Acute coronary syndrome (ACS), comprising unstable angina (UA) and acute myocardial infarction (AMI), is the leading cause of death worldwide. Currently, lacking effective strategies for classifying ACS hinders the prognosis improvement of ACS patients. Disclosing the nature of metabolic disorders holds the potential to reflect disease progress and high‐throughput mass spectrometry‐based metabolic analysis is a promising tool for large‐scale screening. Herein, a hollow crystallization COF capsuled MOF hybrids (UiO‐66@HCOF) assisted serum metabolic analysis is developed for the early diagnosis and risk stratification of ACS. UiO‐66@HCOF exhibits unrivaled chemical and structural stability as well as endowing satisfying desorption/ionization efficiency in the detection of metabolites. Paired with machine learning algorithms, early diagnosis of ACS is achieved with the area under the curve (AUC) value of 0.945 for validation sets. Besides, a comprehensive ACS risk stratification method is established, and the AUC value for the discrimination of ACS from healthy controls, and AMI from UA are 0.890, and 0.928. Moreover, the AUC value of the subtyping of AMI is 0.964. Finally, the potential biomarkers exhibit high sensitivity and specificity. This study makes metabolic molecular diagnosis a reality and provided new insight into the progress of ACS. A hollow crystallization COF capsuled MOF hybrids assisted LDI‐MS serum metabolic analysis platform is proposed. The high‐throughput serum metabolic profiling coupled with machine learning algorithms successfully achieves early diagnosis and risk stratification of acute coronary syndrome. Also, the subtyping of acute myocardial infarction is accomplished. This study provides a new insight into the high‐throughput metabolic molecular diagnosis. Further, AMI including ST‐segment elevation myocardial infarction (STEMI) and non‐ST‐segment elevation myocardial infarction (NSTEMI) is more fatal since it is accompanied by myocardial necrosis. The release of extensive myocardial necrosis markers such as cardiac troponin I (cTnI) and creatine kinase isoenzyme (CK‐MB) in AMI was caused by the arterial blockage devouring lives in minutes under delayed rescue conditions, and the most recent data estimated that more than 7 million new instances of AMI occurred globally in 2021. However, the diagnosis and risk stratification of the ACS at present are performed by the comprehensive assessment of issues involving patients' symptoms, eletrocardiogram (ECG), together with myocardial necrosis markers including cTnI, CK‐MB, etc., which is both time consuming and tiresome in emergency conditions. Besides, as the early symptom of ACS, UA is difficult to diagnose ascribed to the normal ECG results and no increase in cTnI and CK‐MB that can hardly be recognized even by experienced physicians. Moreover, the classification of UA and NSTEMI is another obstacle since patients with either of the disease present comparable physical symptoms and ECG results. Despite cTnI and CK‐MB have been widely used as the main biomarker to distinguish UA and AMI; however, more recently, studies have revealed that cTnI and CK‐MB disruption may be associated with other disorders, such as tachy‐ or brady‐arrhythmias and renal failure, which are not specific to AMI. At this point, developing a precise diagnosis and risk stratification of ACS is of great significance.Acute coronary syndrome (ACS), encompassing unstable angina (UA) and acute myocardial infarction (AMI), is defined as comprehensive clinical symptoms of severe myocardial ischemia and is regarded as the most lethal disease.1 UA i Metabolites, as the end products of life activities including genetic information and protein macromolecule expression, reflect the most natural state of the organism, thus great efforts have been devoted to the discovery of biomarkers for early diagnosis. Mass spectrometry featuring high sensitivity, high throughput, and molecular identification ability with the matched database has developed into a powerful tool for metabolic analysis. In particular, liquid chromatography–mass spectrometry (LC–MS), and gas chromatography–mass spectrometry (GC–MS) have achieved huge success in disease diagnosis, especially for disease‐related metabolic pathway analysis and biomarkers discovery. However, for large‐scale screening, the application of these methods is limited by their time‐consuming analysis, and developing ultra‐high throughput mass spectrometry techniques is in urgent need. Matrix‐assisted laser desorption/ionization–mass spectrometry (MALDI–MS) seems to perfectly meet such disease screening requirements due to its advantages of nanoscale sample loading, seconds‐like analysis speed, and the high throughput result readout. Nevertheless, although MALDI–MS has significantly promoted the advancement in identifying large molecules such as proteins and peptides, its applications in small molecular detection profiling are hampered by the overlapping low‐molecular region produced by the traditional organic substrates. In the past decades, inorganic substrates such as carbon nanomaterials, titanium oxide and noble metal have inspired the detection of small molecules by MALDI–MS. The stable chemical structure and efficient ionization efficiency promise the detection of the key metabolites which are generally at ultralow concentration. Metal–organic frameworks (MOFs) and covalent organic frameworks (COFs), exhibiting large surface area, high crystallinity, and controlled nanopores structures, have attracted considerable attention in photocatalysis, molecular sensing, gas storage, and diagnosis of diseases. Notably, MOFs have been considered promising substrates for numerous affinity sites for the metabolites, and the metal ions in MOFs have been elucidated to be beneficial to charge transfer during the ionization process. Comparatively, the covalent bonds between organic monomers endow COFs with enhanced chemical stability than MOFs. Besides, the extended π–π structures in COFs are conducive to improving ionization efficiency as the substrate of MALDI–MS. More recently, composites with an advanced structure like core‐shell or yolk‐shell structures enjoy enhanced ion efficiency, and the integration of MOFs and COFs holds impressive forward. However, the traditional harsh synthesis conditions of COFs and the grand challenges in the design of yolk–shell structure‐like structure templates limit the application of yolk–shell MOF@COF.Molecular diagnosis is a promising tool for clinical screening that avoids misdiagnosis due to the physicians' empirical judgments and provides a rational interpretation of disease progression from a molecular perspective.8 MetaScheme 2 was adopted as the core and initially assembled with a covalent organic polymer coating layer. After facile aging treatment, a hollow COF capsuled UiO‐66‐(OH)2 was constructed and donated as UiO‐66@HCOF. Compared to the traditional core–shell structure, a yolk–shell structure like UiO‐66@HCOF retains the intrinsic properties of the MOFs and COFs to the maximum degree and fully exposes the large active sites of both. Only 800 nL of serum are required, and the metabolic profiling is obtained as Scheme Herein, a novel hollow crystallization COF capsuled MOF hybrid (UiO‐66@HCOF) was proposed as an alternative substrate of LDI‐MS. Then, we developed a UiO‐66@HCOF‐assisted LDI‐MS metabolic profiling analysis system for precise early diagnosis and risk stratification of ACS Scheme 1. As di22.1Figure 2 was synthesized according to previous work with slight modification and employed as the core component. Following this, 1,3,5‐benzenetricarboxaldehyde (BTCA) and 1,3,5‐tris(4‐aminophenyl)benzene (TAPB) assembled to covalent organic polymer (COP) coating on UiO‐66‐(OH)2 to fabricate UiO‐66@COP. Eventually, UiO‐66@COP was aged at a higher temperature to generate UiO‐66@HCOF hybrids. The morphology of the three products is studied by transmission electron microscope (TEM). As Figure 2 presented a uniform distribution with a diameter of about 120 nm which is consistent with the previously reported literature. Then, a COP coating layer around 30 nm thick can be observed on the surface of UiO‐66‐(OH)2 from Figure 2, and the crystallization COF layer as thin hollow spheres wrapped around the UiO‐66‐(OH)2  of —N—H of TAPB disappeared, and the —C=O (1700 cm−1) of BTCA can still be observed in UiO‐66@COP and UiO‐66@HCOF that probably belongs to the remaining aldehyde end‐group of COP or COF. Notably, the signal of —C=N—(1620 cm−1) generated between the —C=O carboxylic acid functional group (1650 cm−1) of UiO‐66‐(OH)2 and the —C=C (1590 cm−1) of the stretching vibration from benzene skeleton. These results verifying most of the —N—H and —C=O groups are successfully reacted to form an ordered imine chemical bond structure. The crystalline structures of the prepared materials were studied using powder X‐ray powder diffraction (PXRD). As illustrated in Figure 2, and the coated COP layer maintained the high crystallization UiO‐66‐(OH)2. However, different from UiO‐66@COP, UiO‐66@HCOF exhibited representative diffraction peaks of HCOF at 5.7° that demonstrated the formation of crystallization after the aging. The PXRD patterns of UiO‐66@HCOF aging for 12, 24, 36, and 48 h were also recorded, and as Figure 2 maintained the original diffraction peaks without being affected.The Fourier transform‐infrared spectra (FT‐IR) were tested to demonstrate the changes in the composition of the material products at each synthesis step. As displayed in 2‐adsorption and desorption isotherms, as shown in Figure 2 showed type I isotherm and Brunauer–Emmett–Teller (BET) surface area is 436 m2 g−1. As Figure 2 g−1 that illustrated the enhancement of specific surface area with the formation of the HCOF layer. Moreover, in Figure 2 that indicates UiO‐66‐(OH)2 is not being affected during the aging treatments. As Figure Besides, the specific surface area was estimated by N2.2 Based on the reported literature, three major factors including UV–vis light absorption, thermal effects, and charge transfer have been studied in the D/I process to investigate the mechanism in the LDI–MS process. First, the UV–vis light absorption capability of UiO‐66@HCOF, HCOF, and UiO‐66‐(OH)2 at 355nm (the wavelength of the MALDI‐equipped laser) was estimated. As shown in Figure 2 and HCOF, indicating that UiO‐66@HCOF can utilize enhanced UV laser light energy from substrate to analytes in the D/I process. The thermal effects in the D/I process were then investigated, and the chemical thermometer benzyl pyridinium ion (BP+) was used as a probe to estimate the extent of thermal transfer from substrate to analytes. Under thermal effects, BP+ can dissociate into a benzyl cation [BP‐ Pyridine]+ and pyridine, as shown in Figure + intensity, which includes the intensity of [BP]+ and [BP‐ Pyridine]+, summarizes thermal and non‐thermal effects of D/I. Survival yield (SY) is defined as BP+/(BP++[BP‐ Pyridine]+) and has an inverse relationship with thermal effects absorption. The mass spectra of BP+ using UiO‐66@HCOF, UiO‐66‐(OH)2, and HCOF are displayed in Figure + intensity and SY are also displayed in Figure + intensity follows UiO‐66@HCOF> HCOF >UiO‐66‐(OH)2. However, the SY presents UiO‐66@HCOF< HCOF < UiO‐66‐(OH)2. The results show that UiO‐66@HCOF has the best thermal effects D/I, and HCOF also has satisfying thermal effects of D/I. Charge transfer is another key factor in the D/I process that cannot be ignored. Nanomaterials excited by UV lasers can generate electron–hole pairs, and the separated electron–hole in nanomaterials is expected to facilitate ionization in analytes through enhanced charge transfer. However, the visualization and evaluation of electron–hole separation are always of great interest to researchers. We performed the photocurrent‐time curves of UiO‐66@HCOF, HCOF, and UiO‐66‐(OH)2 to character the capability of electron‐hole separation (EHS). Figure 2. More intriguingly, the HCOF exhibits low EHS compared to UiO‐66‐(OH)2, which may be due to the absence of metal nodes in the structures. To further illustrate the EHS capability, we used tetrabutylammonium cation (TBA+) as a sensitive probe to the generation of electrons and holes. As shown in Figure + intensity also demonstrated the efficient charge transfer in the D/I process. Besides, the TBA+ intensity of UiO‐66‐(OH)2 is higher than that of HCOF which is consistent with the photocurrent‐time results. Based on the