Characteristics and phylogenetic distribution of megaplasmids and prediction of a putative chromid in Pseudomonas aeruginosa.

Research on megaplasmids that contribute to the spread of antimicrobial resistance (AMR) in Pseudomonas aeruginosa strains has grown in recent years due to the now widely used technologies allowing long-read sequencing. Here, we systematically analyzed distinct and consistent genetic characteristics of megaplasmids found in P. aeruginosa. Our data provide information on their phylogenetic distribution and hypotheses tracing the potential evolutionary paths of megaplasmids. Most of the megaplasmids we found belong to the IncP-2-type, with conserved and syntenic genetic backbones carrying modules of genes associated with chemotaxis apparatus, tellurite resistance and plasmid replication, segregation, and transmission. Extensively variable regions harbor abundant AMR genes, especially those encoding ß-lactamases such as VIM-2, IMP-45, and KPC variants, which are high-risk elements in nosocomial infection. IncP-2 megaplasmids act as effective vehicles transmitting AMR genes to diverse regions. One evolutionary model of the origin of megaplasmids claims that chromids can develop from megaplasmids. These chromids have been characterized as an intermediate between a megaplasmid and a chromosome, also containing core genes that can be found on the chromosome but not on the megaplasmid. Using in silico prediction, we identified the "PABCH45 unnamed replicon" as a putative chromid in P. aeruginosa, which shows a much higher similarity and closer phylogenetic relationship to chromosomes than to megaplasmids while also encoding plasmid-like partition genes. We propose that such a chromid could facilitate genome expansion, allowing for more rapid adaptations to novel ecological niches or selective conditions, in comparison to megaplasmids.

A Review of Print Heads for Fused Filament Fabrication of Continuous Carbon Fiber-Reinforced Composites.

The print head is one of the most critical components in an additive manufacturing (AM) system. It can significantly affect the quality of printed parts. Recently, because continuous carbon fiber-reinforced composites can have excellent mechanical properties, a relevant AM technique, fused filament fabrication (FFF), has been attracting increasing attention. This has extended the requirements demanded of print heads. To this end, different FFF extrusion methods have been rapidly developed based on various methods of impregnating fibers into the matrix for the corresponding print heads. Generally, these extrusion methods are of three types: single extrusion, in situ extrusion, and dual extrusion. All these methods face substantial challenges, such as the nozzle clogging and damage to the continuous carbon fibers during extrusion. These common issues still need to be fully addressed. This study's aim is to summarize and discuss the different extrusion methods and their FFF specific components in terms of their advantages and disadvantages for continuous carbon fiber-reinforced composites.

A pilot clinical assessment of biphasic asymmetric pulsed field ablation catheter for pulmonary vein isolation.

Pulsed field ablation (PFA) is a new treatment for atrial fibrillation (AF), and its selective ablation characteristics give it a significant advantage in treatment. In previous cellular and animal experiments, we have demonstrated that biphasic asymmetric pulses can be used to ablate myocardial tissue. However, small-scale clinical trials are needed to test whether this approach is safe and feasible before extensive clinical trials can be performed. Therefore, the purpose of this experiment is to determine the safety and feasibility of biphasic asymmetric pulses in patients with AF and is to lay the foundation for a larger clinical trial. Ablation was performed in 10 patients with AF using biphasic asymmetric pulses. Voltage mapping was performed before and after PFA operation to help us detect the change in the electrical voltage of the pulmonary veins (PV). 3-Dimensional mapping system showed continuous low potential in the ablation site, and pulmonary vein isolation (PVI) was achieved in all four PV of the patients. There were no recurrences, PV stenosis, or other serious adverse events during the 12 months follow-up. The results suggest that PFA using biphasic asymmetric waveforms for patients with AF is safe, durable, and effective and that a larger clinical trial could begin. Clinical Trial Registration: https://www.chictr.org.cn/, identifier, ChiCTR2100051894.

Genomic epidemiology and ceftazidime-avibactam high-level resistance mechanisms of Pseudomonas aeruginosa in China from 2010 to 2022.

