Disturbance of fetal growth by azithromycin through induction of ER stress in the placenta.

AIM: Azithromycin (AZM) is widely used to treat mycoplasma infection in pregnancy. However, there is no adequate evaluation of its side effect on the placenta. Here, by using human placental syncytiotrophoblasts and a mouse model, we investigated whether AZM use in pregnancy might adversely affect placental function and pregnancy outcome. RESULTS: Transcriptomic analysis of AZM-treated human placental syncytiotrophoblasts showed increased expression of ER stress-related genes and decreased expression of genes for hormone production and growth factor processing. Verification studies showed that AZM increased the abundance of ER stress mediators (phosphorylated eIF2α, ATF4 and CHOP), and decreased the abundance of enzymes involved in progesterone and estradiol synthesis (STS, CYP11A1 and CYP19A1) and IGFBP cleavage (PAPPA and ADAM12) in human placental syncytiotrophoblasts. Inhibition of ER stress blocked AZM-induced decreases in the expression of CYP19A1, CYP11A1, PAPPA and ADAM12, suggesting that the inhibition of AZM on those genes' expression was secondary to AZM-induced ER stress. Further mechanism study showed that increased ATF4 in ER stress might repressively interact with C/EBPα to suppresstheexpression ofthose genes including CEBPAitself. Mouse studies showed that AZM administration decreased fetal weights along with increased ER stress mediators and decreased levels of insulin-like growth factor, estrogen and progesterone in the maternal blood, which could be alleviated by inhibition of ER stress. INNOVATION AND CONCLUSION: These findings firstly support AZM, often used during pregnancy, may affect fetal growth by inhibiting crucial enzymes for estrogen and progesterone synthesis and disrupting crucial proteases for IGFBP cleavage via inducing ER stress in placental syncytiotrophoblasts.

Morphological analysis of posterior-medial intertrochanteric fracture patterns using fracture-mapping technique.

Introduction: The purpose of this study was to analyze the fracture patterns of different posterior-medial wall types of intertrochanteric fractures by 3-D fracture-mapping technique and to further assess their clinical utility. Methods: In a retrospective analysis of interochanteric fractures treated in a large trauma center, fractures were classified into predesigned groups based on 3D-CT imaging techniques, and a 3-D template of the intertrochanteric region was graphically superimposed on the fracture line. Fracture characteristics were then summarized based on fracture-mapping. Finally, radiographic parameters, function, and range of motion were recorded in different fracture classification states. Results: A total of 348 intertrochanteric fractures were included. There were 111 patients (31.9%) in the posterolateral + posteromedial + medial group, with the most severe fracture displacement (typically characterized by fragmentation of the posteromedial wall into three isolated fragments). There were 102 cases (29.3%) in the posterolateral + posteromedial + simple medial group, and the most common fracture feature was a complete fragment posteromedially. A total of 81 cases (23.3%) were classified into the posterolateral + medial group, with the medial fracture line extending the anterior fracture line but leaving the lesser trochanter intact. In the isolated medial group of 33 cases (9.5%), the fracture type was similar to type IV, but the integrity of the greater trochanter was ensured. In the posteromedial + medial group of 12 cases (3.4%), the fracture was characterized by an interruption when the fracture line of the anterolateral wall extended to the posteromedial wall, often resulting in a complete isolated fragment posteromedially and medially. There were nine patients (2.6%) in the isolated posterolateral group. In addition, we found significantly different radiographic scores and range of motion scores between groups. Discussion: This morphometric study helps us to further characterize posterior-medial fracture patterns of intertrochanteric fractures, which may be closely related to different clinical outcomes. Further studies are needed to verify the reliability of this classification scheme in clinical application.

Metallacage-based enhanced PDT strategy for bacterial elimination via inhibiting endogenous NO production.

