Heart failure with preserved ejection fraction (HFpEF) is thought to be highly prevalent yet remains underdiagnosed. Evidence-based treatments are available that increase quality of life and decrease hospitalization. We sought to develop a data-driven diagnostic model to predict from electronic health records (EHR) the likelihood of HFpEF among patients with unexplained dyspnea and preserved left ventricular EF.The derivation cohort comprised patients with dyspnea and echocardiography results. Structured and unstructured data were extracted using an automated informatics pipeline. Patients were retrospectively diagnosed as HFpEF (cases), non-HF (control cohort I), or HF with reduced EF . The ability of clinical parameters and investigations to discriminate cases from controls was evaluated by extreme gradient boosting. A likelihood scoring system was developed and validated in a separate test cohort. The derivation cohort included 1585 consecutive patients: 133 cases of HFpEF (9%), 194 non-HF cases (Control cohort I) and 1258 HFrEF cases (Control cohort II). Two HFpEF diagnostic signatures were derived, comprising symptoms, diagnoses and investigation results. A final prediction model was generated based on the averaged likelihood scores from these two models. In a validation cohort consisting of 269 consecutive patients [with 66 HFpEF cases (24.5%)], the diagnostic power of detecting HFpEF had an AUROC of 90% (P < 0.001) and average precision of 74%.This diagnostic signature enables discrimination of HFpEF from non-cardiac dyspnea or HFrEF from EHR and can assist in the diagnostic evaluation in patients with unexplained dyspnea. This approach will enable identification of HFpEF patients who may then benefit from new evidence-based therapies.The online version contains supplementary material available at 10.1186/s12872-022-03005-w. Heart Failure with preserved ejection fraction (HFpEF) is a highly prevalent yet under-diagnosed clinical syndrome , 2. The From a patient perspective, a diagnosis of HFpEF confers a high degree of morbidity as well as mortality rates equivalent to many forms of cancer . Rates oUntil recently, no effective therapies were available for HFpEF –11, howeIn previous epidemiological studies, identification and extraction of HFpEF cases from Electronic Health Records (EHR) has typically relied on diagnostic codes, additional medical record abstraction, and/or adjudication based on various expert criteria e.g. European Society of Cardiology criteria . The EHRThe aim of this study was to extract and analyze multi-modality data from the EHR using a machine learning approach to develop an automated prediction tool to identify patients likely to have HFpEF.We performed a retrospective study using de-identified data of patients attending King’s College Hospital NHS Foundation Trust (KCH) in London (UK) between 2000 and 2019. We focused on patients who had undergone echocardiography as part of their inpatient or outpatient evaluation. With this starting point, a number of different patient cohorts were derived based on the LVEF, confirmed or possible HF, symptoms of dyspnea, and NT-proBNP (or BNP) level  , 20. In BNP or NT-proBNP results were obtained from samples drawn at any time in the study period and the maximum value for each subject was used.All cases in the derivation dataset that were identified by the data pipeline as HFpEF were validated by manual review of the EHR by a cardiologist.A binary diagnostic outcome indicating the presence or absence of HFpEF was considered for modeling. Potential predictors to be included in a diagnostic signature included those used in previous HFpEF epidemiological studies , 22. In We made a second level predictor grouping according to whether the variables were initially recorded as (a) structured data: demographic and physiological parameters, and laboratory and echocardiography measurements; or (b) unstructured text in the EHR, extracted via the NLP platform. We adopted the bag-of-words  approachWe used the tree-based multivariable extreme gradient boosting  algorithSHAP  analysis2FPEF score [Model validation was undertaken in the test cohorts described earlier, using clinical assessment criteria from the HEF score  as a comData are presented as mean and standard deviation (SD) or median and interquartile range (IQR) as appropriate. Differences between cases and controls were evaluated by the Mann–Whitney U test or unpaired t test, as appropriate. The area under the receiver-operating characteristic curve (AUROC), F1-score (macro and weighted) and average precision (AP) were used as performance metrics. The F1 score measures the performance of a classifer as the harmonic mean of precision  and recall , placing equal importance on both. Average precision is the weighted mean of precision scores obtained as the classification threshold is adjusted , with the change in recall used as the weight.A stratified fivefold cross-validation scheme  was utilized for feature selection and derivation set validation. As such, the derivation data was divided into five subsets, four of which were used for training the model and the final one for validation/testing. The derivation and test subsets were shuffled until all five subsets were evaluated. The final performance was then reported as mean and standard deviation of all 5 tests see Fig. .Fig. 1FeThe AUROC and AP were used as performance metrics and the Kappa statistic was used to measure the inter-rater agreement of proposed models. All tests were 2-sided, with P < 0.05 considered significant.2FPEF scoring system using the Random Forest (predecessor to XGBoost). We used a score of 3 points indicating > 50% probability of HFpEF.To evaluate the generalizability of the model to a new sample, Harrell optimism was calculated with 1000 boot-strap replicates . To evalStatistical analyses were performed in Python 3 using SciPy and Scikit-learn packages (v0.22).1854 patients were included in the study of whom 1585 were in the derivation cohort  and 0.85 (95% CI  ± 0.001) in HFpEF vs non-HF and HFpEF versus HFrEF models, respectively and an enhanced aggregate performance of 0.90 (95% CI  ± 0.002) in our final aggregate model Fig. .Fig. 3Pe2FPEF model. The AUROC and average precision of both the aggregate model and the individual baseline models was higher than the H2FPEF model  did not feature in the selected predictors. Indeed, we note that several indices from a standard echocardiographic dataset that are typically used to identify HFpEF do not feature as predictors differentiating HFpEF from HFrEF. These include LV cavity dimensions; LV wall thickness and mass; and E/e’ as mentioned above. However, given the defining features of HFpEF versus HFrEF, it is perhaps not surprising that the top differentiating features are variations of quantifying LVEF.Analysis of the signatures that distinguished HFpEF from non-cardiac causes of dyspnea (non-HF) revealed anticipated predictors such as atrial fibrillation, hypertension, diabetes mellitus, kidney failure and obesity, in accordance with previous literature . In addi2FPEF. Our signature is ideally suited to rapidly identify a large number of possible HFpEF cases from EHR whereas H2FPEF is better suited for use by the clinician evaluating an individual patient who is suspected to have HFpEF.A major underlying problem in efforts to develop or test new treatments for HFpEF is the difficulty in consistently diagnosing the syndrome . Many diThis study is the first to use SHAP analysis for feature selection in this context. We comprehensively validated all variations of the derived models in multiple datasets with underlying variational distributions. We demonstrated a significant improvement in HFpEF diagnostic performance when discriminating the patients with HFpEF from those with HFrEF or no HF history. A key strength of our approach is that modeling numerical assessment data (structured results signature) and EHR concept references separately makes the models applicable in scenarios where one of these sources of data may be scarce. Moreover, the dual modeling of HFpEF separation from non-HF and HFrEF subjects increases the utility of the proposed pipeline in distinguishing among a wider group of clinical conditions.The UMLS clinical concept encoding that was used to extract unstructured observations does not support distinct encoding of different disease stages and could therefore cause some inaccuracy. In a more general aspect, the a priori assumptions that we made to identify definite HFpEF cases in the derivation dataset influenced the characterisation of the cohort. For example, we utilised ICD-10 diagnostic codes in the identification of patients with heart failure. Previous studies have demonstrated inaccuracy in identifying incident heart failure using ICD-10 coding as the sole source . It is p2FPEF score [2FPEF should be confirmed in a separate validation cohort.The inclusion of a raised BNP criterion restricts the cohort to a subgroup of HFpEF subjects , which was evident in test cohorts where many of the subjects did not have BNP measurements. This issue could be successfully handled through transfer learning techniques but would require some labelled data from a new domain to facilitate such a feedback training loop. The choice of data imputation technique could be another source of minor but systematic error. The discriminant power of the model to detect HFpEF is lower in test subsets where the missing data rate is higher and HFpEF cases are a small proportion of the overall number. Finally, the applicability of our model in patients with HFpEF who have never required hospital evaluation or admission is unknown. However, a strength of our approach is that a dedicated specialist assessment for HF is not required to assess the probability of HFpEF among patients undergoing general hospital evaluation , even in the absence of commonly used diagnostic data such as NTproBNP levels. The lack of independent validation is a limitation of this study. Evaluation of the derived model’s performance in independent datasets from other centres and in community-based datasets will be informative in future studies. Although we compared performance of the model with the HEF score , due to EF score . FurtherThe HFrEF group (Control Cohort II) comprised patients with a diagnosis of HF and reduced LVEF on echocardiogram using a cut-off value of < 50%. As such, this cohort combines HFrEF and HFmrEF cases as decribed in ESC guidelines . FinallyIn this study, we have developed a rapid and automated data-driven approach that is effective at identifying patients from EHR who are likely to have HFpEF. This algorithm affords significant potential to rapidly identify patients for more detailed analyses and access to evidence-based therapies that are known to improve quality of life and decrease rates of hospitalisation. The approach that we report could in principle be readily applied to other diseases and conditions that are similarly difficult to diagnose.Additional file 1. Supplementary Methods.


