The evolution of autonomy from two cooperative specialists in fluctuating environments.

From microbes to humans, organisms perform numerous tasks for their survival, including food acquisition, migration, and reproduction. A complex biological task can be performed by either an autonomous organism or by cooperation among several specialized organisms. However, it remains unclear how autonomy and cooperation evolutionarily switch. Specifically, it remains unclear whether and how cooperative specialists can repair deleted genes through direct genetic exchange, thereby regaining metabolic autonomy. Here, we address this question by experimentally evolving a mutualistic microbial consortium composed of two specialists that cooperatively degrade naphthalene. We observed that autonomous genotypes capable of performing the entire naphthalene degradation pathway evolved from two cooperative specialists and dominated the community. This evolutionary transition was driven by the horizontal gene transfer (HGT) between the two specialists. However, this evolution was exclusively observed in the fluctuating environment alternately supplied with naphthalene and pyruvate, where mutualism and competition between the two specialists alternated. The naphthalene-supplied environment exerted selective pressure that favors the expansion of autonomous genotypes. The pyruvate-supplied environment promoted the coexistence and cell density of the cooperative specialists, thereby increasing the likelihood of HGT. Using a mathematical model, we quantitatively demonstrate that environmental fluctuations facilitate the evolution of autonomy through HGT when the relative growth rate and carrying capacity of the cooperative specialists allow enhanced coexistence and higher cell density in the competitive environment. Together, our results demonstrate that cooperative specialists can repair deleted genes through a direct genetic exchange under specific conditions, thereby regaining metabolic autonomy.

GTPBP8 modulates mitochondrial fission through a Drp1-dependent process.

Mitochondrial fission is a tightly regulated process involving multiple proteins and cell signaling. Despite extensive studies on mitochondrial fission factors, our understanding of the regulatory mechanisms remains limited. This study shows the critical role of a mitochondrial GTPase, GTPBP8, in orchestrating mitochondrial fission in mammalian cells. Depletion of GTPBP8 resulted in drastic elongation and interconnectedness of mitochondria. Conversely, overexpression of GTPBP8 shifted mitochondrial morphology from tubular to fragmented. Notably, the induced mitochondrial fragmentation from GTPBP8 overexpression was inhibited in cells either depleted of the mitochondrial fission protein Drp1 (also known as DNM1L) or carrying mutated forms of Drp1. Importantly, downregulation of GTPBP8 caused an increase in oxidative stress, modulating cell signaling involved in the increased phosphorylation of Drp1 at Ser637. This phosphorylation hindered the recruitment of Drp1 to mitochondria, leading to mitochondrial fission defects. By contrast, GTPBP8 overexpression triggered enhanced recruitment and assembly of Drp1 at mitochondria. In summary, our study illuminates the cellular function of GTPBP8 as a pivotal modulator of the mitochondrial division apparatus, inherently reliant on its influence on Drp1.

Production of the antidepressant orcinol glucoside in Yarrowia lipolytica with yields over 6,400-fold higher than plant extraction.

Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herb Curculigo orchioides Gaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production in Yarrowia lipolytica was improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield from C. orchioides roots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.

Interaction network of human early embryonic transcription factors.

Embryonic genome activation (EGA) occurs during preimplantation development and is characterized by the initiation of de novo transcription from the embryonic genome. Despite its importance, the regulation of EGA and the transcription factors involved in this process are poorly understood. Paired-like homeobox (PRDL) family proteins are implicated as potential transcriptional regulators of EGA, yet the PRDL-mediated gene regulatory networks remain uncharacterized. To investigate the function of PRDL proteins, we are identifying the molecular interactions and the functions of a subset family of the Eutherian Totipotent Cell Homeobox (ETCHbox) proteins, seven PRDL family proteins and six other transcription factors (TFs), all suggested to participate in transcriptional regulation during preimplantation. Using mass spectrometry-based interactomics methods, AP-MS and proximity-dependent biotin labeling, and chromatin immunoprecipitation sequencing we derive the comprehensive regulatory networks of these preimplantation TFs. By these interactomics tools we identify more than a thousand high-confidence interactions for the 21 studied bait proteins with more than 300 interacting proteins. We also establish that TPRX2, currently assigned as pseudogene, is a transcriptional activator.

