Clinical applications of metagenomic next-generation sequencing in the identification of pathogens in periprosthetic joint infections: a retrospective study.

BACKGROUND: This study aimed to evaluate the application of metagenomic next-generation sequencing (mNGS) technology to identify pathogens in periprosthetic joint infection (PJI). METHODS: A retrospective analysis was conducted on 65 patients suspected of having PJI between April 2020 and July 2023. The patients were categorized into PJI (46 patients) and non-PJI (19 patients) groups based on the 2018 International Consensus Meeting criteria. Clinical data were collected, and both conventional bacterial culture and mNGS were performed. The diagnostic performance of the two methods was compared and analyzed. RESULTS: mNGS exhibited a sensitivity of 89.13%, a specificity of 94.74%, a positive predictive value of 97.62%, a negative predictive value of 78.26%, and an overall diagnostic accuracy of 90.77%. Compared to microbial culture, mNGS demonstrated superior diagnostic sensitivity while maintaining similar specificity. A total of 48 pathogens were successfully identified using mNGS, with Coagulase-negative staphylococci, Streptococci, Staphylococcus aureus, and Cutibacterium acnes being the most common infectious agents. Notably, mNGS was used to identify 17 potential pathogens in 14 culture-negative PJI samples, highlighting its ability to detect rare infectious agents, including Cutibacterium acnes (n = 5), Granulicatella adiacens (n = 1), Mycobacterium tuberculosis complex (n = 1), and Coxiella burnetii (n = 1), among others, which are not detectable by routine culture methods. However, mNGS failed to detect the pathogen in 4 culture-positive PJI patients, indicating its limitations. Among the 46 PJI patients, 27 had positive culture and mNGS results. The results of mNGS were concordant with those of culture at the genus level in 6 patients with PJI and at the species level in 18 patients. Furthermore, the present study revealed a significantly greater proportion of Staphylococcus aureus in the sinus tract group (45.45%) than in the non-sinus tract group (14.29%), indicating the association of this pathogen with sinus formation in PJI (P = 0.03). Additionally, there was no significant difference in the occurrence of polymicrobial infections between the sinus tract group (27.27%) and the non-sinus tract group (33.33%) (P = 0.37). CONCLUSIONS: Metagenomic next-generation sequencing can serve as a valuable screening tool in addition to traditional culture methods to improve diagnostic accuracy through optimized culture strategies.

Diagnostic Value of the Blood Neutrophil-to-Lymphocyte Ratio and Monocyte-to-Lymphocyte Ratio in Tibia Fracture-Related Infection.

Objective: The diagnostic value of neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR) in predicting fracture-related infection (FRI) in tibia fracture patients remains to be explored. Methods: A retrospective controlled study was carried out with 170 tibia FRI patients and 162 control subjects. The following information was evaluated at admission: age, gender, clinical features, number of white blood cells (WBCs), neutrophils, lymphocytes, monocytes, red blood cells (RBCs), platelets, level of hemoglobin, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR), as well as NLR, MLR, and PLR. Results: The number of lymphocytes, RBCs, and platelets in the FRI group was higher than those in the control group, while the number of neutrophils and ESR level was lower (P < 0.05). The level of NLR and MLR was significantly lower in patients with tibia FRI than in control subjects (P < 0.05). Both indicators were positively correlated with WBCs, CRP level, and ESR level (P < 0.001). The results of logistic regression analysis showed that five variables including NLR, MLR, platelets, fracture pattern (closed or open fracture), and site pattern (single or multiple site) were used to construct the FRI risk predictor. The ROC curve analysis result showed that FRI risk predictor yielded the highest AUC, with a sensitivity of 91.2% and a specificity of 90.1%, and made the distinction efficiently between tibia FRI patients and non-FRI patients. Conclusion: NLR and MLR were decreased in tibia FRI patients compared to non-FRI patients. Both indicators had a positive correlation with WBCs, CRP level, and ESR level. FRI risk predictor constructed based on five variables including NLR and MLR had a high diagnostic value for tibia FRI.

The Underlying Molecular Basis and Mechanisms of Venous Thrombosis in Patients with Osteomyelitis: A Data-Driven Analysis.