above results, both HCOF and UiO‐66‐(OH)2 enjoyed favorable UV–vis light absorption. HCOF characterizes a satisfying thermal effect, and UiO‐66‐(OH)2 is more efficient in charge transfer. As a result, UiO‐66@HCOF combines the benefits of UiO‐66‐(OH)2, and HCOF. The D/I process of UiO‐66@HCOF is co‐promoted with the comprehensive interactions of UV–vis light absorption, thermal effects, and charge transfer.The mechanism study of nanomaterials in the desorption/ionization (D/I) process is significant for component selection as well as advanced structural design.32 Bas2, UiO‐66@HCOF, and traditionally organic matrix of 2,5‐dihydroxybenzoic acid (DHB) evidenced that both UiO‐66‐(OH)2 and UiO‐66@HCOF have a clean background. Afterward, the detection of typical metabolites as promising serum biomarkers like glucose (Glc), l‐phenylalanine (Phe), and histidine (His) were performed to determine the performance of the substrates. The mass spectra of these metabolites 2 exhibited some matrix noise around the 100 m/z region which is probably caused by the non‐covalent binding between metal ions and organic ligands. In Figure 2, HCOF showed nearly ionization ability to UiO‐66‐(OH)2, and UiO‐66@HCOF still shows superior detection performance to HCOF. Hence, on the one hand, the HCOF layer coated on the UiO‐66‐(OH)2 imparts higher structure stability to the hybrid, which maintains thermal and chemical stability during the LDI‐MS process. On the other hand, UiO‐66@HCOF endows excellent D/I ability that is consistent with the UV–vis light absorption, thermal effects, and charge transfer evidence.As the advanced hybrids of MOFs and COFs as well as the highly crystalline COP, the performance of UiO‐66@HCOF as LDI–MS substrate for metabolite analysis was comprehensively investigated. First, to ensure less background interference generated by self‐desorption of the substrate under laser irradiation, the background interference was estimated. The results of direct LDI‐MS detection  were successfully detected in 500 mm NaCl solution. Likewise, the abundant biomolecules such as proteins and peptides pose a grand challenge to the detection of metabolites. In this work, despite concentrations of bovine serum albumin (BSA) up to 5 mg mL−1, the clear m/z features, and the excellent signal‐to‐noise ratios of adducts belonging to Glc, Phe, and Asp are obtained  in the three groups. However, there exists a significant difference in gender between AMI (p < 0.05) and UA/CON indicating that AMI more probably occurs in males that is consistent with the reported literature. Therefore, in this work, the gender difference in the AMI sample collection is relatively reasonable. Following this, 800 nL of diluted serum were analyzed by the UiO‐66@HCOF‐assisted LDI‐MS metabolic analysis system, and the presentative metabolic profiling spectra are shown in Figure  We initially applied OPLS‐DA to preliminary characterize the metabolic difference between the four groups. The results yielded by the OPLS‐DA model  in Figure y‐axis are lower than the upper right value, illustrating the high reliability of this model. It further demonstrated that though the patients with AMI or UA suffered similar symptoms, metabolic profiling can tap into the changes of different ACS subtypes at the molecular level and provide the chance to discover the biomarkers. Random forest (RF) algorithm has been proven and widely employed as a credible machine‐learning approach for screening biomarkers in massive metabolic features. This work employed RF algorithm for the screening of the key features between the various groups according to the displayed criteria in Figure p‐value less than 0.05. The key features between different groups were displayed in Tables Extracting high‐quality metabolic fingerprints is an essential step in subsequent metabolic diagnosis as well as metabolic biomarker discovery. We validated the satisfying metabolic profiling detection ability of the established UiO‐66@HCOF‐assisted LDI‐MS system in practical bio‐samples by employing an ACS patient serum and a CON individual serum. Also, the same analysis was carried out with UiO‐66‐(OH)terature.39 TheFigure R2Y = 0.918, Q2 = 0.803) classification and the permutations in Figure As already mentioned, UA is the early type of ACS that is difficult to diagnose by conventional clinical diagnostic methods. However, in this work, 8 screened‐out key features were employed to discriminate UA from CON and the receiver operating characteristic curve (ROC) is displayed in Figure l‐carnitine  and taurine  presented up‐regulated in UA compared with CON that is consistent with previous studies that higher l‐carnitine has recognized to be relevant to cardiovascular disease and widely discovered as the biomarkers for the diagnosis of AMI and the risk stratification of ACS. Moreover, studies have suggested that taurine may have neuro‐ and cardio‐protective functions, and the upregulated taurine in UA may be relevant to the neurological outcomes. Additionally, fumaric acid , succinic acid , and taurine  were detected down‐regulated in AMI in comparison to UA in Figure Based on the above analysis, the screened key features were identified according to the human metabolome database (HMDB), and the promising biomarkers were assigned to metabolites in Table r disease42 andm/z 592.312, HMDB0010403) was considered to be upregulated in patients  for the discovery cohort. Likewise, the AUC values for the discovery cohort of AMI and UA, NST, and ST, and AMI and CON are 0.840 (95% CI: 0.729–0.953), 0.923 (95% CI:0.826–1.000), and 0.895 (95% CI:0.728–0.953), respectively. These results illustrated the high accuracy and specificity of this method for the early diagnosis and risk stratification of ACS. Finally, these potential biomarkers were integrated to explore metabolic pathway disorders in the occurrence of ACS. As displayed in Figure More interestingly, as displayed in Figure s Figure . Afterwa32 and the hollow crystallization COF layer whose chemical structure brings robust stability and high ionization ability to the hybrids. Benefitting from the synergy effect of UiO‐66(OH)2 and hollow crystallization COF layer, metabolic profiling spectra were successfully extracted from 221 serum samples assigned to ST, NST, UA, and CON groups. Remarkably, OPLS‐DA intuitively classified the various groups, and the key metabolite features were screened through random forests machine learning algorithms. Notably, the early diagnosis of ACS was achieved from the discrimination between UA and CON, with the AUC value of the validation sets being 0.945. Besides, a complete ACS risk stratification system was established, and the discrimination of ACS and CON, and AMI versus UA were accomplished with satisfying AUC values of 0.890, and 0.928 for validation sets, respectively. Similarly, for precise AMI subtype stratification, the corresponding AUC values of validation sets demonstrated excellent prediction capability that the AUC value for NST versus ST is 0.985 with 100% sensitivity and 86.7% specificity. Moreover, the AUC value for NST and UA is 1 with 100% sensitivity and 100% specificity. Additionally, referred to the HMDB, the key features are assigned to the metabolites which were employed as the biomarker for the diagnosis of types of ACS. Finally, the potential biomarkers anticipant the significant metabolic pathways, and the metabolic disorders in the TCA cycle are regarded as the main cause of ACS. This work reveals the great promising advanced nanostructure‐based metabolic profiling analysis in disease diagnosis and stratification and demonstrates the great significance of metabolic analysis in the molecular subtyping of diseases.In conclusion, this work developed a UiO‐66@HCOF‐assisted LDI‐MS metabolic analysis system for precise diagnosis and risk stratification of ACS. The UiO‐66@HCOF combined the advantages of UiO‐66‐(OH)The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Haptoglobin is the body’s first line of defence against the toxicity of extracellular haemoglobin released following a subarachnoid haemorrhage (SAH). We investigated the haptoglobin response after SAH in cerebrospinal fluid (CSF) and serum. Paired CSF and serum samples from 19 controls and 92 SAH patients were assayed as follows: ultra-performance liquid chromatography for CSF haemoglobin and haptoglobin, immunoassay for serum haptoglobin and multiplexed CSF cytokines, and colorimetry for albumin. There was marked CSF haptoglobin deficiency: 99% of extracellular haemoglobin was unbound. The quotients for both CSF/serum albumin (qAlb) and haptoglobin (qHp) were used to compute the CSF haptoglobin index (qHp/qAlb). CSF from SAH patients had a significantly lower haptoglobin index compared to controls, especially in Haptoglobin-1 allele carriers. Serum haptoglobin levels increased after SAH and were correlated with CSF cytokine levels. Haptoglobin variables were not associated with long-term clinical outcomes post-SAH. We conclude that: (1) intrathecal haptoglobin consumption occurs after SAH, more so in haptoglobin-1 allele carriers; (2) serum haptoglobin is upregulated after SAH, in keeping with the liver acute phase response to central inflammation; (3) haptoglobin in the CSF is so low that any variation is too small for this to affect long-term outcomes, emphasising the potential for therapeutic haptoglobin supplementation. Central to the toxicity of extravascular blood is the presence of haemoglobin ,2. FolloHaptoglobin (Hp) is an abundant acute-phase glycoprotein—mainly produced by the liver—that binds to extracellular haemoglobin with high affinity . IntrathWhile Hp is an abundant protein in plasma, it is present at much lower levels in the CSF of both healthy individuals and SAH patients ,19. CSF HP alleles: HP1 and HP2  had a significantly higher level of CSF Hp compared to controls (p = 0.047). There was no significant difference in CSF Hp levels between SAH patients  and controls with the Hp 2-1 phenotype (p = 0.641).Among those with the Hp 1-1 phenotype, SAH patients  had a significantly lower level of CSF Hp compared to controls  or WFNS grade (p = 0.187). Considering just those patients with no intracerebral haemorrhage, the Hp index was still not significantly associated with blood volume (p = 0.982). This confirmed that the Hp index value on day 7 was not dependent on the initial blood volume entering the subarachnoid space.After SAH, several factors may affect CSF Hp concentration . Since tp = 0.016), and this was influenced by the Hp phenotype, such that the Hp index decreased in proportion to HP1 allele number (p = 0.025). SAH patients with the Hp 2-1 phenotype  also had a significantly lower Hp index compared to controls  (p = 0.012), but the decrease was not as marked as in Hp 1-1 patients. There was no significant difference in Hp index between SAH patients  and controls  with the Hp 2-2 phenotype (p = 0.760).The Hp index was significantly lower in SAH patients  compared to controls  (e number . SAH patp < 0.001).The median level of Hp in the serum of patients 7 days after SAH was 3.07 g/L (IQR: 2.41–3.60 g/L). The median level of Hp in the serum of 15 controls was 1.39 g/L (IQR: 1.14–1.94 g/L). This was significantly lower compared to SAH patients (p = 0.023) and patients with the Hp 1-1 phenotype  (p = 0.090) (p = 0.045).SAH patients with the Hp 2-2 phenotype had lower day 7 serum Hp level  compared to patients with Hp 2-1 phenotype  (= 0.090) . This sip = 0.098) and participants with Hp 1-1 phenotype  (p = 0.019). Serum Hp levels were significantly greater in SAH patients compared to controls for those with Hp 2-1 phenotype (p < 0.001) or Hp 2-2 phenotype (p = 0.006), but not in those with Hp 1-1 phenotype (p = 0.119).Controls with the Hp 2-2 phenotype had lower serum Hp levels  compared to participants with the Hp2-1 phenotype   when considering all Hp phenotypes. The association between CSF cytokines and serum Hp levels varied between Hp phenotypes  , Extendep = 0.872). Per patient, there was only sufficient Hp present in the CSF to bind a maximum of 1.08% (IQR: 0.38–3.82%) of the total haemoglobin in the CSF 7 days after SAH, and this did not differ by Hp phenotype (p = 0.325). This marked CSF Hp deficit may underlie the lack of association between Hp dynamics and clinical outcomes.Since Hp was not associated with outcome, we next wanted to determine whether Hp was present in sufficient amounts to bind CSF haemoglobin levels after SAH. The median level of day 7 CSF