OPIA: an open archive of plant images and related phenotypic traits.

High-throughput plant phenotype acquisition technologies have been extensively utilized in plant phenomics studies, leading to vast quantities of images and image-based phenotypic traits (i-traits) that are critically essential for accelerating germplasm screening, plant diseases identification and biotic & abiotic stress classification. Here, we present the Open Plant Image Archive (OPIA, https://ngdc.cncb.ac.cn/opia/), an open archive of plant images and i-traits derived from high-throughput phenotyping platforms. Currently, OPIA houses 56 datasets across 11 plants, comprising a total of 566 225 images with 2 417 186 labeled instances. Notably, it incorporates 56 i-traits of 93 rice and 105 wheat cultivars based on 18 644 individual RGB images, and these i-traits are further annotated based on the Plant Phenotype and Trait Ontology (PPTO) and cross-linked with GWAS Atlas. Additionally, each dataset in OPIA is assigned an evaluation score that takes account of image data volume, image resolution, and the number of labeled instances. More importantly, OPIA is equipped with useful tools for online image pre-processing and intelligent prediction. Collectively, OPIA provides open access to valuable datasets, pre-trained models, and phenotypic traits across diverse plants and thus bears great potential to play a crucial role in facilitating artificial intelligence-assisted breeding research.

Small mitochondrial protein NERCLIN regulates cardiolipin homeostasis and mitochondrial ultrastructure.

Cardiolipin (CL) is an essential phospholipid for mitochondrial structure and function. Here, we present a small mitochondrial protein, NERCLIN, as a negative regulator of CL homeostasis and mitochondrial ultrastructure. Primate-specific NERCLIN is expressed ubiquitously from the GRPEL2 locus on a tightly regulated low level. NERCLIN overexpression severely disrupts mitochondrial cristae structure and induces mitochondrial fragmentation. Proximity labeling and immunoprecipitation analysis suggested interactions of NERCLIN with CL synthesis and prohibitin complexes on the matrix side of the inner mitochondrial membrane. Lipid analysis indicated that NERCLIN regulates mitochondrial CL content. Furthermore, NERCLIN is responsive to heat stress ensuring OPA1 processing and cell survival. Thus, we propose that NERCLIN contributes to the stress-induced adaptation of mitochondrial dynamics. Our findings add NERCLIN to the group of recently identified small mitochondrial proteins with important regulatory functions.

Mapping protein-protein interactions by mass spectrometry.

Protein-protein interactions (PPIs) are essential for numerous biological activities, including signal transduction, transcription control, and metabolism. They play a pivotal role in the organization and function of the proteome, and their perturbation is associated with various diseases, such as cancer, neurodegeneration, and infectious diseases. Recent advances in mass spectrometry (MS)-based protein interactomics have significantly expanded our understanding of the PPIs in cells, with techniques that continue to improve in terms of sensitivity, and specificity providing new opportunities for the study of PPIs in diverse biological systems. These techniques differ depending on the type of interaction being studied, with each approach having its set of advantages, disadvantages, and applicability. This review highlights recent advances in enrichment methodologies for interactomes before MS analysis and compares their unique features and specifications. It emphasizes prospects for further improvement and their potential applications in advancing our knowledge of PPIs in various biological contexts.

LUZP1 regulates the maturation of contractile actomyosin bundles.

Contractile actomyosin bundles play crucial roles in various physiological processes, including cell migration, morphogenesis, and muscle contraction. The intricate assembly of actomyosin bundles involves the precise alignment and fusion of myosin II filaments, yet the underlying mechanisms and factors involved in these processes remain elusive. Our study reveals that LUZP1 plays a central role in orchestrating the maturation of thick actomyosin bundles. Loss of LUZP1 caused abnormal cell morphogenesis, migration, and the ability to exert forces on the environment. Importantly, knockout of LUZP1 results in significant defects in the concatenation and persistent association of myosin II filaments, severely impairing the assembly of myosin II stacks. The disruption of these processes in LUZP1 knockout cells provides mechanistic insights into the defective assembly of thick ventral stress fibers and the associated cellular contractility abnormalities. Overall, these results significantly contribute to our understanding of the molecular mechanism involved in actomyosin bundle formation and highlight the essential role of LUZP1 in this process.

