these small molecules against JAK1, JAK2, and JAK3, which are target proteins implicated in the pathophysiology of RA, have been assessed. Subsequent to identifying the ligands with the highest affinity for these target proteins, a ligand-based virtual screening was performed utilizing SwissSimilarity, starting with the chemical structures of the previously identified small molecules. ZINC252492504 had the highest binding affinity  for JAK1, followed by ZINC72147089  for JAK2, and ZINC72135158  for JAK3. Using SwissADME, an in silico pharmacokinetic evaluation showed that oral administration of the three small molecules may be feasible. Based on the preliminary results of the present study, additional extensive research is required for the most promising candidates to be conducted so their efficacy and safety profiles can be thoroughly characterized, and they can become medium- and long-term pharmacotherapeutic solutions for the treatment of RA. Rheumatoid arthritis (RA) is a chronic, inflammatory disorder with major implications for the immune system that predominantly harms the synovial joint lining and is correlated with financial burdens, premature mortality, and gradual disability . FurtherThe associations between genetic factors  and environmental factors  are the most significant risk factors with implications for the development of RA . Various mechanistic concepts have been proposed, even though the pathophysiological processes underlying RA are still not completely understood. According to experimental assessments, autoimmune responses might actually occur years before manifestations of joint inflammation become noticeable, referred to as the pre-RA phase . CitrullMultiple pathways are activated, and a variety of proteins are involved throughout the pathophysiological mechanisms of RA, including cytokines, adhesion molecules, matrix metalloproteinases, interferon signaling, receptor activators of nuclear factor kappa B ligand, B cells, etc. ,8. Among the most relevant molecular targets are Janus kinases (JAKs), a family of intracellular enzymes that have the potential to function as therapeutic targets due to the fact that they contribute significantly to controlling the JAK-signal transducer and activator of transcription proteins pathway, which regulates signal transduction pathways associated with inflammation and immune responses ,10.JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2) constitute the JAK family. Multiple studies have revealed that synovial cells and synovial tissue express various JAK isoforms as well as downstream signal transducers and activators of transcription proteins ,11. Pharmacological approaches to investigating numerous potential molecules that could improve the current treatment for RA have advanced significantly because of continuous development in the strategies and methods used in drug design processes. According to the European League Against Rheumatism and American College of Rheumatology recommendations, available treatments address RA from two viewpoints: management of the symptoms by using nonsteroidal anti-inflammatory drugs  and glucConventional synthetic DMARDs (csDMARDs), biologic DMARDs (bDMARDs), and targeted synthetic DMARDs (tsDMARDs) are various types of designed DMARDs . For recFive JAKis  have currently been authorized by several agencies for the treatment of RA and are considered to be tsDMARDs. All the JAKis that are officially approved are competitive antagonists. Reportedly, it is considered that tofacitinib inhibits JAK1, JAK2, and JAK3 to variable degrees in vivo . UpadaciOther JAKis, such as VX-509 (decernotinib), ruxolitinib (INCB018424), and itacitinib, are in various phases of clinical trials evaluating their potential in the treatment of RA. Decernotinib has been synthesized as a selective JAK3 inhibitor and has been observed to have a reduced action on other JAKs, ruxolitinib inhibits JAK1 and JAK2 to a greater extent, while itacitinib selectively inhibits JAK1 .To improve the medical applications of JAKis, especially if they are considered in the context of cytokine signaling, it is crucial to comprehend how they can modulate or ameliorate the autoimmune response in RA. By targeting JAK signaling and modulating cytokine responses in human leukocyte subpopulations, JAKis reduced inflammation, attenuated joint injury, and alleviated the signs and symptoms of RA, according to experimental studies ,20. ThesTofacitinib was designed to attach to the ATP-binding site of JAK3 in a competitive manner. Initially believed to inhibit JAK3 phosphorylation only, tofacitinib is now shown to inhibit JAK1, JAK2, and JAK3 to variable degrees in vitro and in vivo. Tofacitinib is an effective inhibitor of JAK1 and JAK3, but it is less effective against JAK2 and TYK2, according to in vitro kinase studies . In addiIn an experimental study, the in vitro effect of tofacitinib was compared to that of other JAKis using healthy donors’ peripheral blood mononuclear cells. The investigators measured the levels of phosphorylated signal transducer and activator of transcription (STAT) in the presence of various JAKis following cytokine stimulation. This allowed them to evaluate the effect of each JAKi on cytokine signaling pathways. In addition, the researchers calculated the half-maximal inhibitory concentration (IC50) values for each JAKi in distinct leukocyte subpopulations. By comparing the calculated mean concentration–time profiles of JAKi-treated subjects over a period of 24 h, the therapeutic significance of the in vitro analysis was determined. For each JAKi, cytokine, and cell type, they determined the duration above IC50 and the average daily percent inhibition of pSTAT formation. The results demonstrated that various JAKis had distinct in vitro pharmacological profiles. Tofacitinib and upadacitinib were the most potent inhibitors of IL-2, IL-4, IL-15, and IL-21, which are JAK1- and 3-dependent cytokines. They had lower IC50 values and longer times above IC50, indicating a higher inhibition of STAT signaling over the course of the dosing interval .Variations in biomarkers of inflammation, levels of cytokines, immune cell populations, or specific proteins associated with the illness pathway can be used to measure the in vivo effect of tofacitinib. Single-dose tofacitinib data in a collagen-induced arthritis mouse model revealed immediate effects of tofacitinib on plasma inflammatory mediators and, in some cases, a sustained impact on chemotactic proteins, but no immediate impact on the disease severity index. It has been demonstrated that IL-6 and interferon gamma-inducible protein-10 can be inhibited rapidly. Other mechanistic assessments have shown that tofacitinib inhibition of cytokine receptor signaling in T cells can ultimately result in suppression of IL-12 as well as decreased production of inflammatory T helper 17 cells generated in response to IL-6 and IL-23 . In addiJAKis have emerged as a significant therapeutic approach for RA patients and other inflammatory disorders . DespiteAlthough valuable, current RA therapeutic management has limitations. Therefore, more advanced treatments are required for addressing specific unmet needs. The latest research is aimed at developing novel and improved JAKis ,32, imprMolecular docking studies are valuable because they can provide insights into the potential binding affinity of small drug molecules to JAK proteins with implications for RA, but experimental endorsement is needed to validate the results and determine their genuine therapeutic potential as RA therapies. By modulating the interaction between the drug candidate and the JAK target protein, compounds with strong binding affinities and specificities for JAK can be identified, making them potential candidates for further investigation. Furthermore, the medical applicability of these studies in their final form is found in the design of novel JAKis that are efficient, secure, and cost-effective, offering patients with RA better therapy alternatives .Therefore, the aim of this study was to apply molecular docking, virtual screening, and pharmacokinetic in silico strategies for identifying compounds with the highest affinity to the target protein (JAKs), converting compounds not used in current medical practice into substances with potential use in the management of RA. The contribution made to the field of study emerges from the need to find and establish novel compounds with therapeutic action in RA to address the current limitations, with the present study addressing the possible interactions with JAK1, JAK2, and JAK3 through a distinct design.The grid box size was set to 60 × 60 × 60 Å and its coordinates were set to X = 10.79, Y = 13.83, and Z = −15.33. The Protein Data Bank (PDB) ID of JAK1 is 3EYG.The calculated