haemoglobin was 4.65 µM (IQR: 0.58–18.42 µM) and did not differ by Hp phenotype  following SAH. Moreover, there was a significant effect of Hp phenotype on CSF Hp turnover, such that the decrease in Hp index varied in proportion to the HP1 alleles. This finding is consistent with studies that have been conducted in a multitude of populations: European , East A, Africa, and Me) in the liver following the intracerebral injection of IL-1beta, and neutralisation of CINC-1 in the periphery reverses neutrophil mobilisation and recruitment to the brain . Given lp = 0.002, and serum Hp still higher, p < 0.001, after SAH compared to controls). We used a sensitive ultra-performance liquid chromatography (UPLC) technique to measure Hp and extracellular haemoglobin in the CSF. Missing data for some patients could potentially introduce bias. While CSF samples were collected via an external ventricular drain (EVD) or lumbar puncture (LP), and this may be seen to introduce heterogeneity due to the rostro-caudal gradient in CSF protein content [This study has several strengths and limitations. The sample size was substantial, with paired CSF and serum sampling on the same day after SAH. Day 7 is sufficiently distant from ictus to avoid any interference from the Hp entering the brain with the initial bleed—indeed the day 7 CSF Hp index was not associated with blood volume. On the other hand, limiting the analysis to one timepoint may not capture the dynamics of Hp. Roughly half the SAH patients on this study received SFX-01, which contains sulforaphane and has been shown in experimental studies to upregulate Hp transcription . However content ,63, this content .The samples and data used in this study originate from a prospective, multicentre, randomised, double-blind, placebo-controlled study designed to investigate the safety and efficacy of SFX-01 (a synthetic sulforaphane stabilized within an α-cyclodextrin complex) in patients with SAH (NCT02614742). The SFX-01 after SAH (SAS) trial  was apprOf the 92 SAH patients included for this study, 82 had CSF samples and 90 had serum samples taken. Of the 82 patients with CSF samples, 32 had CSF samples obtained at day 7 (±1 day) via an EVD sited for clinical hydrocephalus. The first 3 mL of CSF (representing dead space) was discarded to ensure fresh CSF was obtained. 50 patients without an EVD had an LP at day 7 (±1 day) and had CSF available for analysis. Day 7 follows the acute rise in Hp following SAH as a result of injection of blood into the subarachnoid space, after which it subsequently declines within the first few days to a relatively steady state by day 7 .Cytokines—interferon-gamma (IFN-γ), IL-1beta, IL-10, IL-12p70, IL-13, IL-2, IL-4, IL-6, IL-8, and tumour necrosis factor alpha (TNF-α)—were quantified using the Meso Scale Discovery (MSD) Multi-Spot Assay System  . All reaAlbumin in matched CSF and serum samples was measured on a Beckman Coulter  clinical chemistry analyser using a timed endpoint method assay which relies on the reaction of albumin with Bromocresol Purple to form a coloured complex; absorbance was measured at 600 nm. The CSF/serum albumin quotient (qAlb) was calculated as a proxy measure for BBB impairment ,68.Hp phenotype was determined by non-denaturing Western blot using 1:5000 polyclonal rabbit anti-Hp antibody . CSF Hp and haemoglobin were assayed using an in-house UPLC technique combining size exclusion chromatography with eluate absorbance measurement at 415 nm . CSF wasThe following baseline variables were prospectively collected and available for analysis for all SAH participants: age, sex, race, premorbid history of hypertension, location of aneurysm, method of securing aneurysm, initial blood volume on the computed tomography scan, location of blood, and WFNS grade.Whilst in hospital, patients were monitored clinically for delayed cerebral ischemia (DCI). Functional outcomes were assessed at days 90, and 180 with the CLCE-24 , GOSE 3, mRS 3232, and Sp < 0.05 was deemed to be statistically significant. All analyses were performed in STATA/IC 16.1.Descriptive statistics were used to summarise demographic and clinical characteristics and outcomes. Variables were tested for normality with the Shapiro–Wilks test. Means and confidence intervals are reported for parametric data. Medians and interquartile ranges (IQR) are reported for non-parametric data. The Kruskal–Wallis test and Dunn’s post hoc test were used to compare medians between independent groups. A comparison of proportions between groups was made with Fisher’s exact test. Linear regressions were used to determine the relationship between continuous variables. Principal component analysis was used to reduce the dimensionality of the CSF cytokines and examine the primary central inflammatory components. Moderation analysis was conducted to determine whether the relationship between serum Hp and cytokine levels varied as a function of Hp phenotype. Univariate logistic regression was used to examine the association between each outcome and Hp levels or type. mRS and SAHOT were analysed using ordinal logistic regression. In summary, this study of the Hp response after SAH has three important and novel findings. First, SAH patients have a significantly lower Hp index compared to controls, suggesting that intrathecal Hp consumption occurs following SAH. Second, we have provided evidence that the Hp phenotype significantly affects intrathecal Hp turnover. Third, the significant association between CSF cytokine levels and serum Hp upregulation following SAH provides human evidence of a systemic acute phase response to a central inflammatory stimulus. These findings have important implications for our understanding of the pathophysiology of SAH and may inform the development of novel therapeutic interventions to improve outcomes for SAH patients, including intrathecal haptoglobin administration and therapies targeting EVs.


BRAF and its downstream mediator MEK, and immune checkpoint inhibitors (ICI), have revolutionized the treatment of metastatic melanoma, improving patient outcomes. However, both TT and ICI have their limitations. Although TTs are associated with high initial response rates, these are typically short‐lived due to resistance. Conversely, although ICIs provide more durable responses, they have lower initial response rates. Due to these distinct yet complementary response profiles, it has been proposed that sequencing ICI with TT could lead to a high frequency of durable responses whilst circumventing the toxicity associated with combined ICI + TT treatment. However, several questions remain unanswered, including the mechanisms underpinning this synergy and the optimal sequencing strategy. The key to determining this is to uncover the biology of each phase of the therapeutic response.Cutaneous melanoma is a lethal form of skin cancer with morbidity and mortality rates highest amongst European, North American and Australasian populations. The developments of targeted therapies (TTs) directed at the oncogene early response, minimal residual disease (MRD) and disease progression. We explore the effects of ICI and TT on melanoma cells and the tumour immune microenvironment, with a particular focus on MRD which is predicted to underpin the development of acquired resistance in the third phase of response.In this review, we show that melanoma responds to TT and ICI in three phases: In doing so, we provide a new framework which may inform novel therapeutic approaches for melanoma, including optimal sequencing strategies and agents that target MRD, thereby ultimately improving clinical outcomes for patients. We demonstrate that melanoma responds to targeted therapies (TTs) and immune checkpoint inhibitors (ICIs) in three phases: early response, minimal residual disease (MRD) and disease progression.The minimal residual disease of both TT and ICI is a nidus for the development of therapeutic resistance and, thus, warrants additional characterization of both tumour cells and their surrounding tumour immune microenvironment.Emerging new technologies, such as spatial transcriptomic, clonal evolution and high‐throughput single‐cell profiling, can be utilized to uncover novel therapeutic opportunities to target the minimal residual disease. Notably, however, this improvement in efficacy comes at the price of higher toxicity rates (55%–58%) compared to TT (25%–33%) monotherapy.TTs include BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) which inhibit the commonly over‐activated mitogen‐activated protein kinase (MAPK) pathway, responsible for cell proliferation and survival in melanoma.2BRAF mutant melanoma. Genomic studies reveal that approximately 65%–90% of cutaneous melanoma harbour genetic events resulting in overactivation of the MAPK pathway.BRAF somatic missense mutation where a valine amino acid is substituted for glutamic acid at exon 6 (V600E) is identified to occur in 50% of melanoma cases.BRAF, the second most common mutation in MAPK pathway is the small GTPase NRAS (25%), and the third is the tumour suppressor and the negative regulator of RAS, neurofibromin 1 (NF1) (14%); both these mutations lead to the activation of the MAPK pathway and increased ERK signalling.BRAF mutant melanoma, the combination of BRAFi and MEKi has shown improvements in median OS from 6–9 to 22–33 months.TTs have revolutionized the treatment landscape of advanced first phase is characterized by early response whereby ∼80% of patients undergo an initial phase of tumour regression. Conversely, ∼20% of patients fail to respond to TT and ultimately undergo tumour progression.second phase is characterized by minimal residual disease (MRD) whereby a portion of cancer cells undergo genetic and non‐genetic changes allowing them to survive therapeutic pressure in a state of tolerance/persistence.third phase is characterized by drug resistance whereby MRD persister cells regain proliferative capacity and expand, ultimately driving disease progression. These changes are accompanied by a transformation in the TIME.second phase is the nidus for the development of acquired resistance in the third phase. This highlights the importance of characterizing the genetic, non‐genetic and TIME changes during MRD to facilitate the creation of therapeutic strategies that circumvent the development of resistance.BRAF mutant melanoma is known to respond to TT in three phases Figure . The fir2.1The disease of responders is highly dependent on the MAPK pathway for its growth and survival. As such, through inhibition of BRAF and its downstream modulator MEK, TTs mediate tumour regression in responders through MAPK blockade.Cyclin D1 overexpression, especially when coupled with CDK4 overexpression/activating mutations, has been found to confer intrinsic resistance to BRAFi.RAC1, which mediates cell proliferation through MAPKConversely, non‐responders harbour loss of tumour suppressor genes or activation of oncogenes which enables cell cycle entry through the upregulation of alternate signalling pathways such as phosphoinositide 3‐kinase (PI3K) or overactivation of MAPK pathways through alternate means.2.2The drug‐tolerant MRD phase is enabled by non‐genetic and genetic changes which confer a survival advantage.MITF but higher expression levels of AXL, nerve growth factor receptor (NGFR) and SRY‐Box Transcription Factor 10 (SOX10). Both SOX10 and NGFR are key markers of neural crest lineage stem cells essential for melanocyte development.MITF and SOX10, though AXL levels remain high.Non‐genetic changes involve remodelling of tumour cells to adapt to stressors in their environment such as drug exposure. These changes result in the establishment of distinct melanoma phenotypes that have the capacity for adaptive switching between phenotypes Figure .16 SinOther persister cells within the MRD harbour genetic mutations resulting in the establishment of molecularly distinctive subclones.2.3BRAF mutant melanoma demonstrated that NCSC depletion from MRD prevented the development of non‐genetic mechanisms of acquired resistance, leaving persistent cells dependent on de novo genetic mechanisms.The changes that occur during MRD ultimately enable the acquisition of resistance to TT and melanoma relapse. In particular, the NCSC and invasive phenotypes that emerge during MRD due to non‐genetic changes expand and subsequently drive disease progression in the third phase.BRAF, MEK1, MEK2 and NRAS) or downregulation of negative regulators such as NF1,AKT1 has also been found in