GWAS Atlas: an updated knowledgebase integrating more curated associations in plants and animals.

GWAS Atlas (https://ngdc.cncb.ac.cn/gwas/) is a manually curated resource of genome-wide genotype-to-phenotype associations for a wide range of species. Here, we present an updated implementation of GWAS Atlas by curating and incorporating more high-quality associations, with significant improvements and advances over the previous version. Specifically, the current release of GWAS Atlas incorporates a total of 278,109 curated genotype-to-phenotype associations for 1,444 different traits across 15 species (10 plants and 5 animals) from 830 publications and 3,432 studies. A collection of 6,084 lead SNPs of 439 traits and 486 experiment-validated causal variants of 157 traits are newly added. Moreover, 1,056 trait ontology terms are newly defined, resulting in 1,172 and 431 terms for Plant Phenotype and Trait Ontology and Animal Phenotype and Trait Ontology, respectively. Additionally, it is equipped with four online analysis tools and a submission platform, allowing users to perform data analysis and data submission. Collectively, as a core resource in the National Genomics Data Center, GWAS Atlas provides valuable genotype-to-phenotype associations for a diversity of species and thus plays an important role in agronomic trait study and molecular breeding.

Integrative analysis of single-cell embryo data reveals transcriptome signatures for the human pre-implantation inner cell mass.

As the source of embryonic stem cells (ESCs), inner cell mass (ICM) can form all tissues of the embryo proper, however, its role in early human lineage specification remains controversial. Although a stepwise differentiation model has been proposed suggesting the existence of ICM as a distinct developmental stage, the underlying molecular mechanism remains unclear. In the present study, we perform an integrated analysis on the public human preimplantation embryonic single-cell transcriptomic data and apply a trajectory inference algorithm to measure the cell plasticity. In our results, ICM population can be clearly discriminated on the dimension-reduced graph and confirmed by compelling evidences, thus validating the two-step hypothesis of lineage commitment. According to the branch probabilities and differentiation potential, we determine the precise time points for two lineage segregations. Further analysis on gene expression dynamics and regulatory network indicates that transcription factors including GSC, PRDM1, and SPIC may underlie the decisions of ICM fate. In addition, new human ICM marker genes, such as EPHA4 and CCR8 are discovered and validated by immunofluorescence. Given the potential clinical applications of ESCs, our analysis provides a further understanding of human ICM cells and facilitates the exploration of more unique characteristics in early human development.

Retinal primary cilia and their dysfunction in retinal neurodegenerative diseases: beyond ciliopathies.

Primary cilia are sensory organelles that extend from the cellular membrane and are found in a wide range of cell types. Cilia possess a plethora of vital components that enable the detection and transmission of several signaling pathways, including Wnt and Shh. In turn, the regulation of ciliogenesis and cilium length is influenced by various factors, including autophagy, organization of the actin cytoskeleton, and signaling inside the cilium. Irregularities in the development, maintenance, and function of this cellular component lead to a range of clinical manifestations known as ciliopathies. The majority of people with ciliopathies have a high prevalence of retinal degeneration. The most common theory is that retinal degeneration is primarily caused by functional and developmental problems within retinal photoreceptors. The contribution of other ciliated retinal cell types to retinal degeneration has not been explored to date. In this review, we examine the occurrence of primary cilia in various retinal cell types and their significance in pathology. Additionally, we explore potential therapeutic approaches targeting ciliopathies. By engaging in this endeavor, we present new ideas that elucidate innovative concepts for the future investigation and treatment of retinal ciliopathies.

A Lateral Flow Biosensor Based on Isothermal Amplification for Visual Identification of Species and Sex from Bloodstain.