root-mean-square deviation (RMSD) value (1.371 Å) fit within the accepted literature values (<2 Å), indicating that the structure of the docked tofacitinib overlapped to a large extent with the structure of the co-crystallized tofacitinib and that the docking method adequately simulates the ligand–protein interaction.Following the docking process, the co-crystallized tofacitinib in its best conformation showed an affinity of −7.5 kcal/mol towards JAK1. In The binding affinities of the ligands studied for their potential to interact with JAK1 are shown in It has been shown that itacitinib has the highest binding affinity for JAK1 of all the ligands studied, signifying that itacitinib has the highest potential for a strong interaction with JAK1, which may lead to its inhibition and subsequent expression of its therapeutic effects. Filgotinib, with a docking score of −9.1 kcal/mol, has the highest affinity for JAK1 of the JAKis approved by regulatory agencies for the treatment of RA. The type and potency of interactions between JAKis and amino acids in JAK1 determines the JAKi’s binding affinity for JAK1. The JAKis’ effectiveness and specificity for JAK1 are significantly influenced by the particular amino acids that they interact with in JAK1. Following the analysis of the 2D diagrams, it has been observed that the control ligand, tofacitinib, interacts with GLU883, VAL889, and LEU1010.Baricitinib interacts with LEU881, LEU959, LEU1010, GLU957, ALA906, VAL889, SER963, GLU883, GLY882, and ARG1007, and upadacitinib, having the lowest docking score , interacts with SER961, PRO960, LYS970, GLU966, LEU881, VAL889, GLU883, LEU1010, and ASN1008. Ruxolitinib has a binding affinity of −9.0 kcal/mol and binds to the following amino acids: GLY962, ARG1007, LEU881, LEU1010, LEU959, ALA906, GLY1020, VAL889, and GLY884.Peficitinib has an affinity of −6.5 kcal/mol towards JAK1 and interacts with the following amino acids: LYS941, PHE870, and HIS869. Filgotinib showed an affinity of −9.1 kcal/mol and interacted with the following amino acids: GLY962, LEU881, ALA906, LEU1010, VAL889, and SER963.Itacitinib, the compound showing the highest affinity for JAK1 , binds to the following amino acids: MET956, VAL938, GLU957, ALA906, LEU1010, LEU959, LEU881, VAL889, ARG1007, ASN1008, GLU883, HIS885, GLY884, and GLU966.The increase in the number of interactions between the amino acids of JAK1 protein and the tested compounds may be associated with higher binding affinities and greater potential for JAK1 inhibition, but this hypothesis needs to be confirmed by further research.Similar to the protocol for JAK1, the native PDB protein ligand with ID 6VNE was redocked, and the RMSD was calculated. In the case of JAK2, the protein was co-crystallized with fedratinib. The calculated RMSD value for docked versus co-crystallized fedratinib is 1.271 Å. The size of the grid box is unchanged (60 × 60 × 60 Å) and the coordinates are X = 36.19, Y = −1.51, and Z = 10.55.In Subsequently, other ligands, including baricitinib , ruxolitOf the examined ligands, peficitinib has the highest affinity for JAK2, with −9.5 kcal/mol, and interacts with the following amino acids: ARG980, GLY993, LEU983, ALA880, LEU932, LEU855, and VAL863. Ruxolitinib, which has a lower affinity than peficitinib  interacts with ASP994, ASN981, ARG980, ALA880, LEU983, LEU855, and VAL863, while baricitinib has an affinity of −8.3 kcal/mol and interacts with GLY858, SER936, VAL863, LEU855, MET929, GLU930, ALA880, LEU932, and LEU983. The target protein (PDB ID: 5TTV) was co-crystallized with the compound entitled covalent inhibitor 6. The grid box size remained 60 × 60 × 60 Å, as with the other proteins tested, and its coordinates were set to X = −0.04, Y = 17.28, and Z = −5.04. The RMSD value of the co-crystallized covalent inhibitor 6 versus the docked one is 1.634. In Ligands with demonstrated high affinity for JAK3 and potential in RA therapy are tofacitinib , decernoDecernotinib interacts with the following amino acids: CYS909, LEU828; MET902; LEU905; LEU956; ALA853; GLU903; VAL884; and VAL836.Tofacitinib interacts with MET902, VAL836, LEU956, ALA828, ALA853, GLU903, LEU905, and VAL884; peficitinib, the ligand with the highest affinity for JAK3, interacts with LEU905, LEU828 ALA853, LEU956, GLU903, MET902, and VAL836.http://www.swisssimilarity.ch/, accessed on 22 March 2023), ligand-based virtual screening was performed to identify compounds with similar chemical structures, thus presenting potential for use in the management of RA, but requiring confirmation based on in-depth studies.In order to identify new compounds with potential use in the treatment of RA, the data obtained from the molecular docking study on the chemical structure of the compounds that showed the highest affinity for JAK1, JAK2, and JAK3 were processed. By inputting and rendering the data into the SwissSimilarity online platform  was performed using the ECFP.A search for target compounds in the ZINC online database  compounds not used in medical practice are shown in The compound ZINC000252492504 presented the highest binding affinity for JAK1 in the present virtual screening study, suggesting that it may be a potential candidate for developing a drug to target JAK1. The binding affinity quantifies the interaction intensity between a ligand and its target protein. It assesses the ligand–protein complex’s stability, and a more negative value suggests a greater affinity for binding. The scientific literature contains values ranging from −6.28 kcal/mol to −18.70 kcal/mol for compounds with known action against JAK1 and those investigated for their potential impact on this protein. This range encompasses the binding affinity of ZINC000252492504 . Comparing ZINC000252492504 favorably with reported values suggests that its binding affinity is consistent with that of compounds known to target JAK1 ,38.Like the protocol for JAK1, molecular docking evaluation suggested that peficitinib  had the highest affinity for the amino acids of JAK2 protein. Based on the structure of peficitinib, 29 ligands were identified. The name assigned by the ZINC database, the 2D structure, the similarity score, and the affinity towards JAK2 resulting from the docking process for the five most promising compounds not used in pharmacotherapies are shown in The compound ZINC000072147089 presented the highest binding affinity for JAK2 in the present virtual screening study, suggesting that it may be a potential candidate for developing a drug to target JAK2 due to a possible effective interaction with the target. The binding affinities of compounds targeting JAK2 have been reported in the literature from in silico studies to range from −2.93 kcal/mol to −10.13 kcal/mol. Furthermore, drugs with known activity show a binding affinity typically starting at −6.84 kcal/mol ,40. In tPeficitinib also showed the highest affinity for JAK3. Based on the structure of peficitinib, 29 ligands with structural similarities were identified in the platform. The name assigned by the ZINC database, the 2D structure, the similarity score, and the affinity towards JAK3 resulting from the docking process for the five most promising compounds not used in pharmacotherapies are shown in In the scientific literature, the reported binding affinities of compounds targeting JAK3, as determined through in silico studies, range from −5.64 kcal/mol to −9.97 kcal/mol ,41. In tBased on substances with pharmacological activity on JAK1, JAK2, and JAK3 demonstrated in the medical literature, itacinib and peficitinib showed the highest binding affinities for JAK isoforms. Structural similarity studies obtained from the assays identified 65 compounds whose affinity for target proteins was tested. The compound ZINC000252492504 showed the highest affinity for JAK1 among the ligands tested. From the 2D analysis, it has been identified that it interacts with GLU883, VAL889, LEU1010, PHE958, ALA906, LEU959, MET956, ALA881, ARG1007, GLY882, and GLU966. The 6,7-dihydro-5H-pyrazolooxazine group interacts with the following amino acids: GLU883 and GLU966. The piperidine group is involved in interaction with GLU966, GLY882, and ARG107. The piperazine group only interacts with LEU881. Most of the interactions between the molecule and the protein occur through the pyridine group and the fluorine atoms, which are frequently used to improve the properties of chemical compounds. These functional groups interact with the following amino acids: LEU881, MET956, LEU959, ALA906, PHE958, LEU1010, and VAL889.For JAK2, the compound showing the highest affinity is ZINC000072147089, which binds to the following amino acids: LEU855, ALA880, LEU983, TYR931, GLU930, and LEU932. The cyclopentane group present in the molecule does not contribute to any interactions with the protein. On the other