Virus-like nanoparticles (VLPs) are natural polymer-based nanomaterials that mimic viral structures through the hierarchical assembly of viral coat proteins, while lacking viral genomes. VLPs have received enormous attention in a wide range of nanotechnology-based medical diagnostics and therapies, including cancer therapy, imaging, and theranostics. VLPs are biocompatible and biodegradable and have a uniform structure and controllable assembly. They can encapsulate a wide range of therapeutic and diagnostic agents, and can be genetically or chemically modified. These properties have led to sophisticated multifunctional theranostic platforms. This article reviews the current progress in developing and applying engineered VLPs for molecular imaging, drug delivery, and multifunctional theranostics in cancer research. A wide range of liposome, synthetic and natural polymer, and inorganic nanoparticle (NP)-based carriers have been developed for tumor imaging and therapy . PropertConsidering that VLPs possess critical properties required for use in biomedical applications, such as water solubility, biocompatibility, and high cellular efficiency with minimal toxicity , they arVLPs are self-assembling protein-based capsular nanoparticles, 20–200 nm in size, composed of capsid proteins without genetic material. VLPs are non-infectious in nature, with the added benefit of being biocompatible and biodegradable. Furthermore, the size and morphology of VLPs can be controlled by manipulating the terminal amino acid of the capsid proteins and altering the pH of the buffer solution and the thermal conditions . TherefoVLPs are divided into enveloped VLPs (eVLPs) and non-enveloped VLPs (non-eVLPs) according to their structure . eVLPs, In contrast, non-eVLPs can be produced in both eukaryotic and prokaryotic expression systems owing to their relatively easy production and purification processes . TherefoDepending on the morphology of the viral capsid, eVLPs are classified as isometric or helical structures . Non-eVL2+-His tag interaction, SpyTag-SpyCatcher interaction, and streptavidin-biotin interaction , cisplatin, paclitaxel (PTX), and 5-fluorouracil (5-FU) to damage DNA, which leads to cell death . However50) than free DOX in cancer cells, but a higher IC50 than free DOX in normal cells.Hepatitis B core protein virus-like particles (HBc VLPs) have been used to deliver DOX . HBc VLP50 in HT29 and Caco-2 cells and an approximately 2-fold higher IC50 in CCD-112 cells than that of free DOX.HBc VLPs have also been used as a carrier for the delivery of 5-fluorouracil-1-acetic acid (5-FA), a 5-FU derivative . The 5-Fvia Zn2+  VLPs have been used as a carrier to deliver DOX . PhMV VLvia a thiol-maleimide reaction (Pt-PhMV). Cisplatin prodrugs are the most effective anti-cancer drugs for solid tumors, such as breast cancer. The pH-sensitive cisplatin might be reduced to a DNA-reactive Pt (II) complex in the acidic extracellular environment of a tumor. The outer surface of Pt-PhMVs was modified with polyethylene glycol (PEG) and cyanine (Cy) 5.5 to increase their biocompatibility and track them in vivo. Compared to free cisplatin and cisplatin-maleimide, these VLPs significantly prolonged the survival of cancer-affected mice by effectively inhibiting the growth of xenograft MDA-MB-231 breast tumors in vivo.PhMV VLPs have also been used to deliver a cisplatin prodrug containing platinum (Pt) IV  Figure . A maleiPicornaviridae family. The capsid consists of 60 copies of each of the four structural proteins (VP1 to VP4), which self-assemble into 30 nm icosahedral particles (Foot-and-mouth disease virus-like particles (FMDV VLPs) have been proposed as a carrier for DOX . Considearticles . DOX wasBromoviridae family of plant viruses and is composed of 180 capsid proteins. CCMV VLPs have a 30 nm icosahedral shell with T = 3 quasi-symmetry (Cowpea chlorotic mottle virus (CCMV) VLPs have been used to deliver DOX due to their high stability in acidic conditions . CCMV issymmetry . The RNASalmonella typhimurium bacteriophage P22 VLPs have been used to deliver DOX due to their large interior cavity (r cavity . The P22r cavity . For actOligodeoxynucleotides (ODNs) with cytosine-guanine dinucleotide (CpG) motifs have been considered potent immunostimulatory drugs . NeverthCowpea chlorotic mottle virus (CCMV) VLPs were used for the targeted delivery of ODN 1826, which induces the phagocytic activity of TAMs by activating the Toll-like receptor (TLR) 9 signaling pathway . ODN1826CCMV VLPs have also been explored as a carrier for siRNAs . CCMV VLBacteriophage MS2 virus-like particles (MS2 VLPs) have been used to deliver microRNA (MiR-122) to target hepatocellular carcinoma (HCC) . MS2 VLPCancer-specific motifs have been introduced for the active targeting of VLPs. HER2 Afb was genetically introduced on HBc VLPs to deliver siRNA for the Polo-like kinase 1 gene (siPLK1) . PLK1 isE. coli. When treated with intratumoral injection of the JCPy VLPs in combination with ganciclovir (GCV), the nude mouse model with orthotopic tumors exhibited prolonged survival by inhibiting cell growth of the human malignant glioblastoma U87 cells compared to GCV and JCPy VLPs without GCV. Furthermore, the tail vein-injected VLPs could deliver a suicide gene to implanted U87 cells via the circulatory system in this mouse model.Neurotropic JC polyomavirus (JCPyV) VLPs have significant potential as a carrier for gene therapy in human glioblastoma because human glioblastoma cells are highly susceptible to the virus . These VCombinations of chemo- and gene-therapeutic agents allow for cancer treatment with increased efficacy. HBc VLPs have been used as a carrier for simultaneous chemotherapy and gene therapy . The braVLPs have been considered an attractive vaccine platform because they have a high density of repetitive antigenic epitopes on their surface that elicit humoral and cell-mediated responses . To dateB peptide and OVAT peptide) have been developed as therapeutic cancer vaccines (B and OVAT peptides are tumor-specific neoantigens produced by somatic mutations in tumor cells and can stimulate the cytotoxic lymphocyte (CTL) response, resulting in a strong anti-tumor immune response  VLPs have been modified into a tumor vaccine by genetically fusing them with murine topoisomerase IIα (TopIIα) and survivin . RHDV VLSeveral VLP-based cancer vaccines target cell surface glycoproteins. Mucin-1 (MUC1), a highly O-glycosylated glycoprotein, is overexpressed in various cancer cells. It plays a substantial role in cancer progression and development . TherefoAdjuvants assist cancer vaccines by triggering an immune response, however, they can induce an autoimmune response due to their toxicity . TherefoA delayed cancer diagnosis can increase the mortality risk ; therefo18F, 64Cu, and 68Ga, are used for PET; however, many of these have a short half-life and low specificity. Owing to their high biocompatibility and easy surface functionalization, VLPs have been used to avoid blood clearance and extend the half-life of radioisotopes by increasing the tracer size (Positron emission tomography (PET) is a highly sensitive and non-invasive clinical modality. It is an important imaging tool for early cancer diagnosis and staging due to its high sensitivity and spatial resolution . Variouscer size .18F-fluorobenzaldehyde (FDG) via amino acid-specific conjugation. 18F-fluoride has a short half-life (110 min) and is quickly cleared by the blood circulatory system  of 64Cu-DOTA-MS2 VLPs than that of free 64Cu was present in the blood. These data indicate that the VLPs improved the time 64Cu circulated in the blood in the form of 64Cu-DOTA-MS2, when compared to free 64Cu. However, a sufficient concentration of 64Cu-DOTA-MS2 VLP was not detected in the tumor site.PEG-functionalized MS2 VLPs have been developed to increase the blood circulation time of bacteriophage MS2-based PET tracers . MS2 cap68Ga-DOTA-NHS were conjugated to the exterior surface of Qβ VLP. CPP-Qβ-VLPs had a cell uptake efficiency of 72.9% in U87-MG cells after 5 h incubation, which was approximately twice as high as that of non-conjugated Qβ-VLPs. The high cellular uptake of 68Ga-DOTA-labeled CPP-Qβ-VLPs enabled the PET tracer to be detected in the tumor site.The ability of CPP-functionalized Qβ VLPs (CPP-Qβ-VLPs) to increase the cell internalization of VLP-based PET tracers has been explored  Figure . Cys-CPP64Cu-DOTA-MS2 VLP to actively target EGFR-overexpressing HCC1954 breast cancer  unnatural amino acid was introduced at position 19 of the MS2 capsid proteins, followed by reacting with aminophenol-antibody conjugates. The specific targeting ability of 64Cu-DOTA-MS2-αEGFR was confirmed in vitro using flow cytometry and confocal live imaging with Oregon Green 488 (OG488)-labeled MS2-αEGFR as a control. MS2-αEGFR showed increased cellular internalization compared to the control in both EGFR-positive breast cell lines MDA-MB-231 and HCC 1954. Live imaging of a HCC1954-bearing breast cancer mouse model using PET revealed the in vivo biodistribution of 64Cu-DOTA-MS2-αEGFR. At 24 h after injection, the intensity of the signal was analyzed in the tumor site and a 4.9 tumor-to-muscle ratio (T/M) was detected, indicating a higher accumulation in the tumor region than in muscle.The introduction of CPP and PEG improved the cell internalization of VLP-based PET tracers and increased the length of time they remained in the blood. However, these VLPs utilized the enhanced permeability and retention (EPR) effect-based passive targeting strategy, which is limited to tumors with low growth rates . Thus, it cancer ; p-aminoin vitro and in vivo compared to conventional contrast agents, demonstrating that VLP-based MRI contrast agents are potential candidates for cancer bioimaging.Magnetic resonance imaging (MRI) is an example of non-invasive imaging technology. In contrast to PET, MRI has excellent spatial resolution without the need for radioisotopes . However3O4-NTA-Ni2+) T2 MRI agent (3O4 nanoparticle (Fe3O4-HBc VLPs) owing to the interaction between the His6-tag on the N-terminal HBc capsid and the nickel-NTA chelate of Fe3O4 nanoparticles. Fe3O4-HBc VLPs exhibited three-fold higher cellular uptake efficiency than pure Fe3O4 nanoparticles in HeLa cells. Furthermore, the VLPs showed signal enhancement in HeLa cells with varying T2 relaxivity depending on the size of the encapsulated Fe3O4 nanoparticles .Genetically modified HBc VLPs were chosen as a carrier for the iron oxide nanoparticle (FeRI agent . During 3+(DOTA)) for both ultrahigh field magnetic resonance imaging (UHFMRI) and near-infrared fluorescence (NIFR) imaging (3+(DOTA) imaging moieties were conjugated to the inner surface of PhMV VLPs using thiol-maleimide click chemistry. DGEA peptides were attached to the exterior of the PhMV VLPs via an NHS-PEG-maleimide linker. The synthesized Gd-Cy5.5-PhMV-mPEG NPs had a longitudinal relaxivity (r1) of 31.0 and 8.2 mM−1s−1 at 1.5 and 7 T, respectively, which is approximately 7-fold higher than that of a Gd3+(DOTA)-based commercial T1 MRI contrast agent. PC-3 tumor-bearing mouse models injected with Gd-Cy5.5-PhMV-mPEG NPs had an