To investigate the effects of clesmatine on the myelination and neural oscillation-related firing activities during sleep in the hippocampus of adult APP/PS1 mice.

Oral administration of clemastine in drinking water was given to APP/PS1 mice for 3 months to enhance myelination in adulthood. Based on the treatment regimens, the mice were randomly assigned into APP/PS1 group, APP/PS1+clemastine group, and wild-type (WT) group. Local field potentials combined with three-dimensional acceleration of the head and neck were employed to determine the brain states, that is, wakefulness, non-rapid eye movement (NREM) sleep, REM sleep. In vivo multi-electrode arrays were utilized to continuously monitor neuronal firing activities in the dorsal hippocampus across the 3 sleep states.

① Oral clemastine administration via drinking water significantly increased MBP⁺ protein expression levels in APP/PS1+Clemastine mice than the APP/PS1 controls (P<0.001). ② Clemastine administration did not alter the time proportions of NREM sleep, REM sleep and wakefulness during the daytime phase in APP/PS1 mice (P_s>0.05). ③ Clemastine administration obviously reduced the mean firing rate of hippocampal pyramidal cells (P<0.05) while increasing that of hippocampal interneurons (P<0.05) in APP/PS1 mice. ④ Clemastine administration increased the occurrence rate of hippocampal sharp wave-ripple (SWR) oscillations during NREM sleep and specifically reduced the firing rate of pyramidal cells during SWR oscillations in APP/PS1 mice (P<0.05). ⑤ Clemastine administration had no significant effect on the hippocampal neuronal firing activity during theta oscillation in REM sleep in APP/PS1 mice (P_s >0.05).

Clemastine administration not only enhances the adult myelination, but also rescues both the SWRs and associated hippocampal neuronal firing during NREM sleep in APP/PS1 mice.

The incidence of Alzheimer's disease (AD) in postmenopausal women is significantly increased with ageing. Menopause induced ovarian estrogen deficiency has been regarded as one of the causative risk factor of AD, but currently the detail mechanisms underlying estrogen regulation on the hippocampus remain unclear. This study aimed to uncover the potential pathways and key molecules through which estrogen deficiency induced by ovariectomy promotes AD pathogenesis by integrating transcriptomic and proteomic analyses.

The ovariectomy (OVX) mouse model was established to simulate postmenopausal estrogen decline. Transcriptomic (n=3) and proteomic (n=3) differences in the hippocampal tissues of OVX mice were analyzed using RNA sequencing and protein mass spectrometry. Co-trend molecules identified from both omics datasets were subjected to GO/KEGG enrichment and cluster analyses. These molecules were further compared with the human AD hippocampal database (AlzDate) for expression trend consistency; and online resources such as String, GeneCards/Uniports were employed to analyze protein interactions and networks of the co-trend genes to identify key molecules.

A total of 139 differentially expressed genes (DEG) and 248 differentially expressed proteins (DEP) were identified, with 18 molecules showing consistent expression trends in both omics. Functional enrichment analysis revealed that these molecules were primarily involved in axonogenesis, cytoskeleton dynamics regulation, and long-chain fatty acid transport. After comparison with the human AD database, 11 of the 18 co-trend molecules exhibited expression inhibition consistent with AD-associated genes in the human hippocampal databases. Interaction network analysis identified Aldoc (involved in glycolysis), Aldh6a1 (involved in ROS scavenging), Etnppl (involved in mitochondrial energy metabolism), and Itih3 (involved in hyaluronic acid-mediated extracellular matrix remodeling) as key genes contributing to AD pathogenesis. Additionally, 3 co-trend molecules (Prxl2a, Set, Plin4) were not detected in human AD databases.

Postmenopausal estrogen deficiency decreases the expression levels of hippocampal Aldoc, Etnppl and Aldh6a1 then affects mitochondrial energy metabolism; and decreases levels of Itih3 expression then disables extracellular matrix remodeling. These effects collectively impair hippocampal axonal regenerative capacity, destabilize the cytoskeleton, and hinder the transport of long-chain fatty acids, ultimately driving the progression of AD pathology.

To investigate the effect of pramlintide, a pancreatic amyloid peptide analog, on learning and memory of Alzheimer’s disease (AD) mice through antioxidant stress, and to determine the expression of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.