Antibiotics are among the most used weapons in fighting microbial infections and have greatly improved the quality of human life. However, bacteria can eventually evolve to exhibit antibiotic resistance to almost all prescribed antibiotic drugs. Photodynamic therapy (PDT) develops little antibiotic resistance and has become a promising strategy in fighting bacterial infection. To augment the killing effect of PDT, the conventional strategy is introducing excess ROS in various ways, such as applying high light doses, high photosensitizer concentrations, and exogenous oxygen. In this study, we report a metallacage-based PDT strategy that minimizes the use of ROS by jointly using gallium-metal organic framework rods to inhibit the production of bacterial endogenous NO, amplify ROS stress, and enhance the killing effect. The augmented bactericidal effect was demonstrated both in vitro and in vivo. This proposed enhanced PDT strategy will provide a new option for bacterial ablation.

Evaluating Risk-Adjusted Hospital Performance Using Large-Scale Data on Mortality Rates of Patients in Intensive Care Units: A Flexible Semi-Nonparametric Modeling Approach.

BACKGROUND AND OBJECTIVE: Significant variability in the quality of healthcare supplied by hospitals is drawing broad attention from the United States Centers for Medicare and Medicaid Services. The primary issue is to evaluate hospital performance based on patient outcomes. Generalized linear random-effects models are a promising analytical tool for evaluating hospital performance. However, hospital compare data often violate the classical assumptions of normality on random effects and linearity representation on transformed conditional mean structures in these models. METHODS: In this article, we proposed and tested the performance of a class of hospital compare models that embraces nonparametric mean structures with semi-nonparametric hospital random effects. Such models were further improved and integrated into a zero-inflated model. [Formula: see text] programs to implement these newly proposed hospital compare models were thoroughly developed. The [Formula: see text] programs are freely available via a GitHub (https:\\www.GitHub.com) repository. RESULTS: We demonstrate the robustness of the proposed hospital compare models by conducting intensive empirical studies. Flexible semi-nonparametric random effects and functional fixed-effects mean structure were used to analyze patient mortality in a large-scale intensive care unit data set. After applying the proposed models to assess standardized modality rates and address patient-mix variability across hospitals, we detected those underperforming hospitals with higher mortality rates. CONCLUSIONS: Our research findings highlight how constructing advanced assessment tools for hospital performance could support better decision-making at the administrative and public levels. The proposed hospital compare models are comprehensive in their capacity to identify patterns of hospital random effects and to convey the variability in healthcare quality with powerful accuracy and interpretability.

A polydopamine coated nanoscale FeS theranostic platform for the elimination of drug-resistant bacteria via photothermal-enhanced Fenton reaction.

Infections caused by drug-resistant bacteria pose a great threat to human health. Non-antibiotic-dependent antibacterial strategies have become the focus of research. Among them, chemical dynamic treatment-based (CDT) therapeutic systems, which catalyze the production of hydroxyl radicals by enzymes, have achieved tremendous success for antibacterial purposes. However, limited kinetics of the Fenton reaction, poor permeability, and short half-life of hydroxyl radicals compromise the antibacterial effects of CDT. In addition, difficulties in the early diagnosis of infection lead to drug abuse and delayed treatment. Herein, a polydopamine coated ferrous sulfide theranostic platform adsorbing a hypochlorite responsive probe with photothermal treatment (PTT) enhanced CDT was synthesized. The probe component was used for the early diagnosis of infection. PTT not only inactivated bacteria by hyperthermia but also accelerated the Fenton reaction to produce more ·OH. In vitro antibacterial experiments demonstrated that the multifunctional theranostic platform has a broad antibacterial spectrum, including methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Escherichia coli (DR E. coli), and Pseudomonas aeruginosa (P. aeruginosa). In addition, in vivo antibacterial experiments demonstrated that nanoparticles could effectively rescue S. aureus-infected full-thickness skin defects with negligible cytotoxicity. This study proposes an efficient and multifunctional theranostic platform for bacterial infection, providing an effective synergistic antibacterial strategy for the treatment of antibiotic resistance. STATEMENT OF SIGNIFICANCE: An infection responsive theranostic platform (ClO- probe@FeS@PDA) is prepared. ·CDT is enhanced prominently by PTT at a relative low temperature. · FeS@PDA exhibits good antibacterial performance against drug resistant bacteria in vitro and in vivo.

Structure of Pseudomonas aeruginosa spermidine dehydrogenase: a polyamine oxidase with a novel heme-binding fold.