approximately 2-fold higher concentration of Gd3+ in the tumor site 240 h later than those injected with VLPs without DGEA ligands. This performance surpassed that of other VLP-based contrast agents (A cancer-targeting ligand was applied to VLP-based MRI contrast agents to enable their target-specific delivery. Prostate cancer-specific Asp-Gly-Glu-Ala (DGEA) peptides-modified PhMV VLPs were developed with a multifunctional contrast agent (Cy5.5 and Gd imaging . The Cy5t agents . These d−1s−1) performed better at 0.47 T in vivo than the current T2 MR contrast agents used clinically. Dark contrast was detected only in the SPARC-expressing prostate cancer, demonstrating that M13-SBP-MNP is able to specifically target prostate cancer. The signal-amplifying ability of M13 as a carrier was investigated using Alexa Fluor-modified peptide-functionalized MNPs (SBP-MNPs) and M13-SBP-MNPs. M13-SBP-MNPs exhibited an 11-fold higher fluorescence intensity than SBP-MNPs. These results indicated that M13-SBP-MNP could amplify MRI signal intensity owing to improved delivery efficiency of MNPs per carrier by cancer-specific targeting.Signal intensity in MRI is highly related to the concentration of the contrast agents . Most ofin vivo imaging. Cyanines are one of the most frequently used NIR fluorescent dyes with diverse clinical applications  fluorescence imaging has improved tissue penetration and reduced background signal compared to visible fluorescence imaging . Owing tications . Howeverications .Escherichia coli were disassembled into virus particle (VP1) pentamers to encapsulate fluorescent molecules. During the reassembly process, successful encapsulation of CH1 (CH1-SV40) occurred owing to the non-covalent interactions between multiple amino acids on the VP1 pentamers and the NIR-II fluorescent molecule CH1. CH1-SV40 was highly stable under different conditions, particularly at low pH, suggesting that CH1-SV40 is suitable for in vivo bio-imaging of cancer cells with a slightly acidic extracellular microenvironment. The in vivo cancer imaging capability of CH1-SV40 was also demonstrated using a HeLa tumor-bearing mouse model; fluorescence signal remained at the tumor site for 5 h after the intra-tumor injection.VLP-based nanocarriers have been proposed to improve the stability of fluorescent dyes. The fusogenic spike glycoprotein of vesicular stomatitis virus (VSV-G) was used as a protein nanocage for NIR bio-imaging . VSV-G ein vitro. CP dimers can be reassembled into smaller structures than native CCMV, depending on the pH or ionic strength. Owing to this unique characteristic, CCMV is an attractive protein cage for various cargos  dimers s cargos . Viral Rs cargos . The asss cargos . NegativBromoviridae family (N-hydroxysuccinimide (BrCy106-NHS) was applied to the OVGs , an icosahedral RNA plant virus belonging to the e family , can sele family . The devthe OVGs . BromineE-Cy5 and PhMV-CI-Cy5.5). The conjugation condition was optimized by changing the reaction time and the molar ratios of the dyes to PhMV to minimize self-quenching. By using amine-specific chemical conjugation, the distance between Cy5 molecules conjugated to the surface was approximately 2–4 nm. Only one Cy5.5 was bound by thiol-specific conjugation. PhMV-KE-Cy5 and PhMV-CI-Cy5.5 exhibited high cellular uptake efficiencies in ovarian, breast, and prostate cancer cells. Therefore, PhMV-based VLPs are a potential carrier for cancer bio-imaging due to their high cellular uptake efficiency and spatial distribution of fluorescence probes. 3+ were conjugated to the interior surface of PhMV by a thiol-maleimide reaction. The outer surface was modified by PEGylation using mPEG-NHS or maleimide-mPEG-NHS (Gd-Cy5.5-PhMV-mPEG NPs). DGEA peptides were also conjugated to the PEG terminal end of Gd-Cy5.5-PhMV-mPEGs in order to target prostate cancer cells (Gd-Cy5.5-PhMV-mPEG/DGEA). The cellular uptake of Gd-Cy5.5-PhMV-mPEG/DGEA was evaluated in the α2β1 integrin-overexpressing prostate cancer cell line PC-3. The fluorescence image showed strong signal intensity in PC-3 cells, suggesting that Gd-Cy5.5-PhMV-mPEG/DGEA might be able to specifically target prostate cancer cells. Additionally, fluorescent signal from the conjugated Cy5.5 was detected in some CD45-and CD68-expressing immune cells, indicating that Gd-Cy5.5-PhMV-mPEG/DGEA could be delivered by immune cells to the tumor region, resulting in a prolonged signal in the tumor.In addition to encapsulation, a fluorescent dye can be specifically labeled on the interior or exterior surface of VLPs by chemical conjugation whilst maintaining an appropriate distance and concentration to minimize the quenching effect . The cyaChemical conjugation is a way to label imaging probes with a suitable spatial arrangement to reduce the quenching effect, however, it is not easy to control the site-specific conjugation precisely. Genetic conjugation has been proposed to overcome this limitation. Three types of fluorescent protein-inserted hepatitis B virus capsid proteins (FP-HBcs) have been developed  Kim et . The fluTheragnosis is an effective strategy for the management of specific diseases and combines diagnosis and therapy . General68Ga and epirubicin (EPI) were used for theragnosis as imaging and therapy agents, respectively. EPI was loaded into the GFP-Qβ VLPs by diffusion, and 57.3% of EPI was released in slightly acidic conditions. This result suggests that EPI could be released in the acidic tumor microenvironment. Owing to its slow release, it was also possible to minimize the damage to normal tissue. The 68Ga radioisotope was conjugated to the exterior of the VLPs using an amine-reactive NHS-ester. The biodistribution of the VLPs in a glioblastoma animal model was analyzed using both PET/CT and fluorescence imaging. After 2 h post-injection, the VLPs remained in the tumor region with minimal diffusion into surrounding normal tissues. Moreover, the tumor was eradicated after 8 days with 2 doses of the VLPs, in contrast to mice treated with free EPI that died after 3 days. These results indicated that the developed VLP carrier was highly efficient at delivering its cargo in a localized manner and was less toxic than EPI. E. coli. RNAic-MET is known to induce the degradation of c-MET mRNA, which is associated with tumor cell proliferation and survival  and photodynamic therapy (PDT), has been introduced to simplify theragnostic particle synthesis . PTT is in vivo efficiency  have been utilized as MRI agents in HBc VLPs (SPAuNCs-HBc VLPs)  Kwon et. HBc cap3O4) nanoparticles -conjugated iron oxide (Fearticles . These nIn this review, we focused on current developments in the successful application of engineered VLPs in cancer diagnosis and therapy. However, most VLPs have been delivered to tumors through passive targeting based on the EPR effect. Only a few studies have used the RGD peptide or folic acid to actively deliver VLPs. There are some limitations to the practical application of VLPs in cancer therapy and diagnosis, and currently there are only a few VLPs being considered for theragnosis. Research on ensuring the structural stability of VLPs is ongoing. Continuous developments in VLP-based therapy and diagnosis strategies will be needed to overcome these limitations.


Many patients with symptoms and signs of heart failure have a left ventricular ejection fraction ≥50%, termed heart failure with preserved ejection fraction (HFpEF). HFpEF is a heterogeneous syndrome mainly affecting older people who have many other cardiac and non-cardiac conditions that often cast doubt on the origin of symptoms, such as breathlessness, or signs, such as peripheral oedema, rendering them neither sensitive nor specific to the diagnosis of HFpEF. Currently, management of HFpEF is mainly directed at controlling symptoms and treating comorbid conditions such as hypertension, atrial fibrillation, anaemia, and coronary artery disease.HFpEF is also characterized by a persistent increase in inflammatory biomarkers. Inflammation may be a key driver of the development and progression of HFpEF and many of its associated comorbidities. Detailed characterization of specific inflammatory pathways may provide insights into the pathophysiology of HFpEF and guide its future management. There is growing interest in novel therapies specifically designed to target deregulated inflammation in many therapeutic areas, including cardiovascular disease. However, large-scale clinical trials investigating the effectiveness of anti-inflammatory treatments in HFpEF are still lacking. In this manuscript, we review the role of inflammation in HFpEF and the possible implications for future trials.  This manuscript was handled by Guest Editor Carolyn S.P. Lam.This article is part of the Spotlight Issue on Heart Failure.1.1 Nevertheless, making a diagnosis of HFpEF is challenging. Most patients are elderly, with a high proportion of women and several cardiovascular and non-cardiovascular comorbidities.1 Outpatients with HFpEF have a better overall prognosis and a much lower rate of cardiovascular events than those with HF with reduced LVEF (HFrEF), but a higher proportion of non-cardiovascular deaths.2 However, among patients with decompensated HF, the outcome is similar for HFpEF and HFrEF, although the prognosis for HFpEF might often be driven by comorbid disease rather than HF itself.3 Also, HFpEF and HFrEF are not entirely distinct entities. Indeed, impaired myocardial contraction is expected in both, e.g. circumferential for HFrEF and long axis for HFpEF.5 However, measurement of LVEF is prone to substantial error—hence the introduction of the HFmrEF phenotype, which acts as a buffer zone to reduce classification error between HFrEF and HFpEF in research and clinical practice. HFrEF may recover, especially for younger patients with little myocardial scar (e.g. dilated cardiomyopathy) who receive guideline-recommended therapy; this should not be considered HFpEF but rather HF with recovered LVEF.7 For patients with HFpEF, LVEF may also drop over time due to measurement error [increased with the onset of atrial fibrillation (AF)]8 or additional myocardial insults .5Many patients diagnosed with heart failure (HF) have a left ventricular ejection fraction (LVEF) ≥50%, i.e. HF with preserved LVEF (HFpEF).1 Recently, a RCT on a factor Xa antagonist demonstrated a substantial reduction in mortality for patients with HFpEF and coronary artery disease (CAD),9 while two others found that sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce hospitalizations for worsening HF.11 These trials showing that the natural history of HFpEF can be favourably modified encourage exploring other therapeutic avenues, including inflammation, which might have a key role in the pathophysiology underlying HFpEF.12 A thorough characterization of inflammatory pathways involved in HFpEF might help identify therapeutic targets and interventions. Herein, we provide an overview of the role of inflammation in the pathogenesis of HFpEF, summarize the available evidence for anti-inflammatory treatments, and discuss the potential implications for the design of future HFpEF trials. We deliberately