approximately 20% of patients on progression.Significant intra‐ and inter‐tumour heterogeneities have been found in the mechanisms of acquired genetic resistance.Hence, this heterogeneity highlights the challenge in developing a uniform therapeutic strategy for patients upon progression, thus emphasizing the importance of treatment before the acquisition of resistance.2.4In addition to the effects of TTs on the intrinsic biology of melanoma cells, preclinical and clinical studies also indicate that TTs influence the composition of the TIME at different phases of treatment response Figure .2.4.1The early phase of TT response is characterized by an immune‐stimulatory TIME. TTs increase MHC class I expression and tumour antigen presentation2.4.2V600EBRAF, −/−Pten and −/−Cdkn2a  1.1‐OVA‐Low (YOVAL1.1)2.4.3Pre‐clinical and clinical studies indicate that the resistance phase is defined by an immunosuppressive TIME characterized by increased infiltrates of immunosuppressive T‐reg cellsOverall, these studies demonstrate that although initial treatment with TT induces an immunostimulatory TIME, the resistance phase is characterized by an immunosuppressive TIME. This highlights the importance of additional studies that characterize the TIME of MRD given the important role this phase may play in dictating the subsequent immunosuppressive TIME. Immunotherapeutic agents may also be used to increase the durability of TT by enhancing the initial immunostimulatory TIME and/or circumventing the immunosuppressive TIME.3BRAF‐mutant melanoma; however, treatment was accompanied by high rates of toxicities, whereby grade 3 or 4 toxicities were observed in 53% of patients.Cutaneous melanoma is regarded as an immunogenic cancer typically with a high neoantigen burden. However, immunosuppressive mechanisms, including T‐reg cell infiltrates and inhibitory immune checkpoint molecules, limit the anti‐cancer immune response. Hence, targeting checkpoints through ICI, including α‐CTLA4 and α‐PD1, increases immune‐mediated clearance of melanoma cells.first phase is characterized by early drug response whereby tumours with primary sensitivity to ICI show early disease control. Conversely, some tumours fail to respond to ICI due to primary resistance. It can be postulated that the second phase is defined by therapeutic tolerance, whereby residual tumour cells persist whilst under therapeutic pressure, establishing MRD. This is akin to the ‘equilibrium’ phase proposed by Schreiber, a latent period in which a balance is struck between tumour clearance and tumour growth.third phase is characterized by disease progression secondary to the development of acquired resistance to ICI.Analogous to TT, the response of melanoma to ICI can also be conceptualized in three distinct phases Figure . The fir3.1During the first phase, patients can be divided into ‘primary responders’ or ‘primary non‐responders’ depending on their sensitivity to ICI Figure . This isBRCA2 that further increased the neoantigen load.MHC I/II59,60 and IFN‐γ/α, resulting in increased cytotoxic activity.Multiple studies have demonstrated a positive correlation between ICI responsiveness and OS with tumour mutational burden (TMB) and neoantigen load.Furthermore, differences in the composition of TIME have been identified in primary responders compared to non‐responders. Responsive tumours have an immunostimulatory TIME characterized by high levels of monocytesThis suggests that certain biomarkers, including a high TMB, an immunostimulatory TIME and an activation of immunostimulatory signalling pathways, predict primary sensitivity to ICI. Conversely, patients with primary resistance to ICI have an immunosuppressive TIME and upregulate immunosuppressive signalling pathways.3.2Based on the frequency of patients who progress after partial or complete response to ICI, we propose that a portion of patients who initially respond to ICI go on to establish MRD.hiNGFR clone characterized by 15q deletion (including beta 2 macroglobulin [B2M]) acquired additional genetic alternations, including the deletion of CDKN2A and phosphatase and tensin homolog (PTEN) genes.hiNGFR clone was found to dominate tumour biopsies during the third progression phase. The authors proposed that the immune system may have played a role in pruning susceptible clones given that cells of the dominant lineage acquired immune‐evasive adaptations.One study that conducted serial biopsies of a patient during ICI demonstrated, through whole exome sequencing and RNA sequencing, the presence of seven dominant tumour clones on initiation of treatment. However, during the MRD phase, an Interestingly, this study, similar to studies with TT, suggested that during treatment with ICI, a genetic clone is selected, which undergoes additional transcriptional changes driving therapeutic resistance and disease progression. Additional studies, however, are required to characterize the mechanisms at play during MRD after treatment with ICI to better understand how this phase may be targeted therapeutically.3.3B2M, a key component of MHC Class I, which results in loss of antigen presentation; LOH of JAK1 and JAK2 proteins resulting in loss of sensitivity to IFN‐mediated growth inhibition and immune‐mediated deathPTEN associated with decreased T‐cell infiltration and increased expression of immunosuppressive cytokines.hiPDL1hi signature is responsible for driving therapeutic resistance and disease progression. During phase 3, an immunosuppressive TIME has been found to surround these cells characterized by a high influx of T‐reg cells.Following MRD or partial response, some patients acquire resistance to ICI, with 12%–38% of those who initially respond eventually progressing on therapy.Given these diverse mechanisms of acquired resistance, no uniform biomarkers predictive of therapeutic sensitivities have been identified to date. To achieve this, longitudinal studies characterizing MRD and acquired resistance in patient cohorts are needed. This requires the multicentre coordination of recruitment and methodologies to ensure adequate patient numbers.4Both TT and ICI have their own therapeutic advantages and disadvantages. Although TTs generate outstanding initial ORR, their effects are usually relatively short‐lived due to acquired resistance. Conversely, although ICIs have lower initial ORR, they induce more durable responses. Given these complementary effects, sequential treatment has been proposed to maximize clinical outcomes whilst circumventing potential toxicities associated with combinatorial treatment. The question remains, however, of which therapy to initiate first. As such, prospective clinical trials have sought to compare therapeutic responses of ICI and TT as first‐ or second‐line treatments in BRAF‐mutant melanoma.4.1p = .054), with a 20% improvement in 2‐year OS (p = .0095)Several prospective clinical trials are investigating various treatment sequences with the view to determining optimal front‐line treatment. The DREAMseq clinical trial NCT02224781) has randomly assigned patients to two treatment sequences: Sequence 1 comprises patients commenced on ICI (Arm A) followed by a switch to TT (Arm C) on progression; sequence 2 comprises patients commenced on TT (Arm B) followed by a switch to ICI (Arm D) on progression Figure . Althoug224781 haIn another clinical trial (SECOMBIT: NCT02631447), patients will be randomized to one of three treatment arms: Arm A (TT first line followed by ICI upon progression); Arm B (ICI first line followed by TT upon progression); Arm C . Preliminary results from SECOMBIT show an ORR for first‐line treatment of 87% and 47% in Arms A and B, respectively. Upon progression, the ORR for second‐line treatment was 25% and 61%, respectively. This suggests an inferior ORR for ICI when given after TT compared to ICI in therapy naïve patients. The 3‐year ‘total’ PFS, or time to second progression, was 41% and 53% in Arms A and B, respectively, and the OS at 3 years was 54% and 62% in Arms A and B, respectively. Arm C, an approach that reflects switching therapy with MRD gave a 3‐year PFS and OS of 54% and 60%, respectivelyHence, akin to DREAMseq, data from SECOMBIT suggested improved clinical outcomes with first‐line ICI. These clinical trials also suggest that melanoma progression on first‐line TT may promote relative resistance to second‐line ICI, referred to as ‘cross‐resistance’. However, the biology of cross‐resistance remains to be adequately characterized.4.2A recent study utilizing a mouse model showed that tumours with acquired resistance to TT are also subsequently resistant to ICI due to an overall immunosuppressive TIME Figure . These chi phenotype predicts resistance to TT and ICI, highlighting yet another mechanism of cross‐resistance Although not investigated experimentally, multiple gene expression signatures are shared between melanomas resistant to TT with those resistant to ICI, such as IPRES.Therefore, although sequencing may assist in maximizing the therapeutic potential of TT and ICI, the emergence of cross‐resistance remains an issue. Additional biological studies are, thus, needed to investigate the mechanisms of cross‐resistance and how they may be overcome to re‐sensitize patients to sequential therapy.5As discussed in this paper, we propose that the MRD phase plays a critical role in determining the ultimate therapeutic response to TT and ICI, by providing a nidus for the development of acquired resistance. Additionally, melanoma may be at its most vulnerable during MRD due to a low tumour load. Therefore, additional therapeutic interventions during MRD may enable long‐term disease control, thereby ultimately achieving a progression‐free state or clinical cure. In doing so, these therapeutics would help reduce the risk of disease reoccurrence under TT and ICI pressure, which differs markedly from current adjuvant therapies, which seek to combat reoccurrence secondary to surgical resection under immune‐system pressure.NCSC phenotype, which, as outlined, is responsible for non‐genetic mechanisms of acquired resistance and immune evasion.Data from Arm C of the SECOMBIT trial, in which patients were switched to ICI during the MRD phase of TT, showed similar OS and total PFS to Arm B (ICI until progression followed by TT), which was superior to Arm A (TT until progression followed by ICI). This highlights that both switching during the MRD phase or ICI followed by TT may be effective therapeutic approaches for melanoma. Results from SECOMBIT's ongoing biomarker analysis will help provide an understanding of the biological mechanisms at play during the response, MRD and resistance phases, by defining genetic, non‐genetic and changes in the TIME. Particular attention should be directed towards the emergence of the 6Over the last decade, the introduction of BRAF/MEK‐TT and ICI has revolutionized the treatment landscape of cutaneous melanoma, dramatically improving patient outcomes. Although TTs have an outstanding ORR, ICIs have durable PFS owing to a robust clinical response. Unfortunately, however, both intrinsic and acquired resistances remain a significant concern for ICI. Both upfront combinatorial treatment and sequencing upon progression have been explored as possible treatment approaches for maximizing the therapeutic impact of ICI and TT. Although retrospective studies and clinical trials show the benefit of triplet therapy (BRAFi + MEKi + ICI) over single‐modality therapies, and upfront ICI followed by TT upon progression, additional mechanistic studies are required to study the biology underpinning these approaches to maximize the benefit.Both TT and ICI have an MRD phase from which disease relapse emerges through a host of genetic and non‐genetic alterations. We suggest that further studies are needed to adequately characterize the origin and evolution of resistance during the MRD phase, for example the emergence of NCSCs, with the view to developing potentially curative treatment options. The clonal evolution of melanoma following therapeutic intervention could be assessed through single cell‐RNA sequencing or clonal mapping of serial tumour biopsies. Importantly, given that the emergence and expansion of NCSCs have been shown to be independent of a tumour's initial driver mutations, these studies may also have implications for BRAF‐non‐mutant melanoma.There are no conflicts of interest to disclose.This article does not contain any studies with human or animal subjects performed by any of the authors.