The opportunistic pathogen Pseudomonas aeruginosa can utilize polyamines (including putrescine, cadaverine, 4-aminobutyrate, spermidine, and spermine) as its sole source of carbon and nitrogen. Spermidine dehydrogenase (SpdH) is a component of one of the two polyamine utilization pathways identified in P. aeruginosa, but little is known about its structure and function. Here, we report the first crystal structure of SpdH from P. aeruginosa to 1.85 Å resolution. The resulting core structure confirms that SpdH belongs to the polyamine oxidase (PAO) family with flavin-binding and substrate-binding domains. A unique N-terminal extension wraps around the flavin-binding domain of SpdH and is required for heme binding, placing a heme cofactor in close proximity to the FAD cofactor. Structural and mutational analysis reveals that residues in the putative active site at the re side of the FAD isoalloxazine ring form part of the catalytic machinery. PaSpdH features an unusual active site and lacks the conserved lysine that forms part of a lysine-water-flavin N5 atom interaction in other PAO enzymes characterized to date. Mutational analysis further confirms that heme is required for catalytic activity. This work provides an important starting point for understanding the role of SpdH, which occurs universally in P. aeruginosa strains, in polyamine metabolism.

Structure and mechanism of the γ-glutamyl-γ-aminobutyrate hydrolase SpuA from Pseudomonas aeruginosa.

Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation-putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

Ubiquitination Flow Repressors: Enhancing Wound Healing of Infectious Diabetic Ulcers through Stabilization of Polyubiquitinated Hypoxia-Inducible Factor-1α by Theranostic Nitric Oxide Nanogenerators.

Current treatments for diabetic ulcers (DUs) remain unsatisfactory due to the risk of bacterial infection and impaired angiogenesis during the healing process. The increased degradation of polyubiquitinated hypoxia-inducible factor-1α (HIF-1α) compromises wound healing efficacy. Therefore, the maintenance of HIF-1α protein stability might help treat DU. Nitric oxide (NO) is an intrinsic biological messenger that functions as a ubiquitination flow repressor and antibacterial agent; however, its clinical application in DU treatment is hindered by the difficulty in controlling NO release. Here, an intelligent near-infrared (NIR)-triggered NO nanogenerator (SNP@MOF-UCNP@ssPDA-Cy7/IR786s, abbreviated as SNP@UCM) is presented. SNP@UCM represses ubiquitination-mediated proteasomal degradation of HIF-1α by inhibiting its interaction with E3 ubiquitin ligases under NIR irradiation. Increased HIF-1α expression in endothelial cells by SNP@UCM enhances angiogenesis in wound sites, promoting vascular endothelial growth factor (VEGF) secretion and cell proliferation and migration. SNP@UCM also enables early detection of wound infections and ROS-mediated killing of bacteria. The potential clinical utility of SNP@UCM is further demonstrated in infected full-thickness DU model under NIR irradiation. SNP@UCM is the first reported HIF-1α-stabilizing advanced nanomaterial, and further materials engineering might offer a facile, mechanism-based method for clinical DU management.

Carbohydrate Metabolism Affects Macrophage-Mediated Killing of Enterococcus faecalis.

