DNA Origami-Enabled Gene Localization of Repetitive Sequences.

Repetitive sequences, which make up over 50% of human DNA, have diverse applications in disease diagnosis, forensic identification, paternity testing, and population genetic analysis due to their crucial functions for gene regulation. However, representative detection technologies such as sequencing and fluorescence imaging suffer from time-consuming protocols, high cost, and inaccuracy of the position and order of repetitive sequences. Here, we develop a precise and cost-effective strategy that combines the high resolution of atomic force microscopy with the shape customizability of DNA origami for repetitive sequence-specific gene localization. "Tri-block" DNA structures were specifically designed to connect repetitive sequences to DNA origami tags, thereby revealing precise genetic information in terms of position and sequence for high-resolution and high-precision visualization of repetitive sequences. More importantly, we achieved the results of simultaneous detection of different DNA repetitive sequences on the gene template with a resolution of ∼6.5 nm (19 nt). This strategy is characterized by high efficiency, high precision, low operational complexity, and low labor/time costs, providing a powerful complement to sequencing technologies for gene localization of repetitive sequences.

Ultrasensitive Electrochemiluminescence Immunosensor for Bladder Marker Human Complement Factor H-Related Protein Detection.

The development of noninvasive and sensitive detection methods for the early diagnosis and monitoring of bladder cancer is critical but challenging. Herein, an ultrasensitive electrochemiluminescence (ECL) immunosensor that uses Ru(bpy)32+-metal-organic framework (Ru-MOF) nanospheres and a DNA tetrahedral (TDN) probe was established for bladder cancer marker complement factor H-related protein (CFHR1) detection. The synthesized Ru(bpy)32+-metal-organic frameworks (Ru-MOFs) served as a linked substrate for immobilization of AuNPs and antibody (Ab2) to prepare the ECL signal probe (Ru-MOF@AuNPs-Ab2), exhibiting a stable and strengthened ECL emission. At the same time, the inherent advantages of TDN probes on the electrode as the capture probe (TDN-Ab1) improve the accessibility of targets to probes. In the presence of CFHR1, the signal probe Ru-MOF@AuNPs-Ab2 was modified on the electrode through immune binding, thereby obtaining an outstanding ECL signal. As expected, the developed ECL immunosensor exhibited splendid performance for CFHR1 detection in the range of 0.1 fg/mL to 10 pg/mL with a quite low detection limit of 0.069 fg/mL. By using the proposed strategy to detect CFHR1 from urine, it showed acceptable accuracy, which can effectively distinguish between bladder cancer patients and healthy samples. This work contributes to a novel, noninvasive, and accurate method for early clinical diagnosis of bladder cancer.

Self-assembly of DNA origami for nanofabrication, biosensing, drug delivery, and computational storage.

Since the pioneering work of immobile DNA Holliday junction by Ned Seeman in the early 1980s, the past few decades have witnessed the development of DNA nanotechnology. In particular, DNA origami has pushed the field of DNA nanotechnology to a new level. It obeys the strict Watson-Crick base pairing principle to create intricate structures with nanoscale accuracy, which greatly enriches the complexity, dimension, and functionality of DNA nanostructures. Benefiting from its high programmability and addressability, DNA origami has emerged as versatile nanomachines for transportation, sensing, and computing. This review will briefly summarize the recent progress of DNA origami, two-dimensional pattern, and three-dimensional assembly based on DNA origami, followed by introduction of its application in nanofabrication, biosensing, drug delivery, and computational storage. The prospects and challenges of assembly and application of DNA origami are also discussed.

A DNA origami nanostructure embedded with NQO1-activated prodrugs for precision drug delivery.

A rectangle DNA origami nanostructure equipped with doxorubicin-derived prodrugs targeting a tumor cell-specific enzyme (NQO1) is constructed. Combining the high prodrug payload of DNA origami and NQO1-activated chemotherapy, this nanosystem presents therapeutic selectivity for NQO1-overexpressing MCF-7 cells over healthy L02 cells, offering a potent strategy for precision cancer therapy.

