Upregulation of HAS2 promotes glioma cell proliferation and chemoresistance via c-myc.

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant human brain tumor. Although comprehensive therapies, including chemotherapy and radiotherapy following surgery, have shown promise in prolonging survival, the prognosis for GBM patients remains poor, with an overall survival rate of only 14.6 months. Chemoresistance is a major obstacle to successful treatment and contributes to relapse and poor survival rates in glioma patients. Therefore, there is an urgent need for novel strategies to overcome chemoresistance and improve treatment outcomes for human glioma patients. Recent studies have shown that the tumor microenvironment plays a key role in chemoresistance. Our study demonstrates that upregulation of HAS2 and subsequent hyaluronan secretion promotes glioma cell proliferation, invasion, and chemoresistance in vitro and in vivo through the c-myc pathway. Targeting HAS2 sensitizes glioma cells to chemotherapeutic agents. Additionally, we found that hypoxia-inducible factor HIF1α regulates HAS2 expression. Together, our findings provide insights into the dysregulation of HAS2 and its role in chemoresistance and suggest potential therapeutic strategies for GBM.

Structural insights into the substrate binding sites of O-carbamoyltransferase VtdB from Streptomyces sp. NO1W98.

The O-carbamoyltransferase VtdB catalyzes the carbamoylation of venturicidin B, which is essential for the biosynthesis of the antibiotic venturicidin A. Here, the crystal structures of VtdB and VtdB in complex with the intermediate carbamoyladenylate (VtdBCAO) were determined at resolutions of 2.99 Å and 2.90 Å, respectively. The structures resemble the conserved YrdC-like and specific Kae1-like domains. A magnesium ion and the intermediate carbamoyladenylate were also observed in the Kae1-like domain of VtdB. The structure of VtdBCAO in complex with the substrate venturicidin B was modeled by a molecular docking method to better understand the substrate binding mode, revealing a novel venturicidin B binding pocket.

Finite element analysis of femoral neck system in the treatment of Pauwels type III femoral neck fracture.

The optimal treatment strategy for femoral neck fractures remained controversial, especially the Pauwels type III femoral neck fracture of young patients was a challenge. Femoral neck system (FNS) was a newly developed internal fixation for treating femoral neck fracture and this study aimed to compare the biomechanical advantages and disadvantages between FNS and 3 cannulated configuration screws (CCS) with or without an additional medial buttress plate (MBP). In this study, Pauwels type III femoral neck fracture model with an angle of 70° was constructed and 3 different fixation models, FNS, CCS + MBP, CCS alone, were developed. A vertical force of 2100N was applied on the femoral head, then the maximum von Mises stress of whole model, distal femur, femoral head, and internal fixation was recorded, as well as the stress distribution of whole model, proximal fracture section, and internal fixation of the 3 models. Moreover, the maximum displacement of the whole model, distal femur, femoral head, internal fixation, and the relative displacement of the proximal and distal portion was also compared. The maximum von Mises stress value was 318.302 MPa in FNS, 485.226 MPa in CCS + 1/3 plate, and 425.889 MPa in CCS. The FNS showed lowest maximum von Mises stress values in distal part, femoral head, and internal implant. All fixation configurations were observed stress concentrated at the posteroinferior area of cross-section of femoral head and at the fracture section area of implant; however, FNS had more uniform stress distribution. For displacement, the maximum displacement value was 8.5446 mm in FNS, 8.2863 mm in CCS + 1/3 plate, and 8.3590 mm in CCS. However, FNS had higher maximum displacement in femoral head and internal implant, but lower maximum displacement in the distal part of fracture model. The FNS represented a significantly higher relative displacement between the femoral head and distal femur when compared with the other 2 fixation configurations. The newly developed FNS could achieve the dual effect of angular stability and sliding compression for the treatment of Pauwels type III femoral neck fractures, which provided superior biomechanical stability than CCS alone and CCS with additional MBP.

Structural insights into a new substrate binding mode of a histidine acid phosphatase from Legionella pneumophila.

MapA is a histidine acid phosphatase (HAP) from Legionella pneumophila that catalyzes the hydroxylation of a phosphoryl group from phosphomonoesters by an active-site histidine. Several structures of HAPs, including MapA, in complex with the inhibitor tartrate have been solved and the substrate binding tunnel identified; however, the substrate recognition mechanism remains unknown. To gain insight into the mechanism of substrate recognition, the crystal structures of apo-MapA and the MapAD281A mutant in complex with 5'-AMP were solved at 2.2 and 2.6 Å resolution, respectively. The structure of the MapAD281A/5'-AMP complex reveals that the 5'-AMP fits fully into the substrate binding tunnel, with the 2'-hydroxyl group of the ribose moiety stabilized by Glu201 and the adenine moiety sandwiched between His205 and Phe237. This is the second structure of a HAP/AMP complex solved with 5'-AMP binding in a unique manner in the active site. The structure presents a new substrate recognition mechanism of HAPs.