hand, the 1H-pyrrolopyridine group exhibits interactions with the amino acid LEU855. LEU855, ALA880, LEU983, TYR931, GLU930, and LEU932 interact with the protein through the fluorobenzamide group. In the case of JAK3, ZINC000072135158 shows the highest affinity and binds to the following amino acids: LEU956, LEU828, ALA853, LYS855, ASP967, VAL836, and CYS909. The interaction of the compound with CYS909 is facilitated by the presence of a cyclopentane group. Additionally, the molecule has the ability to interact with LEU828, LEU956, and ALA853 due to the presence of a 1H-pyrrolopyridine ring. Furthermore, interaction with ASP967, VAL836, and LYS855 is made possible by the 3-pyridinecarboxamide group.http://www.swissadme.ch/, accessed on 9 April 2023) online platform. Important parameters were identified that can facilitate the design of a framework for future possible uses of the three newly identified compounds.Evaluations of the pharmacokinetic profile of the newly discovered compounds were performed in silico using the SwissADME  less than 5, and number of rotatable bonds less than 5. Small, lipophilic, or hydrophobic molecules with a limited number of hydrogen bond donors and acceptors are more likely to achieve good oral bioavailability because they can easily pass through the cellular membrane in the intestines and reach the bloodstream, according to the Lipinski principles . The action on cytochrome P450 results from their inductive or inhibitory action on some of the most important isoforms. Future research should confirm these characteristics because such actions may involve in vivo interactions or reduce or exacerbate the pharmacological effect . Interpretation of pharmacokinetic data includes newly discovered compounds in the category of small drug molecules with the potential to be administered orally and to offer the advantages that come with this route of administration.SwissADME employs computational models and algorithms to estimate crucial ADME parameters, including solubility, lipophilicity, permeability, and pharmacokinetics. Utilizing molecular descriptors and machine learning techniques, it provides estimates based on the chemical structure of the molecule. Despite the fact that in silico methods for assessing drug ADME properties can be beneficial in the early stages of drug development, it is essential to emphasize that they do not replace in vivo investigations; rather, these studies aim to provide a cost-effective and time-efficient method for analyzing the pharmacokinetic properties of small molecules .Virtual screening, pharmacokinetic studies, and molecular docking are effective computational technologies that are currently applied in the research and development of new drugs. Using precise molecular simulation models and analyses to predict the binding affinity of potential active molecules to their target proteins, emphasizing knowledge of drug–protein interactions, and enhancing the ability to develop and optimize new molecules are among the most relevant advantages of in silico studies. They enable the quick assessment of a large number of potential active molecule candidates while minimizing costs and time compared to traditional wet-lab experiments ,46,47.The advantages produced by these techniques were also exploited in the present study, in which in silico studies on the interaction with three JAK isoforms were conducted, providing, through a comprehensive approach, three compounds with potential input for extensive laboratory studies regarding RA for accurately determining their safety and efficacy profile.The present study has a few limitations related to the types of evaluations conducted. Although useful insights on compound–target interactions have been obtained through molecular docking simulations, these findings still require experimental confirmation. The experimental validation method can be time- and money-consuming, and the outcomes might not always agree with the simulations’ predictions. Molecular docking evaluations focus on a single static interaction between the compound and its target protein, whereas proteins are highly dynamic in the biological milieu and can significantly alter their conformation during compound binding. Numerous approximations have been made in molecular docking simulations, including the exclusion of other proteins from the cellular environment and the neglect of solvent effects. These assumptions may reduce the simulations’ accuracy and result in inaccurate estimations.The presence of fluorine atoms in the structure of some compounds identified in ligand-based virtual screening analyses may present a number of limitations due to their pronounced electronegative character and the likelihood of forming very strong bonds with any protein, hence the need for verification of the parent compound used as a ligand as well as experimental optimization and validation in future studies. Although fluorine can enhance chemical stability, the introduction of fluorine atoms can also impact the metabolic fate and toxicity of a compound. Some fluorinated compounds may have longer half-lives or different metabolic profiles, which could impact their safety and suitability as drug candidates. Furthermore, the presence of fluorine in a molecule may complicate the synthesis process, as fluorination reactions may be more challenging and require specialized reagents and conditions.These limitations enable improvements in the study design by opening research directions through the application of network pharmacology and molecular dynamics, which address the dynamic nature of proteins and possible interactions, the effect of solvents and ions, as well as simulating the presence of other cellular structures that may influence the interaction.Clinical evidence demonstrated that JAKs play significant roles in specific pathogenic RA pathways, and research on JAKis resulted in their approval as RA treatments.https://www.rcsb.org/, accessed on 25 April 2023) IDs 3EYG, 6VNE, and 5TTV were selected as target proteins for the present molecular docking study. For all three target JAKs, ligands selected based on affinity for these structures have been docked.The complexity of the role of JAKs in the pathophysiology of RA, as well as the promising results obtained from the use of JAKis, renders the above-mentioned proteins highly essential in the overall management of RA and makes them suitable for in silico studies. Therefore, JAK1, JAK2, and JAK3 with PDB . Since .SDF formatting is not compatible with the molecular docking software used , these files were converted to the specific AutoDock Tools 1.5.7 format , and Atom Type (T) format). Furthermore, ligands were imported into the AutoDockTools software using the ligand input function to add charges and determine rotable bonds [A total of 8 chemical compounds—5 approved  and 3 in studies —were selected and processed in Simulation Description Format (SDF) using the PubChem database  online platform.The data required for the JAK1 (3EYG), JAK2 (6VNE), and JAK3 (5TTV) studies were obtained from the Research Collaboratory for Structural Bioinformatics PDB  has been used in this regard. Furthermore, polar hydrogens and Gasteiger charges were added using AutoDockTools 1.5.7 [To perform the molecular docking assay, a ligand-free and co-crystallized water-free protein is required, and Molegro Molecular Viewer 7.0 software . Furthermore, Molegro Molecular Viewer 7.0 software  was used in the analysis of the protein preparation workflow and Open Babel GUI 3.0.1 in the ligand preparation process [Molecular docking studies were performed using AutoDockTools 1.5.7, and 2D and 3D images were generated using Discovery Studio Visualizer 4.5 . The ligands were downloaded in .SDF format from the ZINC database, converted with OpenBabelGUI 3.0.1  [https://vina.scripps.edu/, accessed on 20 March 2023), and with the Autodock Vina 1.2.0 software the necessary charges were added, and the rotatable bonds were determined [For finding new compounds with potential in the treatment of RA, the chemical structure of the compounds that showed the highest affinity for JAK1, JAK2, and JAK3 following docking studies was used to identify compounds with similar chemical structure and therapeutic potential using the online platform SwissSimilarity (ch 2023)  into a ftermined .http://www.swissadme.ch/, accessed on 15 April 2023).Predictions of the pharmacokinetic profile of compounds that were identified as most promising from molecular docking and virtual screening analyses were performed using the SwissADME online tool  not used in medical practice. These compounds may represent a medium-to-long-term solution if these results are coupled with further research on the extensive study of the efficacy and safety profiles of these molecules.