WWTR1 and CAMTA1, are broken and fused together in 90% of cases. Alternatively, in approximately 10% of cases, the genes that are broken and fused together are YAP1 and TFE3. We analysed an Australian cohort of EHE patients to look for associations between genetic changes and clinical characteristics. EHE cases are typically refractory to therapies, and no anticancer agents are reimbursed for EHE in Australia; therefore, this detailed study of EHE adds important information to advance our understanding and help aid the design of treatment regimens in the future.Epithelioid haemangioendothelioma (EHE) is an ultra-rare malignant vascular tumour with a prevalence of 1 per 1,000,000. It develops from endothelial cells, which are the cells that line all blood vessels in the body. Therefore, it typically expresses endothelial cell markers. It can also be identified through analysis of the genes. Two genes, WWTR1::CAMTA1 gene fusion in approximately 90% of cases, or a YAP1::TFE3 gene fusion in approximately 10% of cases. EHE cases are typically refractory to therapies, and no anticancer agents are reimbursed for EHE in Australia. Methods: We report a cohort of nine EHE cases with comprehensive histologic and molecular profiling from the Walter and Eliza Hall Institute of Medical Research Stafford Fox Rare Cancer Program (WEHI-SFRCP) collated via nation-wide referral to the Australian Rare Cancer (ARC) Portal. The diagnoses of EHE were confirmed by histopathological and immunohistochemical (IHC) examination. Molecular profiling was performed using the TruSight Oncology 500 assay, the TruSight RNA fusion panel, whole genome sequencing (WGS), or whole exome sequencing (WES). Results: Molecular analysis of RNA, DNA or both was possible in seven of nine cases. The WWTR1::CAMTA1 fusion was identified in five cases. The YAP1::TFE3 fusion was identified in one case, demonstrating unique morphology compared to cases with the more common WWTR1::CAMTA1 fusion. All tumours expressed typical endothelial markers CD31, ERG, and CD34 and were negative for pan-cytokeratin. Cases with a WWTR1::CAMTA1 fusion displayed high expression of CAMTA1 and the single case with a YAP1::TFE3 fusion displayed high expression of TFE3. Survival was highly variable and unrelated to molecular profile. Conclusions: This cohort of EHE cases provides molecular and histopathological characterisation and matching clinical information that emphasises the molecular patterns and variable clinical outcomes and adds to our knowledge of this ultra-rare cancer. Such information from multiple studies will advance our understanding, potentially improving treatment options.Background: Epithelioid haemangioendothelioma (EHE) is an ultra-rare malignant vascular tumour with a prevalence of 1 per 1,000,000. It is typically molecularly characterised by a  Epithelioid haemangioendothelioma (EHE) is a rare, low-to-intermediate grade malignant tumour of vascular origin that may develop in the extremities, soft tissue, lung, bone, and liver. The name was first coined in 1982 by Weiss and Enzinger due to its overlapping features between a haemangioma and angiosarcoma. It is a very rare tumour with a prevalence of 1 per 1,000,000 in the general population [The World Health Organisation (WHO) has recommended that EHE be grouped with malignant tumours . It has EHE has no standard treatment regimen, and very few therapeutic options are available. Historically, liver resection and transplantation has been the only curative option for patients with hepatic EHE because of tumour multifocality, but is associated with variable outcomes . HoweverRadiologic findings are often nonspecific and vary according to the site. Two characteristic computed tomography (CT) and magnetic resonance imaging (MRI) findings in hepatic EHE include the “lollipop sign”, in larger lesions (>5 cm) due to bridging vein thrombosis with a rounded “head” and a tapering “tail” , and theYAP1::TFE3 fusion has several distinct morphologic features, including voluminous cytoplasm, well-formed vascular lumens, solid growth, and minimal or no stroma [Morphologically, EHE is most often composed of epithelioid cells, organised in nests or cords, with eosinophilic to vacuolated cytoplasm, set in a distinctive myxohyaline stroma, and occasionally associated with haemorrhagic foci . Blistero stroma . A minoro stroma . WWTR1::CAMTA1 fusion, which results from a t translocation, and the less common YAP1::TFE3 fusion are considered diagnostic fusion genes [WWTR1::CAMTA1 (90%) or YAP1::TFE3 (10%) gene fusions [The WHO classification of sarcomas and the European Society of Medical Oncology (ESMO) consensus have set two gene translocations, both involving the Hippo pathway, as disease-defining fusion genes for EHE ,14. The on genes ,14,17. MWWTR1 gene, regulates the activity of various transcription factors, including the Transcriptional enhancer associated domain (TEAD) transcription factors, which play roles in cell proliferation and apoptosis [Transcriptional co-activator with PDZ-binding motif (TAZ), encoded by the poptosis . Nuclearpoptosis . The fuspoptosis ,20 whichpoptosis .YAP1 is a transcriptional coactivator, also controlled by the Hippo signalling pathway, which also interacts with transcription factors, such as TEADs, to promote growth and inhibit apoptosis . TFE3 beThese different fusion genes give rise to diverse biologic behaviour. Some propose that YAP1-TFE3 fusion tumours should be classified as a distinct entity given their unique clinical and histopathologic characteristics in comparison to conventional EHE ,23. In aXRCC1/2, ERCC1, RB1, APC, FANCA, CDKN2A and CKDN2B and ATRX loss [Next-generation DNA sequencing (IMPACT) and targeted RNA sequencing (Archer FusionPlex Custom Solid Panel) on cohorts of 18 and 49 patients have uncovered additional drivers; at least 22% of cases had alterations in cell cycle and epigenetic pathways, and/or loss-of-function alterations in the DNA damage response pathways. These included pathogenic variants in TRX loss . More thTRX loss . Notably, EHE has a variable clinical behaviour: patients with either solitary soft tissue or multifocal lung/liver disease may follow a relatively indolent course, whereas those with either pleural or lymph node involvement, regardless of their primary site or pathologic grade, follow a highly aggressive clinical course similar to a high-grade sarcoma . In geneSeveral sets of predictors of clinical outcomes have been proposed. Clinical factors associated with shorter survival include: clinical baseline tumour-related pain (TRP), development of TRP during follow-up, baseline temperature, and development of fatigue during follow-up ; multifoThere is no universally agreed treatment strategy for EHE due to its rarity and lack of comprehensive molecular characterisation. However, a recent consensus strategy by experts and consumers has been published. Treatment strategies include liver transplantation (44.8%), surgical resection for isolated tumours (9.4%) ,30,31,32Based on analysis of the World Sarcoma Network database, all systemic treatments for sarcoma have limited activity in EHE. Seventy-three patients  were included, and none showed meaningful activity . SystemiThere are currently no reported cases of delivering genomically guided therapies against any of the identified molecular targets. However, novel small molecules targeting the Hippo pathway are in early-phase trials (NCT04665206) . The fusInformed consent was obtained from all patients in accordance with the National Statement of Ethical Conduct in Human Research 2007. Subjects included in this analysis provided consent directly or were consented to the WEHI Stafford Fox Rare Cancer Program . Clinical follow-up of patient outcome was obtained from the medical record. Additional human ethics approval was obtained from the Walter and Eliza Hall Institute of Medical Research (HREC #10/05 and #G16/02).FFPE tumour samples were sectioned and stained with haematoxylin and eosin (H&E), or the following antibodies: anti-pan-cytokeratin , anti-CD31 , anti-CD34 , anti-ERG , anti-CAMTA1 , and anti-TFE3 . H&E and IHC slides were scanned digitally at 20× magnification using the Pannoramic 1000 scanner . High-definition images were uploaded into CaseCenter (3DHISTECH Ltd.), and images were processed using FIJI image analysis software 2.14.0 .DNA extracted from the FFPE tumour samples of two patients (#162 and #521) was sequenced using the TruSight Oncology 500 Panel. RNA extracted from FFPE tumour samples of five patients  was sequenced using the TruSight RNA Fusion Panel. Whole exome sequencing (WES) was carried out on DNA extracted from the FFPE tumour sample of one patient (#368), and whole genome sequencing (WGS) was carried out on DNA extracted from one snap-frozen tumour sample (patient #130). These tumour samples were matched with patient whole blood. TruSight RNA Fusion Panel data were analysed by aligning the FASTQ files to the human reference assembly (GRCh37) using STAR aligner13 version 2.7.2b. Arriba . Sample-WES data were analysed using a BioNix pipeline  which alWGS data were analysed by aligning to GRCh38 using BWA mem. Small, copy number and structural variants were called using a consensus of at least two of Mutect2 , StrelkaSee Through the WEHI-SFRCP, we report a cohort of nine EHE cases with comprehensive histologic and molecular profiling. The diagnoses of EHE were made based on the histopathological examination and confirmed by IHC and, whenever possible, molecular profiling. See In October 2003, case #368, a man in his forties, presented with abdominal pain and weight loss (BMI 18) and was found to have deranged liver function tests and multi-focal, hypodense, ill-defined nodules in the right lobe of the liver as well as small bilateral pulmonary metastases on CTCAP. Liver biopsy demonstrated an infiltrating tumour, composed of strands of polygonal epithelioid and spindle cells, set within a fibrous stroma. The tumour cells showed moderate nuclear pleomorphism, hyperchromasia and scanty cytoplasm. Occasional tumour cells showed intra-cytoplasmic lumina that contained red blood cells. IHC revealed strong and diffuse positivity for the endothelial cell markers CD34, CD31 and ERG, while it was negative for the epithelial marker pan-cytokeratin . A diagnWWTR1::CAMTA1 fusion identified using the TruSight RNA Fusion Panel. The breakpoints occurred in the third intron of WWTR1  and the ninth exon of CAMTA1  generating a fusion protein containing the WW domain of TAZ and the IPT/TIG domain and IQ calmodulin-binding motif of CAMTA1   .Due to extensive hepatic disease and extrahepatic involvement, neither radical resection nor liver transplantation was feasible. Review of the literature suggested his disease may follow an indolent course, thus he underwent close clinical surveillance without intervention. Two years later, in 2005, he developed progressive right hypochondrial pain with a confirmed radiologic progression. Chemotherapy (carboplatin and etoposide for six cycles) was started. Although the radiologic response was not impressive, he achieved durable pain relief. Subsequently, he developed secondary portal hypertension complicated by oesophageal varices which were effectively controlled by endoscopic banding. Radiological surveillance demonstrated ongoing stable disease c, and heCase #130 was diagnosed in 2009 with metastatic EHE. The right medial calf was identified as the primary tumour, with metastatic deposits in the spine, lungs, liver and right humerus. Pathological review indicated the tumour comprised large epithelioid eosinophilic cells, some of which had intracytoplasmic