avoid a detailed discussion of antifibrotic therapies because fibrosis is a non-specific downstream consequence of myocardial damage.13A series of randomized control trials (RCTs) of neurohormonal antagonists failed to show clear cardiovascular benefits in patients with HFpEF.2.15 Chronic, low-grade, systemic inflammation might have detrimental effects on myocardial structure and function . Experimental models suggest that increased production of pro-inflammatory cytokines enhances oxidative stress, drives the differentiation of fibroblasts into collagen-secreting myofibroblasts, and induces extracellular matrix degradation, leading to increased myocardial stiffness and coronary microvascular dysfunction (CMD).17 Local inflammation also reduces nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) availability, resulting in the hypo-phosphorylation of the giant sarcomeric protein titin, which further increases myocardial stiffness and worsens diastolic function.16 Oxidative stress might also be implicated in the development of metabolic heart disease, indicating bidirectional links between inflammation and cardiac dysfunction.18Inflammatory biomarkers, including tumour necrosis factor (TNF)-α and its receptors (TNFR1 and TNFR2), interleukin (IL)-6 and IL-8, high-sensitivity C-reactive protein (hsCRP), pentraxin-3 and the chemokine (C–C motif) ligand 2 (CCL2), also referred to as monocyte chemoattractant protein-1 (MCP-1), are often elevated in patients with HFpEF.16 Indeed, inflammation is frequently associated with unhealthy ageing and several cardio-metabolic comorbidities , e.g. obesity and altered adiposity, chronic kidney disease (CKD), hypertension, CAD, Type 2 diabetes mellitus (T2DM), AF, elevated serum uric acid (SUA) concentrations, and chronic obstructive pulmonary disease (COPD).19–22 Chronic low-grade inflammation might also contribute to the development of sarcopaenia and frailty, which are very common among outpatients with HFpEF, with a prevalence varying from 30 to 52%.23 All these features, particularly when combined, might lead to a poorer quality of life and drive cardiovascular and non-cardiovascular outcomes.25 Recently, proteomic bioprofiles have been investigated to identify potential mechanistic pathways associated with HF development. Systemic inflammation might mediate the association between several comorbidities and cardiac dysfunction, promoting the progression of disease.25–27Whether the persistent inflammation that characterizes HFpEF represents a causal factor or an epiphenomenon due to one or more pro-inflammatory comorbid conditions is not well understood.28 Biomarkers reflecting systemic congestion, inflammation, and fibrosis predict clinically overt AF as well as HFpEF and are associated with abnormal diastolic filling and reduced exercise capacity.29 Endothelial inflammation can cause CMD and myocardial fibrosis, which can induce both atrial and ventricular myopathy.30 In addition, systemic inflammatory and metabolic disorders have been linked to an expansion and pro-inflammatory transformation of epicardial adipose tissue (EAT)31–35 EAT accumulation may also impair atrial distension and contraction by its proximity to the myocardium, leading to mechanical dysfunction, electroanatomical fragmentation, and ultimately AF.37AF and HFpEF frequently co-exist. Chronically elevated left atrial pressures might produce adverse structural atrial remodelling and dysfunction that increase the risk of developing AF. On the other hand, AF, especially with a rapid ventricular response, leads to a decreased LV filling time and the loss of atrial contribution to LV filling; therefore, it might precipitate the onset or worsening of HFpEF.39 On the other hand, the inflammatory milieu of obese patients is primarily due to adipokines, i.e. cytokines secreted by both adipocytes and macrophages resident in adipose tissue.40 Patients with HFpEF who are obese have higher serum concentrations of many pro-inflammatory proteins .41 This heterogeneity in inflammatory phenotypes could lead to the application of precision medicine in HF treatment, with different therapeutic approaches according to the pattern of comorbidity.42Patients with HFpEF may exhibit different inflammation patterns according to their comorbidities. Inflammation in hypertension is often driven by renin–angiotensin–aldosterone system activation.43 Amyloid deposition might be common in patients with HFpEF,44 but, until recently, the diagnosis of amyloidosis has seldom been considered and rarely investigated. Heart valve diseases, even if not severe, can worsen cardiac structure and function and contribute to HF symptoms in the presence of a normal LVEF: it is noteworthy that degenerative valve disease is the most frequent valvular disorder in Western countries and is characterized by calcification, which might, in turn, be related to inflammation.45 Chronic inflammatory disorders, such as inflammatory bowel disease and rheumatoid arthritis, have been linked to an increased risk of cardiovascular disease (CVD) and HF, especially during flares in disease activity.47 Periodontal disease is extremely common and associated with increased cardiovascular risk.48 The more severe the periodontitis, the higher the risk of developing HF; conversely, good oral hygiene reduces it.50Other non-metabolic diseases cause a chronic, low-grade, systemic inflammatory response, and produce a clinical picture that fits the HFpEF definition. Amyloidosis is a systemic disorder characterized by the progressive deposition of fibrillary proteins that cause immune cell infiltration into tissues and pro-inflammatory cytokine production in various organs, eventually resulting in their failure.3.3.151The inflammatory profile of patients with HFpEF can be investigated by measuring plasma concentrations of inflammatory biomarkers. However, plasma concentrations reflect a steady-state between production and disposal and are not necessarily evidence of changes in inflammation. In the presence of renal or hepatic dysfunction, elevated plasma concentrations may reflect reduced clearance rather than increased production.Figure 1), with the subsequent cleavage and activation of IL-1β, IL-6, IL-12, IL-18 and, finally, the production of CRP in the liver.52 This pathway is driven by endogenous stimuli and defined as ‘sterile’ inflammation instead of exogenous-induced inflammation due to infection. In routine clinical practice, hsCRP is widely available, relatively stable in peripheral blood and the most commonly measured inflammatory biomarker. High concentrations of hsCRP are associated with more comorbidity and greater disease severity in HFpEF and HFrEF,54 and predict a worse outcome, although for HFpEF this is mainly for non-cardiovascular events.53The most explored inflammatory pathway involves the nucleotide oligomerization domain-like receptor family, pyrin domain-containing (NLRP3) inflammasome  should report soon.61In several population-based longitudinal registries including patients with cardiovascular risk factors, SUA levels are associated with an increased risk of all-cause and cardiovascular mortality at serum concentrations substantially lower than those used to define hyperuricaemia in clinical practice.62 In the Aldo-DHF trial, baseline serum concentrations of galectin-3 were inversely correlated with functional capacity and directly associated with NYHA class in HFpEF patients.62 Data from multiple RCTs and registries suggest that galectin-3 is an independent predictor of adverse outcomes in chronic and acutely decompensated HF, regardless of LVEF.63 Thus, galectin-3 has been proposed as a marker of fibrotic activity,64 potentially mediated by inflammation. However, it is excreted by the kidney, and not all studies have corrected for renal function.Galectin-3 is a lectin expressed by activated cardiac macrophages and induces the secretion of IL-6. Elevated serum concentrations of galectin-3 are associated with adverse LV remodelling, cardiomyocyte hypertrophy, and myocardial fibrosis.65–67 Many other markers of inflammation, metabolic dysfunction, and extracellular matrix remodelling , adrenomedullin, cystatin C, and resistin) have prognostic significance in HFpEF68–70 but have yet to find a role in clinical practice.High serum concentrations of the soluble suppression of tumourigenicity 2  are also associated with increased myocardial fibrosis and inflammation as well as with poorer outcomes in patients HFpEF.71 Iron deficiency is common in HF, irrespective of LVEF and associated with adverse outcomes.72 Inflammation can also inhibit iron absorption and iron mobilization and/or utilization even when iron stores are not depleted. Pro-inflammatory cytokines  upregulate the protein hepcidin, leading to reduced intestinal iron absorption and decreased iron mobilization from bone marrow stores.73 Intravenous iron administration in patients with systolic HF improves symptoms and clinical outcomes, but evidence of benefit is still lacking for those with HFpEF.74Ferritin binds to iron in cells. In the absence of other diseases, iron deficiency is associated with low release of ferritin, and low serum concentrations signify iron deficiency. However, in the presence of inflammation, serum ferritin leaks from cells rendering it unreliable as a marker of iron deficiency, which might mask a diagnosis of iron deficiency in HFpEF.75 and the diversity of immune cells implicated in the development of CVD.76 Multi-omics approaches may soon uncover novel inflammatory cardiac pathways that offer new therapeutic opportunities.Omics techniques such as single-cell RNA sequencing are an emerging tool for studying the transcriptional heterogeneity in both healthy and diseased hearts3.2E/e’ ratio is a widely used surrogate marker of LV filling pressures, but its clinical utility remains controversial, and it has not been used to select patients for any of the successful landmark trials of HFpEF.77 Left atrial volume and function might be the best marker to integrate left ventricular systolic and diastolic dysfunction, including the mitral apparatus and heart rhythm79 but will be agnostic to the underlying pathophysiology. Echocardiography can also evaluate EAT accumulation,80 which is associated with greater inflammatory activity, impaired haemodynamics, worse symptoms, and a poorer prognosis in patients with HF.81Echocardiography is the most widely available imaging technique and provides valuable information regarding structural and functional alterations in HFpEF. The 82 which is central to the diagnosis and prognosis of HF and is also associated with increased inflammatory activity.83 In experimental conditions, the development of venous congestion activates the innate immune system and the secretion of pro-inflammatory cytokines in healthy individuals.84 Therefore, treatment targeted at congestion might improve inflammation. Intensification of therapy with loop diuretics had a mixed effect on biomarkers of immune activation and inflammation in one small study of HFrEF, but it normalized endotoxin, which is often increased in congestive HF due to altered gut permeability