A protein encoded by circular ZNF609 RNA induces acute kidney injury by activating the AKT/mTOR-autophagy pathway.

Autophagy plays a crucial role in the development and progression of ischemic acute kidney injury (AKI). However, the function and mechanism of circular RNAs (circRNAs) in the regulation of autophagy in ischemic AKI remain unexplored. Herein, we find that circ-ZNF609, originating from the ZNF609 locus, is highly expressed in the kidney after ischemia/reperfusion injury, and urinary circ-ZNF609 is a moderate predictor for AKI in heart disease patients. Overexpression of circ-ZNF609 can activate AKT3/mTOR signaling and induce autophagy flux impairment and cell apoptosis while inhibiting proliferation in HK-2 cells, which is blocked by silencing circ-ZNF609. Mechanistically, circ-ZNF609 encodes a functional protein consisting of 250 amino acids (aa), termed ZNF609-250aa, the overexpression of which can activate AKT3/mTOR signaling and induce autophagy flux impairment and cell apoptosis in HK-2 cells in vitro and in AKI kidneys in vivo. The blockade of AKT and mTOR signaling with pharmacological inhibitors is capable of reversing ZNF609-250aa-induced autophagy flux impairment and cell apoptosis in HK-2 cells. The present study demonstrates that highly expressed circ-ZNF609-encoded ZNF609-250aa induces cell apoptosis and AKI by impairing the autophagy flux via an AKT/mTOR-dependent mechanism. These findings imply that targeting circ-ZNF609 may be a novel therapy for ischemic AKI.

An FeP/carbon composite derived from a phytic acid-Fe3+ complex for sulfathiazole degradation through peroxymonosulfate activation.

Peroxymonosulfate (PMS) activation-based advanced oxidation technology possesses great potential for antibiotic-containing wastewater treatment. Herein, we developed an iron phosphide/carbon composite and verified its capability and superiority towards a model antibiotic pollutant (sulfathiazole, STZ) degradation through PMS activation. Benefiting from the chelating ability of phytic acid (PA) with metal ions and its abundance on phosphorous element, a PA-Fe3+ complex was firstly formed and then served as sole precursor for iron phosphide formation by anoxic pyrolysis. Well crystalized FeP particle were found loading on the simultaneously formed thin layer carbon structure. Catalytic activity evaluation showed that FeP/carbon composite could remove over 99% of STZ (20 mg L-1) in 20 min adsorption and 30 min catalysis process under the reaction conditions of catalyst dosage 0.2 g L-1, PMS loading 0.15 g L-1. A pseudo-first-order reaction rate constant of 0.2193 min-1 was obtained, which was among the highest compared with reported studies. Further investigations indicated that the developed FeP/carbon composite worked well in a wide solution pH range of 3-9. Reaction mechanism study showed that reactive species of SO4-• and 1O2 generated from PMS activation played major roles for STZ degradation. Based on liquid chromatography-mass spectroscopy (LC-MS) analysis, a few STZ degradation intermediate products were identified, which facilitated the proposal of STZ degradation pathways. The possible ecological risk of STZ and related degradation intermediates were also considered by toxicity assessment using the Ecological Structure Activity Relationships (ECOSAR) Class Program. The obtained acute and chronic toxicity values implied the relatively low ecological risk of FeP/carbon-PMS reaction system for STZ treatment.

Efficacy, Safety and Pharmacokinetics of IL-17 Monoclonal Antibody Injection (AK111) in Patients with Moderate-to-Severe Plaque Psoriasis: A Randomized, Double-Blinded, Placebo-Controlled Phase Ib Multidose Escalation Clinical Study.