Enterococcus faecalis, an opportunistic pathogen that causes severe community-acquired and nosocomial infections, has been reported to resist phagocyte-mediated killing, which enables its long-term survival in the host. Metabolism, especially carbohydrate metabolism, plays a key role in the battle between pathogens and hosts. However, the function of carbohydrate metabolism in the long-term survival of E. faecalis in phagocytes has rarely been reported. In this study, we utilized transposon insertion sequencing (TIS) to investigate the function of carbohydrate metabolism during the survival of E. faecalis in RAW264.7 cells. The TIS results showed that the fitness of carbohydrate metabolism-related mutants, especially those associated with fructose and mannose metabolism, were significantly enhanced, suggesting that the attenuation of carbohydrate metabolism promotes the survival of E. faecalis in macrophages. The results of our investigation indicated that macrophages responded to carbohydrate metabolism of E. faecalis and polarized to M1 macrophages to increase nitric oxide (NO) production, leading to the enhancement of macrophage-mediated killing to E. faecalis. Meanwhile, E. faecalis automatically decreased carbohydrate metabolism to escape from the immune clearance of macrophages during intracellular survival. The shift of primary carbon resources for macrophages affected the ability to clear intracellular E. faecalis. In summary, the results of the present study demonstrated that carbohydrate metabolism affects the macrophage-mediated killing of E. faecalis. IMPORTANCE E. faecalis has become a major pathogen leading to a variety of infections around the world. The metabolic interaction between E. faecalis and its host is important during infection but is rarely investigated. We used transposon insertion sequencing coupled with transcriptome sequencing to explore the metabolic interaction between E. faecalis and macrophages and uncovered that the shift of carbohydrate metabolism dramatically affected the inflammatory response of macrophages. In addition, E. faecalis attenuated carbohydrate metabolism to avoid the activation of the immune response of macrophages. This study provides new insights for the reason why E. faecalis is capable of long-term survival in macrophages and may facilitate the development of novel strategies to treat infectious diseases.

Structural characterization and Kemp eliminase activity of the Mycobacterium smegmatis Ketosteroid Isomerase.

The Kemp elimination reaction, involving the ring-opening of benzoxazole and its derivatives under the action of natural enzymes or chemical catalysts, has been of interest to researchers since its discovery. Because this reaction does not exist in all currently known metabolic pathways, the computational design of Kemp eliminases has provided valuable insights into principles of enzymatic catalysis. However, it was discovered that the naturally occurring promiscuous enzymes ydbC, xapA and ketosteroid isomerase also can catalyze Kemp elimination. Here, we report the crystal structure of ketosteroid isomerase (KSI) from Mycobacterium smegmatis MC2 155. MsKSI crystallizes in the P212121 space group with two molecules in an asymmetric unit, and ultracentrifugation data confirms that it forms a stable dimer in solution, consistent with the 1.9 Å-resolution structure. Our assays confirm that MsKSI accelerates the Kemp elimination of 5-nitrobenzoxazole (5NBI) with an optimal pH of 5.5. A 2.35 Å resolution crystal structure of the MsKSI-5NBI complex reveals that the substrate 5NBI is bound in the active pocket of the enzyme composed of hydrophobic residues. In addition, the Glu127 residue is proposed to play an important role as a general base in proton transfer and breaking weak O-N bonds to open the five-membered ring. This work provides a starting point for exploring the artificial modification of MsKSI using the natural enzyme as the backbone.

Cerium Oxide Nanoparticles Regulate Osteoclast Differentiation Bidirectionally by Modulating the Cellular Production of Reactive Oxygen Species.

BACKGROUND: Cerium oxide nanoparticles (CeO2NPs) are potent scavengers of cellular reactive oxygen species (ROS). Their antioxidant properties make CeO2NPs promising therapeutic agents for bone diseases and bone tissue engineering. However, the effects of CeO2NPs on intracellular ROS production in osteoclasts (OCs) are still unclear. Numerous studies have reported that intracellular ROS are essential for osteoclastogenesis. The aim of this study was to explore the effects of CeO2NPs on osteoclast differentiation and the potential underlying mechanisms. METHODS: The bidirectional modulation of osteoclast differentiation by CeO2NPs was explored by different methods, such as fluorescence microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. The cytotoxic and proapoptotic effects of CeO2NPs were detected by cell counting kit (CCK-8) assay, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and flow cytometry. RESULTS: The results of this study demonstrated that although CeO2NPs were capable of scavenging ROS in acellular environments, they facilitated the production of ROS in the acidic cellular environment during receptor activator of nuclear factor kappa-Β ligand (RANKL)-dependent osteoclast differentiation of bone marrow-derived macrophages (BMMs). CeO2NPs at lower concentrations (4.0 µg/mL to 8.0 µg/mL) promoted osteoclast formation, as shown by increased expression of Nfatc1 and C-Fos, F-actin ring formation and bone resorption. However, at higher concentrations (greater than 16.0 µg/mL), CeO2NPs inhibited osteoclast differentiation and promoted apoptosis of BMMs by reducing Bcl2 expression and increasing the expression of cleaved caspase-3, which may be due to the overproduction of ROS. CONCLUSION: This study demonstrates that CeO2NPs facilitate osteoclast formation at lower concentrations while inhibiting osteoclastogenesis in vitro by inducing the apoptosis of BMMs at higher concentrations by modulating cellular ROS levels.