and subsequent translocation of lipopolysaccharide into the circulation.86Echocardiography and, more generally, ultrasound represent a valuable tool to estimate congestion,88 CMR can also be used to estimate the extracellular volume and myocardial oedema.89 In HFpEF, the former correlates with myocardial stiffness and the extent of interstitial collagen deposition evaluated at the histopathological level,89 while the latter could be related to increased microvascular permeability or impaired lymphatic function.90Cardiac magnetic resonance (CMR) represents the gold standard for evaluating chamber geometry and structure, as well as quantifying EAT volume with great precision.91Localized myocardial inflammation can be assessed by positron emission tomography using [18F]fluorodeoxyglucose, which accumulates in activated inflammatory cells  due to increased glucose uptake. Mechanistic studies suggest that [18F]fluorodeoxyglucose imaging after myocardial infarction (MI) and pressure-overload HF may provide additional prognostic information.4.Table 1).92 Most of these trials excluded patients with moderate or severe HF, did not always include HF-related endpoints and often did not provide data on LVEF,105 limiting extrapolation of their results to patients with HFpEF. Nevertheless, IHD is frequently undiagnosed and sub-optimally treated in patients with HFpEF.1 Not many patients with HFpEF undergo coronary angiography due to their advanced age, the high number of comorbidities, particularly renal dysfunction, and the lack of evidence that revascularization is beneficial in the absence of acute ischaemia. Of those who are investigated, many (>50%) have obstructive epicardial CAD, and most (85%) have evidence of CMD.111 The presence of CAD is not surprising, given the high proportion of patients with HFpEF who have hypertension, T2DM, obesity, and CKD. These comorbidities also account for the high prevalence of CMD, which may play a key pathophysiological role in the development of HFpEF, independent of atherosclerotic burden.113 Inflammation promotes all stages of atherosclerosis, from plaque formation to rupture, leading to macrovascular and microvascular ischaemic events.114 Also, chronic inflammation, even in the absence of epicardial stenoses and traditional coronary risk factors, is associated with CMD.115Many large RCTs have investigated the effect of anti-inflammatory agents on cardiovascular events in patients with atherosclerosis, hypertension, and other cardiovascular disorders , but, until now, most of them were focused on the HFrEF phenotype.116–118Inflammation has been the therapeutic target for many RCTs that enrolled only patients with HF  (roughly <1000 ng/L) had less severe disease and a better prognosis but appeared to benefit from a statin.136 Further analyses of the Heart Protection Study confirmed this finding, showing that as NT-proBNP and absolute cardiovascular risk increase, the relative risk reduction with statins shrinks, reaching a point where statin therapy is futile.137 A recent collaborative meta-analysis of unpublished data from major primary and secondary prevention RCTs showed that statins modestly reduced the risks of non-fatal HF hospitalization but not HF death.138 However, the authors did not have data on LVEF; relatively few were likely to have had HFpEF.This drug class has several anti-inflammatory properties, including induction of endothelial NO synthase, inhibition of adhesion molecules expression, and reduction of immune cells chemotaxis.4.2139 as NO inhibits platelet aggregation and promotes vascular smooth muscle cell relaxation.140 NO regulates myocardial stiffness and diastolic function in healthy myocardium through the cGMP-protein kinase G pathway.142 Moreover, NO can also balance the functional activity, growth, and death of many immune and inflammatory cell types, including macrophages, T lymphocytes, antigen-presenting cells, mast cells, neutrophils, and natural killer cells.143 NO signalling could represent a therapeutic target for myocardial stiffness and inflammation, which are typical stigmata of HFpEF. In an inflammatory milieu, NO bioavailability is reduced by reactive oxygen species142 and the inactivation of endothelial NO synthase.144 However, in the RELAX trial, 216 patients with chronic HFpEF were randomized to a phosphodiesterase-5 inhibitor known to prolong NO half-life (sildenafil) or placebo. Sildenafil did not improve exercise performance.24 Likewise, in the NEAT-HFpEF120 and INDIE-HFpEF122 trials, nitrates/nitrites failed to improve daily activity level and exercise capacity in patients with HFpEF. Cimlanod, a nitroxyl donor, has been studied in patients with HFrEF in the STAND-UP AHF trial: compared with placebo, this drug led to decreased NT-proBNP plasma concentrations during infusion.145 In patients with chronic HFrEF, cimlanod showed haemodynamic effects similar to those of nitroglycerin, i.e. venodilatation and preload reduction without additional inotropic or lusitropic effects.146 Ongoing trials of cimlanod will further define its potential role in the treatment of HF.NO is an intercellular messenger synthesized and released into the endothelial cells by NO synthases while converting arginine into citrulline. Impaired NO signalling is one of the cardinal features of endothelial dysfunction and atherosclerosis,123 However, for patients with HFpEF , vericiguat failed to reduce plasma NT-proBNP concentrations, left atrial volume, or physical activity.124Vericiguat, an oral soluble guanylate cyclase stimulator, enhances the cGMP pathway by acting synergistically with NO. For patients with HFrEF, the VICTORIA trial suggested a reduction in morbidity and mortality, except in those with a very high NT-proBNP (>5314 ng/L) who appeared to be harmed.4.3147 decreasing neutrophil L-selectin expression, thereby inhibiting diapedesis148 and inhibiting activation of the NLRP3 inflammasome, which is indirectly responsible for the cleavage of pro-IL-1β to active IL-1β.149 Colchicine has been extensively studied with encouraging findings in patients with IHD96–99 but with no apparent benefit for patients with chronic HFrEF.125 Two ongoing trials could provide valuable insights into the use of colchicine for HFpEF: the COLCOT-T2D will recruit 10 000 patients with T2DM but without known CAD and evaluate whether colchicine reduces cardiovascular risk and progression to HF; the COLpEF (NCT04857931) will enrol 426 patients with HFpEF to assess the effects of colchicine on hsCRP concentrations (primary outcome), symptoms, and other secondary outcomes.Colchicine interferes with cytosolic microtubule assembly inhibiting immune cells chemotaxis and cytokines secretion,4.4et al.151 described the significant association of serum concentrations of inflammatory biomarkers (e.g. IL-6 and CRP) with an increased risk of cardiovascular events. Recently, IL-6 has been associated with new-onset HFpEF in community-dwelling individuals.152 IL-1β is a key determinant of IL-6 production and has adverse effects on myocardial function in animal models, impairing systolic function, interfering with mitochondrial energy production and uncoupling β-adrenergic receptors, and L-type calcium channels.153More than 20 years ago, Ridker 154 In the CANTOS trial, 10 061 patients with a history of MI and hsCRP ≥2 mg/L were randomized to receive canakinumab in three different doses or placebo. After a median follow-up of 4 years, patients treated with the 150 mg and 300 mg dose had a lower incidence of the primary endpoint, a composite of non-fatal MI, non-fatal stroke, or cardiovascular death.105 In a prespecified analysis, the use of canakinumab led to a lower incidence of HF hospitalization and HF-related mortality; patients who achieved a hsCRP of <2 mg/L with treatment appeared to derive more benefit.106 However, there were no data on LVEF, precluding an analysis by LVEF phenotype.Canakinumab is a human monoclonal antibody targeting IL-1β, approved for treating many auto-immune diseases.155 The VCU-ART-3 trial enrolled 99 patients admitted for ST-elevation MI with a mean LVEF of 51%; anakinra reduced hsCRP compared with placebo.107 The D-HART enrolled 12 patients with HFpEF (median BNP 32 pg/mL) and hsCRP >2 mg/L were assigned to receive anakinra vs. matching placebo. After 14 days, patients assigned to anakinra had a lower hsCRP and greater peak VO2.126 However, the subsequent D-HART2, which included 32 obese patients with HFpEF and hsCRP >2 mg/L , did not confirm an effect on VO2, but anakinra reduced hsCRP and NT-proBNP concentrations after 4 weeks.127Anakinra is a recombinant antagonist of the IL-1 receptor, developed as a disease-modifying intervention for rheumatoid arthritis.4.5156 They were primarily developed to treat patients with T2DM, but recent landmark RCTs of HFrEF demonstrated that SGLT2i also reduce HF hospitalizations and cardiovascular death, whether or not the patients have T2DM.157 In the EMPEROR-Preserved trial, nearly 6000 patients with chronic HF and a LVEF >40% were randomized to empagliflozin or placebo. Empagliflozin reduced the incidence of the composite primary endpoint , mostly due to a reduction in hospitalization. Haematocrit increased, NT-proBNP fell, and the decline in the estimated glomerular filtration rate was slowed. The benefit was observed for both HFmrEF (LVEF 40–49%) and HFpEF (LVEF ≥50%) regardless of a diagnosis of T2DM.10 The ongoing DELIVER trial will assess the effects of another SGLT2i, dapagliflozin, in HFpEF, probably establishing this class of drugs as a cornerstone for treating HFpEF.159SGLT2i, or gliflozins, inhibit the kidney reabsorption of glucose but also appear to have anti-inflammatory effects.160–167 Applying artificial intelligence to a cohort of patients with HFpEF, Bayes-Genis et al.168 recently proposed that SGLT2i act at a molecular level, reducing systemic inflammation by lowering the plasma concentration of NO synthase Type 2 and NLRP3 inflammasome. Improvement in cardiac function with administration of empagliflozin to nondiabetic patients with HFrEF has been linked to a reduction in serum markers of inflammation and EAT volume.170SGLT2i cause a diuresis, resulting in a reduction in plasma volume and interstitial fluid, an increase in haematocrit and a reduction in body weight. Longer-term, SGLT2i might cause a further rise in haematocrit due to stimulation of erythropoietin and improved iron absorption and further loss in weight due to glycosuria and glucose wasting. However, diverse other potential mechanisms have been proposed, including an anti-inflammatory effect, reduction in oxidative stress and fibrosis, reduced deposition of advanced-glycation end-products, inhibition of the sodium/hydrogen exchanger-1 expressed on cardiomyocyte sarcolemma, and an increase in ketone body production as an energy substrate.4.6171 The generation of thrombin can amplify the effects of other stimuli on inflammatory pathways, which might be ameliorated by reducing its production. The COMPASS trial randomized >27 000 patients with CAD in sinus rhythm to a combination of rivaroxaban 2.5 mg bd and aspirin compared with aspirin and rivaroxaban