Objective: Osteomyelitis (OM) is one of the most risky and challenging diseases. Emerging evidence indicates OM is a risk factor for increasing incidence of venous thromboembolism (VTE) development. However, the mechanisms have not been intensively investigated. Methods: The OM-related dataset GSE30119 and VTE-related datasets GSE19151 and GSE48000 were downloaded from the Gene Expression Omnibus (GEO) database and analyzed to identify the differentially expressed genes (DEGs) (OMGs1 and VTEGs1, respectively). Functional enrichment analyses of Gene Ontology (GO) terms were performed. VTEGs2 and OMGs2 sharing the common GO biological process (GO-BP) ontology between OMGs1 and VTEGs1 were detected. The TRRUST database was used to identify the upstream transcription factors (TFs) that regulate VTEGs2 and OMGs2. The protein-protein interaction (PPI) network between VTEGs2 and OMGs2 was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database and then visualized in Cytoscape. Topological properties of the PPI network were calculated by NetworkAnalyzer. The Molecular Complex Detection (MCODE) plugin was utilized to perform module analysis and choose the hub modules of the PPI network. Results: A total of 587 OMGs1 and 382 VTEGs1 were identified from the related dataset, respectively. GO-BP terms of OMGs1 and shared DGEs1 were mainly enriched in the neutrophil-related immune response process, and the shared GO-BP terms of OMGs1 and VTEGs1 seemed to be focused on cell activation, immune, defense, and inflammatory response to stress or biotic stimulus. 230 VTEGs2, 333 OMGs2, and 13 shared DEGs2 were detected. 3 TF-target gene pairs (SP1-LSP1, SPI1-FCGR1A, and STAT1-FCGR1A) were identified. The PPI network contained 1611 interactions among 467 nodes. The top 10 hub proteins were TP53, IL4, MPO, ELANE, FOS, CD86, HP, SOCS3, ICAM1, and SNRPG. Several core nodes (such as MPO, ELANE, and CAMP) were essential components of the neutrophil extracellular traps (NETs) network. Conclusion: This is the first data-mining study to explore shared signatures between OM and VTE by the integrated bioinformatic approach, which can help uncover potential biomarkers and therapeutic targets of OM-related VTE.

Antenna effect of pyridoxal phosphate on the fluorescence of mitoxantrone-silicon nanoparticles and its application in alkaline phosphatase assay.

As a kind of sensing and imaging fluorescent probe with the merit of low toxicity, good stability, and environment-friendly, silicon nanoparticles (SiNPs) are currently attracting extensive research. In this work, we obtained mitoxantrone-SiNPs (MXT-SiNPs) with green emission by one-pot synthesis under mild temperature condition. The antenna based on pyridoxal phosphate (PLP) was designed for light-harvesting to enhance the luminescence of MXT-SiNPs and to establish a novel sensing strategy for alkaline phosphatase (ALP). PLP transfers the absorbed photon energy to MXT-SiNPs by forming Schiff base. When PLP is dephosphorized by ALP, the released free hydroxyl group reacts with aldehyde group to form internal hemiacetal, which leads to the failure of Schiff base formation. Based on the relationship between antenna formation ability and PLP hydrolysis degree, the activity of ALP can be measured. A good linear relationship was obtained from 0.2 to 3.0 U/L, with a limit of detection of 0.06 U/L. Furthermore, the sensing platform was successfully used to detect ALP in human serum with recovery of 97.6-106.2%. The rational design of antenna elements for fluorescent nanomaterials can not only provide a new pathway to manipulate the luminescence, but also provide a new direction for fluorescence sensing strategy.

Highly reproducible and sensitive electrochemical biosensor for Chlamydia trachomatis detection based on duplex-specific nuclease-assisted target-responsive DNA hydrogels and bovine serum albumin carrier platform.

16S ribosomal-RNA (16S rRNA) is often used as an ultrasensitive marker for Chlamydia trachomatis (CT) detection because of its species specificity and high copy number in CT. Robust methods for 16S rRNA detection must be developed to realize the early diagnosis of CT infections. In this work, a highly reproducible and sensitive electrochemical biosensor based on duplex-specific nuclease (DSN)-assisted target-responsive DNA hydrogels and bovine serum albumin (BSA) carrier platform for CT detection was developed. Target rRNA can trigger the DNA hydrogel response, which causes it to be repeatedly cleaved by DSN, ultimately leading to the release of a large amount of horseradish peroxidase-labelled streptavidin (SA-HRP) embedded in the hydrogel beforehand. The released SA-HRP was stably captured by the capture probes that were orderly loaded at the gold electrode with the help of a BSA layer. Then, SA-HRP catalyzed the redox reaction of 3,3',5,5'-tetramethylbenzidine and H2O2, producing a current signal that can be detected. The current signal was proportional to the concentration of CT 16S rRNA from 10 fM to 25 pM with a detection limit of 5.8 fM (S/N = 3). The signal conversion function of the DNA hydrogel avoids the instability of nonhomogeneous nucleic acid hybridization on the gold electrode surface, and combined with optimization by BSA for capture probe modification, this electrochemical biosensor is highly reproducible with a relative standard deviation of 4.3% for the detection of 10 samples of the same concentration. The proposed strategy provides a highly reproducible and sensitive detection method for the extensive screening of CT.