vacuoles. In some areas, the cells were clustered around blood vessels, and some intracytoplasmic red blood cells were noted a. AccompYAP1::TFE3 fusion was identified  and exon 3 of TFE3  so that a fusion protein containing half of the TEAD-interaction domain of YAP1 and the activation domain (AD), basic helix-loop-helix (bHLH) and leucine zipper (LZ) DNA-binding domains of TFE3 was generated (YAP1::TFE3 fusion. This fusion gene was likely to be a passenger. This novel gene fusion (CBX3::HECW1) would give rise to a fusion protein containing the Chromatin Organisation Modifier (Chromo) domain of CBX3 and the ubiquitin transferase (HECT) domain of HECW1 (Whole genome sequencing (WGS) was carried out on DNA extracted from the primary tumour, indicating a stable genome with very low TMB (0.12 mutations/Mb) with very few chromosomal rearrangements c. No patentified . The breenerated d. Intereof HECW1 .These two cases exemplify the potential for EHE to follow an indolent course, with both having been followed closely for over 10 years without the need for subsequent intervention. Six subsequent cases of EHE, #104, #154, #162, #455, #503 and #521, were pathologically assessed. A range of key pathologic features were observed, including plump epithelioid cells and/or spindle cells with enlarged irregular hyperchromatic nuclei, a dense fibrous stroma, and in some cases, apparent intravascular growth and associated mixed inflammatory infiltrate. The cellularity was higher at the periphery of the lesion, with a relatively pauci-cellular and hyalinised central zone. Scattered stromal calcifications were present a. The imWWTR1::CAMTA1 fusion was identified using RNA extracted from a pleural biopsy for #154, liver biopsy for #455 and liver lesions for #503 and #521 (The and #521 . These gand #521 . WWTR1::CAMTA1 fusion (FBN1::WWTR1) would give rise to a fusion protein containing the WW domain of TAZ only, and its function is unknown  Assay. Both cases had low TMBs, and no pathogenic variants were detected. A VUS in  in #521 . AnalysiWWTR1::CAMTA1 fusion, exhibited positive CAMTA1 staining. The only case harbouring a YAP1::TFE3 fusion (#130) exhibited positive TFE3 staining. Interestingly, case #154, which did not harbour a rearrangement of TFE3, also exhibited positive TFE3 staining  components of the SCF E3 ubiquitin ligase. FBXW7 is a critical tumour suppressor that is frequently mutated in human cancers [ADRBK1 gene, which encodes the G-protein-couple receptor kinase 2 (GRK2). Altered GRK2 expression has been observed in many human cancers and may play a role in angiogenesis [WWTR1::CAMTA1, these additional molecular features may indicate a more aggressive tumour phenotype. Indeed, the presence of secondary genomic variants has previously been proposed to indicate more aggressive EHE [The se #130; d, and th Network ,74. FBN1 cancers . Importa cancers . If thisogenesis . Of partogenesis . This vasive EHE .TFE3 rearrangement. This has been observed previously, indicating that TFE3 staining may occur irrespective of TFE3 rearrangement [YAP1::TFE3 fusion did appear to have relatively indolent disease compared to most of the cases with WWTR1::CAMTA1 fusions.The immunophenotype in all cases was consistent with the known character of this vascular tumour, displaying positivity for the typical endothelial cell markers CD31, CD34 and ERG. In addition, tumours displayed CAMTA1 positivity if they harboured a CAMTA1 fusion and TFE3 positivity in the one tumour harbouring a TFE3 fusion. The tumour for case #154 also displayed positive TFE3 staining despite lacking a angement ,79. In oIn our cohort, case #368 reported clinically significant symptomatic pain relief after combination carboplatin and etoposide chemotherapy. Pinet et al. reported a complete response to the same regimen in a patient with metastatic pleural EHE disease . By contWWTR1::CAMTA1 and YAP1::TFE3, supports previous reports that these abnormalities define different biologic entities. This group exemplifies the need to characterise the natural history of rare cancers, as well as identifying underlying genomic drivers. Advancing therapeutic progress for rare tumours requires a precision medicine approach using genomic sequencing and basket trial designs. In the case of EHE, a watch-and-wait approach should be considered, to spare patients with indolent disease from potentially unnecessary treatments. Further work is warranted to determine if the YAP1::TFE3 fusion predicts a less aggressive course than the WWTR1::CAMTA1 fusion. EHE is an ultra-rare, vascular tumour that often presents incidentally and has an unpredictable clinical course. Histologically, it can be mistaken for other benign and malignant vascular-epithelial tumours. However, it can be differentiated by IHC staining for CD31, CD34 and ERG, as well as by molecular testing. Our analysis of nine EHE cases harbouring the two gene-defining molecular abnormalities, Analysis of this cohort of EHE was achieved through the WEHI-SFRCP, facilitated through enrolment in the ARC Portal, providing matched clinical, molecular, and histopathological characterisation of rare cancers in the hope of improving treatment options. Cohort analyses such as this, achieved through national collaboration, are critical to advancing our knowledge of ultra-rare cancers. An obvious limitation of any study involving a rare cancer will always be cohort size. Considering the incidence of EHE is 1 per 1,000,000 population, our cohort of nine patients is quite impressive. While a small cohort size makes it difficult to draw significant associations, it is extremely important to make the results widely available so they can be added to the already presented data on this rare cancer. By combining the results of many small studies, we are able to advance our knowledge of this ultra-rare cancer.


Ser450 is a crucial residue for the interactions between BoGGT and donors. BoGGT forms more hydrogen bonds with L-glutamine than γ-GpNA, promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. The BoGGT intermediate forms more hydrogen bonds with Val-Gly than L-methionine and L-leucine, which can promote the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the critical residues responsible for the interactions of donors and acceptors with the BoGGT and provides a new understanding of the substrate selectivity and catalytic mechanism of GGT.Bovine milk γ-glutamyltransferase (BoGGT) can produce γ-glutamyl peptides using L-glutamine as a donor substrate, and the transpeptidase activity is highly dependent on both γ-glutamyl donors and acceptors. To explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, molecular docking and molecular dynamic simulations were performed with L-glutamine and L-γ-glutamyl- Because of its ability to catalyze the transpeptidase reaction to produce γ-glutamyl peptides, γ-glutamyl transferase from bovine milk (BoGGT) has recently received increasing attention ,2,3. γ-GEscherichia coli GGT has stereoselectivity for γ-glutamyl acceptors, suggesting that the transpeptidase activity of E. coli GGT depends on the acceptor substrate [p-nitroanilide (γ-GpNA) as a donor [pNA as the γ-glutamyl donor than that of a L-glutamine donor; further, the transpeptidase activity of BoGGT is highly dependent on the acceptor with both L-glutamine and γ-GpNA donors [The types of γ-glutamyl donors and acceptors affect the transpeptidase activity of GGT. A recent study documented that ubstrate . Hillmanubstrate  proposed a donor ,12,13,14However, no previous study has been conducted into the interaction mechanism between BoGGT and its γ-glutamyl donor, as well as the interaction between the BoGGT intermediate and γ-glutamyl acceptor. It is well-established that human γ-glutamyl transferase (GGT) works by sequential mechanisms to catalyze a bi-substrate reaction ,15. FirsTo explore the binding mechanism between BoGGT and substrates, molecular docking and molecular dynamics (MD) simulations have been utilized ,18. The Our previous study identified BoGGT as the bovine enzyme (G3N2D8) . In the pNA as a γ-glutamyl donor than the L-glutamine donor [The catalytic nucleophile of human GGT has already been reported to be Thr381 ,16; multne donor . HoweverpNA. These hydrogen bonds are responsible for glutamine binding at the active site. This might explain why BoGGT has a higher transferase activity with L-glutamine as a γ-glutamyl donor than γ-GpNA. Molecular docking results showed eight hydrogen bonds between BoGGT and L-glutamine, whereas there were four hydrogen bonds between BoGGT and γ-GpNA is presented in pNA. The hydrogen atom on Tyr402, Ser450, and Gly472 formed H-bonds with the oxygen atom on γ-GpNA. Based on the interactions between BoGGT and the γ-glutamyl donor, it could be concluded that Ser450 is a crucial residue for donor substrate recognition.An H-bond occurs between the oxygen atom on Thr398 and the amide hydrogen on L-glutamine. It is universally accepted that the formation of H-bonds between enzyme and substrates greatly affects the specificity of the enzyme and thus facilitates the enzymatic reaction. PyMol visualization of the interaction between BoGGT and γ-Gα of BoGGT and the donor substrate) was used to assess the system stability of BoGGT–donor complexes during an 80 ns simulation. As demonstrated in pNA donor were tightly bound to BoGGT. The RMSDs of the donor substrates and backbone of BoGGT were calculated to evaluate the stability of the donor substrate and backbone of BoGGT further (pNA) from complexes stabilized, indicating that the protein had equilibrated. Nevertheless, the RMSDs of the complexes were notably greater than that of the BoGGT backbone, which might be due to the opening and closing motion of a lid from BoGGT observed during the simulations. The above results indicate that the BoGGT–glutamine and BoGGT–GpNA complexes reached a steady state after 60 ns of simulations. For each BoGGT–donor substrate complex, the root-mean-square fluctuations (RMSFs) of its constituent amino acids were determined, as well. Amino acids 121–124 and 431–434 of the BoGGT–Glutamine complexes were found to have higher fluctuation values than those of BoGGT–GpNA, suggesting these residues might be involved in the conformational alteration of the BoGGT–Glutamine complexes  represents how structures change over time compared to the starting point. The RMSD of the complexes (C further B. After omplexes C.pNA during the 60–80 ns simulation (p < 0.05), which was consistent with the docking results. This suggests that BoGGT has a higher interaction with L-glutamine than γ-GpNA.H-bonds between the enzyme and substrate are widely considered to be the primary factor that facilitate the enzymatic reaction . Consequ1) on Thr380 and the amide carbon (C) on the γ-glutamyl donor was inspected to evaluate the possibility of the interaction between BoGGT and the donor. As displayed in 1 on Thr380 and the amide carbon on L-glutamine (0.514 ± 0.031 nm) was significantly (p < 0.05) lower than that for BoGGT and γ-GpNA (0.749 ± 0.042 nm) during the 60–80 ns simulations.GGT first forms γ-glutamyl–GGT intermediate during the transpeptidase reaction when the hydroxyl oxygen on the active site attacks the carbonyl carbon on the donor substrate . ConsequpNA is a bigger molecule than L-glutamine, allowing L-glutamine to access the active site easily. This could also explain why BoGGT has higher transpeptidase activity with L-glutamine as a donor than γ-GpNA. The H-bond numbers between L-glutamine and BoGGT were significantly (p < 0.05) greater than those of γ-GpNA, which can decrease the OG1-C distance and thus promote the binding affinity between BoGGT and L-glutamine.Previous research has reported that Pro427-Ser438 of human GGT form a lid-loop, which may affect the substrate binding in the active site ,25,26. SpNA donors [Our previous study revealed that the transpeptidase activity of BoGGT is highly dependent on the acceptor with both L-glutamine and γ-GA donors . In the Our previous study demonstrated that the transpeptidase activity of BoGGT with L-methionine as an acceptor substrate was significantly lower than that of Val-Gly, whereas it was significantly higher than that of L-leucine . HillmanAs shown in The hydrogen atom on Ile399 and Asn400 formed one H-bond with the same hydroxyl oxygen atom on L-methionine, respectively. The oxygen atom on Ser81 formed an H-bond with the amide hydrogen atom on L-methionine, and carboxyl oxygen on Gly379 formed an H-bond with the hydroxyl hydrogen atom on L-methionine. Regarding the interaction between the intermediate and L-leucine, the hydrogen atom on Ile399 and Asn400 formed one H-bond with the same hydroxyl oxygen atom on L-leucine, respectively. The carboxyl oxygen on Gly379 formed an H-bond with the amide hydrogen atom on L-leucine. It could be concluded that Gly379, Ile399, and Asn400 are key residues responsible for the interactions between the intermediate and acceptors, thereby regulating the transfer of the γ-glutamyl moiety from the γ-glutamyl–BoGGT intermediate to the acceptor.