Research progress of treating hyperuricemia in rats and mice with traditional Chinese medicine.

Hyperuricemia (HUA) is a common chronic metabolic disease caused by abnormal purine metabolism and uric acid excretion. Despite extensive research on HUA, no clear treatment has been found so far. Improving purine metabolism and promoting uric acid excretion is crucial for the effective treatment of HUA. In recent years, traditional Chinese medicine and traditional Chinese medicine prescriptions have shown good effects in treating HUA. This article summarizes the latest progress in treating HUA in rats and mice using traditional Chinese medicine and prescriptions, elaborates on the pathogenesis of HUA, explores the application of commonly used traditional Chinese medicine treatment methods and prescriptions, and discusses the previous pharmacological mechanisms. In general, our research indicates that traditional Chinese medicine can effectively relieve the symptoms related to elevated uric acid levels in HUA rats and mice. However, further exploration and research are needed to verify its efficacy, safety, and feasibility.

Correction: A ternary system of meloxicam with matching hydrophilic polymer and cyclodextrin for improved stability in liquid preprations.

[This corrects the article DOI: 10.1039/D4RA02811B.].

MetaPredictor: in silico prediction of drug metabolites based on deep language models with prompt engineering.

Metabolic processes can transform a drug into metabolites with different properties that may affect its efficacy and safety. Therefore, investigation of the metabolic fate of a drug candidate is of great significance for drug discovery. Computational methods have been developed to predict drug metabolites, but most of them suffer from two main obstacles: the lack of model generalization due to restrictions on metabolic transformation rules or specific enzyme families, and high rate of false-positive predictions. Here, we presented MetaPredictor, a rule-free, end-to-end and prompt-based method to predict possible human metabolites of small molecules including drugs as a sequence translation problem. We innovatively introduced prompt engineering into deep language models to enrich domain knowledge and guide decision-making. The results showed that using prompts that specify the sites of metabolism (SoMs) can steer the model to propose more accurate metabolite predictions, achieving a 30.4% increase in recall and a 16.8% reduction in false positives over the baseline model. The transfer learning strategy was also utilized to tackle the limited availability of metabolic data. For the adaptation to automatic or non-expert prediction, MetaPredictor was designed as a two-stage schema consisting of automatic identification of SoMs followed by metabolite prediction. Compared to four available drug metabolite prediction tools, our method showed comparable performance on the major enzyme families and better generalization that could additionally identify metabolites catalyzed by less common enzymes. The results indicated that MetaPredictor could provide a more comprehensive and accurate prediction of drug metabolism through the effective combination of transfer learning and prompt-based learning strategies.

Aspirin exposure coupled with hypoxia interferes energy metabolism, antioxidant and autophagic processes and causes liver injury in estuarine goby Mugilogobius chulae.

Toxicity assessments of pollutants often overlook the impact of environmental factors like hypoxia, which can alter chemical toxicity with unexpected consequences. In this study, Mugilogobius chulae, an estuarine fish, was used to investigate the effects of hypoxia (H), aspirin (ASA), and their combination (H_ASA) exposure over 24, 72, and 168 h. We employed RNA-seq analysis, expression of key gene expression profiling, enzymatic activity assays, and histopathological and ultrastructural examinations of liver tissue to explore the effects and mechanisms of ASA-coupled hypoxia exposure in fish. Results showed that glycolysis was inhibited, and lipolysis was enhanced in ASA/H_ASA groups. The PPAR signaling pathway was activated, increasing fatty acid ß-oxidation and lipophagy to mitigate energy crisis. Both ASA and H_ASA exposures induced p53 expression and inhibited the TOR pathway to combat environmental stress. However, a greater energy demand and heightened sensitivity to ASA were observed in H_ASA compared to ASA exposure. Disruptions in energy and detoxification pathways led to increased stress responses, including enhanced antioxidant activities, autophagy, and apoptotic events, as observed in organelle structures. Overall, sub-chronic H_ASA exposure caused liver injury in M. chulae by affecting energy metabolism, antioxidant regulation, and autophagy processes. This study highlights the influence of hypoxia on ASA toxicity in fish, providing valuable insights for ecological risk assessment of NSAIDs.

Antithrombotic therapy at discharge and prognosis in patients with chronic coronary syndrome and atrial fibrillation who underwent PCI: a real-world study.

BACKGROUND: This study aimed to describe the status of antithrombotic therapy at discharge and prognosis in patients with atrial fibrillation (AF) and chronic coronary syndrome (CCS) who underwent percutaneous coronary intervention (PCI). METHODS: This was an observational, prospective study. The primary endpoint was major adverse cardiovascular events (MACE), including all-cause death, myocardial infarction, stroke/transient ischemic attach (TIA), systemic embolism or ischemia-driven revascularization. Bleeding events were collected according to the Thrombolysis in Myocardial Infarction (TIMI) criteria. RESULTS: Between 2017 and 2019, a cohort of 516 patients (mean age 66, [SD 9], of whom 18.4% were female) with AF and CCS who underwent PCI were evaluated, with a median followed-up time of 36 months (Interquartile range: 22-45). MACE events occurred in 13.0% of the patients, while the TIMI bleeding events were observed in 17.4%. Utilization of TAT (triple antithrombotic therapy) (P < 0.001) and oral anticoagulation (OAC) therapy (P < 0.001) increased through years. History of heart failure (HF) (Hazard ratio [HR], 1.744; 95% confidence interval [CI], 1.011-3.038) and TAT (HR, 2.708; 95%CI, 1.653-4.436) had independent associations with MACE events. OAC (HR, 10.378; 95%CI, 6.136-17.555) was identified as a risk factor for bleeding events. A higher creatine clearance (HR, 0.986; 95%CI, 0.974-0.997) was associated with a lower incidence of bleeding events. CONCLUSIONS: Antithrombotic therapy has been improved among patients with AF and CCS who underwent PCI these years. History of HF and TAT were independently associated with MACE events. Higher creatine clearance was protective factor of bleeding events, while OAC was a risk factor for TIMI bleeding events.

NREM sleep brain networks modulate cognitive recovery from sleep deprivation.

Decrease in cognitive performance after sleep deprivation followed by recovery after sleep suggests its key role, and especially non-rapid eye movement (NREM) sleep, in the maintenance of cognition. It remains unknown whether brain network reorganization in NREM sleep stages N2 and N3 can uniquely be mapped onto individual differences in cognitive performance after a recovery nap following sleep deprivation. Using resting state functional magnetic resonance imaging (fMRI), we quantified the integration and segregation of brain networks during NREM sleep stages N2 and N3 while participants took a 1-hour nap following 24-hour sleep deprivation, compared to well-rested wakefulness. Here, we advance a new analytic framework called the hierarchical segregation index (HSI) to quantify network segregation across spatial scales, from whole-brain to the voxel level, by identifying spatio-temporally overlapping large-scale networks and the corresponding voxel-to-region hierarchy. Our results show that network segregation increased in the default mode, dorsal attention and somatomotor networks during NREM sleep compared to wakefulness. Segregation within the visual, limbic, and executive control networks exhibited N2 versus N3 sleep-specific voxel-level patterns. More segregation during N3 was associated with worse recovery of working memory, executive attention, and psychomotor vigilance after the nap. The level of spatial resolution of network segregation varied among brain regions and was associated with the recovery of performance in distinct cognitive tasks. We demonstrated the sensitivity and reliability of voxel-level HSI to provide key insights into within-region variation, suggesting a mechanistic understanding of how NREM sleep replenishes cognition after sleep deprivation.

Perturbations in gut microbiota composition in schizophrenia.

Schizophrenia is a severe, complex and long-term psychiatric disorder with unclear etiology. Gut microbes influence the central nervous system via the gut-brain axis. Consequently, investigations of the relationship between gut microbes and schizophrenia are warranted. This study involved 29 patients with schizophrenia and 30 age-matched normal controls. After 16S rRNA gene sequencing and whole-genome shotgun metagenomic sequencing, we analyzed microbial diversity, composition, and function. According to 16S rRNA and metagenomic gene sequencing results, patients with schizophrenia had higher abundances of Clostridium and Megasphaera. Functional analysis showed that sphingolipid, phosphonates and phosphinates, as well as glutamine metabolism were associated with the occurrence and development of schizophrenia. Our data suggest that the gut microbiota exerts an effect on patients with schizophrenia, providing valuable insights into the potential regulation of in the context of this disorder.

Isovaleryl Sucrose Esters from Atractylodes japonica and Their Cytotoxic Activity.

Cancer represents one of the most significant health challenges currently facing humanity, and plant-derived antitumour drugs represent a prominent class of anticancer medications in clinical practice. Isovaleryl sucrose esters, which are natural constituents, have been identified as having potential antitumour effects. However, the mechanism of action remains unclear. In this study, 12 isovaleryl sucrose ester components, including five new (1-5) and seven known compounds (6-12), were isolated from the roots of Atractylodes japonica. The structures of the compounds were elucidated using 1D and 2D-NMR spectroscopy, complemented by HR-ESI-MS mass spectrometry. The cytotoxic activities of all the compounds against human colon cancer cells (HCT-116) and human lung adenocarcinoma cells (A549) were also evaluated using the CCK8 assay. The results demonstrated that compounds 2, 4, and 6 were moderately inhibitory to HCT-116 cells, with IC50 values of 7.49 ± 0.48, 9.03 ± 0.21, and 13.49 ± 1.45 µM, respectively. Compounds 1 and 6 were moderately inhibitory to A549, with IC50 values of 8.36 ± 0.77 and 7.10 ± 0.52 µM, respectively. Molecular docking revealed that compounds 1-9 exhibited a stronger affinity for FGFR3 and BRAF, with binding energies below -7 kcal/mol. Compound 2 exhibited the lowest binding energy of -10.63 kcal/mol to FGFR3. We screened the compounds with lower binding energies, and the protein-ligand complexes already obtained after molecular docking were subjected to exhaustive molecular dynamics simulation experiments, which simulated the dynamic behaviour of the molecules in close proximity to the actual biological environment, thus providing a deeper understanding of their functions and interaction mechanisms. The present study provides a reference for the development and use of iso-valeryl sucrose esters in the antitumour field.

A ternary system of meloxicam with matching hydrophilic polymer and cyclodextrin for improved stability in liquid preprations.

The purpose of the study is to investigate the effect of ternary systems consisting of meloxicam with cyclodextrins (HP-ß-CD or SBE-ß-CD) and different polymers (HA, HPMC and PVP) on the stability of meloxicam. The t 0.9 values of meloxicam were determined within all the aforementioned systems and the influence of various polymers on the alteration in meloxicam's stability was evaluated. All three polymers altered the stability of meloxicam to varying degrees, with the extent of the effect being related to hydrophilicity, concentration of components, and the interaction of the newly formed ternary system. Among them, meloxicam demonstrated its highest degree of stabilization within the ternary system formed by SBE-ß-CD&HPMC and HP-ß-CD&HA. We characterized the ternary system of meloxicam using differential scanning calorimetry (DSC), X-ray diffraction, and scanning electron microscopy analysis, which determined the presence of ternary system inclusions. In addition, we investigated the optimized prescription of eye drops of meloxicam using the ternary system and further determined that the ternary system improved the stability of the drug in liquid formulations.

Cadmium contamination decreased bacterial network complexity and stability in coastal reclamation areas.

Cadmium(Cd) contamination can exert significantly adverse effects on soil microbiota in reclaimed areas, however, its effects on bacterial network structure are still limitedly understood. Here we collected soil samples from typical reclaimed wetlands (RW) and ditch wetlands (DW) in coastal reclamation areas and examined the effects of Cd contamination on the bacterial network complexity and stability. The results showed that the bacterial networks were destabilized by the Cd contamination, while bacteria in DW soils showed robust invulnerability characterized by higher node constancy and compositional stability compared with RW soils. Soil bacteria resisted Cd stress by forming a network with intensive connections in the module but sparser connections among the modules. Especially, network modularity was higher in DW soils than in RW soils, but made it more vulnerable to nodes removal. In addition, Cd contamination promoted bacterial positive cohesion but decreased negative cohesion in RW soils. Flavobacteriaceae, Xanthomonadaceae, and Alcaligenaceae were identified as core phylotypes, which played pivotal roles in regulating interspecies interactions due to higher contributions to cohesion and significant correlations with soil nutrients. The findings of this work indicate the changes of bacterial network structure and the indispensable role of core phylotypes in regulating interactions and maintaining network sustainability under Cd contamination.

Percutaneous Kyphoplasty via Transverse Process-Rib-Pedicle Approach for Upper and Middle Thoracic Osteoporosis Fracture with Pedicle Stenosis.