Upon admission to our hospital, her pertinent physical examination revealed a newborn girl with dysmorphic facial features, including widely separated eyes, downward slanting of the palpebral fissure, microphthalmia, retrognathia, and low-set ears. She had an area of loss of scalp skin and skull bone with seen brain tissue and an exposed sagittal sinus that was 6 by 5 cm in size. She had a clenched fist, overlapping fingers, and rocker bottom feet. Precordium auscultation revealed medium-pitched high-grade continuous murmur heard best at the pulmonary position with a harsh machinelike quality that often radiated to the left clavicle. Laboratory investigations include basic labs, and the TORCH screen was negative. On the 9th day of life, a chromosomal analysis showed a female karyotype with three copies of chromosome number 13 (trisomy 13) in all 20 metaphase cell counts. The patient was managed with a moist gauze dressing, topical antibiotic ointment, and povidone-iodine. However, a multidisciplinary team agreed on a do-not-resuscitate (DNR) order with no further surgical intervention as the survival rate of trisomy 13 is poor. In this article, we report a case of aplasia cutis congenita of the scalp with dura and bone defect and an exposed sagittal sinus in a newborn diagnosed with trisomy 13. It emphasizes the importance of ACC-associated syndrome, which has high mortality prior to surgical intervention.Aplasia cutis congenita (ACC) is a heterogeneous disorder with a rarely reported incidence of 0.5–1 in 10,000 births. ACC can be associated with physical defects or syndromes that may help in the diagnosis, prognosis, and further evaluation of the patient. Trisomy 13 is one of the most common fetal life-limiting diagnoses associated with ACC of membranous-type scalp. The patient was born at 35 weeks of gestation  ACC Diagnosis of ACC is clinical with appearance variability. Physical examinations may show ulcerations or erosions of the skin, which may extend to a deeper tissue, such as muscle or bone. Additionally, it may appear as an atrophic scar. Approximately 86% of aplasia cutis congenita cases involve the scalp, particulary the vertex region. Bone abnormalities in ACC are found in approximately 15–20% of cases, and the remaining majority involve the scalp  due to respiratory distress, which initially required continuous positive airway pressure (CPAP) and then intubation on the 4th day of life. She was transferred to our hospital on the 5th day of life due to limited other services and financial issues. Upon admission to our hospital, her physical examination revealed a newborn girl under a radiation warmer and intubated with connection to mechanical ventilation. Her vital signs were within acceptable values, and she had pinkish skin color and was not pale, jaundiced, or cyanosed. Her growth parameters based on age and sex were in the 10th percentile for weight, 10th percentile for length, and 10th percentile for head circumference. She had dysmorphic facial features, including widely separated eyes, downward slanting of the palpebral fissure, microphthalmia, retrognathia, and low-set ears. She had an area of loss of scalp skin and skull bone with visible brain tissue and an exposed sagittal sinus that measured 6 by 5 cm in size, as shown in Laboratory investigations at the age of 5 days included basic workups within the normal range for her age. The patient’s blood group was O-positive, and the direct Coombs test (DCT) was negative. A liver function test showed total bilirubin of 13.7 mg/dl, direct bilirubin of 1.1 mg/dl, SGOT of 75 U/L, SGPT of 14 U/L, lactate dehydrogenase (LDH) of 889 U/L, GGTP of 118 U/L, total protein of 4.1 g/dl, and albumin of 2.7 g/dl. Virology screening, including the TORCH test , was negative. Hepatitis B surface antigen and antibody were non-reactive and the core antibody was negative. The blood culture did not show bacterial growth. On the 9th day of life, chromosomal analysis showed a female karyotype with three copies of chromosome number 13 (trisomy 13) in all 20 metaphase cell counts. Radiology investigations included head magnetic resonance imaging (MRI) and revealed absence of scalp and calvarium at the parietal region with no herniation visualized, as shown in The patient was managed with a moist gauze dressing, topical antibiotic ointment, and povidone-iodine. A multidisciplinary team meeting was held that involved a neurosurgeon, a neonatologist, and pediatric genetics consultants who agreed on a do-not-resuscitate (DNR) order with no further surgical intervention as the survival rate of trisomy 13 is poor along with associated skull bone defect.3.In 1767, Gordon was the first to report aplasia cutis congenita . Such anSkull or dura defect in ACC results in brain and sagittal sinus exposure. Such exposure increases the risk of hemorrhage, infection, and sagittal sinus thrombosis. The reported fatality of ACC is 20–50% . The preSurgical management complications include hemorrhage, graft or flap loss, infection, donor site morbidity, and anesthesia-related complications , 6. HoweIn conclusion, the history and physical examination of ACC can guide healthcare providers to the underlying pathophysiology and further evaluations and treatments can be performed if needed. Family counseling is important to provide understandable family values and goals, as well as to achieve the expected outcomes and disease trajectories . The cas


Diffusion Tensor Imaging (DTI) is the most employed method to assess white matter properties using quantitative parameters derived from diffusion MRI, but it presents known limitations that restrict the evaluation of complex structures. The objective of this study was to validate the reliability and robustness of complementary diffusion measures extracted with a novel approach, Apparent Measures Using Reduced Acquisitions (AMURA), with a typical diffusion MRI acquisition from a clinical context in comparison with DTI with application to clinical studies. Fifty healthy controls, 51 episodic migraine and 56 chronic migraine patients underwent single-shell diffusion MRI. Four DTI-based and eight AMURA-based parameters were compared between groups with tract-based spatial statistics to establish reference results. On the other hand, following a region-based analysis, the measures were assessed for multiple subsamples with diverse reduced sample sizes and their stability was evaluated with the coefficient of quartile variation. To assess the discrimination power of the diffusion measures, we repeated the statistical comparisons with a region-based analysis employing reduced sample sizes with diverse subsets, decreasing 10 subjects per group for consecutive reductions, and using 5,001 different random subsamples. For each sample size, the stability of the diffusion descriptors was evaluated with the coefficient of quartile variation. AMURA measures showed a greater number of statistically significant differences in the reference comparisons between episodic migraine patients and controls compared to DTI. In contrast, a higher number of differences was found with DTI parameters compared to AMURA in the comparisons between both migraine groups. Regarding the assessments reducing the sample size, the AMURA parameters showed a more stable behavior than DTI, showing a lower decrease for each reduced sample size or a higher number of regions with significant differences. However, most AMURA parameters showed lower stability in relation to higher coefficient of quartile variation values than the DTI descriptors, although two AMURA measures showed similar values to DTI. For the synthetic signals, there were AMURA measures with similar quantification to DTI, while other showed similar behavior. These findings suggest that AMURA presents favorable characteristics to identify differences of specific microstructural properties between clinical groups in regions with complex fiber architecture and lower dependency on the sample size or assessing technique than DTI. It has been widely used in the study of the brain and neurological disorders  is an imaging modality employed to assess diverse The most relevant feature of dMRI is its ability to measure directional variance, i.e., anisotropy, which, inside the brain, is related to structural connectivity between areas. The most common methodology to estimate the anisotropy is via the diffusion tensor (DT)  were defined and widely employed to characterize damaged tissues in multiple neurological and psychiatric disorders , return-to-plane probabilities (RTPP), return-to-axis probabilities (RTAP) and the propagator anisotropy (PA)  with several (moderate to high) b-values, which may be difficult to acquire in many commercial MRI scanners; and (3) heavy computational burdens with very long processing times. These three issues have hindered the general adoption of EAP-related metrics in the clinical routine, despite the growing interest in the exploration of their potential applicability  long acquisition times; (2) several shells with large To overcome these limitations and facilitate the widespread use of advanced diffusion metrics in clinical studies, a new approach called Apparent Measures Using Reduced Acquisitions (AMURA) has been recently proposed . A common distinction when studying migraine is made between episodic migraine (EM), in which patients suffer from headache less than 15 days per month, and chronic migraine (CM), in which patients suffer from headache at least 15 days per month.A recent study identified statistically significant differences in migraine using advanced diffusion measures calculated with AMURA  if appropriate multi-compartment models of diffusion are employed.AMURA was proposed in Aja-Fernández et al.  as a metnd Jones , in the b-value, the measures derived this way must be seen as