Intravenous versus oral acetaminophen as an adjunct on pain and recovery after total knee arthroplasty: A systematic review and meta-analysis.

BACKGROUND: Total knee arthroplasty (TKA) is gradually emerging as the treatment of choice for end-stage osteoarthritis. In the past, intravenous (IV) versus oral acetaminophen (APAP) treatment is still a controversial subject in TKA. Therefore, we write this systematic review and meta-analysis to evaluate the efficacy of IV versus oral APAP on pain and recovery after TKA. METHODS: Embase, Pubmed, and Cochrane Library were comprehensively searched. Randomized controlled trials, cohort studies were included in our meta-analysis. Five studies that compared IV APAP groups with oral APAP groups were included in our meta-analysis. The research was reported according to the preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines to ensure the reliability and verity of results. RESULTS: Pooled results indicated that no significant difference between the IV APAP groups and oral APAP groups in term of VAS score at 24 hours (P = .67), 48 hours (P = 0.08), and total morphine consumption at 24 hours (P = .07), but there was a significant difference in terms of length of hospital stay (LOS) (P = .0004). CONCLUSION: IV APAP was not found to be superior to oral APAP in patients undergoing TKA in terms of VAS scores at 24 hours, 48 hours, and total morphine consumption at 24 hours. However, it can significantly reduce the LOS. We still need a large of high-quality research to verify the relationship between the oral and the IV APAP to give the conclusion.

Dynamic Obstacle Avoidance for Unmanned Underwater Vehicles Based on an Improved Velocity Obstacle Method.

In view of a dynamic obstacle environment with motion uncertainty, we present a dynamic collision avoidance method based on the collision risk assessment and improved velocity obstacle method. First, through the fusion optimization of forward-looking sonar data, the redundancy of the data is reduced and the position, size and velocity information of the obstacles are obtained, which can provide an accurate decision-making basis for next-step collision avoidance. Second, according to minimum meeting time and the minimum distance between the obstacle and unmanned underwater vehicle (UUV), this paper establishes the collision risk assessment model, and screens key obstacles to avoid collision. Finally, the optimization objective function is established based on the improved velocity obstacle method, and a UUV motion characteristic is used to calculate the reachable velocity sets. The optimal collision speed of UUV is searched in velocity space. The corresponding heading and speed commands are calculated, and outputted to the motion control module. The above is the complete dynamic obstacle avoidance process. The simulation results show that the proposed method can obtain a better collision avoidance effect in the dynamic environment, and has good adaptability to the unknown dynamic environment.

The efficacy and safety of epinephrine for postoperative bleeding in total joint arthroplasty: A PRISMA-compliant meta-analysis.

BACKGROUND: Total joint arthroplasty (TJA) usually results in postoperative bleeding. Some randomized controlled trials (RCTs) and nonrandomized controlled trials (non-RCTs) have been performed to evaluate the effects of epinephrine on postoperative bleeding after TJA. However, this remained controversial about the efficacy and safety of epinephrine for postoperative bleeding in TJA. The objective of our meta-analysis was to compare the overall effect and safety of epinephrine and placebo for postoperative bleeding in TJA. METHODS: PubMed, Embase, and the Cochrane Library were searched to identify potentially relevant articles. RCTs or non-RCTs involving epinephrine and placebo for blood loss in total knee arthroplasty or total hip arthroplasty were included. Our study was performed based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. RevMan v5.3 was used to analyze the relevant data. RESULTS: Four RCTs and 1 non-RCT involving 646 participants met the inclusion criteria. The overall pooled results from meta-analysis demonstrated that compared with control groups, epinephrine groups could significantly reduce the postoperative bleeding volume (mean difference [MD] = -168.42, 95% confidence interval [CI]: -272.37 to -64.47, P = 0.001). There was no significant difference in intraoperative bleeding volume between epinephrine and control groups (MD = -12.89, 95% CI: -53.45 to 27.69, P = 0.53). No significant difference was found between 2 groups in terms of postoperative hemoglobin loss (MD = -0.28, 95% CI: -0.66 to 0.10, P = 0.15). Compared with the control groups, no statistically significant difference was found in terms of postoperative transfusion rate in epinephrine groups (relative risk [RR] 0.86, 95% CI: 0.64-1.15, P = 0.31). In addition, the results of the meta-analysis also indicated no significant difference in terms of the incidence rate of deep venous thrombosis (DVT) between 2 groups (RR 0.28, 95% CI: 0.05-1.64, P = 0.16). CONCLUSION: The meta-analysis showed that epinephrine could significantly reduce postoperative bleeding volume in TJA without increasing the incidence of DVT. However, there was no significant reduction in intraoperative bleeding volume, postoperative hemoglobin loss, and transfusion rate after the administration of epinephrine. LIMITATIONS: In this study, a higher heterogeneity and a risk of selection bias may be present in postoperative hemoglobin loss. In addition, the sample size of the included studies was too small, so our findings need to be further validated with more high-quality and larger scale RCTs in the future. SYSTEMATIC REVIEW REGISTRATION NUMBER: None.