The APP/PS1 mice were divided into a pramlintide treatment group (intraperitoneal injection of 0.5 μmol/L per day for 10 weeks) and an AD group (same dose of PBS), with 5 mice in each group. The learning and memory abilities were detected with water maze test, the pathological changes of the hippocampus were observed with HE staining and immunohistochemistry, the morphological characteristics of dendritic spines in hippocampus were observed after Golgi staining, and the ultrastructure of hippocampal neurons was observed through transmission electron microscopy (TEM). The content of malondialdehyde (MDA) and the level of superoxide dismutase (SOD) in the hippocampal tissue were detected by biochemical assay, and the levels of inflammatory factors IL-6, TNF-α and IL-1β were determined with ELISA. Western blotting was applied to measure the expression of PI3K/Akt signaling pathway related proteins in the hippocampus. In the cell experiment, SH-SY5Y cells were added with Aβ 1-42 to establish a cell model of AD. After the cells were treated with pramlintide, the levels of oxidative stress and inflammatory response were detected, and cell apoptosis was detected by immunofluorescence.

The animal experiments showed that pramlintide treatment resulted in significantly shortened escape latency (P<0.01), increased platform crossings (P<0.01), and prolonged time to exploring hidden platform (P<0.01). In the hippocampal tissue of the pramlintide treatment group, HE staining displayed hippocampal neurons in high density and neat arrangement (P<0.05), immunohistochemical results showed significantly reduced Aβ protein (P<0.01), Golgi staining results demonstrated more dendritic spines (P<0.05), TEM revealed almost intact neuronal mitochondrial structure, with reduced vacuolization and clear and identifiable morphology. When compared with the AD group, the levels of oxidative stress and inflammatory response were decreased (P<0.01), and the relative expression of p-PI3K/PI3K and p-Akt/Akt proteins was increased (P<0.01) in the treatment group. In cell experiments, the levels of oxidative stress and inflammatory response were decreased in AD cell model after pramlintide treatment (P<0.01), and the results of immunofluorescence showed that cell apoptosis was declined (P<0.01).

Pramlintide can improve the cognitive function, reduce the hippocampal deposition of Aβ, reduce oxidative stress and inflammatory response, alleviate the pathological changes of neuronal ultrastructure, and enhance the expression of PI3K/Akt signaling pathway in AD mice.

To investigate the expression level of circular RNA circ-Tns3 in Alzheimer’s disease (AD) mice and its role in Aβ-induced neuronal damage.

Five APP/PS1 transgenic AD mice and 5 wild-type (WT) mice, weighting of 23~26 g and aged 6 months were subjected in the study. Morris water maze test was used to assess learning and memory abilities, and immunohistochemical staining was performed to observe the number of Aβ plaques in the hippocampal tissue. Subsequently, total RNA was extracted from the brains to detect the differential expression of circRNAs between AD and WT mice, and the results were further analyzed with Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The top 6 differentially expressed circRNAs were validated by real-time quantitative PCR (RT-qPCR). In in vitro experiments, Aβ1-42 was used to treat neuronal cells to establish AD cell model, and si-circ-Tns3 was transfected into Aβ1-42-treated neuronal cells to knock down circ-Tns3. RT-qPCR was used to determine the expression levels of circ-Tns3 and miR-671-5p. Cell viability and apoptotic rate were detected by CCK-8 assay and TUNEL staining, respectively. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured using corresponding kits, and the levels of inflammatory factors IL-6, IL-1β, and TNF-α were detected with ELISA. The interaction between circ-Tns3 and miR-671-5p was verified by dual-luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay. The expression level of Sirt1 protein was detected by Western blotting.

The 6-month-old AD mice exhibited significant cognitive impairment and Aβ deposition (P<0.01). There were 269 differentially expressed circRNAs identified between AD and WT mice, of which 159 were up-regulated and 110 down-regulated. GO and KEGG enrichment analyses showed that these differentially expressed circRNAs were mainly involved in synaptic transmission, memory, and cholinergic synapse signaling pathways. The expression of circ-Tns3 was significantly increased not only in the brain tissue of AD mice but also in neuronal cells after Aβ1-42 treatment. In cellular experiments, knockdown of circ-Tns3 significantly reduced cell viability and number of apoptotic cells in Aβ1-42-treated neuronal cells, decreased MDA content, increased SOD activity, and reduced the levels of IL-6, IL-1β, and TNF-α (P<0.01). The starBase database predicted that circ-Tns3 and miR-671-5p have complementary sequences, and dual-luciferase reporter and RIP assays confirmed their interaction. The bioinformatics database predicted that miR-671-5p and sirt1 have complementary sequences. Western blotting indicated that in neuronal cells treated with Aβ1-42, the expression of sirt1 was increased after knockdown of circ-Tns3 (P<0.01). In Aβ1-42-treated neuronal cells, after knockdown of circ-Tns3, addition of miR-671-5p inhibitor significantly decreased the expression level of sirt1 protein (P<0.01).

irc-Tns3 is highly expressed in AD mice and cell model of AD. Knocking circ-Tns3 down improves neuronal damage. circ-Tns3 may be involved in the neuronal damage through regulating sirt 1 protein by binding to miR-671-5p.

To explore the neuroprotective effects of osteocalcin (OCN) on an Alzheimer’s disease (AD) cell model and its potential mechanisms, providing a scientific basis for new therapeutic targets for AD.

Human neuroblastoma cell line SH-SY5Y was treated with 40 nmol/L okadaic acid (OA) for 24 h to establish an AD cell model. The cells were divided into a normal group (untreated SH-SY5Y cells), a model group (40 nmol/L OA intervention), and an OCN intervention group (intervention with various concentrations of OCN in the AD cell model), and AKT knockout/overexpression groups( AKT-KO group and AKT-OE group), and AKT-KO OCN group and AKT-OE OCN group. CCK-8 assay was used to detect the changes in cell viability. Wright's staining was employed to observe the morphological changes of AD cells. Western blotting was utilized to detect the protein levels of Tau, p-Tau, Bax, Bcl-2, Caspase-3 and their lytic types, as well as the expression of Tau, p-Tau, mTOR, AKT and p-AKT in each group after AKT knockout/overexpression. TUNEL staining and flow cytometry were applied to detect the changes in early and late apoptotic cells and the apoptotic rate in the OCN-treated AD cell model.

①Compared to the normal group, the model group exhibited a significant decrease in cell viability, noticeable morphological and structural damage, upregulation of p-Tau and Caspase-3, increased early and late apoptosis, and a significantly higher apoptotic rate (P<0.05). ②After treatment of different concentrations of OCN for 24 h, cell viability was increased to varying degrees compared to the AD model group, with the 100 pg/mL OCN group showing a significant increase in cell viability (P<0.01) and marked improvement in cell number and morphology (P<0.01). ③ Compared to the AD cell model group, the p-Tau/Tau ratio was decreased in all OCN treatment groups, particularly in the 100 pg/mL OCN intervention group, where the p-Tau/Tau ratio was significantly lower than that of the model group (P<0.01). ④ Compared to the model group, a significant concentration-dependent decrease in the Cleaved Caspase-3/Caspase-3 ratio was observed when OCN concentrations ranged from 1 to 100 pg/mL, with a significant reduction in the Bax/Bcl-2 ratio in the 100 pg/mL group (P<0.000 1). ⑤ The results of TUNEL staining and flow cytometry showed that, compared to the model group, all concentrations of OCN effectively inhibited the apoptosis in the AD model cells, with a significant reduction in early and late apoptotic cells and apoptotic rate in the 100 pg/mL OCN group. ⑥ Compared with the control group and the model group, the P-AKT was significantly increased in the AKT-OE group after AKT overexpression (P<0.05). The expression level of AKT protein was decreased in the AKT-KO group after AKT knockout (P<0.05). When the AKT pathway was inhibited, the expression level of p-Tau was higher in the AKT-KO group than the control group (P<0.05), and when the AKT was overexpressed, the expression level was significantly inhibited (P<0.05).

OCN may inhibit cell apoptosis and reduce p-tau protein level by regulating the ratio of Caspase-3/Caspase-3 and Bax/Bcl-2, and thereby improve the morphology of AD model cells and effectively protect nerve cells, which may be related to the regulation of the AKT/mTOR pathway.

To explore the role and mechanism of mechanically sensitive ion channel Piezo2 in mechanical allodynia of rats with low back pain induced by simulating the low-frequency vibration of a helicopter.

Low-frequency vibration (LFV) model with 3-dimensional 6 degrees of freedom was used to induce low back pain in awake rats in a sitting position. Twenty-four male SD rats (8 weeks old) were randomly divided into control (Ctrl) group and LFV group. HE staining was used to evaluate the injury of the multifidus muscle. Von Frey test was carried out to detect pain sensitivity. Open field test was employed to assess the spontaneous activity and anxiety. ELISA, Western blotting and immunofluorescence staining were performed to detect the expression of NGF, TrkA and downstream molecule Piezo2. Dorsal root ganglia (DRG) neurons was isolated from SD rats and primarily cultured. After identified with immunofluorescence staining, the neurons were divided into the Ctrl group, the LFV group, and the LFV+ D-GsMTx4 (D-G4, an Piezo2 channel antagonist) group. Western blotting was used to detect the protein expression of Piezo2, and a calcium ion fluorescent probe was utilized to detect the intracellular Ca²⁺. The DRG neurons were pretreated with 50 ng/mL NGF for 1 h. Calcium ion fluorescent probe was used to observe the changes in intracellular Ca²⁺ in the LFV group, the LFV+NGF group, and the LFV+NGF+D-G4 group.

The rats of the LFV group showed abnormal morphology in multifidus muscles, accompanied with inflammatory cell infiltration, decreased paw withdrawal reflex threshold (P<0.05), and shortened total active time and active time in the centre, and decreased distance traveled in the centre (P<0.05), while prolonged total stationary time, stationary time in the periphery, and increased distance traveled in the periphery (P<0.05), and moreover, enhanced expression of Piezo2, NGF and TrkA in the DRG tissues (P<0.05). Cell experiments showed that compared with the Ctrl group, the expression of Piezo2 in the neurons was increased (P<0.05), and the intracellular Ca²⁺ level was significantly elevated in the LFV group (P<0.05). Compared with the LFV group, the Ca²⁺ level was higher in the LFV+NGF group (P<0.05), and the sensitization effect of NGF on Piezo2 was reversed after D-G4 treatment (P<0.05).

Sustained low-frequency vibration induces low back pain and mechanical allodynia in rats through the NGF-TrkA/Piezo2 pathway.

To analyze the composition characteristics and biological functions of tumor cell subpopulations in glioblastoma (GBM) through multi-transcriptomics sequencing technology, and explore the hypoxia characteristics and spatial localization features of the mesenchymal-like (MES-like) tumor cell subpopulation in GBM and the influence on malignant biological behaviors.

Multi-transcriptomics sequencing data, including single-cell RNA sequencing (scRNA-seq) data (18 patients), bulk RNA sequencing (bulk RNA-seq) and spatial transcriptomics (ST) data of GBM, were employed to define cell subpopulations in GBM, and Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) were utilized to analyze their functions. The proportions and locations of cell subpopulations in bulk RNA-seq data were evaluated with BayesPrism deconvolution. Immunofluorescence assay was conducted for verification on 12 paraffin samples of GBM from patients who visited the neurosurgical department of our hospital from 2015 to 2023 and met the pathological diagnostic criteria for GBM (10 males and 2 females, at an average age of 53.50 years and a median age of 54.50 years). pySCENIC was applied to predict specific transcription factors of tumor cell subpopulations.

Tumor cells in GBM were highly heterogeneous, and could be mainly divided into 4 subpopulations: astrocyte-like (AC-like), neural progenitor-like (NPC-like), oligodendrocyte progenitor-like (OPC-like) and MES-like. Differential gene analysis found that the MES-like tumor cells highly expressed vascular endothelial growth factor A (VEGFA), adrenomedullin (ADM), N-myc downstream regulated 1 (NDRG1), insulin like growth factor binding protein 5 (IGFBP5), and A-kinase anchoring protein 12 (AKAP12) (P<0.001). pySCENIC transcription factor prediction found that the high-active transcription factors of the MES-like tumor cells were AT-rich interaction domain 3A (ARID3A), FOS like 2, AP-1 transcription factor subunit (FOSL2), endothelial PAS domain protein 1 (EPAS1), CCAAT enhancer binding protein delta (CEBPD), and CCAAT enhancer binding protein beta (CEBPB) (P<0.05). GO and GSEA enrichment analyses found that the MES-like tumor cells were enriched in hypoxia-related pathways, especially the pathway of cell responses to hypoxia levels (NES=2.437, P<0.001). BayesPrism deconvolution showed that the MES-like tumor cells mainly existed in PAN(Pseudopalisading cells around necrosis) and perinecrotic zone. Immunofluorescence assay confirmed CD44⁺ (CD44 antigen) MES-like tumor cells were mainly located in hypoxia areas with highly expression of hypoxia inducible factor 1 subunit alpha (HIF1α) (P<0.01). Multivariate Cox regression analysis indicated that the MES-like tumor cells were significantly correlated with the adverse prognosis of GBM patients (HR=1.71, 95%CI: 1.38~2.11, P<0.001).

Tumor cells in GBM are of highly heterogeneity. They could be mainly divided into 4 subpopulations: AC-like, NPC-like, OPC-like and MES-like. MES-like tumor cells, mainly locating in PAN and perinecrotic zone, are characterized by hypoxia, which can promote the malignant progression of GBM.

To investigate the multi-target mechanisms of quercetin in treating endometriosis (EMT) through integrative network pharmacology analysis.

Active targets of quercetin were collected from the TCMSP database, while EMT-related differentially expressed genes (DEGs) were identified through the Gene Expression Omnibus (GEO) dataset. A comparative analysis was conducted to pinpoint potential therapeutic targets of quercetin for EMT treatment. Functional enrichment analyses were employed to investigate the biological functions associated with these targets, and a protein-protein interaction (PPI) network was conducted to identify core targets. Molecular docking and dynamics simulations were performed to validate the binding characteristics between quercetin and the core targets. The top 2 target protein pairs, HSP90AB1 and AR, exhibiting the lowest binding energy, were selected for subsequent cellular experimental validation. Human EMT-immortalized ectopic endometrial epithelial cell line 12Z (n=6, independent replicates) was subjected, and CCK-8 assay was used to determine ehe effects of quercetin on cell viability and proliferation, and the half-maximal inhibitory concentration (IC50) was calculated at 48 h after treatment. Then the 12Z cells were treated with quercetin at a concentration gradient of 0, 30, 60 and 90 μmol/L, the migration and invasion abilities were assessed with cell scratch and cell invasion assays. Western blotting was conducted to detect the changes in the expression of HSP90AB1 and AR proteins after different doses of treatment.

There were 49 potential EMT-related therapeutic targets and 10 core targets identified. Functional enrichment analyses revealed that these targets were significant enriched in inflammation-related signaling pathways, including AGE-RAGE, ErbB and TNF; immune-related pathways, such as Th17 cell differentiation, T/B cell receptor signaling; angiogenesis-related pathways like VEGF; and hormonal regulatory pathways involving estrogen and GnRH. Molecular docking demonstrated that quercetin exhibited favorable binding activity (binding energy <-5 kcal/mol) with all core target proteins, with particularly strong binding energies (<-7 kcal/mol) observed for AR, EGFR, FOS, ERBB2, and HSP90AB1. Molecular dynamics simulations revealed that quercetin forms sustained hydrogen bond interactions with AR and HSP90AB1, facilitating the formation of stable complexes. CCK-8 assay, cell scratch assay, and transwell invasion assay indicated that quercetin inhibited the proliferative activity, and migrative and invasive abilities of 12Z cells in a concentration-dependent manner, with more pronounced inhibitory effects observed at 60 and 90 μmol/L quercetin (P<0.001); Western blotting revealed that treatment of 12Z cells with varying quercetin concentrations for 48 h up-regulated the expression of HSP90AB1 and AR, with the most significant increase observed at 90 μmol/L quercetin (HSP90AB1, P<0.05; AR, P<0.001). The restored expression levels of HSP90AB1 and AR showed positive correlations with the proliferative activity, migrative and invasive abilities of ectopic endometrial cells.

Quercetin effectively addresses endometriosis through multiple molecular targets and signaling pathways, and stabilization of the HSP90AB1/AR complex and subsequent protein upregulation represents a key therapeutic mechanism.

To investigate risk factors for residual lesions after initial transurethral resection of bladder tumors (TURBT) and risk factors for tumor recurrence after second TURBT in patients with non-muscle-invasive bladder cancer (NMIBC) in order to provide reference for clinical management.

A case-control study design was adopted to include 120 NMIBC patients who underwent initial TURBT and then second surgery within 2~8 weeks in our department from January 2017 to January 2025. Based on the presence of residual lesions after the initial TURBT or not, the patients were divided into a residual lesion group (n=34) and a non-residual lesion group (n=86). Chi-square test and multivariate logistic regression analysis were performed to identify potential risk factors for residual lesions following the initial TURBT. Univariate and multivariate Cox regression models were used to analyze potential risk factors for tumor recurrence after the second TURBT.

The residual lesion rate after initial TURBT was 28.33%. Chi-square test analysis revealed that tumor stage T1 (Chi-square=5.756, P=0.016) and broad tumor base (Chi-square=4.331, P=0.037) were factors influencing residual lesions after initial TURBT. Multivariate logistic regression analysis identified tumor stage T1 (OR=3.047, 95%CI: 1.128~8.226, P=0.028) as an independent risk factor for residual lesions after initial TURBT. The tumor recurrence rate after second TURBT was 17.5%. Multivariate Cox regression analysis identified tumor stage T1 (OR=4.258, 95%CI: 1.248~14.532, P=0.021), intravesical chemotherapy instillation after second TURBT (OR=3.539, 95%CI: 1.284~9.752, P=0.015), history of urinary system tumors (OR=3.002, 95%CI: 1.145~7.873, P=0.025) and high platelet-to-lymphocyte (PLR) ratio (OR=2.798, 95%CI: 1.115~7.023, P=0.028) as independent risk factors for tumor recurrence after second TURBT.

Tumor stage T1 and broad tumor base are risk factors for residual lesions after initial TURBT, while tumor stage T1, intravesical chemotherapy instillation after second TURBT, history of urinary system tumors and high PLR ratio are risk factors for tumor recurrence after second TURBT. Comprehensive analysis on above 4 indicators can effectively assess the risk of tumor recurrence in NMIBC patients following second TURBT, and timely early medical intervention is beneficial for improving patient outcomes.

Patients with chronic liver diseases often exhibit unique hemodynamic abnormalities and metabolic disorders. Postoperative fluid management presents numerous challenges, especially for critically ill patients admitted to the intensive care unit (ICU) after surgery. This study aimed at exploring the relationship between postoperative fluid therapy and prognosis.

Based on 2 414 patients with chronic liver diseases who underwent surgical treatment and were subsequently transferred to the ICU in the MIMIC-IV database, a retrospective cohort study was conducted on the final 2 143 patients after our inclusion and exclusion criteria. Multivariate adjusted logistic regression model was used to analyze the association between fluid therapeutic regimens on the first day after ICU admission and the risk of 7-day death after surgery. Restricted cubic spline (RCS) was applied to analyze the dose-response relationship.

Multivariate analysis indicated that restrictive fluid resuscitation was an independent protective factor. Compared with the non-restrictive fluid resuscitation group, restrictive fluid resuscitation significantly reduced the 7-day postoperative mortality rate (6.4% vs 12.4%, OR=0.44, 95%CI: 0.22~0.88, P=0.021), decreased the use of mechanical ventilation (42.9% vs 72.3%, OR=0.29, 95%CI: 0.24~0.35, P<0.001), and shortened the ICU stay (1.86 vs 3.47 d, OR=0.81, 95%CI: 0.78~0.84, P<0.001). RCS curve showed that the fluid intake on the first postoperative day and the 7-day postoperative mortality risk presented a J-shaped curve relationship, with an inflection point at 1 850 mL. Beyond this threshold, the 7-day postoperative mortality risk was increased. Subgroup analysis results indicated that the protective effect of restrictive fluid resuscitation was consistent across different age and comorbidity groups.

For patients with chronic liver diseases, adopting a restrictive fluid therapy on the first day after surgery can effectively reduce the risk of short-term death. Moreover, there is a non-linear dose-effect relationship between the fluid intake and the 7-day mortality risk. When the fluid intake exceeds 1 850 mL, the risk of death significantly increases.