apparent values at a given b-value, related to the original ones but dependent on the selected shell. The AMURA metrics used in this work are , also known as probability of zero displacement, it is related to the probability density of water molecules that minimally diffuse within the diffusion time τ.Return-to-plane probability (RTPP), which is a good indicator of restrictive barriers in the axial orientation.Return-to-axis probability (RTAP), an indicator of restrictive barriers in the radial orientation.Apparent Propagator Anisotropy (APA), an alternative anisotropy metric. It quantifies how much the propagator diverges from the closest isotropic one.Diffusion Anisotropy (DiA), an alternative derivation of APA.1/2).Generalized Moments, specifically we will consider the full moments of order 2  and 1/2  that participated in previous studies , stable clinical situation, and first screening related to migraine just before the recruitment. Exclusion criteria were use of preventive treatments before the MRI acquisition, migraine onset in people older than 50 years, recently developed migraine (less than 1 year), frequent painful conditions, psychiatric and neurological disorders different to migraine, and pregnancy. Further details are available at Planchuelo-Gómez et al. .The local Ethics Committee of Hospital Clínico Universitario de Valladolid approved the study (PI: 14-197). Additionally, all participants read and signed a written consent form prior to their participation.The detailed demographic and clinical features of the three groups are shown in For patients with migraine, the images were acquired at least 24h after the last migraine attack and before 2 weeks after the clinical visit to the headache unit. High resolution 3D T1-weighted followed by DWI were acquired using a Philips Achieva 3T MRI unit  with a 32-channel head coil.o, 256 × 256 matrix size, spatial resolution of 1 × 1 × 1 mm3 and 160 sagittal slices covering the whole brain.The acquisition of T1-weighted images was carried out using a Turbo Field Echo sequence with the following parameters: repetition time (TR) = 8.1 ms, echo time (TE) = 3.7 ms, flip angle = 8o, 61 diffusion gradient orientations, one baseline volume, b-value = 1,000 s/mm2, 128 × 128 matrix size, spatial resolution of 2 × 2 × 2 mm3 and 66 axial slices covering the whole brain.The acquisition parameters for DWI were TR = 9,000 ms, TE = 86 ms, flip angle = 90All the images were acquired in the same session with a total acquisition time of 18 min.dwidenoise, dwipreproc, and dwibiascorrect tools  denoising based on the Marchenko-Pastur Principal Component Analysis procedure , and radial diffusivity (RD). We considered only these measurements as they are the ones employed in most previous studies, particularly in the literature migraine, with no studies applying other measurements excluding the one carried out with this sample or the use of kurtosis . The simplest case is a 2-compartment model in which we considered that the main anisotropic diffusion was ruled by a zeppelin-shaped compartment , d|| (μm2/s) and d⊥ (μm2/s) are the parallel and perpendicular diffusivities that model the zeppelin and (1−f) is the free-water fraction.where f were considered, ranging from 0.3 to 1. The value of d|| was fixed and d⊥ was changed as a function of f for two different casesFor the experiment, different values of S(b) is constant.The FA obtained after estimating the DT from S(b) is constant.The MD obtained after estimating the DT from b = 1,000 s/mm2 were considered. DTI and AMURA metrics were calculated from the synthetic signal.Sixty-one gradient directions and In comparison with previous studies that assessed EAP-derived measures  assessment. For both approaches, statistical differences between EM, CM, and HC were assessed with two-by-two comparisons. Forty-eight different regions of interest (ROIs) were identified using the Johns Hopkins University ICBM-DTI-81 White Matter Atlas (JHU WM)  for every measure and ROI.For the ROI-based analysis, to obtain more robust measures, the average value of the metrics for each subject was obtained using voxels exclusively included in the white matter skeleton within the 2% and 98% percentiles of the corresponding skeleton values. Then we carried out a two-sampled-two-tailed, pooled variance randomize tool from FSL  was progressively decreased from the original number to 10 subjects in each group, reducing five subjects for each iteration. For each iteration, 5,001 different subsamples were randomly obtained following a bootstrapping procedure. For each subsample, the ROI-based approach described in Section 2.5.1, i.e., the uncorrected The coefficient of quartile variation (CQV) was used to measure the stability across groups. The CQV is a measure of homogeneity  was set taking the 2.5 and 97.5 percentiles of the whole CQV values for each group of values. This 95% CI was compared between the diverse measures and regions for each sample size.f, presenting qMSD, ϒ1/2 and RTOP similar but higher slopes of opposite sense compared to MD, which decreases with f, begin the change of the RTAP almost identical to the one shown by the MD. On the other hand, when MD is set to constant, Results for the experiment with synthetic data are gathered in 1/2, APA, and DiA). p-value scheme for the 48 ROIs considered for each of the measures. Those ROIs that exhibit differences with statistical significance above 95% (p < 0.05) are highlighted in green and above 99% (p < 0.01) in amber. The size of the effect (Cohen's D) is shown for those ROIs with significant differences .Eleven different measures were considered for the analysis: four DT-based measures  and seven AMURA-based , the equivalent AMURA metrics showed a smaller p-value and higher effect sizes. RTOP, qMSD, and DiA were the metrics providing a higher number of statistically significant differences with the higher significance (see amber ROIs) and the greater effect size.Regarding the other two sets of comparisons (CM-EM and CM-HC), AMURA metrics showed no clear higher number of differences compared to DTI metrics. In fact, AD and MD were able to detect more differences in the comparisons between CM and EM, coherently with previous studies  and the right external capsule (EC-R) for being the ones with the highest number of differences and the greatest effect sizes in 1/2 Regarding the other two sets of comparisons (CM-EM and CM-HC), AMURA metrics showed no clear higher number of differences compared to DTI metrics. In fact, AD and MD were able to detect more differences in the comparisons between CM and EM. This would mean that AMURA better discriminates EM in this ROI. According to In the PCT, regarding DTI, the statistical analysis found differences between EM and HC for MD and AD, and between CM and HC for AD, but no differences were found between both migraine groups. In For the ICP-R, according to A similar effect can be observed in the EC-R, where no differences were found for DTI parameters, but for AMURA in the comparisons between HC and the two migraine groups. If we see In D-values. Specifically, qMSD, RTOP, and DiA were consistently getting values over 0.5 (the threshold for medium effect) and, in some cases, near 0.8. In the right external capsule (EC-R), for instance, most AMURA metrics showed a moderate-large effect size while DTI metrics did not get to 0.5. Even in those regions where DTI values showed statistical differences and a moderate effect , AMURA outperformed them. There is only one case, the MD in the ICP-R, where a DTI metric showed a moderate effect size. However, if we check The comparison between EM and HC, the AMURA metrics showed the largest effect sizes as measured by larger Cohen's D-values over 0.5 for the AD, while AMURA only achieved medium effects in the pontine crossing tract (PCT).Regarding the comparison between CM and EM , most me1/2 showed values over 0.5 in the right inferior cerebellar peduncle (ICP-R).Finally, in the comparison between CM and HC , the rigD-values close to 0.6, and, respectively, 27 and 22 ROIs with significant differences for the ROI analysis, 43 and 41 in TBSS. On the other hand, the comparison between CM and HC presented the lowest Cohen's D-values, none of them reaching 0.5. Regarding the comparison between CM and EM, the AD, MD were the measures with greatest Cohen's D-values, over 0.5.It is also interesting to analyze the behavior of each measure over the whole WM. N = 25), most AMURA metrics still were able to find differences between groups, while only a few differences remained for the DTI case.Results showed a stable behavior of AMURA measures in relation to the sample size, which can be understood as a linear dependence between the group sample size and the number of statistically significant ROIs. In N = 50, 5 for MD and 5 for AD (see ROIs marked in amber). Then, we look at the results for those specific ROIs for a reduced sample size of N = 25. Note that, in that case, when the number of subjects is reduced to half, the FA was only able to detect one ROI (out of 7), MD only one (out of 5), and AD none (ROIs marked in red). When we look to the AMURA metrics, we see that they were able to still keep most of those differences even for a reduced sample size (see ROIs marked in green): DiA and APA, anisotropy measures similar to the FA, were able to, respectively find 4 and 7 out of the original 7 FA ROIs. RTAP, and ϒ1/2 succeed in finding 2 of the 5 MD ROIs, while RTOP finds 3 and qMSD 4 of them. In addition, with RTPP, 2 out of the 5 AD ROIs were identified for the reduced sample size. All in all, for this comparison, AMURA outperformed DTI in keeping the differences even for a smaller sample size.In order to better understand this effect, we now analyze the behavior of the measures in selected ROIS. We have chosen, according to results