OBJECTIVES: To evaluate the safety, tolerability, immunogenicity, and induced expression of skin biomarkers of AK111 injection after multiple administrations in subjects with moderate-to-severe plaque psoriasis. METHODS: This study is a randomized, double-blinded, placebo-parallel-controlled study using a dose escalation mode of multiple doses. A total of 48 subjects were sequentially randomized to receive each AK111 dose regimen (75 mg, 150 mg, 300 mg, 450 mg) or the corresponding placebo. All subjects were treated with the study drug at weeks 0, 1, 4, and 8 and were unblinded at week 12, with the placebo group ending and the AK111 group being followed up to 20 weeks. RESULTS: At week 12, compared with placebo, the percentage of subjects achieving Psoriasis Area and Severity Index 75 (PASI75) and static Physician Global Assessment (sPGA) 0/1 in the AK111 75 mg-450 mg dose groups was significantly increased, and higher PASI90 was achieved in the 150 mg, 300 mg, and 450 mg dose groups than in the 75 mg group. All efficacy indicators were maintained at week 20. The incidence of treatment-emergent anti-drug antibodies (ADAs) was 0% (0/48). Neutralizing antibodies (NAbs) were not detected in any subject. The proportion of subjects who reported any treatment-emergent adverse event (TEAE) was 75.0% in the AK111 group, similar to the 66.7% in the placebo group. The most commonly reported adverse events were hyperglycemia, elevated blood pressure, and hypokalemia. The AK111 pharmacokinetics showed approximate dose proportionality with regard to the maximum observed concentration (Cmax) and area under the curve from 0 to the time of the last quantifiable concentration (AUC0-t) following subcutaneous injection doses of 150-450 mg. CONCLUSIONS: After moderate-to-severe plaque psoriasis subjects received multiple subcutaneous AK111 injections of 150-450 mg, AK111 exposure increased in a roughly dose-proportional relationship. AK111 was safe and tolerable. In subjects with moderate-to-severe plaque psoriasis, AK111 demonstrated encouraging preliminary efficacy, which was sustained for a relatively long time after the last dose administration. CLINICAL TRIAL REGISTRATION: The clinical trial identification number is NCT05504317.

Population pharmacokinetic/pharmacodynamic analysis of AK111, an IL-17A monoclonal antibody, in subjects with moderate-to-severe plaque psoriasis.

AK111 is an innovative IL-17A antibody, presenting high affinity to IL-17A and showing similar pharmacokinetic (PK) characteristics to those of typical immunoglobulin (Ig) G1 antibodies. To optimize the dosage regimen for phase 2/3 clinical trials, PK and pharmacodynamics (PD) of AK111 were first characterized in Chinese moderate-to-severe plaque psoriasis patients in a phase 1b study. AK111 PK serum sample and Psoriasis Area and Severity Index (PASI) score data were collected from 48 moderate-to-severe psoriasis patients in this study. Non-linear mixed-effects modeling was used for the population PK/PD analysis. A one-compartment model with a first-order absorption and a first-order elimination best described the PK behavior of AK111. The apparent systemic clearance was 0.182 L/day, and the central volume was 6.65 L. The exposure-response relationship was characterized using an indirect response model. The pharmacological effect of AK111 was described in the form of inhibiting the formation of psoriatic plaque, whereas placebo was quantified in the form of promoting the degradation of psoriatic skin lesions. The maximum effect of drug effect (Imax) and placebo effect (PLBmax) was 1 and 0.429, respectively. The rate constant for psoriatic plaque production (Kin) was 0.474 PASI/day and psoriatic plaque loss (Kout) was 0.024 day-1. The body surface area (BSA) affected by psoriasis was identified as a significant covariate on K o u t . The simulation results confirmed that all of the predicted PASI90 response rates at week 12 were higher than 60% at 150 and 300 mg dose levels with different regimens and could reach higher than 80% at week 24. We hope this first PK/PD study of AK111 in Chinese moderate-to-severe plaque psoriasis patients will be of help in the further clinical development of AK111 and provide a reference to the dosage optimization for similar antibodies with a long half-life.