Efficient demyristoylase activity of SIRT2 revealed by kinetic and structural studies.

Sirtuins are a class of enzymes originally identified as nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacetylases. Among the seven mammalian sirtuins, SIRT1-7, only SIRT1-3 possess efficient deacetylase activity in vitro, whereas SIRT4-7 possess very weak in vitro deacetylase activity. Several sirtuins that exhibit weak deacetylase activity have recently been shown to possess more efficient activity for the removal other acyl lysine modifications, such as succinyl lysine and palmitoyl lysine. Here, we demonstrate that even the well-known deacetylase SIRT2 possesses efficient activity for the removal of long-chain fatty acyl groups. The catalytic efficiency (kcat/Km) for the removal of a myristoyl group is slightly higher than that for the removal of an acetyl group. The crystal structure of SIRT2 in complex with a thiomyristoyl peptide reveals that SIRT2 possesses a large hydrophobic pocket that can accommodate the myristoyl group. Comparison of the SIRT2 acyl pocket to those of SIRT1, SIRT3, and SIRT6 reveals that the acyl pockets of SIRT1-3 are highly similar, and to a lesser degree, similar to that of SIRT6. The efficient in vitro demyristoylase activity of SIRT2 suggests that this activity may be physiologically relevant and warrants future investigative studies.

The Legionella effector SidC defines a unique family of ubiquitin ligases important for bacterial phagosomal remodeling.

The activity of proteins delivered into host cells by the Dot/Icm injection apparatus allows Legionella pneumophila to establish a niche called the Legionella-containing vacuole (LCV), which is permissive for intracellular bacterial propagation. Among these proteins, substrate of Icm/Dot transporter (SidC) anchors to the cytoplasmic surface of the LCV and is important for the recruitment of host endoplasmic reticulum (ER) proteins to this organelle. However, the biochemical function underlying this activity is unknown. Here, we determined the structure of the N-terminal domain of SidC, which has no structural homology to any protein. Sequence homology analysis revealed a potential canonical catalytic triad formed by Cys46, His444, and Asp446 on the surface of SidC. Unexpectedly, we found that SidC is an E3 ubiquitin ligase that uses the C-H-D triad to catalyze the formation of high-molecular-weight polyubiquitin chains through multiple ubiquitin lysine residues. A C46A mutation completely abolished the E3 ligase activity and the ability of the protein to recruit host ER proteins as well as polyubiquitin conjugates to the LCV. Thus, SidC represents a unique E3 ubiquitin ligase family important for phagosomal membrane remodeling by L. pneumophila.

The N-terminal ß-sheet of peroxiredoxin 4 in the large yellow croaker Pseudosciaena crocea is involved in its biological functions.

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that exhibit peroxidase and peroxynitrite reductase activities involved in the reduction of reactive oxygen species. The peroxiredoxin Prx4 from the large yellow croaker Pseudosciaena crocea is a typical 2-Cys Prx with an N-terminal signal peptide. We solved the crystal structure of Prx4 at 1.90 Å and revealed an N-terminal antiparallel ß-sheet that contributes to the dimer interface. Deletion of this ß-sheet decreased the in vitro peroxidase activity to about 50% of the wild-type. In vivo assays further demonstrated that removal of this ß-sheet led to some impairment in the ability of Prx4 to negatively regulate nuclear factor-κB (NF-κB) activity and to perform its role in anti-bacterial immunity. These results provide new insights into the structure and function relationship of a peroxiredoxin from bony fish.

Crystal structures and putative interface of Saccharomyces cerevisiae mitochondrial matrix proteins Mmf1 and Mam33.