in N = 25 are displayed.As an illustration, in As we have previously stated, the ROI analysis carried out in the previous sections could be an illustrative example of the performance of the different metrics and it gives a valuable insight on the relation among them. However, since no statistical correction was considered, the results could not be acceptable for clinical studies. Thus, in order to mimic an actual clinical study, we have now repeated the analysis using TBSS for the three comparisons.Using the DTI measures , statistically significant differences between CM and EM patients were observed for two parameters. Patients with CM showed lower AD and MD values than EM in 40 and 38 out of 48 regions from the JHU-WM Atlas, respectively. No statistically significant differences were found using DTI measures between EM and HC or between CM and HC.1/2 values in EM compared to HC were found in 41, 39, 43, 27, 29, and 9 ROIs out of 48, respectively. Concerning the comparison between both groups of patients, higher values in CM compared to EM were identified for the RTPP and ϒ1/2 in 4 and 32 regions, respectively.For the AMURA metrics, the comparison between patients with EM and HC showed the highest number of parameters with statistically significant differences. Significant lower RTOP, RTAP, qMSD, APA, DiA, and ϒ1/2 and only DiA for EM vs. HC. On the other hand, DTI and AMURA measures can be distinguished in the last CM vs. EM comparison. For DTI, the merged measures depicted are AD and MD, while for AMURA are RTPP and ϒ1/2. As it can be seen, AMURA measures showed differences in group comparisons where the DTI ones did not, as shown in the green circles. A summary with the previous TBSS results regarding the number of ROIs and the group comparisons can be found in 1/2 and DIA, with CQV average values between 2% and 5%. The remaining DTI and AMURA descriptors , presented a moderate-high stability, with CQV average values between 5% and 10%. The descriptor with the lowest stability was the qMSD, with CQV average values between 15% and 20%.1/2 were significantly higher in HC than CM in 13 regions. The number of regions with CQV differences between CM and EM was lower compared to the comparison between HC and the patient groups. FA and MD were the descriptors with a higher number of regions (nine) that showed higher variance in EM compared to CM, and MD was also the parameter with more regions (eight) with significantly higher variance in CM.Regarding the comparisons between the three groups of interest, after reducing the group sample size to 45 subjects, the assessment of the CQV 95% CI showed that the HC presented a general higher variability than patients with EM and CM. The parameters with a higher number of regions with statistically significant differences between HC and migraine patients according to the 95% CI were the three AMURA measures  and the MD, with 14–22 regions presenting differences. Additionally, in the comparison between HC and CM, the CQV of APA or ϒIn this study, we assessed the viability of advanced diffusion descriptors obtained with a novel approach, AMURA, in comparison with traditional DTI parameters. To this end, their capability to discriminate difference between clinical groups of interest was compared, together with the stability of these results for reduced sample sizes. Using synthetic and real data with a single-shell and low b-value, we observed that AMURA is sensitive to changes of parameters associated with the dMRI signal, showing a higher capability of discrimination between clinical groups, even for decreased sample sizes. Specifically, with AMURA we detected a larger number of ROIs with statistically significant differences between groups, or results complementary to those identified with DTI, presenting higher effect size but lower stability than DTI metrics.b-values, a high number of diffusion gradient directions and very long processing times. This makes them unfeasible for their use in clinical practice or in many commercial MRI scanners. Besides, the use of these metrics in retrospective studies is usually impossible since the acquisition protocols do not allow for it.Advanced diffusion descriptors such as RTOP, RTAP and APA have shown to be useful for the analysis of the WM of the brain . For lower values, these measures show a weaker correlation since the underlying features measured are better visible at higher b-values  are subtle, as studies using small sample sizes have often reported no differences and even contradictory findings have been published , including differences that were not detected using DTI-based measures. Although AMURA showed additional differences between groups in a preliminary previous study  were AMURA-based APA and RTPP, and the DTI-based measures, while AMURA-based qMSD seems to present low stability. This high variability was expected, since qMSD is a quadratic measure, so it must show a greater range of variability. Interestingly, it presented a relatively high number of regions with statistically significant differences in the comparisons of both migraine groups against controls for diverse sample sizes despite their low stability. Therefore, the results of this study suggest that qMSD is able to characterize specific microstructural properties that are particularly difficult to find with other parameters. Moreover, as it has been suggested previously in this section, differences between both groups of patients with migraine and controls may be qualitatively distinct compared to the differences between CM and EM. Furthermore, qMSD is especially sensitive to short diffusion time scales  and cannot be made public. Researchers may request access to them by contacting the corresponding author. Requests to access these datasets should be directed to The studies involving human participants were reviewed and approved by Comité Ético, Hospital Clínico Universitario de Valladolid. The patients/participants provided their written informed consent to participate in this study.CM-M: validation, formal analysis, investigation, writing—original draft, and writing—review and editing. ÁP-G: methodology, validation, formal analysis, investigation, data curation, writing—original draft, and writing—review and editing. ÁG and DG-A: methodology and validation. AT-V: methodology, validation, formal analysis, supervision, project administration, and funding acquisition. RL-G: methodology, validation, formal analysis, investigation, writing—original draft, supervision, project administration, and funding acquisition. SA-F: conceptualization, methodology, software, validation, formal analysis, investigation, writing—original draft, writing—review and editing, supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.


We present a case report of an older patient with aortic stenosis who was managed before and after transcatheter aortic valve implantation by a team of cardiologists but without the support of a geriatrician. We first describe the patient’s post-interventional complications from a geriatric perspective and afterwards, discuss the unique approach that the geriatrician would have provided. This case report was written by a group of geriatricians working in an acute hospital, along with a clinical cardiologist who is an expert in aortic stenosis. We discuss the implications for modifying conventional practice in tandem with existing literature. Although aortic stenosis (AS) patients are predominantly elderly, geriatricians seldom are involved in the decision-making processes of the Heart Team. Moreover, a comprehensive geriatric assessment (CGA) is a key factor contributing to the optimization of care for geriatric patients but is rarely performed. In this clinical case, we discuss the importance of involving a geriatrician in the decision-making processes of the heart-team and highlight the clinical implications of using the CGA to identify the complex needs of older patients with AS. A clinical cardiologist with specific competence in evaluating AS aided us in the discussion of the case report.2 and body surface area (BSA) 1.69 m2. He smoked tobacco for roughly 20 years (1 pk/day), and stopped 30 years ago. He had a past medical history of systemic arterial hypertension, hypercholesterolemia, stage 2 chronic renal disease with an estimated glomerular filtration rate of 60 mL/min, anxiety, and depression. Additionally, his daughter reported mild cognitive disorder, which had not been further investigated. Medications included atorvastatin 40 mg once a day orally, enalapril/hydrochlorothiazide 20 mg + 12.5 mg once a day orally, paroxetine 20 mg once a day orally, and alprazolam 0.50 mg once a day orally (before sleep).An 81-year-old man was referred to our hospital by his general practitioner (GP) for a comprehensive cardiological evaluation due to a severe systolic murmur in the absence of dyspnea, syncope, and angina. The patient weighed 60 kg and was 172 cm tall with a body mass index (BMI) of 20.3 kg/m2) while ejection fraction was 49%, and there was a grade I diastolic dysfunction of the left ventricle. The aortic valve was severely calcified, and hypo mobile with a peak valvular velocity of 4.2 m/s and a mean gradient of 43 mmHg. The aortic valve area was 0.9 cm2 (0.52 cm2/m2). Tricuspid regurgitation was mild with a peak velocity of 2.9 m/s, and the right ventricle was normally sized with tricuspid annular plane systolic excursion of 19 mm, E/e’ was 15. Consistently, the patient was diagnosed as having asymptomatic high-gradient AS with reduced ejection fraction and referred for evaluation for aortic valve replacement.The electrocardiogram showed sinus rhythm and was otherwise unremarkable. Blood pressure was 130/80 mmHg. Echocardiography showed normal left ventricular mass  was assessed, as indicated by the 2021 ESC/EACTS guidelines for