Highly Reproducible and Sensitive Electrochemiluminescence Biosensors for HPV Detection Based on Bovine Serum Albumin Carrier Platforms and Hyperbranched Rolling Circle Amplification.

Most DNA-based electrochemiluminescence (ECL) biosensors are established through the self-assembly of thiolated single-stranded DNA (ssDNA) probes on the Au electrode surface. Because of this random assembly process, a significant discrepancy exists in the distribution of a modified DNA film on different electrodes, which greatly affects the reproducibility of a biosensor. In this study, a porous bovine serum albumin (BSA) layer was first modified on the electrode surface, which can improve the position distribution and spatial orientation of the self-assembly ssDNA probe. It was then coupled with hyperbranched rolling circle amplification to develop the high-reproducibility-and-sensitivity ECL biosensor for human papillomavirus 16 E6 and E7 oncogene detection. In the presence of the target DNA, the surface of the electrode accumulates abundant amplified products through reaction, which contain double-stranded DNA (dsDNA) fragments of different lengths, followed by plentiful dichlorotris (1,10-phenanthroline) ruthenium(II) hydrate (Ru(phen)32+, acting as an ECL indicator) insertion into grooves of dsDNA fragments, and a strong signal can be detected. There is a linear relationship between the signal and the target concentration range from 10 fM to 15 pM, and the detection limit is 7.6 fM (S/N = 3). After the BSA modification step, the relative standard deviation was reduced from 9.20 to 3.96%, thereby achieving good reproducibility. The proposed ECL strategy provides a new method for constructing high-reproducibility-and-sensitivity ECL biosensors.

Gold nanoclusters/graphene quantum dots complex-based dual-emitting ratiometric fluorescence probe for the determination of glucose.

Herein, we report the design of a single-excitation/double-emission ratiometric fluorescence nanosensor for the determination of glucose. The sensing system combines glucose oxidation catalyzed by glucose oxidase, Fenton chemistry, Fe3+-sensitive fluorescent gold nanoclusters (AuNCs), and Fe3+-inert fluorescent graphene quantum dots (GQDs). We used orange-fluorescent AuNCs co-modified with bovine serum albumin and 3-mercaptopropionic acid as the indicator probe, and GQDs with the same excitation wavelength as the BSA/MPA-AuNCs, but with different emission wavelength, as the reference probe. The fluorescence intensity-ratio between 420 nm and 575 nm (F420/F575) was used to quantitatively determine glucose with a low detection limit of 0.18 µM, and the nanosensor was successfully used to detect glucose in human serum. This ratiometric fluorescence sensing system, based on AuNCs and GQDs, ensures sensitive and convenient determination of glucose, and has broad application prospects for biomedical-analysis applications.

New photothermal immunoassay of human chorionic gonadotropin using Prussian blue nanoparticle-based photothermal conversion.

A new near-infrared-based photothermal immunosensing strategy was developed for the sensitive and feasible detection of human chorionic gonadotropin (HCG) by use of a Prussian blue nanoparticle-based photothermal conversion system. Prussian blue nanospheres synthesized by the one-pot method were used for the labeling of anti-HCG detection antibody. A sandwich-type immunoreaction was initially conducted on a monoclonal anti-HCG antibody-coated microplate with a nanoparticle-labeled signal antibody. Accompanying formation of the sandwiched immunocomplex, Prussian blue nanospheres caused photothermal conversion under 980-nm laser irradiation, thereby resulting in an increase of the temperature of the detection system measured by a portable digital thermometer. The properties and factors influencing the analytical performance of the photothermal immunoassay were studied in detail. Under the optimal conditions, the Prussian blue nanoparticle-based photothermal immunoassay exhibited good temperature responses relative to target HCG concentrations within the dynamic range of 0.01-100 ng mL-1 at a low detection limit of 5.8 pg mL-1. This system also displayed good anti-interference behavior with regard to other cancer biomarkers, good reproducibility, and relatively long storage stability. The method accuracy was evaluated for analysis of human serum specimens, giving results that matched well with those obtained with a commercial HCG enzyme-linked immunosorbent assay kit. Importantly, this protocol is promising for advanced development of photothermal immunoassays. Graphical abstract.