Sepsis-Induced Gut Dysbiosis Mediates the Susceptibility to Sepsis-Associated Encephalopathy in Mice.

Sepsis-associated encephalopathy (SAE) is common in septic patients and is associated with adverse outcomes. The gut microbiota has been recognized as a key mediator of neurological disease development. However, the exact role of the gut microbiota in regulating SAE remains elusive. Here, we investigated the role of the gut microbiota in SAE and its underlying mechanisms. Cecal ligation and puncture (CLP) was conducted to induce sepsis in mice. Neurological scores were recorded to distinguish SAE-resistant (SER) (score of >6 at 36 h postoperatively) from SAE-susceptible (SES) (score of ≤6 at 36 h postoperatively) mice. 16S rRNA gene sequencing and metabolomics analyses were used to characterize the gut microbiota in the two groups. Fecal microbiota transplantation was performed to validate the role of the gut microbiota in SAE progression. The gut microbiota was more severely disrupted in SES mice than in SER mice after sepsis modeling. Interestingly, mice receiving postoperative feces from SES mice exhibited more severe cortical inflammation than mice receiving feces from SER mice. Indole-3-propionic acid (IPA), a neuroprotective molecule, was more enriched in feces from SER mice than in feces from SES mice. IPA alleviated CLP-induced anxiety and spatial memory impairment in septic mice. Moreover, IPA markedly inhibited NLRP3 inflammasome activation and interleukin-1ß (IL-1ß) secretion in lipopolysaccharide-stimulated microglia. These responses were attenuated after antagonizing the aryl hydrocarbon receptor. Our study indicates that the variability in sepsis-induced gut dysbiosis mediates the differential susceptibility to SAE in CLP-induced experimental sepsis mice, and microbially derived IPA is possibly involved in SAE development as a neuroprotective compound. IMPORTANCE The bidirectional interactions between the gut microbiota and sepsis-associated encephalopathy (SAE) are not well characterized. We found that the gut microbiota was more severely disturbed in SAE-susceptible (SES) mice than in SAE-resistant (SER) mice after sepsis modeling. Mice gavaged with postoperative feces from SES mice exhibited more severe neuroinflammation than mice gavaged with feces from SER mice. The gut microbiota from SER mice enriched a neuroprotective metabolite, IPA, which appeared to protect mice from SAE. The potential underlying mechanism of the protective effect of IPA may be mediated via the inhibition of NLRP3 inflammasome activation and IL-1ß secretion in microglia. These anti-inflammatory effects of IPA may be regulated by aryl hydrocarbon receptors. These results enhance our understanding of the role of the intestinal microbiota in sepsis. In particular, gut microbiota-derived IPA may serve as a potential therapeutic agent to prevent neuroinflammation in SAE.

Comparative Expression Profiling of Snf2 Family Genes During Reproductive Development and Stress Responses in Rice.

Sucrose non-fermenting 2 (Snf2) protein family, as chromatin remodeling factors, is an enormous and the most diverse protein family, which contributes to biological processes of replication, transcription, and DNA repair using the energy of adenosine triphosphate (ATP) hydrolysis. The members of Snf2 family proteins have been well characterized in Arabidopsis, rice, and tomato. Although this family received significant attention, few genes were identified uniquely for their roles in mediating reproductive development and stress tolerance in rice. In the present study, we comprehensively analyzed the expression profiling of Snf2 genes during reproductive development and biotic/abiotic stresses. Our results showed that five proteins (OsCHR712/715/720/726/739) were mainly localized in the nucleus, while OsCHR715/739 were also slightly expressed in the cell membrane. There were abundant cis-acting elements in the putative promoter of Snf2 genes, including dehydration, MeJA, MYB binding site for drought, ABA-responsive, and stress-responsive element. Most of the genes were induced immediately after Magnaporthe oryzae infection at 12 h post-infection (hpi). About 55% of the total genes were upregulated under salt and drought stresses during the entire time, and 22-35% of the total genes were upregulated at 3 h. It was noteworthy that the seven genes (OsCHR705, OsCHR706, OsCHR710, OsCHR714, OsCHR721, OsCHR726, and OsCHR737) were upregulated, and one gene (OsCHR712) was downregulated under salt and drought stresses, respectively. The deficiency of OsCHR726 mutations displayed a hypersensitive phenotype under salt stress. These results will be significantly useful features for the validation of the rice Snf2 genes and facilitate understanding of the genetic engineering of crops with improved biotic and abiotic stresses.

OsDDM1b Controls Grain Size by Influencing Cell Cycling and Regulating Homeostasis and Signaling of Brassinosteroid in Rice.

Snf2 family proteins are the crucial subunits of chromatin-remodeling complexes (CRCs), which contributes to the biological processes of transcription, replication, and DNA repair using ATP as energy. Some CRC subunits have been confirmed to be the critical regulators in various aspects of plant growth and development and in epigenetic mechanisms such as histone modification, DNA methylation, and histone variants. However, the functions of Snf2 family genes in rice were poorly investigated. In this study, the relative expression profile of 40 members of Snf2 family in rice was studied at certain developmental stages of seed. Our results revealed that OsCHR741/OsDDM1b (Decrease in DNA methylation 1) was accumulated highly in the early developmental stage of seeds. We further analyzed the OsDDM1b T-DNA insertion loss-of-function of mutant, which exhibited dwarfism, smaller organ size, and shorter and wider grain size than the wild type (Hwayoung, HY), yet no difference in 1,000-grain weight. Consistent with the grain size, the outer parenchyma cell layers of lemma in osddm1b developed more cells with decreased size. OsDDM1b encoded a nucleus, membrane-localized protein and was distributed predominately in young spikelets and seeds, asserting its role in grain size. Meanwhile, the osddm1b was less sensitive to brassinosteroids (BRs) while the endogenous BR levels increased. We detected changes in the expression levels of the BR signaling pathway and feedback-inhibited genes with and without exogenous BR application, and the alterations of expression were also observed in grain size-related genes in the osddm1b. Altogether, our results suggest that OsDDM1b plays a crucial role in grain size via influencing cell proliferation and regulating BR signaling and homeostasis.

Metal Azolate Coordination Polymer-Enabled High Payload and Non-Destructive Enzyme Immobilization for Biocatalysis and Biosensing.

The biomineralized metal-organic frameworks (MOFs) as protective layers help enhance the robustness of enzymes for biocatalysis. Despite great efforts, it is still challenging to develop a recyclable system with high payload and tolerance to harsh conditions. Here, we report a facile surface charge-independent strategy based on Zn-based coordination polymer (ZnCP) for nondestructive immobilization of enzyme. The ZnCP outcompetes most of the previously reported MOFs, in terms of high-payload enzyme packaging. Moreover, benefiting from the hydrophilicity of ZnCP, the entrapped enzymes (e.g., positive cytochrome C and negative glucose oxidase) maintained high catalytic activity, resembling their native counterparts. Notably, compared with ZIF-8, such enzyme-incorporated ZnCP (enzyme@ZnCP) is more tolerant to acidic pH, which imparts the enzyme with good recyclability, even in acid species-generated catalytic reactions, thus broadening its application in biocatalysis. The feasibility of enzyme@ZnCP for protein packaging, enzyme cascade catalysis, and biosensing was also validated. Altogether, enzyme@ZnCP demonstrates high enzyme payload, operational stability, and preservation of enzymatic activity, affording a versatile platform to accommodate bioactive enzyme for biocatalysis and biosensing.

Gut Microbiota Mediates the Susceptibility of Mice to Sepsis-Associated Encephalopathy by Butyric Acid.

Purpose: Neuroinflammation plays an important part in the pathophysiology of sepsis-associated encephalopathy (SAE). Gut microbiota and gut brain axis are considered as important mediators in the development of neurological diseases. The aim of this study was to investigate the role of intestinal microbiota in sepsis-related brain injury and to explore the underlying mechanisms. Methods: Mouse model of SAE was established using cecal ligation and puncture (CLP). Based on the mouse mortality and the associated time of death, light SAE (LSAE) and severe SAE (SSAE) were classified. Fecal microbiota transplantation (FMT) was performed to verify the role of intestinal microbiota. Feces of mice in the two groups which collected before operation were sequenced for 16S and targeted short chain fatty acids. Results: Intestinal microbiota from SSAE and LSAE mice displayed diverse functions. Interestingly, LSAE mice produced more butyric acid compared with SSAE mice. In the in vivo experiments, sodium butyrate (NaB) reduced the high oxidative stress levels in mice hippocampus and conferred a marked survival superiority to sepsis mice. In addition, NaB prevented the increase in intracellular reactive oxygen species (ROS) generation and inducible nitric-oxide synthase expression in LPS-stimulated primary microglia. The GPR109A/Nrf2/HO-1 signaling pathway was found to be involved in the activation of antioxidant response of primary microglia induced by sodium butyrate. Conclusion: Our findings indicate a crucial role of gut microbiota in the susceptibility to SAE. Butyrate, a metabolite of intestinal microbiota, may have a neuroprotective effect in the process of sepsis by GPR109A/Nrf2/HO-1 pathway.

Smart Drug Delivery Systems Based on DNA Nanotechnology.

The development of DNA nanotechnology has attracted tremendous attention in biotechnological and biomedical fields involving biosensing, bioimaging and disease therapy. In particular, precise control over size and shape, easy modification, excellent programmability and inherent homology make the sophisticated DNA nanostructures vital for constructing intelligent drug carriers. Recent advances in the design of multifunctional DNA-based drug delivery systems (DDSs) have demonstrated the effectiveness and advantages of DNA nanostructures, showing the unique benefits and great potential in enhancing the delivery of pharmaceutical compounds and reducing systemic toxicity. This Review aims to overview the latest researches on DNA nanotechnology-enabled nanomedicine and give a perspective on their future opportunities.

Gene/drug-embedded nanoscale metal azolate framework-7 for the reversal of P-glycoprotein-mediated multidrug resistance.

We report the straightforward synthesis of ATP-responsive nanoscale metal azolate framework-7 (MAF-7) for gene/drug codelivery. The MAF-7 functions as (i) the armour to preserve DNAzymes, (ii) an ATP scavenger to lower the intracellular ATP level, and (iii) a built-in Zn2+ arsenal to initiate the biocatalysis of DNAzymes, ultimately inhibiting P-gp expression to enhance chemotherapy.

Self-assembled nanomaterials for biosensing and therapeutics: recent advances and challenges.

Self-assembled nanomaterials (SANs) exhibit designable biofunctions owing to their tunable nanostructures and modifiable surface. Various constituent units and multi-dimensional structures of SANs provide unlimited possibilities for numerous applications. This review emphasizes the recent development of SANs in the fields of biosensing, bioimaging, and nano-drug engineering. The unit type, design concepts, material advantages, assembly driving force, nanostructure effects, drug loading performance, etc. are discussed and summarized. Finally, we briefly summarize how to assemble unique nanomaterials and point out the key challenges in this field.

Dysbiosis of intestinal microbiota in critically ill patients and risk of in-hospital mortality.