alone. The combination reduced cardiovascular events and mortality.172 The overall result was driven by a large benefit in a subgroup of 4250 patients with HFpEF and mild symptoms;9 patients with severe symptoms had been excluded. In contrast, the COMMANDER-HF, conducted in patients with HFrEF, CAD and in sinus rhythm who had a recent decompensation, showed no improvement in the primary endpoint  with low-dose rivaroxaban vs. placebo.173 Overall, these data suggest that the efficacy of this intervention, as for many others, depends on the patient profile. In patients with mild, stable HFpEF (and probably HFrEF), low-dose rivaroxaban may be rather effective, but in patients with more severe HF the dose is either too low or too late because other factors are driving progression.174Activation of inflammatory pathways induces microvascular dysfunction and increases the risk of thrombotic events contributing to the progression of HF.4.7176 In a recent double-blind RCT including 2571 patients with a recent MI or severe CAD, influenza vaccination reduced the risk of all-cause mortality, MI or stent thrombosis after 12 months; benefits appeared very early, suggesting a therapeutic effect on the post-MI inflammatory phase.108 Unfortunately, the study was terminated prematurely due to the COVID-19 pandemic, but an observational study on >7000 patients with HF showed that COVID-19 vaccination was associated with a substantial reduction in all-cause hospitalization rates and mortality, irrespective of LVEF.177 RCTs are ongoing in patients with recent MI or HF.178Observational studies suggest that influenza vaccination may reduce mortality in patients with HF, and guidelines recommend considering this intervention in patients with HF.5.Table 2).HFpEF is associated with the activation of many inflammatory pathways; whether any of these are therapeutic targets is uncertain .181 Indeed, IL-6 can increase vascular smooth muscle cell stiffness and mitochondrial dysfunction, which explains the link between inflammation and impaired vascular function.182 Interestingly, Tet-2-mutated macrophages secrete higher amounts of IL-6, and these mutations are frequently seen in clonal haematopoiesis of indeterminate potential (CHIP), which is associated with an increased risk of cardiovascular events. In the BIOSTAT-HF study, more than half of the enrolled population had elevated serum concentrations of IL-6, associated with higher NT-proBNP and TNF-α, more iron deficiency, and poorer cardiovascular outcomes.183 Moreover, IL-6 administration in animal models was associated with myocardial hypertrophy and fibrosis, promoting diastolic dysfunction.184 Thus, IL-6 blockade might reduce the cardiovascular burden in HFpEF, but robust data are lacking.182 A subanalysis of the CANTOS trial showed the beneficial effects of canakinumab were more pronounced in those who achieved on-treatment IL-6 concentrations below the study median value of 1.65 ng/L.179 In patients with rheumatoid arthritis without CVD, inhibition of IL-6 receptor with tocilizumab was associated with improved LV systolic function and reduced LV mass.185 In the ASSAIL-MI trial, tocilizumab increased myocardial salvage as measured by CMR in patients with acute ST-segment elevation MI.186 Ziltivekimab, a fully human monoclonal antibody directed against the IL-6 ligand, reduced biomarkers of inflammation and thrombosis among patients with high cardiovascular risk, elevated hsCRP and CKD187; a RCT is ongoing to evaluate its clinical value in patients with CV and renal disease (NCT05021835).Recent data suggest cross-talk between IL-1 and IL-6 signalling pathways in HF,5.2– macrophages are the most represented subset in a normal heart, showing many cardioprotective properties, like promoting tissue regeneration and coronary angiogenesis. Conversely, only a small amount of inflammatory monocyte-derived CCR2+ macrophages is present in healthy mice. The latter initiate inflammation because they can induce neutrophil and monocyte migration into damaged tissues. Mice that developed chronic HF following coronary artery ligation had an increase in CCR2+/CCR2– ratio188; findings subsequently confirmed in human hearts. CCR2 + macrophage abundance was associated with LV remodelling and more advanced systolic dysfunction in myocardial specimens obtained from HFrEF patients who underwent LV assist device implantation.189 An intense proliferation of CCR2+ macrophages has also been described in murine models of pressure overload obtained by aortic constriction.190 CCR2 modulation could represent a potential target to reduce inflammation and block the development of HFpEF: in murine models of angiotensin-II-induced HFpEF, inhibition of CCR2+ macrophages improved diastolic function,191 while in humans, the anti-CCR2 humanized monoclonal antibody MLN1202  reduced hsCRP in 112 individuals with CV risk factors.192 Ongoing exploratory trials targeting CCR2 in different diseases (e.g. T2DM and COPD) could hopefully pave the way to study this therapy in the whole HF spectrum.12In mouse models, two subsets of cardiac macrophages can be identified according to the surface expression of C–C chemokine receptor 2 (CCR2). Tissue-resident CCR25.3193 The ongoing REGRESS-HFpEF trial (NCT02941705) will assess whether intracoronary administration of cardiosphere-derived cells can reduce pro-inflammatory and pro-fibrotic signalling, as well as improve functional status and haemodynamics in patients with HFpEF.The systemic inflammatory state can be reduced by targeting cytokine pathways and a generalized modulation of the immune system activity. Cardiosphere-derived cells are a population of cardiac progenitor cells with prominent anti-inflammatory properties. In a rat model of hypertensive HFpEF, intracoronary treatment with cardiosphere-derived cells reduced serum concentrations of inflammatory cytokines, improved diastolic function, and decreased myocardial fibrosis, despite persistent hypertension.5.4194 which is likely to be multifactorial, including cardiac dysfunction, skeletal muscle deconditioning, obesity, co-existent lung or joint disease, and psychological factors.195 In patients with HFpEF, endurance exercise training was associated with increased functional capacity (as assessed by peak VO2) and quality of life but no improvement in endothelial function or arterial stiffness.197 Physical training reduces systemic vascular resistance198 and increases skeletal muscle perfusion, peripheral oxygen utilization, and mitochondrial function.200 These effects further modulate inflammatory and oxidative processes201 that may benefit patients with HFpEF.203Impaired exercise capacity is a hallmark of HFpEF,5.5204 The administration of epigenetics regulators in animal models of hypertensive cardiomyopathy reduced TNF-α concentrations and interstitial myocardial fibrosis.205 The BETonMACE trial enrolled 2425 patients with T2DM up to 3 months after an acute coronary syndrome: participants were randomized to placebo and apabetalone, an inhibitor of bromodomain and extra-terminal motif proteins, which are epigenetic modulators of inflammation, thrombogenesis, and lipoprotein metabolism implicated in atherothrombosis.206 Although the trial missed its primary endpoint , in a prespecified secondary analysis, treatment with apabetalone was associated with a lower incidence of HF hospitalization than placebo110 {hospitalization for HF or cardiovascular death }. Unfortunately, there were no data about LVEF; thus, further RCTs are needed to evaluate whether epigenetic modulators represent another promising therapeutic approach to preventing and treating HFpEF.The term ‘epigenetics’ encompasses the changes that affect gene activity and expression without involving alterations in the genome (DNA sequence).207 A single miRNA can act as an epigenetic modulator, regulating the expression of hundreds of different mRNAs without modifying the gene sequences.208 Several cardiovascular conditions seem to be associated with specific miRNAs: for example, serum concentrations of miR-210 and miR-1 correlate with symptom severity in HF.210 Furthermore, inhibition of miRNA-21 prevented the development of HFpEF in an experimental model.211Micro-RNA (miRNA) are small, non-coding RNAs involved in the RNA-induced silencing complex, which binds messenger RNA either inducing its degradation or inhibiting its translation at the ribosomal level.5.6212 Target engagement and safety of AZD4831 have been tested in a Phase 2a study of HFpEF (NCT03756285), supporting further development.Extracellular deposition of granulocyte-derived myeloperoxidase (MPO) can cause oxidative stress, leading to microvascular dysfunction, inflammation, tissue damage, and fibrosis. A novel MPO inhibitor (AZD4831) reduced inflammation and improved microvascular function in preclinical models.5.7213 Adrecizumab is a monoclonal, non-neutralizing antibody that stabilizes adrenomedullin, ‘trapping’ it in the circulation without blocking adrenomedullin receptor signalling. In addition, adrecizumab translocates adrenomedullin from the tissue into the circulation. In animal models of systemic inflammation and septic shock, adrecizumab-induced increases in plasma adrenomedullin improved haemodynamics, renal function, and reduced markers of inflammation.213 A Phase II proof of concept study in patients hospitalized for acute HF is ongoing (NCT04252937).Adrenomedullin is a peptide hormone synthesized by endothelial and vascular smooth muscle cells. Its production is stimulated by volume overload to maintain endothelial barrier function, while the disruption of the adrenomedullin system results in vascular leakage and systemic and pulmonary oedema. Adrenomedullin is markedly elevated in patients with sepsis and in patients with acute HF, probably as a compensatory mechanism against fluid overload and tissue congestion.5.831–33 altered adipogenesis by secretion of pro-inflammatory and pro-atherogenic adipokines,34 and an adverse prognosis.81 Noteworthy, increased biventricular hypertrophy and EAT exacerbate pericardial restraint in HFpEF, resulting in higher LV filling pressure to achieve a given transmural pressure, particularly during exercise.32 Enhanced pericardial restraint in patients with HFpEF partially explains the lower concentration of natriuretic peptides observed in this cohort, showing pathophysiology similar to that observed in constrictive pericarditis.In patients with HFpEF, EAT accumulation is often marked, and it promotes haemodynamic derangements,214 or pharmacological therapies (e.g. SGLT2i and GLP-1 agonists)35 targeting excessive EAT accumulation might decrease inflammation and potentially provide meaningful clinical benefits.Thus, in selected HFpEF patients, non-pharmaceutical interventions 6.Subclinical inflammation is common in patients with HFpEF, regardless of underlying aetiology and associated comorbidities. A deeper understanding and detailed characterization of inflammatory mechanisms responsible for disease onset and progression may lead to new therapeutic opportunities to improve the well-being and outcomes of those with or at risk of developing HFpEF.