Structural characterization of the Pseudomonas aeruginosa dehydrogenase AtuB involved in citronellol and geraniol catabolism.

Pseudomonas aeruginosa can metabolize acyclic monoterpenoids (such as citronellol and geraniol) as the only carbon and energy sources. A total of seven proteins (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG, AtuH) have been identified in Pseudomonas aeruginosa as participating in the acyclic terpene utilization pathway. AtuB is a dehydrogenase enzyme responsible for citronellol and geraniol catabolism in the acyclic terpene utilization (Atu) pathway, although its structure and function have not been characterized to date. Here we report the crystal structure of AtuB from Pseudomonas aeruginosa PAO1 (PaAtuB) to 1.8 Å resolution. PaAtuB crystallizes in the space group F222 with a single monomer in the asymmetric unit. Analytical ultracentrifugation data shows that PaAtuB forms a stable tetramer in solution, which is consistent with the structure. Structural analysis confirms that AtuB belongs to the short-chain dehydrogenase/reductase (SDR) family. AtuB is predicted to bind NADP(H) from the crystal structure, which is confirmed by MicroScale Thermophoresis analysis that shows PaAtuB binds NADP(H) with a Kd value of 258 µM. This work provides a starting point to explore potential biotechnology and pharmaceutical applications of AtuB.

Crystal structure of RecR, a member of the RecFOR DNA-repair pathway, from Pseudomonas aeruginosa PAO1.

DNA damage is usually lethal to all organisms. Homologous recombination plays an important role in the DNA damage-repair process in prokaryotic organisms. Two pathways are responsible for homologous recombination in Pseudomonas aeruginosa: the RecBCD pathway and the RecFOR pathway. RecR is an important regulator in the RecFOR homologous recombination pathway in P. aeruginosa. It forms complexes with RecF and RecO that can facilitate the loading of RecA onto ssDNA in the RecFOR pathway. Here, the crystal structure of RecR from P. aeruginosa PAO1 (PaRecR) is reported. PaRecR crystallizes in space group P6122, with two monomers per asymmetric unit. Analytical ultracentrifugation data show that PaRecR forms a stable dimer, but can exist as a tetramer in solution. The crystal structure shows that dimeric PaRecR forms a ring-like tetramer architecture via crystal symmetry. The presence of a ligand in the Walker B motif of one RecR subunit suggests a putative nucleotide-binding site.

Dissecting the genetic architecture of Fusarium verticillioides seed rot resistance in maize by combining QTL mapping and genome-wide association analysis.

Fusarium verticillioides can be transmitted via seeds and cause systemic infection in maize (Zea mays L.); its mycotoxin has harmful effects on animal and human health. We combined QTL mapping in recombinant inbred line (RIL) populations with a genome-wide association study (GWAS) of 217 diverse maize lines using 224,152 single nucleotide polymorphisms (SNPs) under controlled conditions to determine the genetic architecture of F. verticillioides seed rot (FSR) resistance. Our study identified 8 quantitative trait loci (QTLs) and 43 genes associated with 57 SNPs that were correlated with FSR resistance through linkage mapping and GWAS, respectively. Among these, there were three candidate genes, namely GRMZM2G0081223, AC213654.3_FG004, and GRMZM2G099255, which were detected in both linkage mapping and GWAS. Furthermore, the near-isogenic lines (NILs) containing GRMZM2G0081223, which also had a susceptible parent background, were found to have a significantly improved level of resistance. In addition, the expression profile of the three candidate genes revealed that they all respond to the infection following inoculation with F. verticillioides. These genetic analyses indicate that FSR resistance is controlled by loci with minor effect, and the polymerization breeding of lines with beneficial alleles and candidate genes could improve FSR resistance in maize.