Fabrication of ultra-small monolayer graphene quantum dots by pyrolysis of trisodium citrate for fluorescent cell imaging.

BACKGROUND: The preparation and biological applications of ultra-small graphene quantum dots (GQDs) with accurate-controlled size are of great significance. METHODS: Here in, we report a novel procedure involving pyrolysis of trisodium citrate and subsequent ultrafiltration for fabricating monolayer GQDs with ultra-small lateral size (1.3±0.5 nm). RESULTS: The GQDs exhibit blue photoluminescence with peak position independent of excitation wavelength. The quantum yield of GQDs is measured to be 3.6%, and the average fluorescence lifetime is 2.78 ns. CONCLUSION: Because of high stability and low toxicity, GQDs are demonstrated to be excellent bioimaging agents. The ultra-small GQDs can not only distribute in the cytoplasm but also penetrate into the nuclei. We ensure that this work will add a new dimension to the application of graphene materials for nanomedicine.

Preparation of strongly fluorescent water-soluble dithiothreitol modified gold nanoclusters coated with carboxychitosan, and their application to fluorometric determination of the immunosuppressive 6-mercaptopurine.

Water-soluble and non-aggregating gold nanoclusters (AuNCs) were obtained by modification of the AuNCs with dithiothreitol (DTT) and then coating them with carboxylated chitosan. This process remarkably enhances the dispersibility of DTT-coated AuNCs in water. The resulting AuNCs, on photoexcitation at 285 nm, display strong red emission with a maximum at 650 nm and a 23% quantum yield. Fluorescence is strongly and selectively suppressed in the presence of 6-mercaptopurine (6-MP). Photoluminescence drops linearly in the 0.1-100 µM 6-MP concentration range, and the detection limit of this assay is 0.1 µM. Other features of the modified AuNCs include a decay time of 8.56 µs, a 365 nm Stokes shift, good colloidal stability, ease of chemical modification, and low toxicity. Conceivably, these NCs may find a range of applications in biological imaging and optical sensing. Graphical abstract Highly fluorescent and water-soluble gold nanoclusters (AuNCs) were obtained by modification of the AuNCs with dithiothreitol (DTT) and then coating them with carboxylated chitosan (CC). The resulting CC/DTT-AuNCs were used for sensitive and selective detection of 6-mercaptopurine.

Chitosan-stabilized platinum nanoparticles as effective oxidase mimics for colorimetric detection of acid phosphatase.

Capping molecules on the surface of nanomaterials not only enhance the dispersion and stability of nanomaterials but also greatly facilitate their surface modification and biological applications. However, most capping molecules can severely block the active sites of the catalytic core, thereby decreasing the enzymatic activity of nanomaterial-based enzyme mimics. This work demonstrates the superiority of chitosan (Ch) as a capping molecule for synthesizing catalytic platinum nanoparticles (PtNPs). The experimental results show that Ch simultaneously exhibits an excellent stabilizing effect and enhances the oxidase-like activity of PtNPs. Kinetic studies indicate that Ch-PtNPs have a higher affinity for 3,3',5,5'-tetramethylbenzidine (TMB) than other kinds of oxidase mimics. Furthermore, the TMB chromogenic reaction catalyzed by Ch-PtNPs is found to be much faster in an acidic medium, thus adapting well to the optimal pH for acid phosphatase (ACP). Therefore, a novel colorimetric approach for ACP determination is developed for the first time, which is based on the Ch-PtNP-catalyzed oxidation of TMB, the inhibitory effect of ascorbic acid (AA) on the oxidase-like activity of Ch-PtNPs, and the ACP-catalyzed hydrolysis of AA 2-phosphate (AAP) into AA. The linear range for ACP is 0.25-2.5 U L-1 and the limit of detection is measured to be 0.016 U L-1. This new colorimetric method is utilized to detect ACP in real biological samples and to screen ACP inhibitors. We believe that these new PtNPs, which exhibit high colloidal stability, excellent catalytic performance, good biocompatibility, simple preparation, and easy modification, can be promising candidates for a broad range of applications in optical sensing, environmental monitoring, clinical diagnosis, and drug discovery.

Partially reduced graphene oxide as highly efficient DNA nanoprobe.