The prompt species identification from biological samples at a crime scene can rapidly filter out truly valuable biometric information for subsequent personal identification. Meanwhile, early sex determination can assist in narrowing the pool of suspects. However, the current methods for forensic DNA analysis, particularly in point-of-care scenarios, are often limited by the intricate equipment for signal generation and the laborious procedure for DNA purification. The present study introduces a novel portable lateral flow biosensor that possesses extraction-free and anti-aerosol characteristics for on-site determination of species and sex. The bloodstain can be directly submitted to loop-mediated isothermal amplification (LAMP) for the analysis of both mitochondrial and nuclear DNA. The incorporation of a lateral flow device with gold magnetic nanoparticle probes allows for visual interpretation of results through colorimetric signals while also preventing interference on result judgment from pigments such as hemoglobin. Carryover contamination, which is a disharmonious factor in LAMP, especially as the inherent contradiction derived from uncapping in the lateral flow strategy, has been effectively addressed through the integration of uracil DNA glycosylase without compromising the isothermy throughout the process. As a proof-of-concept experiment, species and sex can be accurately identified within 40 min from trace bloodstains amidst significant background interference by targeting cytochrome b and Y-chromosomal amelogenin. Furthermore, the single-blind study revealed a concordance rate of up to 100% in both simulative degraded and true dated bloodstains. This suggests that this biosensor has the potential to be utilized in forensic DNA analysis at crime scenes.

A computational framework to unify orthogonal information in DNA methylation and copy number aberrations in cell-free DNA for early cancer detection.

Cell-free DNA (cfDNA) provides a convenient diagnosis avenue for noninvasive cancer detection. The current methods are focused on identifying circulating tumor DNA (ctDNA)s genomic aberrations, e.g. mutations, copy number aberrations (CNAs) or methylation changes. In this study, we report a new computational method that unifies two orthogonal pieces of information, namely methylation and CNAs, derived from whole-genome bisulfite sequencing (WGBS) data to quantify low tumor content in cfDNA. It implements a Bayes model to enrich ctDNA from WGBS data based on hypomethylation haplotypes, and subsequently, models CNAs for cancer detection. We generated WGBS data in a total of 262 samples, including high-depth (>20×, deduped high mapping quality reads) data in 76 samples with matched triplets (tumor, adjacent normal and cfDNA) and low-depth (~2.5×, deduped high mapping quality reads) data in 186 samples. We identified a total of 54 Mb regions of hypomethylation haplotypes for model building, a vast majority of which are not covered in the HumanMethylation450 arrays. We showed that our model is able to substantially enrich ctDNA reads (tens of folds), with clearly elevated CNAs that faithfully match the CNAs in the paired tumor samples. In the 19 hepatocellular carcinoma cfDNA samples, the estimated enrichment is as high as 16 fold, and in the simulation data, it can achieve over 30-fold enrichment for a ctDNA level of 0.5% with a sequencing depth of 600×. We also found that these hypomethylation regions are also shared among many cancer types, thus demonstrating the potential of our framework for pancancer early detection.

Internal Vanadium Doping and External Modification Design of P2-Type Layered Mn-Based Oxides as Competitive Cathodes toward Sodium-Ion Batteries.

P2-type layered manganese-based oxides have attracted considerable interest as economical, cathode materials with high energy density for sodium-ion batteries (SIBs). Despite their potential, these materials still face challenges related to sluggish kinetics and structural instability. In this study, a composite cathode material, Na0.67Ni0.23Mn0.67V0.1O2@Na3V2O2(PO4)2F was developed by surface-coating P2-type Na0.67Ni0.23Mn0.67V0.1O2 with a thin layer of Na3V2O2(PO4)2F to enhance both the electrochemical sodium storage and material air stability. The optimized Na0.67Ni0.23Mn0.67V0.1O2@5wt %Na3V2O2(PO4)2F exhibited a high discharge capacity of 176 mA h g-1 within the 1.5-4.1 V range at a low current density of 17 mA g-1. At an increased current density of 850 mA g-1 within the same voltage window, it still delivered a substantial initial discharge capacity of 112 mAh g-1. These findings validate the significant enhancement of ion diffusion capabilities and rate performance in the P2-type Na0.67Ni0.33Mn0.67O2 material conferred by the composite cathode.