α of the BoGGT intermediate–acceptor) stabilized after 20 ns of simulations, indicating that the BoGGT intermediate and the acceptor reached a steady state. After the molecular docking, the BoGGT intermediate–acceptor complexes were used to perform 40 ns MD simulations to explore the interaction between the intermediate and acceptors. As shown in p < 0.05) lower than that of L-methionine (0.917 ± 0.039 nm) and L-leucine (3.244 ± 0.094 nm), indicating that the intermediate has a higher interaction possibility with Val-Gly. It is generally accepted that H-bonds between enzyme and substrate are the main driving force for the enzymatic reaction. Consequently, the number of H-bonds was also employed to evaluate the interactions between the intermediate and acceptors. As shown in p < 0.05), which could decrease the O-N distance and thus promote the transfer of the γ-glutamyl moiety from the BoGGT intermediate to Val-Gly. To further validate the binding affinity between the acceptor substrate and the BoGGT intermediate, MMPBSA analysis was employed to estimate the binding affinity between the complexes during MD simulations. As shown in In the second step, the nitrogen atom on the acceptor will attack the oxygen atom on GGT380 (γ-glutamyl-threonine) to produce γ-glutamyl peptides. Therefore, the distance between the oxygen atom on residue 380 and the nitrogen atom on the acceptor is very important to produce γ-glutamyl peptides. As presented in In the current study, the three-dimensional (3D) structure of bovine GGT (UniProt no. G3N2D8) was produced by AlphaFold (v2.0) on an in-house system equipped with an A40 GPU running Docker ,27. Multp-nitroanilide) were obtained from PubChem, and the 3D structures were converted using ChemSketch . This allowed us to generate models for donor substrate–enzyme complexes by molecular docking using Autodock Vina with default parameters [The 2D structures of the donor substrates . In the first stage, a steep descent approach of 50,000 steps was adopted to minimize energy and achieve a stable conformation. After minimizing the system’s energy, we performed an isochoric–isothermal equilibration (300 K) for 100 ps, followed by an isothermal–isobaric equilibration  for 100 ps. The complexes were subsequently subjected to 80 ns MD simulations, meaning that the simulations were run in blocks of 80 ns until RMSD simulations were stable within this time course.GROMACS (version 2022.2) was utilized to conduct MD simulations for BoGGT–Gln and BoGGT–Gus study . Force fpNA) were calculated after the MD simulations. To evaluate their interactions, the distance between the amide carbon (C) on the donor and hydroxyl oxygen (OG1) on the active site was also calculated.The RMSD, RMSF, and H-bond number between BoGGT and donors . In the first stage, a steep descent approach of 50,000 steps was adopted to minimize energy and achieve a stable conformation. After minimizing the system’s energy, we performed an isochoric–isothermal equilibration (300 K) for 100 ps, followed by an isothermal–isobaric equilibration  for 100 ps. The complexes were subsequently subjected to MD simulations that were run in 40 ns blocks until a time course of relatively stable RMSD was observed. GROMACS was utilized to conduct MD simulations for BoGGT intermediate–acceptor complexes, based on our previous study . Force fRMSDs and H-bond numbers between the intermediate and acceptors were calculated after the MD simulations. To evaluate their interactions, the distance between the amide nitrogen (N) on the acceptor and hydroxyl oxygen on the active site was also calculated. The molecular mechanics Poisson Boltzmann surface area (MMPBSA) was applied to determine the binding free energy using the g_mmpbsa package . Bindingt-test was used. A p-value less than 0.05 (p < 0.05) indicates that there are significant differences between the samples. The figures and ANOVAs were created using GraphPad Prism 9.3.1 and SAS 9.4, respectively.The MD simulations were performed in triplicate. The obtained data are presented as mean ± standard deviation. To test differences among means of multiple groups, one-way analysis of variance (ANOVAs) with Duncan’s analysis was utilized. To determine whether the difference in means between the two groups was statistically significant, a pNA–BoGGT interactions. H-bond numbers between L-glutamine and BoGGT were significantly higher than that of γ-GpNA, which can decrease the distance between the hydroxyl oxygen atom on the active site of BoGGT and the amide carbon on the donor substrate (OG1-C), thereby promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. H-bond numbers between the BoGGT intermediate and Val-Gly were significantly higher than that of L-methionine and L-leucine, which can decrease the O1-N distance, thereby promoting the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the interactions between BoGGT and the donor, as well as the interactions between the intermediate and the acceptor substrate, and provides a meaningful method for selecting donors and acceptors with higher affinity for GGT, which can extend the understanding of the catalytic mechanism and promote the yields of γ-glutamyl peptides.BoGGT is an enzyme that could produce γ-glutamyl peptides with L-glutamine as a donor substrate and has the potential as a catalyst to produce taste enhancers, whereas the transpeptidase activity is highly dependent on the γ-glutamyl donors and acceptors. In summary, to explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, structure prediction of BoGGT, molecular docking, and molecular dynamic simulations were performed. The light subunit of BoGGT  is responsible for forming the majority of the H-bonds with the L-glutamine donor. Ser450 is a crucial residue for both L-glutamine–BoGGT and G


Elateriospermum tapos (E. tapos) seed extract modulates body mass and ameliorates stress hormones in obese dams, while a probiotic bacterial strain can cross the placenta and boost a child’s memory. Thus, we speculate that probiotics are the best medium to integrate plant extract (E. tapos extract) to access the effect on the child’s cognition. Thus, this study aimed to investigate the early intervention of E. tapos yoghurt in obese dams in the cognition and anxiety levels of male offspring. In this study, 40 female rats were fed with a high-fat diet (HFD) to induce obesity before pregnancy, while another 8 rats were fed with standard rat pellets for 16 weeks. Upon successful copulation, treatment was initiated for the obese dams up to the postnatal day (PND) 21. The groups included normal chow and saline (NS), HFD and saline (HS), HFD and yoghurt (HY), HFD and 5 mg/kg E. tapos yoghurt (HYT5), HFD and 50 mg/kg E. tapos yoghurt (HYT50), and HFD and 500 mg/kg E. tapos yoghurt (HYT500). All rats were euthanised on PND 21, and the body mass index (BMI), Lee index, and waist circumference were measured for the male offspring. Hippocampal-dependent memory tests and open field tests were conducted to access for cognition and anxiety status. Fasting blood glucose (FBG), total fat (%), insulin, leptin, lipid profile, and antioxidant parameter on serum and hypothalamus (FRAP and GSH) were accessed on PND 21. The result shows male offspring of 50 mg/kg-supplemented obese dams have comparable total fat (%), lipid profile, insulin level, FBG level, plasma insulin level, recognition index, low anxiety level, and improved hypothalamic FRAP and GSH levels to the normal group. In conclusion, this study highlights that the effect of early intervention of our novel formulation of E. tapos yoghurt in obese dams alleviates cognitive deficits and anxiety in male offspring by modulating metabolic profiles at the dose of 50 mg/kg.Maternal obesity is an intergenerational vicious cycle and one of the primary causes of cognitive deficits and high anxiety levels in offspring, which often manifest independently of sex. It is proven that curbing the intergenerational inheritance of obesity through early intervention during the gestation period has a positive outcome on the body composition, cognitive function, and anxiety level of the offspring. A recent discovery shows that the consumption of  Excessive gestational weight gain is often linked to a neurodevelopmental delay in the offspring . MaternaAlong with this, the intake of an HFD induces oxidative stress in the hypothalamus. This is evident based on the drastic reduction of enzymatic antioxidant activity, such as glutathione (GSH)  and noneElateriospermum tapos (E. tapos) comprises a variety of polyphenol and bioactive molecules that can exhibit an antiobesity effect in obese dams by suppressing the activity of lipoprotein lipase [E. tapos extract can modulate stress hormones [E. tapos is a semideciduous plant that can be found in the deep forest of Southeast Asian countries, such as Borneo, Thailand, Peninsular Malaysia, and Indonesia. The seed of the E. tapos plant contains high levels of proteins, unsaturated fatty acids, white oleic acids, a-linolenic acid [E. tapos seed can cause dizziness [E. tapos seed shows that it is safe for consumption up to 2000 mg/kg [E. tapos yoghurt in the male offspring of obese dams focusing on intergenerational cognitive deficits and anxiety levels.Thusly, natural products are said to be the best and most effective option to curb obesity and its complications. In this context, our preliminary study shows that a local tropical plant known as n lipase . Studieshormones . E. taponic acid , omega-3nic acid . The seeizziness . On the izziness . This isizziness . The saf00 mg/kg . Thus, t00 mg/kg ,29,30. HE. tapos seed was obtained from the Forest Research Institute of Malaysia (FRIM) and was sent for identification and confirmation to Herbarium Biodiversity Unit, University Putra Malaysia (UPM) under the approval code of UPM SK 3154/17.The E. tapos seed was soaked in 2 litre of 95% ethanol for 7 days. On the 7th day, we collected the filtrate and evaporated it using a rotary evaporator, and maltodextrin powder was added to the filtrate at a ratio of 1:1 [E. tapos was collected and stored in the freezer until further usage.Upon confirmation, about 500 g of o of 1:1  and was o of 1:1 . The folStreptococcus thermophilus APC151 and Lactobacillus delbrueckii subsp. Bulgaricus