This work investigates the effect of reduction degree on graphene oxide (GO)-DNA interaction and the fluorescence quenching mechanism. Partial reduced graphene oxide (pRGO), which maintains well water-dispersibility, is synthesized using a mild reduction method by incubating GO suspension under alkaline condition at room temperature. The fluorescence quenching enhances with the restoration degree of sp(2) carbon bonds and follows the static quenching mechanism. The binding constant values imply that pRGO has much stronger affinity with ssDNA than GO. Utilizing this highly efficient nanoprobe, a universal sensing strategy is proposed for homogeneous detection of DNA. Compared with the reported GO-based DNA, this present strategy has obvious advantages such as requirement of low nanoprobe dosage, significantly reduced background, fast fluorescence quenching, and improved sensitivity. Even without any amplification process, the limit of detection can reach as low as 50 pM.

[Chemical constituents of leaves of Panax japonicus var. major].

Seven compounds were isolated from the leaves of Panax japonicus var. major by chromatographic methods including silica gel, Sephadex LH-20, ODS and semi-preparative HPLC. Their structures were elucidated by their physical and chemical properties and spectral data analysis as 5, 7-dihydroxy-8-methoxyl flavone (1), ginsenoside Rs2 (2), quinquenoside R1 (3), ginsenoside Rs1 (4), notoginsenoside Fe (5), ginsenoside Rd2 (6) and gypenosiden IX (7). Among them, compound 1 was obtained from the Panax genus for the first time, and compounds 2-7 were isolated from this plant for the first time.

Fluorescent hydrogen peroxide sensor based on cupric oxide nanoparticles and its application for glucose and L-lactate detection.

A novel fluorescent hydrogen peroxide sensor was developed based on the peroxidase-like activity of cupric oxide nanoparticles. Cupric oxide nanoparticles effectively catalyzed the decomposition of hydrogen peroxide into hydroxyl radicals. Then terephthalic acid was oxidized by hydroxyl radical to form a highly fluorescent product. The linear range of hydrogen peroxide estimated to be 5.0 × 10(-6)-2.0 × 10(-4)M with a detection limit of 3.4 × 10(-7)M. Moreover, this detection system enabled the sensing of analytes which can enzymatically generate hydrogen peroxide. By coupling the oxidation of glucose or L-lactate catalyzed by their corresponding oxidase enzymes with terephthalic acid oxidation catalyzed by cupric oxide nanoparticles, sensitive assays of glucose and l-lactate with detection limits of 1.0 × 10(-6) and 4.5 × 10(-8)M were realized. The successful applications of this approach in human serum samples have also been demonstrated.

Bovine serum albumin-based probe carrier platform for electrochemical DNA biosensing.

A bovine serum albumin (BSA)-monolayer-based probe carrier platform is shown to improve the performance of a conventional thiolated single-stranded DNA probe self-assembled-monolayer-based electrochemical DNA hybridization biosensor. A detection limit of 0.5 fM can be obtained in a very reproducible manner (relative standard deviation <5%), along with high specificity. The performance of the biosensor can be attributed primarily to the enhanced spatial positioning range and accessibility of the probes on the BSA-based platform. Furthermore, the novel biosensor shows high resistance to nonspecific adsorption of nucleic acid and protein and can be directly employed in detection in biological fluids. These advantages give this simple developed methodology great promise for a wide range of nucleic acid testing.

Ultrasensitive and facile electrochemical deoxyribonucleic acid biosensor based on the conformational change of the recognition interface.

An ultrasensitive electrochemical impedance spectroscopic deoxyribonucleic acid biosensor has been developed based on the conformational change of the deoxyribonucleic acid recognition interface with lodging probes. Pairing process leads to desorption of deoxyribonucleic acid bases from the gold surface, leading to a significant change of the interfacial conformation and the charge transfer resistance. Remarkably low detection limits down to 40 fM are thus obtained without any additional amplification step.

A sandwich-type DNA electrochemical biosensor for hairpin-stem-loop structure based on multistep temperature-controlling method.

A highly sensitive and selective method for amplified electrochemical detection for hairpin-stem-loop structured target sequences was developed based on the temperature regulation of DNA hybrids on a sandwich-type electrochemical DNA sensor. Multistep hybridization was applied to promote the hybridization efficiency of each section of sandwich structure. The results showed that both multistep and temperature-controlling hybridization techniques were both especially made to fabricate the sensor for the tendency of internal hybridization of target gene sequences. This strategy provides significantly enhanced hybridization efficiency and sequence specificity of electrochemical detection.