The cytoplasmic localization of ADNP through 14-3-3 promotes sex-dependent neuronal morphogenesis, cortical connectivity, and calcium signaling.

Defective neuritogenesis is a contributing pathogenic mechanism underlying a variety of neurodevelopmental disorders. Single gene mutations in activity-dependent neuroprotective protein (ADNP) are the most frequent among autism spectrum disorders (ASDs) leading to the ADNP syndrome. Previous studies showed that during neuritogenesis, Adnp localizes to the cytoplasm/neurites, and Adnp knockdown inhibits neuritogenesis in culture. Here, we hypothesized that Adnp is localized in the cytoplasm during neurite formation and that this process is mediated by 14-3-3. Indeed, applying the 14-3-3 inhibitor, difopein, blocked Adnp cytoplasmic localization. Furthermore, co-immunoprecipitations showed that Adnp bound 14-3-3 proteins and proteomic analysis identified several potential phosphorylation-dependent Adnp/14-3-3 binding sites. We further discovered that knockdown of Adnp using in utero electroporation of mouse layer 2/3 pyramidal neurons in the somatosensory cortex led to previously unreported changes in neurite formation beginning at P0. Defects were sustained throughout development, the most notable included increased basal dendrite number and axon length. Paralleling the observed morphological aberrations, ex vivo calcium imaging revealed that Adnp deficient neurons had greater and more frequent spontaneous calcium influx in female mice. GRAPHIC, a novel synaptic tracing technology substantiated this finding, revealing increased interhemispheric connectivity between female Adnp deficient layer 2/3 pyramidal neurons. We conclude that Adnp is localized to the cytoplasm by 14-3-3 proteins, where it regulates neurite formation, maturation, and functional cortical connectivity significantly building on our current understanding of Adnp function and the etiology of ADNP syndrome.

Physical and functional interactome atlas of human receptor tyrosine kinases.

Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK-protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.

Assessing the importance of primary care diagnoses in the UK Biobank.

The UK Biobank has made general practitioner (GP) data (censoring date 2016-2017) available for approximately 45% of the cohort, whilst hospital inpatient and death registry (referred to as "HES/Death") data are available cohort-wide through 2018-2022 depending on whether the data comes from England, Wales or Scotland. We assessed the importance of case ascertainment via different data sources in UKB for three diseases that are usually first diagnosed in primary care: Parkinson's disease (PD), type 2 diabetes (T2D), and all-cause dementia. Including GP data at least doubled the number of incident cases in the subset of the cohort with primary care data (e.g. from 619 to 1390 for dementia). Among the 786 dementia cases that were only captured in the GP data before the GP censoring date, only 421 (54%) were subsequently recorded in HES. Therefore, estimates of the absolute incidence or risk-stratified incidence are misleadingly low when based only on the HES/Death data. For incident cases present in both HES/Death and GP data during the full follow-up period (i.e. until the HES censoring date), the median time difference between an incident diagnosis of dementia being recorded in GP and HES/Death was 2.25 years (i.e. recorded 2.25 years earlier in the GP records). Similar lag periods were also observed for PD (median 2.31 years earlier) and T2D (median 2.82 years earlier). For participants with an incident GP diagnosis, only 65.6% of dementia cases, 69.0% of PD cases, and 58.5% of T2D cases had their diagnosis recorded in HES/Death within 7 years since GP diagnosis. The effect estimates (hazard ratios, HR) of established risk factors for the three health outcomes mostly remain in the same direction and with a similar strength of association when cases are ascertained either using HES only or further adding GP data. The confidence intervals of the HR became narrower when adding GP data, due to the increased statistical power from the additional cases. In conclusion, it is desirable to extend both the coverage and follow-up period of GP data to allow researchers to maximise case ascertainment of chronic health conditions in the UK.