OBJECTIVE: To explore the clinical effect of percutaneous kyphoplasty (PKP) via process-rib-pedicle approach for upper and middle thoracic osteoporosis fractures with pedicle stenosis. METHODS: This study is a retrospective observational study. In this study, we retrospectively analyzed the data of 62 patients with upper thoracic vertebral bone loss compression fracture treated via the process-rib-pedicle pathway PKP at the First Affiliated Hospital of Soochow University from January 2020 to December 2022. The patients were divided into group A (unilateral PKP, 38 cases) and group B (bilateral PKP, 24 cases). The aspects of surgical safety, clinical efficacy, and radiological outcome were investigated. RESULTS: All 62 patients successfully completed the surgery without any spinal cord, nerve, or vascular injury, and there were no complications such as infection and vascular embolism. The differences in visual analog scale scores（P < 0.05）, Oswestry disability index functional index（P < 0.05）, and Cobb angle（P < 0.05） were significant when comparing preoperative and postoperative periods, and the differences were not significant when comparing the postoperative periods (P > 0.05). There were no statistically significant differences in days of hospital stay (P = 0.653) and the rate of bone cement leakage (P = 0.537) between the 2 groups. CONCLUSIONS: For upper middle osteoporotic thoracic vertebral fractures with pedicle stenosis, puncture via the process-rib-pedicle path is a safe and reliable puncture route, and more than 2.5 ml of cement can achieve good clinical outcomes, regardless of bilateral or unilateral PKP.

E3 ubiquitin ligase TRIM31 alleviates dopaminergic neurodegeneration by promoting proteasomal degradation of VDAC1 in Parkinson's Disease model.

Mitochondrial dysfunction plays a pivotal role in the pathogenesis of Parkinson's disease (PD). As a mitochondrial governor, voltage-dependent anion channel 1 (VDAC1) is critical for cell survival and death signals and implicated in neurodegenerative diseases. However, the mechanisms of VDAC1 regulation are poorly understood and the role of tripartite motif-containing protein 31 (TRIM31), an E3 ubiquitin ligase which is enriched in mitochondria, in PD remains unclear. In this study, we found that TRIM31-/- mice developed age associated motor defects and dopaminergic (DA) neurodegeneration spontaneously. In addition, TRIM31 was markedly reduced both in nigrostriatal region of PD mice induced by MPTP and in SH-SY5Y cells stimulated by MPP+. TRIM31 deficiency significantly aggravated DA neurotoxicity induced by MPTP. Mechanistically, TRIM31 interacted with VDAC1 and catalyzed the K48-linked polyubiquitination to degrade it through its E3 ubiquitin ligase activity. In conclusion, we demonstrated for the first time that TRIM31 served as an important regulator in DA neuronal homeostasis by facilitating VDAC1 degradation through the ubiquitin-proteasome pathway. Our study identified TRIM31 as a novel potential therapeutic target and pharmaceutical intervention to the interaction between TRIM31 and VDAC1 may provide a promising strategy for PD.

SGLT2 inhibitors as a potential therapeutic option for pulmonary hypertension: mechanisms and clinical perspectives.

Pulmonary arterial hypertension (PAH), one subtype of pulmonary hypertension (PH), is a life-threatening condition characterized by pulmonary arterial remodeling, elevated pulmonary vascular resistance, and blood pressure in the pulmonary arteries, leading to right heart failure and increased mortality. The disease is marked by endothelial dysfunction, vasoconstriction, and vascular remodeling. The role of Sodium-Glucose Co-Transporter-2 (SGLT2) inhibitors, a class of medications originally developed for diabetes management, is increasingly being explored in the context of cardiovascular diseases, including PAH, due to their potential to modulate these pathophysiological processes. In this review, we systematically examine the burgeoning evidence from both basic and clinical studies that describe the effects of SGLT2 inhibitors on cardiovascular health, with a special emphasis on PAH. By delving into the complex interactions between these drugs and the potential pathobiology that underpins PH, this study seeks to uncover the mechanistic underpinnings that could justify the use of SGLT2 inhibitors as a novel therapeutic approach for PAH. We collate findings that illustrate how SGLT2 inhibitors may influence the normal function of pulmonary arteries, possibly alleviating the pathological hallmarks of PAH such as inflammation, oxidative stress, aberrant cellular proliferation, and so on. Our review thereby outlines a potential paradigm shift in PAH management, suggesting that these inhibitors could play a crucial role in modulating the disease's progression by targeting the potential dysfunctions that drive it. This comprehensive synthesis of existing research underscores the imperative need for further clinical trials to validate the efficacy of SGLT2 inhibitors in PAH and to integrate them into the therapeutic agents used against this challenging disease.

Image recognition technology provides insights into relationships between anthocyanin degradation and color variation during jet drying of black carrot.

To identify interesting relationships between anthocyanin degradation and color variation during food processing, black carrot slice (BCS) was dried by air-impingement jet drying (AIJD) and hot air drying (HAD). AIJD was a better technology for drying BCS than HAD. Results of colorimeter determination showed that the color of BCS was significantly changed during AIJD at 50, 60 and 70 °C. UHPLC-QqQ-MS/MS analysis found that AIJD-induced degradations of main BCS anthocyanins, cyanidin-3-xylosyl(feruloylglucosyl)galactoside and cyanidin-3-xylosyl(sinapoylglucosyl)galactoside, belonged to non-spontaneous endothermic reactions, which followed the 0.5- and 1-order kinetic equations, respectively. Anthocyanin content and colors obtained from colorimeter presented strong positive correlation, particularly the a* and chroma values. We further developed a Python script based on image recognition technology to visualize the correlation matrixes between the anthocyanin contents and colors of BSC images. The plots revealed that strong positive correlations between anthocyanins and colors primarily concentrated in the sample's periphery following a concentric pattern.

Label-Free Identification of AML1-ETO Positive Acute Myeloid Leukemia Using Single-Cell Raman Spectroscopy.

Acute myeloid leukemia (AML) is a malignant hematological tumor disease. Chromosomal abnormality is an independent prognostic factor in AML. AML with t(8:21) (q22; q22)/AML1-ETO (AE) is an independent disease group. In this research, a new method based on Raman spectroscopy is reported for label-free single-cell identification and analysis of AE fusion genes in clinical AML patients. Raman spectroscopy reflects the intrinsic vibration information of molecules in a label-free and non-destructive manner, and the fingerprint Raman spectrum of cells characterizes intracellular molecular types and relative concentration information, so as to realize the identification and molecular metabolism analysis of different kinds of cells. We collected the Raman spectra of bone marrow cells from clinically diagnosed AML M2 patients with and without the AE fusion gene. Through comparison of the average spectra and identification analysis based on multivariate statistical methods such as principal component analysis and linear discriminant analysis, the distinction between AE positive and negative sample cells in M2 AML patients was successfully achieved, and the single-cell identification accuracy was more than 90%. At the same time, the Raman spectra of the two types of cells were analyzed by the multivariate curve resolution alternating least squares decomposition method. It was found that the presence of the AE fusion gene may lead to the metabolic changes of lipid and nucleic acid in AML cells, which was consistent with the results of genomic and metabolomic multi-omics studies. The above results indicate that single-cell Raman spectroscopy has the potential for early identification of AE-positive AML.

Perioperative remedial antiviral therapy in hepatitis B virus-related hepatocellular carcinoma resection: How to achieve a better outcome.

BACKGROUND: Although the benefits of antiviral therapy for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) have been proven, researchers have not confirmed the differences in patient outcomes between patients who received preoperative antiviral therapy for a period of time (at least 24 wk) and patients who received remedial antiviral therapy just before radical resection for HBV-related HCC. AIM: To investigate the efficacy of perioperative remedial antiviral therapy in patients with HBV-related HCC. METHODS: A retrospective study of patients who underwent radical resection for HBV-related HCC at the First Affiliated Hospital of Xi'an Jiaotong University from January 2016 to June 2019 was conducted. Considering the history of antiviral therapy, patients were assigned to remedial antiviral therapy and preoperative antiviral therapy groups. RESULTS: Kaplan-Meier analysis revealed significant differences in overall survival (P < 0.0001) and disease-free survival (P = 0.035) between the two groups. Multivariate analysis demonstrated that a history of preoperative antiviral treatment was independently related to improved survival (hazard ratio = 0.27; 95% confidence interval: 0.08-0.88; P = 0.030). CONCLUSION: In patients with HBV-related HCC, it is ideal to receive preoperative long-term antiviral therapy, which helps patients tolerate more extensive hepatectomy; however, remedial antiviral therapy, which reduces preoperative HBV-DNA levels to less than 4 Log10 copies DNA/mL, can also result in improved outcomes.

Downregulation of SMAD4 protects HaCaT cells against UVB-induced damage and oxidative stress through the activation of EMT.

As a member of the SMAD family, SMAD4 plays a crucial role in several cellular biological processes. However, its function in UVB radiation-induced keratinocyte damage is not yet clarified. Our study aims to provide mechanistic insight for the development of future UVB protective therapies and therapeutics involving SMAD4. HaCaT cells were treated with UVB, and the dose dependence and time dependence of UVB were measured. The cell function of UVB-treated HaCaT cells and the activity of epithelial-mesenchymal transition (EMT) after overexpression or silencing of SMAD4 was observed by flow cytometry, quantitative reverse transcription PCR (qRT-PCR) and Western Blots (WB). We found that a significant decrease in SMAD4 was observed in HaCaT cells induced by UVB. Our data confirm SMAD4 as a direct downstream target of miR-664. The down-regulation of SMAD4 preserved the viability of the UVB-treated HaCaT cells by inhibiting autophagy or apoptosis. Furthermore, the silencing of SMAD4 activated the EMT process in UVB-treated HaCaT cells. Down-regulation of SMAD4 plays a protective role in UVB-treated HaCaT cells via the activation of EMT.

admetSAR3.0: a comprehensive platform for exploration, prediction and optimization of chemical ADMET properties.

Absorption, distribution, metabolism, excretion and toxicity (ADMET) properties play a crucial role in drug discovery and chemical safety assessment. Built on the achievements of admetSAR and its successor, admetSAR2.0, this paper introduced the new version of the series, admetSAR3.0, as a comprehensive platform for chemical ADMET assessment, including search, prediction and optimization modules. In the search module, admetSAR3.0 hosted over 370 000 high-quality experimental ADMET data for 104 652 unique compounds, and supplemented chemical structure similarity search function to facilitate read-across. In the prediction module, we introduced comprehensive ADMET endpoints and two new sections for environmental and cosmetic risk assessments, empowering admetSAR3.0 to provide prediction for 119 endpoints, more than double numbers compared to the previous version. Furthermore, the advanced multi-task graph neural network framework offered robust and reliable support for ADMET prediction. In particular, a module named ADMETopt was added to automatically optimize the ADMET properties of query molecules through transformation rules or scaffold hopping. Finally, admetSAR3.0 provides user-friendly interfaces for multiple types of input data, such as SMILES string, chemical structure and batch molecule file, and supports various output types, including digital, chart displays and file downloads. In summary, admetSAR3.0 is anticipated to be a valuable and powerful tool in drug discovery and chemical safety assessment at http://lmmd.ecust.edu.cn/admetsar3/.

Atorvastatin causes developmental and behavioral toxicity in yellowstripe goby (Mugilogobius chulae) embryos/larvae via disrupting lipid metabolism and autophagy processes.

Atorvastatin (ATV) is one of the most commonly prescribed lipid-lowering drugs detected frequently in the environment due to its high use and low degradation rate. However, the toxic effects of residual ATV in the aquatic environment on non-target organisms and its toxic mechanisms are still largely unknown. In the present study, embryos of a native estuarine benthic fish, Mugilogobius chulae, were employed to investigate the developmental and behavioral toxic effects of ATV including environmentally relevant concentrations. The aim of this study was to provide a scientific basis for ecological risk assessment of ATV in the aquatic environment by investigating the changes of biological endpoints at multiple levels in M. chulae embryos/larvae. The results showed that ATV had significantly lethal and teratogenic effects on M. chulae embryos/larvae and caused abnormal changes in developmental parameters including hatch rate, body length, heart rate, and spontaneous movement. ATV exposure caused oxidative stress in M. chulae embryos/larvae subsequently inhibited autophagy and activated apoptosis, leading to abnormal developmental processes and behavioral changes in M. chulae embryos/larvae. The disruptions of lipid metabolism, autophagy, and apoptosis in M. chulae embryos/larvae caused by ATV exposure may pose a potential ecological risk at the population level.