the management of valvular heart disease. Blood test showed hemoglobin 12.8 g/dL, hematocrit 35%, creatinine 0.8 mg/dL, and N-terminal pro-B-type natriuretic peptide 411 pg/mL . The operative risk was low by the Society of Thoracic Surgeons score (2.112%) and by the European System for Cardiac Operative Risk Evaluation II score (4.74%). To assess the eligibility to a transcatheter aortic valve implantation (TAVI), the patient was then evaluated with chest and abdomen computed tomography that showed no significant stenosis of the coronary arteries and confirmed the feasibility of this approach. Therefore, the Cardiology team decided to treat the patient with a balloon-expandable Edwards Sapien 3 via transfemoral access under procedural sedation and analgesia. The procedure was performed successfully 2 months after the referral, with no aortic regurgitation at standardized aortography and by transthoracic echocardiography one day after valve implantation.On the first day after the procedure, while staying in the ICU, the patient developed mild restlessness and reduced awareness of the environment. He was not oriented in place and time. Blood examinations found hyponatremia (Na 131 mmol/L) and a slight increase in C-reactive protein serum levels (CRP 5 mg/dL), without an increase of white cells. At night, agitation became severe, the patient tried to get out of bed, and self-removed both the venous lines and the bladder catheter, provoking hematuria. The cardiologist on call decided to replace the bladder catheter and ordered promazine 50 mg intramuscularly with little benefit to the patient’s restlessness.From day III to day V, there was a close-up fluctuation from drowsiness to hyperactivity, which was treated with promazine. To improve the patient’s behavior, the cardiologist decided to remove the bladder catheter, but acute urinary retention developed, which compelled them to maintain it in situ until discharge. On the sixth day, the patient was discharged from hospital in good clinical status, with a scheduled cardiological checkup in one month and the suggestion to contact a nurse for the management of bladder catheter.3/mL, neutrophils 9.59 × 103/mL), further increased CRP (8.71 mg/dL), thrombocytosis (platelet = 450 × 103/mL), and hyponatremia (Na 130 mmol/L), in addition, NT-proBNP 1223 pg/mL and troponin T 13 ng/L. The electrocardiogram, the bedside echocardiogram, and the chest X-ray showed nothing significant.Eight days after the discharge, the patient was referred by his GP to the emergency department of the same hospital due to persistent agitation, fever, and genitourinary pain. Biochemical examinations showed leukocytosis (12.30 × 10E. coli). The 4AT (The patient was then transferred to the Acute Geriatric Unit (AGU) with the diagnosis of suspected urinary tract infection (UTI), which was then confirmed at the urine culture  and activate both the microglia and astrocytes, leading to transient or persistent neuroinflammation of the brain . Other sHowever, the use of bladder catheters, venous lines, nasogastric tubes, and other devices, as well as inappropriate medications , can exaUnfortunately, despite clear evidence to support their use, CGA is not routinely performed , and non-pharmacological approaches are underused in hospital wards ,20. It iDegenerative AS is a common, age-related condition whose prevalence and incidence are often underestimated. There is an increased risk of mortality across the whole spectrum of AS, independently from many comorbidities and treatments ,22. OverThe progressive introduction of multiple innovative technologies has dramatically impacted the scenario of aortic valve replacement (AVR) with a disruptive role of transcatheter aortic valve replacement in expanding the overall numbers, risk profile, and outcome of patients undergoing AVR ,26.TAVI is usually successful in older patients, as shown by several studies . HoweverThe reported case offers multiple points for discussion. First, it underscores that AS is underdiagnosed and disregarded. Second, in an older patient, a combination of comorbidities and dependence in daily activities may often mask the presentation of the typical symptoms of AS, implying that AS is often diagnosed at an advanced stage ,34,35. ETo date, the patient journey of individuals referred to AVR, mostly consists of a series of tests aimed at assessing appropriateness, choice of procedure for safety and effectiveness, and their optimization to avoid complications, or detecting/treating them appropriately, and to reduce as much as possible the in-hospital stay. The time has come to integrate this process with a brief screening test to identify the patients who may require further CGA. The Essential Frailty Toolset may be particularly appropriate to this aim . Based oAvailable evidence suggests the need for the actual profile of all centers performing aortic valve interventions should be combined with an integrated geriatric approach moving from a traditional bedrest-based hospitalization—possibly resulting in sarcopenia —to one tBased on this case record and on the existing literature, we recommend the involvement of the geriatricians both in the decision-making processes of the heart team and in the co-management of the periprocedural period for older patients with AS. In particular, the use of the CGA by a geriatric team is crucial to provide optimal care and identify the complex needs of these patients before and after the surgical procedures.


Lolium multiflorum Lam.). We observed that a 200 mg/kg coumarin treatment had a negligible effect on the rhizosphere soil bacterial species of the annual ryegrass rhizosphere, though it exhibited a significant effect on the abundance of bacteria in the rhizospheric microbial community. Under coumarin-induced allelopathic stress, annual ryegrass can stimulate the colonization of beneficial flora in the root rhizosphere; however, certain pathogenic bacteria, such as Aquicella species, also multiply in large numbers in such conditions, which may be one of the main reasons for a sharp decline in the annual ryegrass biomass production. Further, metabolomics analysis revealed that the 200 mg/kg coumarin treatment triggered the accumulation of a total of 351 metabolites, of which 284 were found to be significantly upregulated, while 67 metabolites were significantly downregulated in the T200 group (treated with 200 mg/kg coumarin) compared to the CK group (control group) (p < 0.05). Further, the differentially expressed metabolites were primarily associated with 20 metabolic pathways, including phenylpropanoid biosynthesis, flavonoid biosynthesis, glutathione metabolism, etc. We found significant alterations in the phenylpropanoid biosynthesis and purine metabolism pathways (p < 0.05). In addition, there were significant differences between the rhizosphere soil bacterial community and root metabolites. Furthermore, changes in the bacterial abundance disrupted the balance of the rhizosphere micro-ecosystem and indirectly regulated the level of root metabolites. The current study paves the way towards comprehensively understanding the specific relationship between the root metabolite levels and the abundance of the rhizosphere microbial community.Rhizosphere microorganisms can help plants absorb nutrients, coordinate their growth, and improve their environmental adaptability. Coumarin can act as a signaling molecule that regulates the interaction between commensals, pathogens, and plants. In this study, we elucidate the effect of coumarin on plant root microorganisms. To provide a theoretical basis for the development of coumarin-derived compounds as biological pesticides, we determined the effect of coumarin on the root secondary metabolism and rhizosphere microbial community of annual ryegrass ( Umbelliferae, Rutaceae, Leguminosae, and Compositae -2-pentanol, and Coumarin (p < 0.05), while these bacteria were negatively correlated with N-Hydroxydecanamide (p < 0.05).Correlations between the bacterial genera colonized in the rhizosphere soil and root metabolites in the T200 and CK groups were analyzed in the current study, and the The rhizosphere is the shallow zone in the soil surrounding the plant roots that is directly affected by root exudates. The rhizosphere is one of the most complex ecosystems, comprising plant roots, soil, and diverse microbes. It has an important influence on the growth and development of plants and their immunity . SeveralProteobacteria, Actinobacteria, Planctomycetes, and Firmicutes were the dominant flora identified in the rhizosphere soil samples of both the CK and T200 groups, which is consistent with the findings of Cheng et al. [Geobacter, Aquicella, and unidentified_Nitrospiraceae was significantly increased in the T200 group compared to the CK group. Geobacter is an important iron-reducing bacteria. Numerous studies have shown that coumarin compounds and their derivatives can decrease the impact of environmental stress in plants by chelating iron or promoting the uptake of iron by roots [Geobacter species. Since Nitrospiraceae is known to fix atmospheric nitrogen, unidentified_Nitrospiraceae, detected in our study, can also improve the tolerance of annual ryegrass to allelopathic stress. In contrast, Aquicella, a unique pathogenic bacterium, has a negative impact on plant growth and development [Aquicella species propagate in large numbers. From these observations, we speculated that one of the possible reasons for this phenomenon could be that stress exposure leads to alterations in the rhizosphere soil microbial community, which has an inhibitory effect on the reproduction of certain microbial communities. These microorganisms have an antagonistic effect on the growth of Aquicella, which leads to the mass propagation of Aquicella species. In addition, plant roots