BACKGROUND: Despite the essential functions of the intestinal microbiota in human physiology, little research was reported on gut microbiota alterations in intensive care patients. This investigation examined the dysbacteriosis of intestinal flora in critically ill patients and evaluated the prognostic performance of this dysbiosis to predict in-hospital mortality. METHODS: A prospective cohort of patients were consecutively recruited in the Intensive Care Units (ICUs) in Guangdong Provincial People's Hospital from March 2017 through October 2017. Acute Physiology and Chronic Health Evaluation (APACHE) II score and Sequential Organ Failure Assessment (SOFA) score were assessed, and fecal samples were taken for examination within 24 hours of ICU admission. The taxonomic composition of the intestinal microbiome was determined using 16S rDNA gene sequencing. Patients were divided into survival and death groups based on hospital outcomes. The two groups were statistically compared using the Wilcoxon test and Metastats analysis. The genera of bacteria showing significantly different abundance between groups were assessed as predictors of in-hospital death. The prognostic value of bacterial abundance alone and in combination with APACHE II or SOFA score was evaluated using the area under the receiver operating characteristic curve (AUROC). RESULTS: Among the 61 patients examined, 12 patients (19.7%) died during their hospital stay. Bifidobacterium abundance was higher in the survival group than the death group (P = 0.031). The AUROC of Bifidobacterium abundance in identifying in-hospital death at a cut-off probability of 0.0041 was 0.718 (95% confidence interval [CI], 0.588-0.826). The panel of Bifidobacterium abundance plus SOFA (AUROC, 0.882; 95% CI, 0.774-0.950) outperformed SOFA (AUROC, 0.649; 95% CI, 0.516-0.767; P = 0.012) and Bifidobacterium abundance alone (P = 0.007). The panel of Bifidobacterium abundance plus APACHE II (AUROC, 0.876; 95% CI, 0.766-0.946) outperformed APACHE II (AUROC, 0.724; 95% CI, 0.595-0.831; P = 0.035) and Bifidobacterium abundance alone (P = 0.012). CONCLUSIONS: Dysbiosis of intestinal microbiota with variable degrees of reduction in Bifidobacterium abundance exhibited promising performance in the predicting of in-hospital mortality and provides incremental prognostic value to existing scoring systems in the adult intensive care unit (ICU) setting.

C-reactive protein concentration as a risk predictor of mortality in intensive care unit: a multicenter, prospective, observational study.

BACKGROUND: It is not clear whether there are valuable inflammatory markers for prognosis judgment in the intensive care unit (ICU). We therefore conducted a multicenter, prospective, observational study to evaluate the prognostic role of inflammatory markers. METHODS: The clinical and laboratory data of patients at admission, including C-reactive protein (CRP), were collected in four general ICUs from September 1, 2018, to August 1, 2019. Multivariate logistic regression was used to identify factors independently associated with nonsurvival. The area under the receiver operating characteristic curve (AUC-ROC), net reclassification improvement (NRI), and integrated discrimination improvement (IDI) were used to evaluate the effect size of different factors in predicting mortality during ICU stay. 3 -knots were used to assess whether alternative cut points for these biomarkers were more appropriate. RESULTS: A total of 813 patients were recruited, among whom 121 patients (14.88%) died during the ICU stay. The AUC-ROC values of PCT and CRP for discriminating ICU mortality were 0.696 (95% confidence interval [CI], 0.650-0.743) and 0.684 (95% CI, 0.633-0.735), respectively. In the multivariable analysis, only APACHE II score (odds ratio, 1.166; 95% CI, 1.129-1.203; P = 0.000) and CRP concentration > 62.8 mg/L (odds ratio, 2.145; 95% CI, 1.343-3.427; P = 0.001), were significantly associated with an increased risk of ICU mortality. Moreover, the combination of APACHE II score and CRP > 62.8 mg/L significantly improved risk reclassification over the APACHE II score alone, with NRI (0.556) and IDI (0.013). Restricted cubic spline analysis confirmed that CRP concentration > 62.8 mg/L was the optimal cut-off value for differentiating between surviving and nonsurviving patients. CONCLUSION: CRP markedly improved risk reclassification for prognosis prediction.