GTPBP8 is required for mitoribosomal biogenesis and mitochondrial translation.

Mitochondrial translation occurs on the mitochondrial ribosome, also known as the mitoribosome. The assembly of mitoribosomes is a highly coordinated process. During mitoribosome biogenesis, various assembly factors transiently associate with the nascent ribosome, facilitating the accurate and efficient construction of the mitoribosome. However, the specific factors involved in the assembly process, the precise mechanisms, and the cellular compartments involved in this vital process are not yet fully understood. In this study, we discovered a crucial role for GTP-binding protein 8 (GTPBP8) in the assembly of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. GTPBP8 is identified as a novel GTPase located in the matrix and peripherally bound to the inner mitochondrial membrane. Importantly, GTPBP8 is specifically associated with the mt-LSU during its assembly. Depletion of GTPBP8 leads to an abnormal accumulation of mt-LSU, indicating that GTPBP8 is critical for proper mt-LSU assembly. Furthermore, the absence of GTPBP8 results in reduced levels of fully assembled 55S monosomes. This impaired assembly leads to compromised mitochondrial translation and, consequently, impaired mitochondrial function. The identification of GTPBP8 as an important player in these processes provides new insights into the molecular mechanisms underlying mitochondrial protein synthesis and its regulation.

BrainBase: a curated knowledgebase for brain diseases.

Brain is the central organ of the nervous system and any brain disease can seriously affect human health. Here we present BrainBase (https://ngdc.cncb.ac.cn/brainbase), a curated knowledgebase for brain diseases that aims to provide a whole picture of brain diseases and associated genes. Specifically, based on manual curation of 2768 published articles along with information retrieval from several public databases, BrainBase features comprehensive collection of 7175 disease-gene associations spanning a total of 123 brain diseases and linking with 5662 genes, 16 591 drug-target interactions covering 2118 drugs/chemicals and 623 genes, and five types of specific genes in light of expression specificity in brain tissue/regions/cerebrospinal fluid/cells. In addition, considering the severity of glioma among brain tumors, the current version of BrainBase incorporates 21 multi-omics datasets, presents molecular profiles across various samples/conditions and identifies four groups of glioma featured genes with potential clinical significance. Collectively, BrainBase integrates not only valuable curated disease-gene associations and drug-target interactions but also molecular profiles through multi-omics data analysis, accordingly bearing great promise to serve as a valuable knowledgebase for brain diseases.

Clinical Effect of Ultrasound-Guided Adductor Block on Postoperative Analgesia After Total Knee Replacement.

Objective: To explore the clinical effects of ultrasound-guided adductor block (UGAB) on postoperative analgesia after total knee replacement. Methods: From March 2022 to June 2022, 60 patients in the First Affiliated Hospital of Chongqing Medical University were included. They were divided into control (n = 30) and ultrasonic groups (n = 30). They all received total knee arthroplasty. Before total knee arthroplasty, patients in the control and ultrasonic groups underwent general anesthesia and UGAB, respectively. Visual Analogue Scale (VAS) was used to assess the pain. The time of the first straight leg elevation and the first landing time were recorded. Knee joint function was evaluated. Information about the dosage of tramadol intramuscular injection and the number of times patient-controlled analgesia pump pressing was collected. The serum levels of interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hs-CRP) were detected. Results: Compared with the control group, UGAB increased the rate of muscle contraction and relaxation and total and relaxation after total knee replacement in the ultrasonic group (P < .05). UGAB reduced VAS scores of pain during passive activity after operation (P < .05). UGAB also facilitated the first straight leg lifting time after the operation and the time of the first landing after the operation (P < .05). Meanwhile, UGAB reduced the dose of tramadol and press times of the self-control analgesia pump after operation (P < 0.05). UGAB also suppressed postoperative IL-6 and hs-CRP levels and increased postoperative joint range of motion (P < .05). Conclusion: UGAB promotes early recovery of knee function with high safety in patients undergoing total knee replacement, with reduced postoperative pain and inflammatory reaction.