ATCC 11842 was added. The mixture was then incubated in the yoghurt maker (Pensonic PYM-700) for a maximum of 8 h and refrigerated at 4 °C overnight. The following day, the preprepared E. tapos powder was added to the yoghurt at the ratio of 2 g per 100 mL yoghurt and stirred well [The yoghurt was prepared by boiling 100 mL of full cream milk (Dutch Lady Purefarm UHT) at 70 °C for 20–30 min. Then, the milk was allowed to cool down at room temperature, and a starter culture consisting of live bacterial culture of red well .The preparation of a high-fat diet (HFD) was adapted from elsewhere. A total of 68% standard chow pellet (Gold Coin Feedmills (M) Sdn Bhd, Selangor, Malaysia), 6% corn oil , 6% ghee  Sdn Bhd, Kuala Lumpur, Malaysia), and 20% milk powder  were mixed and baked at 100 °C for 1–2 h before being refrigerated overnight at 4 °C .All animal procedures were conducted based on the guideline provided by the Institutional Animal Care and Use Committee (IACUC), UPM under the approval code of UPM/IACUC/AUP-R025/2022. In this study, 48 female Sprague–Dawley (SD) rats weighing from 150 to 200 g were used. All rats were acclimatised for one week at 12/12 light/dark cycles in a temperature-controlled room (23–24 °C). During the acclimatisation period, all rats were supplemented with standard chow pellet (Gold Coin Feedmills (M) Sdn Bhd, Selangor, Malaysia) containing 23.4% protein, 4.5% fat, and 72.1% carbohydrates with free access to water (bottle feeding) .n = 40) were supplemented with HFD pellets for 16 weeks, while the control group (n = 8) received standard chow pellets. Obesity was confirmed in HFD-supplemented group upon confirming a 13% mean body weight increase compared to control groups [Female SD rats (l groups .n = 8) and obese rats (n = 40) proceeded with mating by placing one male rat per 2 female rats in a cage. The next morning, all female rats were subjected to manual palpation, and vaginal smears were collected and observed under a microscope for the presence of sperm. The first day for detection of sperm was recorded as postcoital day 0 [E. tapos yoghurt was initiated in the obese dams. The treatment groups were as follows: normal chow and saline (NS), HFD and saline (HS), HFD and yoghurt (HY), HFD and 5 mg/kg E. tapos yoghurt (HYT5), HFD and 50 mg/kg E. tapos yoghurt (HYT50), and HFD and 500 mg/kg E. tapos yoghurt (HYT500). The treatment was given through oral gavage until postnatal (PND) 21 for the obese dams. No direct treatments were administered to the offspring.Upon successful obesity induction, both the control group 2;BMI = weight (g)/(length (cm))2 [Obesity threshold: BMI > 0.687 g/cm2 .(b)Lee index = (weight (g)/length (cm))^(1/3);Obesity threshold: Lee index > 310 g .On PND 21, all male offspring were sacrificed using carbon dioxide overdose. Body mass index (BMI), Lee index, and waist circumference were determined on the male offspring on PND 21. The waist circumference of the rats was measured in a ventral posture using a flexible tape at the greatest portion of their stomach . To measThe thigmotactic behaviour in male offspring was determined through an open-field test (OFT) on PND 21. In this test, a grey PVC box  measuring 80 cm in width, 80 cm in length, and 50 cm in height was used. The test was conducted during the light illumination cycle. During the test, the rats were placed in one corner of the box, and the time spent close to the wall (thigmotaxis), time spent at the central region, and total distance travelled were recorded using ANY-maze™ Video Tracking System  .The hippocampal-dependent memory tests known as NORT and PRT were performed on all male offspring on PND 21. For both these tests, all male offspring were allowed to acclimatise in a grey PVC box  measuring 80 cm in width, 80 cm in length, and 50 cm in height for 5 min on the first two days. On the 3rd day, for NORT, the rats were allowed to tour around an identical object (1.25-litre plastic bottle) for 5 min  followed by a 5 min retention phase. During the testing phase, a novel object was added (mug) to the PVC box, and the rats were placed into the box. The total time spent on the novel object was recorded using ANY-maze™ Video Tracking System . For PRT, both the objects used were identical during the trial phase. However, during the testing phase, one of the objects was transferred to a new location in the PVC box. Thus, the time the rats spent at the new location was recorded using ANY-maze™ Video Tracking System . All data obtained from NORT and PRT were expressed as recognition index (%) .All rats were fasted for 12 h overnight with free access to water on PND 21 before fasting blood glucose (FBG) analysis. The following morning, the tails of all male offspring were pricked, and blood was sucked using a glucose strip. The FBG levels were recorded using a glucometer  .Upon sacrificing the rats on PND 21, the body fat  was extracted from the rats, and their weights were measured. The weights were determined based on 100 g of body weight .Upon sacrificing the rats on PND 21, blood samples (4–5 mL) were collected using a heparin tube, while the hypothalamus was harvested and stored at −80 °C for further analysis. The blood samples were then subjected to centrifugation at 3500 rpm for 15 min to obtain the plasma. The plasma insulin level was then measured using commercial rat insulin ELISA kit  .The level of low-density lipoprotein (LDH), high-density lipoprotein (HDL), triglycerides, and total cholesterol in blood were measured using a diagnostic reagent test kit  using Hitachi Automatic Analyzer 902  .g for 20 min [The hypothalamus was defrosted at room temperature, minced into small pieces, and diluted with 1:15 w:v of phosphate buffer saline (PBS). The samples were then homogenised  together with protease inhibitor and butylated hydroxytoluene followed by sonication three times, each lasting about 20 s using ultrasonic cell disrupter . The final homogenates were then subjected to centrifugation at 5000× r 20 min . The supr 20 min . A similp-value was less than 0.05, a Bonferroni correction test was employed to adjust the significance level and account for the increased risk of false positives associated with multiple comparisons. Because there were six groups, the adjusted significance level was set to p < 0.0083 (0.05 divided by 6) to maintain an overall family-wise error rate of 0.05. This allowed for a more accurate determination of statistically significant differences among the groups. Different letters in the figures and tables indicate a significant difference.Data were analysed using SPSS version 27.0. Normality tests were performed on all obtained data to ensure normal distribution of the results. All data were expressed as mean ± standard error of the mean (SEM). To test for significant differences among the six groups, a one-way ANOVA was conducted. If the resulting Elateriospermum tapos yoghurt in obese dams to mitigate intergenerational cognitive deficits and thigmotactic behaviour in male offspring, focusing on metabolic parameters and antioxidant changes in the hypothalamus. The key findings from this study show that the male offspring born to the HFD-supplemented group without any treatment (HS group) have a high BMI, Lee index, and waist circumference. They do possess a low level of recognition index in NORT and PRT as well as an increased level of thigmotactic behaviour with a reduced antioxidant profile in the hypothalamus. Their fat percentage in BAT and RpWAT and visceral, and gonadal fat is significantly high with an altered metabolic profile on PND 21. This outcome in this study proves the successful establishment of the intergenerational obese model in male offspring with a poor neurodevelopment condition. This result is similar to the study conducted by O’Reilly et al., 2013 [Maternal obesity greatly affects the growth and behaviour of the child. Preliminary studies show that male offspring are more prone to glucose tolerance and increased levels of adiposity compared to female offspring due to overnutrition during the gestational period . Similarl., 2013 , who notl., 2013 .Lactobacillus strain is able to restore cognitive decline in the offspring of HFD-supplemented dams [E. tapos-integrated yoghurt  in this study. This is because medicinal plant-integrated yoghurt exhibits more beneficial effects similar to the NS in the study. The outcome in HY50 and HY500 is almost similar to the NS group; however, male offspring HY50 exhibit a similar mean value as NS in all of the parameters accessed in this study.On the contrary, the male offspring of the yoghurt-supplemented obese dams (HY) show a slight reduction in body composition and metabolic profile and a slight improvement in memory compared to the HS group. Our result is similar to the study performed by Wiciński et al., 2020, as the child’s obese mum supplemented with probiotics shows normal body mass, metabolic profile, and inflammatory markers just as the negative control group , and theted dams . This isted dams . Similarted dams . HoweverE. tapos extract was proven to inhibit the transgenerational inheritance of obesity in the female offspring [E. tapos extract contains numerous bioactive compounds that have a molecular weight of ≤600 daltons [E. tapos seed contains a high concentration of phenolic and flavonoids, which possess inhibitory activity on α-amylase, α-glucosidase, and pancreatic lipase [E. tapos prevents lipid absorption, thereby manifesting in a decreased level of fat content [Intriguingly, our data are similar to the previous study performed by Balan et al., 2021, who noticed that ffspring  and amelffspring . This is daltons  that couc lipase . The abi content . Meanwhi content . Resulta content . ProportE. tapos extract [E. tapos yoghurt-treated group, which proves that flavonoids could increase the level of antioxidants. Increased levels of antioxidants (FRAP and GSH) could neutralise free radicals and prevent inflammatory responses [E. tapos in obese dams prevents cognitive deficits and thigmotactic behaviour in male offspring via the modulation of the metabolic profile.In the bargain, the presence of flavonoids, such as kaempferol and amentoflavone in  extract , is one  extract ,69. Compesponses  that areesponses . This isesponses , while Fesponses . Hence, E. tapos yoghurt during the gestational period to the HFD-fed obese dams has proven to mitigate intergenerational cognitive deficits and thigmotactic behaviour in male offspring via the modulation of the metabolic profile at the dose of 50 mg/kg/day.An HFD intake during pregnancy is one of the factors for maternal obesity, while maternal obesity is positively correlated with metabolic disturbance, cognitive decline, and anxiety-like behaviour in the offspring. The supplementation of One of the limitations of this study could be that the outcomes from this study may not provide a complete understanding of how the intervention impacts female offspring. There could be significant differences in how the intervention affects the metabolic profiles and cognitive functions of male and female offspring. Thus, from the outcomes of this study, it may not be possible to generalise the findings to female offspring. This is because there may be gender-specific differences in response to the intervention or in the manifestation of cognitive deficits and thigmotactic behaviour. Additionally, the study does not investigate the long-term effects of the intervention on the health outcomes of the male offspring or subsequent generations. Therefore, further research should be conducted to confirm the results and to explore the effects of the intervention on other populations.