secrete a few substances under allelopathic stress that are selective for Aquicella, such as organic acids, amino acids, and phenols, in order to promote its mass reproduction [Aquicella species and the secretion of these specific compounds could be an important reason behind a sharp decline or even death of annual ryegrass biomass.At the phylum level, g et al.  and Wangg et al. . Furtherg et al. . At presg et al. . Therefog et al.  demonstrby roots ,43,44. Telopment . Under aoduction . AltogetArabidopsis thaliana. It was speculated from the observations that in the pentose phosphate pathway, the oxidation phase of NADPH (OxPPP) maintained the activity of glutathione reductase (GR), thereby inducing the preservation and regeneration of GSH in the cells under glyphosate-induced stress. These results were consistent with our previous findings that coumarin has an inhibitory effect on the growth  of annual ryegrass (Lolium multiflorum Lam.), which is directly proportional to the concentration of coumarin, thus explaining the effect of coumarin on the glutathione metabolism pathway in roots [Arabidopsis thaliana [In the current study, 351 metabolites were found to be significantly different between the T200 and CK groups. Further, the KEGG enrichment analysis of the metabolic pathways revealed that the metabolites were involved in the biosynthesis of phenylpropane, as well as in metabolic pathways such as the Pentose phosphate pathway, carbon metabolism, and tryptophan metabolism. The Pentose phosphate pathway not only mediates the oxidative decomposition of glucose but also serves as an important multifunctional metabolic pathway that provides NADPH to maintain plant growth and promote other biosynthetic and metabolic processes . Few stuin roots . Moreovethaliana . In the thaliana  showed tEnvironmental stress leads to the induction of oxidative stress response in plants, which further affects plant growth and metabolism . HoweverBenzene metabolism plays a vital role in the physiological activities of enzymes and intermediate products. Compounds such as lignin and phenol provide plants with protection by regulating cell differentiation, inhibiting pathogen infection, and promoting pigment formation . Both phAquicella, Bdellovibrio, Geobacter, and ten other genera of bacteria were positively correlated with 1,2,4-Benzenetriace, benzaldehyde, cinnamic acid, and 18 metabolites; however, no positive correlation was observed with N-Hydroxydecanamide. The above metabolites are mainly concentrated in plant-resistant substances, such as N-hydroxydecanamide, coumarin, and 7-methoxy-4-methylcoumarin, and plant growth regulators, such as cinnamic acid, N-hydroxyphenylacetylglutamic acid, and benzoyl β-D-glucoside. The rhizosphere is the center of interaction between the plant roots and soil microorganisms and is directly influenced by the physiological and biochemical activities of plant roots. It regulates the compounds involved in these activities. In the current study, we illustrated that under coumarin-induced allelopathic stress, the abundance and composition of soil microbes are altered, which disrupts the balance of the rhizosphere micro-ecosystem, indirectly regulating the concentrations of root metabolites. However, a comprehensive investigation is required to understand how the rhizosphere microbiota regulates root metabolism.In the current study, the correlation analysis between rhizosphere bacterial community and root metabolites revealed that Aquicella species, was also observed under coumarin-induced stress. The metabolome analysis unraveled a total of 284 metabolites that were more significantly upregulated in the T200 group compared to the CK group, while only 67 compounds were downregulated to a lower degree in the T200 group compared to the CK group. These metabolites were enriched with respect to the Pentose phosphate pathway, carbon metabolism, anthocyanin biosynthesis, glutathione metabolism, and flavonoid biosynthesis, which are mainly related to energy metabolism and antioxidant activity in annual ryegrass. Further, some rhizosphere soil bacteria exhibited significant correlations with several root metabolites, although the specific mechanism behind these correlations needs further investigation.In this study, under coumarin-induced allelopathic stress, the relative abundance of rhizosphere-beneficial bacteria was found to be stimulated in order to resist the stress conditions in annual ryegrass. However, the accumulation of a few pathogenic bacteria, such as Lolium multiflorum Lam.) were procured from the Grass Science Laboratory of Yangzhou University. The potting soil was collected from surface cultivation soil (0~20 cm) from the pilot fields of Yangzhou University. The soil texture was sandy loam, and its pH was 7.07. Plastic pots with an upper diameter of 16 cm, a lower diameter of 12 cm, and a height of 12 cm were used to grow the plants.Coumarin was purchased from SIGMA . The seeds of annual ryegrass  and a distilled water treatment (the control treatment) were recorded as T200 and CK groups, respectively, and each treatment was repeated 7 times. A total of 40 annual ryegrass seeds were selected, and after disinfection by HgClRhizosphere soil samples were collected through the shaking method, after removing the non-rhizosphere soil, and then packed in a 15 mL centrifuge tube, frozen in liquid nitrogen, and stored at −80 °C for subsequent experiments. After the collection of rhizosphere soil samples, the roots were rinsed with distilled water and cut with scissors. The roots of each group were divided into 6 parts, wrapped in foil paper, marked, and then frozen in liquid nitrogen quickly and stored at −80 °C for metabolome analysis.®High-Fidelity PCR Master Mix  according to the manufacturer’s instructions. The diluted total DNA was used as a template for PCR amplification, and the primer used in this study was 515F-806R. The purification of the PCR product was performed using a GeneJET gel kit . The Ion Plus Fragment Library Kit 48 reactions  was used to construct the library, and sequencing was performed on an Ion S5TMXL platform.The SDS method was used to extract the total DNA of rhizosphere soil samples. The samples were ground to break their tissues and cells, and then SDS was used to split them. Then, a high concentration of potassium acetate was added, and the samples were stored at 0 °C to remove protein and polysaccharide impurities; finally, ethanol or isopropanol was used to precipitate the DNA. The treated samples were then stored on standby. The purity levels of the DNA were determined by agarose gel electrophoresis. An appropriate amount of the sample DNA was extracted and placed in a centrifuge tube, and the samples were diluted to 1 ng/μL with sterile water. PCR amplification was performed using Phusionhttps://www.lipidmaps.org/ (accessed on 20 June 2022)) was searched to detect the chimeric sequences in the microbial species through Vsearch [p < 0.05 was considered significant.Cutadapt (V1.9.1)  was used Vsearch , and chi Vsearch . UPARSE  Vsearch . Mothur  Vsearch ,66, and  Vsearch . Finallyg and 4 °C for 10 min. Finally, the filtrate was injected into the LC-MS/MS system.Root tissues (100 mg) were individually ground with liquid nitrogen, and the homogenate was re-suspended in pre-chilled 80% methanol and 0.1% formic acid by vortexing. The samples were incubated on ice for 5 min and then centrifuged at 15,000 rpm and 4 °C for 5 min. The supernatant was diluted to a final concentration containing 60% methanol via LC-MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube after filtration with a 0.22 μm filter and then centrifuged at 15,000× LC-MS analysis was performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive series of the mass spectrometer . Samples were injected in a Hyperil Gold column  using a 16 min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in Water) and eluent B (Methanol). The eluents for the negative polarity mode were eluent A  and eluent B (Methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2–100% B, 12.0 min; 100% B, 14.0 min; 100–2% B, 14.1 min; and 2% B, 16 min. Q Exactive mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, capillary temperature of 320 °C, sheath gas flow rate of 35 arb, and aux gas flow rate of 10 arb.https://www.mzcloud.org/ (accessed on 14 May 2022)) and ChemSpider (http://www.chemspider.com/ (accessed on 14 May 2022)) databases to obtain accurate qualitative and relative quantitative results.The raw data files generated by UHPLC-MS were processed using Compound Discoverer 3.0  to perform peak alignment, peak picking, and quantitation for each metabolite. The main parameters were set as follows: retention time tolerance, 0.2 min; actual mass tolerance, 5 ppm; signal intensity tolerance, 30%; signal/noise ratio, 3; and minimum intensity, 100,000. After processing, peak intensities were normalized to the total spectral intensity. The normalized data were used to predict the molecular formulae based on additive ions, molecular ion peaks, and fragment ions. Then, peaks were matched with the mzCloud (http://www.genome.jp/kegg/ (accessedon 14 May 2022)). Principal components analysis (PCA) and Partial least squares discriminant analysis (PLS-DA) were performed using metaX [p-values < 0.05 were considered to be differentially expressed metabolites. Volcano plots were used to filter metabolites of interest, which were based on Log2(FC) and −log10 values of metabolites. For heat map clustering, the data were normalized using z-scores of the intensity areas of differential metabolites and plotted using the Pheatmap package in R language. The functions of these metabolites and metabolic pathways were analyzed using the KEGG database [Metabolites were annotated using the KEGG database (ng metaX ,69. We udatabase ,71. Furt