The results show that WF substitution with TNF increased bread brownness and color saturation and decreased lightness, showing the highest impact on Sb, followed by Tb and Bb. Additionally, bread-specific volume decreased significantly after 20% (Bb), 25% (Tb), and 30% (Sb) TNF substitution. Furthermore, substituting WF with 30% TNF increased crumb hardness from approx. 1.87 N to 3.64 N (Bb), 3.46 N to 8.14 N (Tb), and 6.71 N to 11.39 N (Sb) and caused significant increases to 17.80 N (Tb) and 21.08 N (Sb) after 3 d storage. Only a marginal effect on storage hardness (4.32 N) was observed for Bb. Substituting WF with 10% TNF for Bb or 25% TNF for Tb led to significantly higher consumer (N = 56) scores for all attributes and overall acceptability. However, no significant effect on the overall acceptability of Sb was observed. Flash profiling showed frequently used descriptors for Bb as firm, moist, buttery, smooth, and astringent. After 10% TNF substitution, descriptors were chewy, firm, sweet, porous, dry, and caramel, and that of 30% TNF were grainy, chocolate, brown, nutty, and flaky. Substituting WF with TNF increased the lipids, fiber, and minerals content but decreased the protein and carbohydrate contents of bread. TNF substitution led to different physical and sensory effects depending on bread type, showing that Bb with 10% or Tb with 25% TNF is more comparable with the overall acceptance quality of 100% WF. The study is relevant for utilizing tiger nuts as an ingredient in bread products.Tiger nut is a valuable source of fiber, lipids, minerals, and carbohydrates. However, avenues for incorporating tiger nuts into food remain underexplored, especially in several tropical countries where the plant grows well. The current study investigated the effects of partially substituting wheat flour (WF) with tiger nut flour (TNF) on the physical and sensory properties of different bread types to evaluate the more amenable system for tiger nut incorporation. The substitution was done at WF:TNF ratio of 100 : 0, 90 : 10, 85 : 15, 80 : 20, 75 : 25, and 70 : 30 for butter bread (B Cyperus esculentus L) is a tuberous rhizome belonging to the Cyperaceae family of grass-like plants. The edible, almond-like tuber grows in temperate and several tropical regions and is rich in fiber and lipids comprising high content of mono- and polyunsaturated fatty acids, carbohydrates, and some proteins. The nut-like tuber is also rich in minerals, vitamins C and E, and several bioactive phytochemical compounds [Tiger nut , (Pride of the West) supplied by Iran brothers and others Ltd., Ghana, was obtained from the Kotokoraba Supermarket, Cape Coast, Ghana. Granulated sugar, commercially available bakery shortening (margarine), yeast, nutmeg, and salt commonly used for baking were obtained from the Kotokoraba Supermarket trading center at Cape Coast, Ghana.Tiger nuts were hand-picked to exclude discolored or broken pieces and stones and washed with water to eliminate dirt, adhering root hairs, and soil . To enhab), tea bread (Tb), and sugar bread (Sb) at five (5) levels, namely, 10%, 15%, 20%, 25%, or 30%. Then, the procedure for bread preparation reported by Aryeetey et al. [b and Sb with TNF compositions of 7.7%, 11.6%, 15.4%, 19.3%, and 23.2% and for Tb, 8.5%, 12.7%, 17.0%, 21.2%, and 25.4%. Bread prepared using only WF served as the control. For each type of bread, 1000 g dough was sliced, rolled, molded, and arranged in a baking pan and covered with a clean cloth in an enclosed room to allow for proofing at ambient temperature (28-32°C) for 60 min. Then, the dough was transferred to a Swiss baking oven maintained at 230°C for 30 min [For making tiger nut-substituted bread, a Minitab software was used for a centroid mixture design whereby WF was substituted with TNF for butter bread  using a hand-held portable colorimeter . The instrument was calibrated against the white and black tiles, and the mean values for lightness (L∗), red–green intensity (a∗), and yellow–blue intensity (b∗) were calculated from the color primaries. Chroma was computed using equation (Color properties of the bread crust and the crumb were measured based on the Hunter parameters (equation  accordin3 [v (cm3) is the volume and w (g) is the weight of the bread.The specific volume of bread was determined by measuring the weight and the volume by the displacement method according to the procedure by Ahemen et al.  with a f3 . Specifi3  using eqSurface firmness of bread crumb was measured on the first day of baking and at 24 h intervals for 3 d using a modified penetrometer  as described by Manohar and Rao  with slib, Bb, and Sb. Each type of bread had five WF:TNF substitution levels, namely, 90 : 10, 85 : 15, 80 : 20, 75 : 25, and 70 : 30. Bread prepared with only WF (100 : 0) served as the control. About 30 g samples were given random 3-digit codes and served to a randomly recruited 56-member panel  in a counterbalanced manner.Consumer acceptability was determined by using a 9-point hedonic scale as described by Lawless and Heymann . Attribub with TNF of 10%) and its analogs was chosen and flash-profiled [Based on the results of the hedonic study, the bread that showed the highest overall consumer acceptance (Bprofiled  to deterb with WF:TNF substitution ratios as follows: 100 : 0 (Bb), 90 : 10 (T10Bb), 85 : 15 (T15Bb), 80 : 20 (T20Bb), 75 : 25 (T25Bb), and 70 : 30 (T30Bb). An identical sample from T20Bb was included to assess the discriminatory quality. Samples were given 3-digit random codes and served in small trays. A bottle of water (300 mL) was added to rinse the mouth between samples. Raw data on the attributes and the corresponding intensities were analyzed by principal component and Generalized Procrustes Analyses using the Senstools. Net software . Results on hedonic assessment and flash profiling of bread attributes are based on single and duplicate experiments, respectively.A panel of 12 members  was randomly recruited and presented B10Bb), tea bread with 25% tiger nut flour (T25Tb), and sugar bread with 30% tiger nut flour (T30Sb), were carried out. Additionally, the composition of TNF was analyzed to help determine its contribution to the different bread samples. Proximate analysis was carried out as explained previously [N × 6.25) using the Kjeldahl method. Fat was determined by acid digestion followed by Soxhlet extraction with diethyl ether. Total fiber content was analyzed using the sequential acid and alkaline digestion method. Ash content was analyzed by combusting in a muffle furnace, and carbohydrates were determined by difference [Compositional analyses of WF bread and the TNF-substituted analogs that showed the highest consumer acceptance , namely,eviously . Brieflyfference . Resultsp < 0.05.To determine the effects of partially substituting wheat flour with tiger nut flour at a ratio of 100 : 0, 90 : 10, 85 : 15, 80 : 20, 75 : 25, and 70 : 30 for butter bread, tea bread, and sugar bread on the response parameters, a Minitab software was used. Data were evaluated using a one-way analysis of variance. Tukey HSD or Games-Howell post hoc analysis was used to compare the mean values where it is necessary. SPSS software package version 16.0 was used for performing the analysis . All significance statements refer to b) gave the highest browning index (BI) followed by that of tea bread (Tb) and butter bread (Bb). Different browning intensities of bread could be caused by the variations in sugar composition, which is known to caramelize and also react with amino acids in a Maillard process during baking, contributing golden brown color compounds to the product [r = 0.995 (Bb), r = 0.992 (Tb), and r = 0.989 , and a gradient or magnitude (BI/Unit) of 9.2 ± 0.04 (Bb), 12.5 ± 0.06 (Tb), and 15.8 ± 0.05 (Sb) for the bread crust, showing that tiger nut addition significantly impacts crust of Sb the most, followed by Tb and Bb. The crumb showed lower browning effects compared to the crust, and after adding 25% TNF, a more pronounced effect on the crumb brownness was observed. The results agree with the report according to Oke et al. [Figures  product . Substite et al. , which ie et al. . Here, wL∗) and Chroma of the bread crust and crumb in Figures L∗ and higher values for color saturation (Chroma) when compared to the other bread types without TNF. Crust and crumb lightness of Bb was comparable, whilst significantly lower values of crust lightness than crumb lightness were observed for Tb and Sb. TNF addition diminished L∗, showing more pronounced effects on the crust or the crumb after 20% TNF or 25% TNF addition, respectively. Additionally, TNF improved the redness (a∗) and yellowness (b∗) of bread, causing a more drastic effect on the color saturation of the crust than the crumb after 25% TNF addition. The effects of TNF substitution on the crust and crumb appearance could be relevant for modulating bread color properties. Browning and other color attributes play a major role in the consumer acceptability of bread, besides the contribution to flavour and aroma. However, the browning reactions can adversely affect nutritional value due to amino acid-simple sugar Maillard-type reactions and the possible formation of acrylamide-related compounds [Color primaries  was the highest followed by Tb (1.301 ± 0.003 cm3/g) and Sb (1.105 ± 1.002 cm3/g), which were higher than that reported by Oke et al. [b (5.74 ± 0.08 cm3/g) and Sb (4.14 ± 0.03 cm3/g) by Aryeetey et al. [b, Tb, or Sb, respectively, which was higher than that reported by Oke et al. [Specific volume is an intrinsic property of bread, which immensely influences consumers' choices .  Figuree et al.  but lowey et al. . The spey et al. . Substity et al. . Signifib (1.87 ± 0.01 N), Tb (3.46 ± 0.0 2 N), and Sb (6.71 ± 0.02 N) significantly increased to 2.46 ± 0.01 N, 13.12 ± 0.02 N, and 16.40 ± 0.02 N, respectively, after 3 d storage. Although differences in bread types exist, a similar range of bread hardness and storage-related increases were reported by Oke et al. [b, 2.36-fold for Tb, and 1.70-fold for Sb, showing that TNF contributes to bread texture differently. Furthermore, whilst storing T30Bb for 3 d did not significantly affect the crumb hardness (4.23 ± 0.01 N), that of T30Tb and T30Sb significantly increased to 17.80 ± 0.02 N and 21.08 ± 0.02 N, respectively. This shows that although TNF increases bread hardness as reported by Oke et al. [b and its TNF substituted analogs. Bread hardness is relevant as it affects consumers' perception of anticipated chewing and judgment and strongly influences product acceptability [Plane bread showed different levels of crumb hardness, which varied according to bread type and storage duration . Hardnese et al. , purporte et al. . Substite et al. , the effe et al. , probable et al.  and impae et al. , thus, ctability .b showed reasonably high scores for all attributes with an overall acceptance rating of 6.50 ± 0.33  of the overall acceptance.The impact of substituting WF with TNF on the sensory scores for bread is shown in 0 ± 0.33 . Nonetheb) had low consumer scores for all attributes with an overall acceptance of 4.81 ± 0.27 , 25% TNF for Tb (T25Tb), and 30% TNF for Sb (T30Sb), are shown in b, 21.09 ± 0.13%, Tb, 26.02 ± 0.03%, and Sb, 26.70 ± 0.39%. The moisture values were comparable to that reported by Oke et al. [10Bb, 21.83 ± 0.07%; T25Tb, 26.87 ± 0.4%, and T30Sb, 25.73 ± 0.04%. The low moisture of Bb and T10Bb may be attributed to the high margarine content, which is known to decrease water binding because of the coating effects, rendering a higher propensity to bake-related moisture loss [Nutrient composition of Be et al. , althouge et al. , this sture loss . Low moiure loss .The nutrient composition of the bread was within the range reported by Maietti et al.  and Oke b), tea bread (Tb), and sugar bread (Sb) to determine the bread type that is more amenable to tiger nut incorporation. Generally, substituting WF with TNF increased the brownness and color saturation, decreased bread lightness with a greater influence on the crust than the crumb, and showed the greatest impact on Sb, followed by Tb and Bb. The incorporation of TNF in bread progressively decreased the specific volume. The amount of TNF that was required to significantly impact specific bread volume was the highest for Sb followed by Tb and Bb. Furthermore, bread hardness increased with TNF substitution, showing the greatest impacts on Tb, followed by Bb and Sb. Although ambient storage further increased bread crumb hardness of Tb and Sb, that of Bb did not significantly change. Regarding consumer evaluation, substituting Bb with 10%TNF gave the highest overall acceptance. Consumer scores for Tb were generally low; however, adding TNF significantly improved the scores for all attributes, and substitution using 25% TNF gave the highest scores for overall acceptance. Descriptors for Bb improved after substituting WF with TNF. Thus, WF substitution with TNF showed that Bb with 10% or Tb with 25% TNF is more comparable with the overall acceptance quality of 100% WF. The results are relevant for enhancing the utility of tiger nuts as an ingredient in bread processing to generate fiber-rich, palatable, and probably, more affordable bread alternatives. Future studies would assess the cost of tiger nut bread production and willingness-to-pay among bread consumers.The feasibility of tiger nut utilization for bread processing was experimented by partially substituting wheat flour (WF) with tiger nut flour (TNF) at a ratio of 100 : 0, 90 : 10, 85 : 15, 80 : 20, 75 : 25, and 70 : 30 for butter bread (B


The aim of the study was to distinguish the hypokinetic gait disorder in idiopathic normal pressure hydrocephalus (NPH) patients from the gait decline in the elderly population by quantifying pathological gait parameter thresholds utilizing a multiple condition gait assessment. 55 NPH patients and 55 age-matched healthy subjects underwent a standardized gait assessment with eight gait conditions. Spatiotemporal gait parameters were assessed through a pressure-sensitive carpet. Statistical analysis consisted of a binary logistic regression (BLR) model, logistic curve-fit evaluated by a Chi-square goodness-of-fit-test, receiver operating characteristic models with area under the curves (AUC), and inverse BLR. Most discriminative gait parameter thresholds were observed in pace, gait cycle, and support gait domains. The most distinct gait conditions were preferred walking speed and semantic dual task. During preferred walking speed, the most significant gait parameter thresholds were stride length ≤ 1.02 m (sensitivity 0.93/specificity 0.91/AUC 0.96), gait velocity ≤ 0.83 m/s (0.80/0.91/0.93), double support phase ≥ 27.0% (0.96/0.76/0.91), and stride length coefficient of variation ≥ 3.4% (0.93/0.72/0.90). In conclusion, the hypokinetic gait disorder in NPH can be quantitatively differentiated from gait patterns of the elderly population. In future studies, this approach may be useful to differentiate clinical entities with similar gait disorders utilizing instrumented gait analysis procedures. Impaired circulation of cerebrospinal fluid (CSF) and mostly normal to mildly increased cerebrospinal fluid pressure are considered to play a key role for the emergence of disease, thus, shunt surgery represents a potent and causative treatment option2.Idiopathic normal pressure hydrocephalus (NPH) is a chronic neurological disease characterized by gait disturbance, dementia, and urinary incontinence, which was first described by Adam and Hakim in the 1960s5. Although the overall prevalence appears to be low, the clinical routine often comprises an overestimation of suspected NPH. With the gait disorder being most prominent in NPH7, neurologists and neurosurgeons are challenged to identify and integrate gait features into differential diagnosis for NPH.Even though epidemiological data is sparse, NPH is a disease of the elderly population and is estimated to occur most frequently above the age of 60 years11. However, these features are ambiguous since they also occur as a result of the age-related decline in gait performance14. This may complicate clinical identification of NPH-related gait impairments, particularly during early stages of disease. Furthermore, a comprehensive and objectified quantification of gait performance in NPH is not only crucial for differential diagnosis, but also for the selection of eligible candidates for shunt placement and the monitoring of intervention effects after shunt surgery. There is an ongoing debate, whether the examination of gait performance in NPH during sensory disturbed or motor-cognitive dual tasks might increase the diagnostic power17. As common in patients with motor-cognitive gait disturbances, a decline of walking performance or the “stops walking while talking” phenomenon have been described in several studies18.The gait disorder in NPH typically represents a hypokinetic gait pattern with slow gait velocity, decreased step length, increased double support phase, and stride widthTo analytically evaluate the discriminative power of each collected gait feature with respect to a distinctive threshold with optimal balanced sensitivity and specificity for the identification of NPH in an elderly cohort.To rank each feature with respect to its discriminative validity in order to inform clinicians about the most relevant gait parameters and examination conditions for this clinical decision setting.Therefore, we quantitatively characterized gait performance of patients with NPH and age-matched healthy controls during a comprehensive multiple condition gait assessment including walking with different speeds, sensory perturbations and walking with a second cognitive task . Based on this, the aim of this study was twofold:All study procedures have been approved by the Ethics Committee of the University of Munich (reference number 34-16). The study was in accordance with the Declaration of Helsinki in its newest revision. Informed consent was obtained from all subjects and/or their legal guardian(s).20. Exclusion criteria were secondary forms of hydrocephalus, other primary causes of gait impairment  and the inability to walk independently. Patients were followed up in the course of disease with evaluation of shunt candidacy  and if they subsequently underwent a VP shunt surgery. For the control group, 55 age-matched healthy subjects  were recruited. Exclusion criteria were any morbidities with significant impact on locomotion.One hundred ninety-four adult patients, who presented due to suspected NPH symptoms, were screened in our clinic. Fifty-five patient with NPH  with a mean duration of symptoms of 2.0 ± 1.5 years during the first clinical visit were included in the study according to previously published guidelines and criteria21: (1) Pace: velocity (m/s), stride length (m), stride time (s). (2) Cycle: swing phase (%), double support phase (%). (3) Variability: stride length coefficient of variation (CV) (%), stride time CV (%), swing phase CV (%). (4) Asymmetry: stride length asymmetry (%), stride time asymmetry (%), swing phase asymmetry (%). (5) Support: stride width (m), stride width CV (%). Occurrences of “stops walking while talking” and freezing of gait phenomena were identified by two independent investigators (KM and RS) based on video recordings.Gait performance was investigated using a pressure-sensitive sensor carpet  and a concomitant 2D video recording. Patients and healthy subjects underwent a standardized gait protocol with eight different gait conditions: walking in preferred speed (PS), slow speed (SS), and maximum walking speed (MS); walking during head reclination (HR) and eyes closed (EC); walking during cognitive calculatory dual task , semantic dual task , and motoric dual task . For each condition, spatiotemporal gait parameters were calculated and analyzed. Parameters are summarized in five independent gait domains, which are based on a previous study22. For each collected gait feature a binary logistic regression model (BLR) was performed with respect to the binary outcome variable . The validity of the logistic curve-fit was evaluated using a Chi-square goodness-of-fit test. Classification accuracy of the regression model was evaluated based on a receiver operating characteristic (ROC) procedure with respect to the area under the curve (AUC). The optimal operating point of the ROC curve with balanced sensitivity and specificity levels was determined. This point was then fed into an inverse BLR model to determine the optimal discrimination threshold of each gait feature. The statistical methodology is visualized in Fig. p-value ≤ 0.05.Descriptive statistics are presented as mean ± standard deviation (SD). Statistical analysis was performed in correspondence to a previously proposed procedureNPH patient characteristics are depicted in Table The statistical approach is visualized in Fig. During preferred walking speed, the most distinct gait thresholds were variables in the Pace domain, namely stride length ≤ 1.02 m  and gait velocity ≤ 0.83 m/s . Other significant variables include Cycle variables such as swing phase ≤ 36.5%  and double support phase ≥ 27.0%  as well as the Support variable stride width ≥ 0.13 m . The most distinct variability parameter was stride length CV ≥ 3.4% . Asymmetry parameters showed non-significant to only moderate discriminative power.The most noticeable thresholds were observed during DTS: Stride length ≤ 0.97 m , swing phase ≤ 35.1% , double support phase ≥ 30.2% , and stride width ≥ 0.13 m . During EC, the gait velocity threshold was ≤ 0.69 m/s . The mean AUC values of all gait conditions for each gait parameter ranged from 0.52 (stride time asymmetry) to 0.94 (stride length). Further high overall AUC values were found for velocity (0.92) and for double support phase  to 0.84 (DTM). AUC values for PS and HR were 0.82. Highest single AUC values were found in DTS , DTM , EC , and PS .The most distinct dual task condition throughout all gait parameters was DTM with following noticeable threshold parameters: gait velocity ≤ 0.87 m/s , stride length ≤ 1.03 m , swing phase ≤ 35.5% , double support phase ≥ 28.0% , stride length CV ≥ 3.7% , and stride width ≥ 0.12 m .The results of this study reveal that instrumented gait assessment is able to discriminate hypokinetic gait features of patients with NPH from walking performance of elderly healthy subjects with high accuracy. We found that the most discriminative gait parameters for NPH are present in gait features that are readily and easy to obtain by clinical gait assessment . In addition, we observed that the variation of gait examination conditions affords a higher discriminatory power for single gait features. Most prominent examples were found for verbal fluency and motoric dual tasking.24. Parameters of the Pace gait domain  show the highest discriminatory power and best diagnostic values.Outcomes of spatiotemporal gait parameters are consistent with previous studies of the hypokinetic gait disorder in NPH. Typical features are reduced stride lengths, a decreased walking speed, elongated double support phases, and a broadened base of support26. Some gait parameters of the healthy control group seem to differ slightly among several publication , which might be due to varying baseline characteristics and/or a different gait assessment.The estimated thresholds for spatiotemporal gait parameters lie mostly between previously reported gait parameter mean values of NPH patients and healthy subjects28. Other studies revealed that the evaluation of gait performance during single tasks is sufficient29. In line with this, the present results reveal a good to excellent classification accuracy across all examined conditions with highest mean AUC values for preferred walking and walking with a motoric dual tasking. In accordance to the latter finding, a comparative study between NPH and progressive supranuclear palsy  patients revealed that motor dual task walking is the best condition to differentiate both diseases17. For Pace domain parameters such as velocity and stride length, our results reveal approximately equal to even higher values during dual task in healthy subjects.Expanding the clinical gait assessment by additional examination conditions is useful for selected clinical settings, such as the early identification of patients with mild cognitive impairments. Previous studies emphasized the importance of examining dual task performance during evaluation of shunt candidacy in NPH during CSF tap testFurthermore, single gait parameters show a very high discriminatory power and excellent sensitivity and specificity values in selected examination conditions. Most prominent is the evaluation of stride length during verbal fluency dual task walking. This finding might indicate that the utilization of these features might serve as a key diagnostic test to differentiate NPH from gait patterns of the healthy elderly population.30, further studies are necessary to extend our knowledge regarding clinical meaningful gait thresholds for an older elderly population.This study has several limitations. First, the reported gait parameter thresholds are established during a highly standardized walking examination procedure of steady-state walking. The thresholds cannot reflect parameters of walking in complex environmental situations or off-laboratory measurements. Therefore, it will in future be important to apply analogous ROC analysis procedures to gait data from other instrument-based sources . Second, the NPH cohort consisted of patients with a mean age of 72.6 ± 4.7 years indicating that the calculated thresholds should be applied to patients in this age range. Due to the demographics of NPH with patients older than 80 yearsIn conclusion, the present study provides a comprehensive and systematic overview of clinical thresholds for spatial and temporal gait parameters that are useful for the diagnosis of NPH. We found that parameters of the pace domain during walking with self-selected speed and under dual task paradigms show the highest discriminatory power and excellent sensitivity and specificity for distinguishing the gait of patients with NPH from that of the healthy elderly walkers. By applying these analysis techniques to cohorts of clinical entities with similar gait patterns, future studies might support and promote the clinical status of instrumented gait assessment procedures for differential diagnosis.Supplementary Information.