The yeast Saccharomyces cerevisiae mitochondrial matrix factor Mmf1, a member in the YER057c/Yigf/Uk114 family, participates in isoleucine biosynthesis and mitochondria maintenance. Mmf1 physically interacts with another mitochondrial matrix protein Mam33, which is involved in the sorting of cytochrome b2 to the intermembrane space as well as mitochondrial ribosomal protein synthesis. To elucidate the structural basis for their interaction, we determined the crystal structures of Mmf1 and Mam33 at 1.74 and 2.10 Å, respectively. Both Mmf1 and Mam33 adopt a trimeric structure: each subunit of Mmf1 displays a chorismate mutase fold with a six-stranded ß-sheet flanked by two α-helices on one side, whereas a subunit of Mam33 consists of a twisted six-stranded ß-sheet surrounded by five α-helices. Biochemical assays combined with structure-based computational simulation enable us to model a putative complex of Mmf1-Mam33, which consists of one Mam33 trimer and two tandem Mmf1 trimers in a head-to-tail manner. The two interfaces between the ring-like trimers are mainly composed of electrostatic interactions mediated by complementary negatively and positively charged patches. These results provided the structural insights into the putative function of Mmf1 during mitochondrial protein synthesis via Mam33, a protein binding to mitochondrial ribosomal proteins.

Structural insights into the catalytic mechanism of the yeast pyridoxal 5-phosphate synthase Snz1.

In most eubacteria, fungi, apicomplexa, plants and some metazoans, the active form of vitamin B6, PLP (pyridoxal 5-phosphate), is de novo synthesized from three substrates, R5P (ribose 5-phosphate), DHAP (dihydroxyacetone phosphate) and ammonia hydrolysed from glutamine by a complexed glutaminase. Of the three active sites of DXP (deoxyxylulose 5-phosphate)independent PLP synthase (Pdx1), the R5P isomerization site has been assigned, but the sites for DHAP isomerization and PLP formation remain unknown. In the present study, we present the crystal structures of yeast Pdx1/Snz1, in apo-, G3P (glyceraldehyde 3-phosphate)- and PLP-bound forms, at 2.3, 1.8 and 2.2 Å (1 Å=0.1 nm) respectively. Structural and biochemical analysis enabled us to assign the PLP-formation site, a G3P-binding site and a G3P-transfer site. We propose a putative catalytic mechanism for Pdx1/Snz1 in which R5P and DHAP are isomerized at two distinct sites and transferred along well-defined routes to a final destination for PLP synthesis.

Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate.

2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, the accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed as a putative receptor for 2-OG. Here we present three crystal structures of NtcA from the cyanobacterium Anabaena: the apoform, and two ligand-bound forms in complex with either 2-OG or its analogue 2,2-difluoropentanedioic acid. All structures assemble as homodimers, with each subunit composed of an N-terminal effector-binding domain and a C-terminal DNA-binding domain connected by a long helix (C-helix). The 2-OG binds to the effector-binding domain at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. Whereas catabolite activator protein adopts a transition from off-to-on state upon cAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apoform, and how 2-OG just enhances the DNA-binding activity of NtcA. These findings provided the structural insights into the function of a global transcription factor regulated by 2-OG, a metabolite standing at a crossroad between carbon and nitrogen metabolisms.

Structural basis for the different activities of yeast Grx1 and Grx2.

Yeast glutaredoxins Grx1 and Grx2 catalyze the reduction of both inter- and intra-molecular disulfide bonds using glutathione (GSH) as the electron donor. Although sharing the same dithiolic CPYC active site and a sequence identity of 64%, they have been proved to play different roles during oxidative stress and to possess different glutathione-disulfide reductase activities. To address the structural basis of these differences, we solved the crystal structures of Grx2 in oxidized and reduced forms, at 2.10 A and 1.50 A, respectively. With the Grx1 structures we previously reported, comparative structural analyses revealed that Grx1 and Grx2 share a similar GSH binding site, except for a single residue substitution from Asp89 in Grx1 to Ser123 in Grx2. Site-directed mutagenesis in combination with activity assays further proved this single residue variation is critical for the different activities of yeast Grx1 and Grx2.

Crystal structure and computational analyses provide insights into the catalytic mechanism of 2,4-diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens.

2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C-C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 A resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical alpha/beta-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C-C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr(121), Tyr(229), and Asn(132), which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.

Structural insights into the substrate tunnel of Saccharomyces cerevisiae carbonic anhydrase Nce103.

BACKGROUND: The carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: alpha-, beta-, gamma-, delta-, epsilon-, zeta- CAs, with beta-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of beta-CA Nce103/YNL036W. RESULTS: We determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 A resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His112 and Cys115 in addition to a water molecule. Residues Asp59, Arg61, Gly111, Leu102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. Activity assays of full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable. CONCLUSION: The quaternary structure of Nce103 resembles the typical plant type beta-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket.