A body map of somatic mutagenesis in morphologically normal human tissues.

Somatic mutations that accumulate in normal tissues are associated with ageing and disease1,2. Here we performed a comprehensive genomic analysis of 1,737 morphologically normal tissue biopsies of 9 organs from 5 donors. We found that somatic mutation accumulations and clonal expansions were widespread, although to variable extents, in morphologically normal human tissues. Somatic copy number alterations were rarely detected, except for in tissues from the oesophagus and cardia. Endogenous mutational processes with the SBS1 and SBS5 mutational signatures are ubiquitous among normal tissues, although they exhibit different relative activities. Exogenous mutational processes operate in multiple tissues from the same donor. We reconstructed the spatial somatic clonal architecture with sub-millimetre resolution. In the oesophagus and cardia, macroscopic somatic clones that expanded to hundreds of micrometres were frequently seen, whereas in tissues such as the colon, rectum and duodenum, somatic clones were microscopic in size and evolved independently, possibly restricted by local tissue microstructures. Our study depicts a body map of somatic mutations and clonal expansions from the same individual.

AWANet: Attentive-Aware Wide-Kernels Asymmetrical Network with Blended Contour Information for Salient Object Detection.

Although deep learning-based techniques for salient object detection have considerably improved over recent years, estimated saliency maps still exhibit imprecise predictions owing to the internal complexity and indefinite boundaries of salient objects of varying sizes. Existing methods emphasize the design of an exemplary structure to integrate multi-level features by employing multi-scale features and attention modules to filter salient regions from cluttered scenarios. We propose a saliency detection network based on three novel contributions. First, we use a dense feature extraction unit (DFEU) by introducing large kernels of asymmetric and grouped-wise convolutions with channel reshuffling. The DFEU extracts semantically enriched features with large receptive fields and reduces the gridding problem and parameter sizes for subsequent operations. Second, we suggest a cross-feature integration unit (CFIU) that extracts semantically enriched features from their high resolutions using dense short connections and sub-samples the integrated information into different attentional branches based on the inputs received for each stage of the backbone. The embedded independent attentional branches can observe the importance of the sub-regions for a salient object. With the constraint-wise growth of the sub-attentional branches at various stages, the CFIU can efficiently avoid global and local feature dilution effects by extracting semantically enriched features via dense short-connections from high and low levels. Finally, a contour-aware saliency refinement unit (CSRU) was devised by blending the contour and contextual features in a progressive dense connected fashion to assist the model toward obtaining more accurate saliency maps with precise boundaries in complex and perplexing scenarios. Our proposed model was analyzed with ResNet-50 and VGG-16 and outperforms most contemporary techniques with fewer parameters.

Identification of anti-inflammatory active ingredients from Tumuxiang by ultra-performance liquid chromatography/quadrupole time-of-flight-MSE.

The dried roots of Inula helenium L. (IH) and Inula racemosa Hook f. (IR) are used commonly as folk medicine as 'tumuxiang' (TMX). The mixing and sharing of IH and IR in clinical use is a universal phenomenon. Modern pharmacological studies confirmed that IH and IR display anti-inflammatory activities. However, the difference in anti-inflammatory pharmacodynamic substances between these two herbs is still unknown. In the present study, the fingerprints of 18 IH and nine IR samples were established using UPLC/QTOF-MSE . A dimethylbenzene-induced mouse ear vasodilation model was applied in evaluating the anti-inflammatory properties of all 27 samples. Then, the spectrum-efficacy model between chemical characteristic peaks and anti-inflammatory activities was investigated using principal component regression and partial least squares. Finally, the combination of UNIFI Scientific Information System with a library search of traditional Chinese medicines was employed to automatically characterize the peaks. UNIFI identified a total of 80 chemical components. Among the components, the 53 characteristic peaks showed correlation with anti-inflammatory activities, pointing to phenolic and organic acids as primary anti-inflammatory ingredients of TMX. This approach can efficiently and intelligently facilitate the identification of bioactive components from traditional Chinese medicine.

Genome-wide survey of Aux/IAA gene family members in potato (Solanum tuberosum): Identification, expression analysis, and evaluation of their roles in tuber development.

The Auxin/indole-3-acetic acid (Aux/IAA) genes encode short-lived nuclear proteins that are known to be involved in the primary cellular responses to auxin. To date, systematic analysis of the Aux/IAA genes in potato (Solanum tuberosum) has not been conducted. In this study, a total of 26 potato Aux/IAA genes were identified (designated from StIAA1 to StIAA26), and the distribution of four conserved domains shared by the StIAAs were analyzed based on multiple sequence alignment and a motif-based sequence analysis. A phylogenetic analysis of the Aux/IAA gene families of potato and Arabidopsis was also conducted. In order to assess the roles of StIAA genes in tuber development, the results of RNA-seq studies were reformatted to analyze the expression patterns of StIAA genes, and then verified by quantitative real-time PCR. A large number of StIAA genes (12 genes) were highly expressed in stolon organs and in during the tuber initiation and expansion developmental stages, and most of these genes were responsive to indoleacetic acid treatment. Our results suggested that StIAA genes were involved in the process of tuber development and provided insights into functional roles of potato Aux/IAA genes.

[Progress in research of traditional Chinese medicine Citrus aurantium].

Citrus aurantium is one of the most common traditional Chinese medicines. In this paper, the chemical components, content determination and pharmacological actions of C. aurantium were summarized for the comprehensive utilization of its resources. Because of the complicated resources of C. aurantium, only one single component as index couldn't reflect the quality and effects and comprehensive evaluation which concluding multiple components should be established in the future quality control. In recent years, the pharmacological effects research of C. aurantium has made tremendous progress, and it is important to explore new drugs from the development and utilization of the active ingredient of C. aurantium. In recent years, the pharmacological effects research of C. aurantium has made tremendous progress, and it is important to explore new drugs from the development and utilization of the active ingredient of C. aurantium.

Model-agnostic counterfactual reasoning for identifying and mitigating answer bias in knowledge tracing.

Knowledge tracing (KT) aims to monitor students' evolving knowledge states through their learning interactions with concept-related questions, and can be indirectly evaluated by predicting how students will perform on future questions. In this paper, we observe that there is a common phenomenon of answer bias, i.e., a highly unbalanced distribution of correct and incorrect answers for each question. Existing models tend to memorize the answer bias as a shortcut for achieving high prediction performance in KT, thereby failing to fully understand students' knowledge states. To address this issue, we approach the KT task from a causality perspective. A causal graph of KT is first established, from which we identify that the impact of answer bias lies in the direct causal effect of questions on students' responses. A novel COunterfactual REasoning (CORE) framework for KT is further proposed, which separately captures the total causal effect and direct causal effect during training, and mitigates answer bias by subtracting the latter from the former in testing. The CORE framework is applicable to various existing KT models, and we implement it based on the prevailing DKT, DKVMN, and AKT models, respectively. Extensive experiments on three benchmark datasets demonstrate the effectiveness of CORE in making the debiased inference for KT. We have released our code at https://github.com/lucky7-code/CORE.

Determinants of gastric cancer immune escape identified from non-coding immune-landscape quantitative trait loci.

The landscape of non-coding mutations in cancer progression and immune evasion is largely unexplored. Here, we identify transcrptome-wide somatic and germline 3' untranslated region (3'-UTR) variants from 375 gastric cancer patients from The Cancer Genome Atlas. By performing gene expression quantitative trait loci (eQTL) and immune landscape QTL (ilQTL) analysis, we discover 3'-UTR variants with cis effects on expression and immune landscape phenotypes, such as immune cell infiltration and T cell receptor diversity. Using a massively parallel reporter assay, we distinguish between causal and correlative effects of 3'-UTR eQTLs in immune-related genes. Our approach identifies numerous 3'-UTR eQTLs and ilQTLs, providing a unique resource for the identification of immunotherapeutic targets and biomarkers. A prioritized ilQTL variant signature predicts response to immunotherapy better than standard-of-care PD-L1 expression in independent patient cohorts, showcasing the untapped potential of non-coding mutations in cancer.

A single-cell atlas characterizes dysregulation of the bone marrow immune microenvironment associated with outcomes in multiple myeloma.

Multiple Myeloma (MM) remains incurable despite advances in treatment options. Although tumor subtypes and specific DNA abnormalities are linked to worse prognosis, the impact of immune dysfunction on disease emergence and/or treatment sensitivity remains unclear. We established a harmonized consortium to generate an Immune Atlas of MM aimed at informing disease etiology, risk stratification, and potential therapeutic strategies. We generated a transcriptome profile of 1,149,344 single cells from the bone marrow of 263 newly diagnosed patients enrolled in the CoMMpass study and characterized immune and hematopoietic cell populations. Associating cell abundances and gene expression with disease progression revealed the presence of a proinflammatory immune senescence-associated secretory phenotype in rapidly progressing patients. Furthermore, signaling analyses suggested active intercellular communication involving APRIL-BCMA, potentially promoting tumor growth and survival. Finally, we demonstrate that integrating immune cell levels with genetic information can significantly improve patient stratification.

Sarcolemmal localisation of Na+/H+ exchange and Na+-HCO3- co-transport influences the spatial regulation of intracellular pH in rat ventricular myocytes.

Membrane acid extrusion by Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC) is essential for maintaining a low cytoplasmic [H(+)] (∼60 nm, equivalent to an intracellular pH (pHi) of 7.2). This protects myocardial function from the high chemical reactivity of H(+) ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pHi regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 m formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pHi imaging reveals that NBC stimulation induces spatially uniform pHi recovery from acidosis, whereas NHE1 stimulation induces pHi non-uniformity during recovery (of ∼0.1 units, for 2-3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pHi microdomains is favoured by low cytoplasmic H(+) mobility and long H(+) diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H(+)]i regulation in ventricular myocytes, suggesting that, by guarding pHi, NHE1 preferentially protects gap junctional communication at intercalated discs, while NBC locally protects t-tubular excitation-contraction coupling.

[Retrospective review of airway obstruction in 19 infants with pharyngeal cysts].

Objective:To discuss the clinical characteristic and treatment of laryngeal cysts in infants. Methods:The clinical data of 19 patients diagnosed with laryngeal cysts in Department of Otolaryngology, Sichuan Provincial Maternity and Child Health Care Hospital from November 2017 to April 2023 were retrospectively analyzed. Results:All of the 19 patients were diagnosed as laryngeal cysts, with clinical manifestations included respiratory distress, inspiratory dyspnea, difficulty in feeding and low and weak crying, etc. All of them were cured after surgical treatment. Conclusion:Misdiagnosis and missed diagnosis of laryngeal cysts are prone to occur in infants and young children. After diagnosis, patients should undergo early surgical treatment to remove airway obstruction and improve ventilation.

Multi-level advances in databases related to systems pharmacology in traditional Chinese medicine: a 60-year review.

The therapeutic effects of traditional Chinese medicine (TCM) involve intricate interactions among multiple components and targets. Currently, computational approaches play a pivotal role in simulating various pharmacological processes of TCM. The application of network analysis in TCM research has provided an effective means to explain the pharmacological mechanisms underlying the actions of herbs or formulas through the lens of biological network analysis. Along with the advances of network analysis, computational science has coalesced around the core chain of TCM research: formula-herb-component-target-phenotype-ZHENG, facilitating the accumulation and organization of the extensive TCM-related data and the establishment of relevant databases. Nonetheless, recent years have witnessed a tendency toward homogeneity in the development and application of these databases. Advancements in computational technologies, including deep learning and foundation model, have propelled the exploration and modeling of intricate systems into a new phase, potentially heralding a new era. This review aims to delves into the progress made in databases related to six key entities: formula, herb, component, target, phenotype, and ZHENG. Systematically discussions on the commonalities and disparities among various database types were presented. In addition, the review raised the issue of research bottleneck in TCM computational pharmacology and envisions the forthcoming directions of computational research within the realm of TCM.

Accumulation of dissolved organic matter in the transition from fresh to aged seasonal snow in an industrial city in NE China.

Dissolved organic matter (DOM) plays a significant role in the reduction of snow albedo and the acceleration of snowmelt, but its accumulation in snow remains poorly understood. This study investigated the accumulation of DOM in seasonal snow including its accumulation rate, molecular characteristics, and biological and chemical processing. Sixteen snow samples of both fresh and aged snow were collected at one-day interval in Changchun, a typical industrial city in NE China. The snow DOM contents increased linearly with accumulation time at a rate of 30.3 µg L-1 d-1. The optical properties, including fluorescence intensity and optical absorption coefficient, of snowmelt increased exponentially with time owing to the rapid accumulation of terrestrial humic-like fluorophores through snow-soil exchange and deposition of soil-derived substances. Fourier transform-ion cyclotron resonance-mass spectrometry highlighted the properties of DOM at a molecular level, indicating that compounds derived from underlying soil and vascular plants make the largest contribution to DOM. Microbe-derived compounds contribute 35.5 % to the DOM pool. Degrees of saturation and oxidation increase slightly after accumulation, with the impacts of photo- and bio-chemistry on DOM molecules being non-negligible. This study provides a new perspective concerning the accumulation and fate of organic contaminants in snow ecosystems.

Epithelial cells activate fibroblasts to promote esophageal cancer development.

Esophageal squamous-cell carcinoma (ESCC) develops through multistage epithelial cancer formation, i.e., from normal epithelium, low- and high-grade intraepithelial neoplasia to invasive carcinoma. However, how the precancerous lesions progress to carcinoma remains elusive. Here, we report a comprehensive single-cell RNA sequencing and spatial transcriptomic study of 79 multistage esophageal lesions from 29 patients with ESCC. We reveal a gradual and significant loss of ANXA1 expression in epithelial cells due to its transcription factor KLF4 suppression along the lesion progression. We demonstrate that ANXA1 is a ligand to formyl peptide receptor type 2 (FPR2) on fibroblasts that maintain fibroblast homeostasis. Loss of ANXA1 leads to uncontrolled transformation of normal fibroblasts into cancer-associated fibroblasts (CAFs), which can be enhanced by secreted TGF-ß from malignant epithelial cells. Given the role of CAFs in cancer, our study underscores ANXA1/FPR2 signaling as an important crosstalk mechanism between epithelial cells and fibroblasts in promoting ESCC.

Comprehensive Analyses Reveal Effects on Tumor Immune Infiltration and Immunotherapy Response of APOBEC Mutagenesis and Its Molecular Mechanisms in Esophageal Squamous Cell Carcinoma.

The apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) mutagenesis is prevalent in esophageal squamous cell carcinoma (ESCC). However, the functional role of APOBEC mutagenesis has yet to be fully delineated. To address this, we collect matched multi-omics data of 169 ESCC patients and evaluate characteristics of immune infiltration using multiple bioinformatic approaches based on bulk and single-cell RNA sequencing (scRNA-seq) data and verified by functional assays. We find that APOBEC mutagenesis prolongs overall survival (OS) of ESCC patients. The reason for this outcome is probably due to high anti-tumor immune infiltration, immune checkpoints expression and immune related pathway enrichment, such as interferon (IFN) signaling, innate and adaptive immune system. The elevated AOBEC3A (A3A) activity paramountly contributes to the footprints of APOBEC mutagenesis and is first discovered to be transactivated by FOSL1. Mechanistically, upregulated A3A exacerbates cytosolic double-stranded DNA (dsDNA) accumulation, thus stimulating cGAS-STING pathway. Simultaneously, A3A is associated with immunotherapy response which is predicted by TIDE algorithm, validated in a clinical cohort and further confirmed in mouse models. These findings systematically elucidate the clinical relevance, immunological characteristics, prognostic value for immunotherapy and underlying mechanisms of APOBEC mutagenesis in ESCC, which demonstrate great potential in clinical utility to facilitate clinical decisions.

Flavokawain B induces apoptosis of non-small cell lung cancer H460 cells via Bax-initiated mitochondrial and JNK pathway.

Flavokawain B (FKB) possesses strong anti-neoplastic activity against many cancer cells. Here we assessed its antitumor activity and molecular mechanisms in lung cancer H460 cells in vitro. FKB significantly inhibited cell proliferation and caused arrest of the cell cycle G2-M of H460 cells in a dose-dependent manner. FKB also inducted apoptosis, which was associated with cytochrome c release, caspase-7 and caspase-9 activation and Bcl-xL/Bax dys-regulation. FKB significantly down-regulated survivin and XIAP, and the inhibitory effect induced by FKB was greatly attenuated by through over-expression of survivin or Bax(-/-) MEFs. Furthermore, FKB activated the mitogen-activated protein kinases and the JNK inhibitor SP600125 significantly decreased the growth-inhibitory and apoptotic effects of FKB. Together, these results suggest the anti-lung cancer potential of flavokawain B for the prevention and treatment of lung cancer.

Metabolomic Analysis Reveals Changes of Bioactive Compounds in Mung Beans (Vigna radiata) during γ-Aminobutyric Acid Enrichment Treatment.

Soaking together with Heat and Relative Humidity (HRH) treatment has been applied successfully to enrich γ-aminobutyric acid (GABA) in mung beans. However, whether and how the above GABA enrichment processing influences the other bioactive molecules is elusive. In the present study, mung beans were soaked and then treated by HRH for 5 or 7 h. By using metabolomics techniques, the changes of 496 metabolites were determined. The relative content of flavonoids and phenolic acids increased during soaking but slightly decreased during HRH. Intriguingly, soaking and HRH had the opposite effects on the glycosylation of polyphenols. The relative content of glycosylated or un-glycosylated polyphenols increased during soaking or HRH, respectively. The relative content of α-ketoglutaric acid increased more than 20 times after 5 h HRH treatment. Bioactive molecules could be enriched during GABA enrichment processing. Depending on the desired bioactive compounds, soaking and different duration of HRH treatment could be selected.

Preparation of formate-free PMA@MOF-808 catalysts for deep oxidative desulfurization of model fuels.

Due to the increasingly serious environmental problems caused by the combustion of sulfides in fuel, deep desulfurization of fuel became particularly urgent. Herein, the catalyst (PMA@MOF-808) of the Zr-based metal-organic framework (MOF-808) encapsulating phosphomolybdic acid (PMA) was prepared via a one-pot hydrothermal method. Besides, the formate ions of PMA@MOF-808 were removed by posttreatment with methanol, resulting in formate-free PMA@MOF-808-H catalysts with unsaturated open metal sites. The as-synthesized catalysts were systematically characterized by XRD, FT-IR, SEM, BET, TGA, 1H NMR and XPS. The catalysts were also applied in catalytic oxidation desulfurization of fuel. The results indicated that the introduction of PMA and the removal of formate ions can improve the desulfurization performance of catalysts. Formate-free 0.2-PMA@MOF-808-H catalyst can reach 100% desulfurization rate for DBT. Besides, the kinetic properties were studied, and the apparent activation energy was 29.34 kJ/mol.

Tri-Branch Convolutional Neural Networks for Top-k Focused Academic Performance Prediction.

Academic performance prediction aims to leverage student-related information to predict their future academic outcomes, which is beneficial to numerous educational applications, such as personalized teaching and academic early warning. In this article, we reveal the students' behavior trajectories by mining campus smartcard records, and capture the characteristics inherent in trajectories for academic performance prediction. Particularly, we carefully design a tri-branch convolutional neural network (CNN) architecture, which is equipped with rowwise, columnwise, and depthwise convolutions and attention operations, to effectively capture the persistence, regularity, and temporal distribution of student behavior in an end-to-end manner, respectively. However, different from existing works mainly targeting at improving the prediction performance for the whole students, we propose to cast academic performance prediction as a top-k ranking problem, and introduce a top-k focused loss to ensure the accuracy of identifying academically at-risk students. Extensive experiments were carried out on a large-scale real-world dataset, and we show that our approach substantially outperforms recently proposed methods for academic performance prediction. For the sake of reproducibility, our codes have been released at https://github.com/ZongJ1111/Academic-Performance-Prediction.

Effect of an Environment Friendly Heat and Relative Humidity Approach on γ-Aminobutyric Acid Accumulation in Different Highland Barley Cultivars.

In this study, heat and relative humidity (HRH) treatment was applied in highland barley for γ-aminobutyric acid (GABA) accumulation. Tibetan highland barley cultivars (25) were selected for comparison and analysis. HRH treatment could accumulate GABA in several hours with low moisture content and high temperature, and the grains were treated for 2.5 h at 65 °C in this study. The GABA content of processed grains under HRH optimal condition ranged from 26.91 to 76.28 mg·100 g−1, which was significantly higher than the initial content (12.78−43.00 mg·100 g−1). The highest GABA accumulation capacity was observed in two-row yellow cultivars (YT1), increasing from 36.52 to 76.28 mg·100 g−1. Correlation analysis showed that the accumulation of GABA after HRH treatment was positively and significantly (p < 0.05) correlated with the contents of protein (0.52), total free amino acids (0.68), threonine (0.53), serine (0.51), glutamate (0.69), glycine (0.49), alanine (0.46), cysteine (0.57), tyrosine (0.50), lysine (0.53), proline (0.40), and glutamate decarboxylase (GAD) activity (0.62), which were closely related to GABA-shunt pathway. The polyamines contents, diamine oxidase (DAO) and polyamine oxidase (PAO) activities, as the substrates and critical enzymes of polyamine degradation pathway, showed no significant correlation with GABA accumulation. The results suggested that the main pathway of GABA accumulation in highland barley under HRH treatment was GABA-shunt pathway.

Characterization of genomic alterations and neoantigens and analysis of immune infiltration identified therapeutic and prognostic biomarkers in adenocarcinoma at the gastroesophageal junction.

Objectives: Adenocarcinoma at the gastroesophageal junction (ACGEJ) refers to a malignant tumor that occurs at the esophagogastric junction. Despite some progress in targeted therapies for HER2, FGFR2, EGFR, MET, Claudin 18.2 and immune checkpoints in ACGEJ tumors, the 5-year survival rate of patients remains poor. Thus, it is urgent to explore genomic alterations and neoantigen characteristics of tumors and identify CD8+ T-cell infiltration-associated genes to find potential therapeutic targets and develop a risk model to predict ACGEJ patients' overall survival (OS). Methods: Whole-exome sequencing (WES) was performed on 55 paired samples from Chinese ACGEJ patients. Somatic mutations and copy number variations were detected by Strelka2 and FACETS, respectively. SigProfiler and SciClone were employed to decipher the mutation signature and clonal structure of each sample, respectively. Neoantigens were predicted using the MuPeXI pipeline. RNA sequencing (RNA-seq) data of ACGEJ samples from our previous studies and The Cancer Genome Atlas (TCGA) were used to identify genes significantly associated with CD8+ T-cell infiltration by weighted gene coexpression network analysis (WGCNA). To construct a risk model, we conducted LASSO and univariate and multivariate Cox regression analyses. Results: Recurrent MAP2K7, RNF43 and RHOA mutations were found in ACGEJ tumors. The COSMIC signature SBS17 was associated with ACGEJ progression. CCNE1 and VEGFA were identified as putative CNV driver genes. PI3KCA and TP53 mutations conferred selective advantages to cancer cells. The Chinese ACGEJ patient neoantigen landscape was revealed for the first time, and 58 potential neoantigens common to TSNAdb and IEDB were identified. Compared with Siewert type II samples, Siewert type III samples had significant enrichment of the SBS17 signature, a lower TNFRSF14 copy number, a higher proportion of samples with complex clonal architecture and a higher neoantigen load. We identified 10 important CD8+ T-cell infiltration-related Hub genes (CCL5, CD2, CST7, GVINP1, GZMK, IL2RB, IKZF3, PLA2G2D, P2RY10 and ZAP70) as potential therapeutic targets from the RNA-seq data. Seven CD8+ T-cell infiltration-related genes (ADAM28, ASPH, CAMK2N1, F2R, STAP1, TP53INP2, ZC3H3) were selected to construct a prognostic model. Patients classified as high risk based on this model had significantly worse OS than low-risk patients, which was replicated in the TCGA-ACGEJ cohort. Conclusions: This study provides new neoantigen-based immunotherapeutic targets for ACGEJ treatment and effective disease prognosis biomarkers.