Ceftazidime-avibactam (CZA) resistance is a huge threat in the clinic; however, the underlying mechanism responsible for high-level CZA resistance in Pseudomonas aeruginosa (PA) isolates remains unknown. In this study, a total of 5,763 P. aeruginosa isolates were collected from 2010 to 2022 to investigate the ceftazidime-avibactam (CZA) high-level resistance mechanisms of Pseudomonas aeruginosa (PA) isolates in China. Fifty-six PER-producing isolates were identified, including 50 isolates carrying blaPER-1 in PA, and 6 isolates carrying blaPER-4. Of these, 82.1% (46/56) were classified as DTR-PA isolates, and 76.79% (43/56) were resistant to CZA. Importantly, blaPER-1 and blaPER-4 overexpression led to 16-fold and >1024-fold increases in the MICs of CZA, respectively. WGS revealed that the blaPER-1 gene was located in two different transferable IncP-2-type plasmids and chromosomes, whereas blaPER-4 was found only on chromosomes and was carried by a class 1 integron embedded in a Tn6485-like transposon. Overexpression of efflux pumps may be associated with high-level CZA resistance in blaPER-1-positive strains. Kinetic parameter analysis revealed that PER-4 exhibited a similar kcat/Km with ceftazidime and a high (∼3359-fold) IC50 value with avibactam compared to PER-1. Our study found that overexpression of PER-1 combined with enhanced efflux pump expression and the low affinity of PER-4 for avibactam contributes to high-level resistance to CZA. Additionally, the Tn6485-like transposon plays a significant role in disseminating blaPER. Urgent active surveillance is required to prevent the further spread of high-level CZA resistance in DTR-PA isolates.

CarsiDock: a deep learning paradigm for accurate protein-ligand docking and screening based on large-scale pre-training.

The expertise accumulated in deep neural network-based structure prediction has been widely transferred to the field of protein-ligand binding pose prediction, thus leading to the emergence of a variety of deep learning-guided docking models for predicting protein-ligand binding poses without relying on heavy sampling. However, their prediction accuracy and applicability are still far from satisfactory, partially due to the lack of protein-ligand binding complex data. To this end, we create a large-scale complex dataset containing ∼9 M protein-ligand docking complexes for pre-training, and propose CarsiDock, the first deep learning-guided docking approach that leverages pre-training of millions of predicted protein-ligand complexes. CarsiDock contains two main stages, i.e., a deep learning model for the prediction of protein-ligand atomic distance matrices, and a translation, rotation and torsion-guided geometry optimization procedure to reconstruct the matrices into a credible binding pose. The pre-training and multiple innovative architectural designs facilitate the dramatically improved docking accuracy of our approach over the baselines in terms of multiple docking scenarios, thereby contributing to its outstanding early recognition performance in several retrospective virtual screening campaigns. Further explorations demonstrate that CarsiDock can not only guarantee the topological reliability of the binding poses but also successfully reproduce the crucial interactions in crystalized structures, highlighting its superior applicability.

Investigating the ID3/SLC22A4 as immune-related signatures in ischemic stroke.

BACKGROUND: Ischemic stroke (IS) is a fearful disease that can cause a variety of immune events. Nevertheless, precise immune-related mechanisms have yet to be systematically elucidated. This study aimed to identify immune-related signatures using machine learning and to validate them with animal experiments and single cell analysis. METHODS: In this study, we screened 24 differentially expressed genes (DEGs) while identifying immune-related signatures that may play a key role in IS development through a comprehensive strategy between least absolute shrinkage and selection operation (LASSO) regression, support vector machine (SVM) and immune-related genes. In addition, we explored immune infiltration using the CIBERSORT algorithm. Finally, we performed validation in mouse brain tissue and single cell analysis. RESULTS: We identified 24 DEGs for follow-up analysis. ID3 and SLC22A4 were finally identified as the better immune-related signatures through a comprehensive strategy among DEGs, LASSO, SVM and immune-related genes. RT-qPCR, western blot, and immunofluorescence revealed a significant decrease in ID3 and a significant increase in SLC22A4 in the middle cerebral artery occlusion group. Single cell analysis revealed that ID3 was mainly concentrated in endothelial_2 cells and SLC22A4 in astrocytes in the MCAO group. A CIBERSORT finds significantly altered levels of immune infiltration in IS patients. CONCLUSIONS: This study focused on immune-related signatures after stroke and ID3 and SLC22A4 may be new therapeutic targets to promote functional recovery after stroke. Furthermore, the association of ID3 and SLC22A4 with immune cells may be a new direction for post-stroke immunotherapy.

Photoinduced reductive Reformatsky reaction of α-haloesters and aldehydes or ketones by cooperative dual-metal catalysis.

A photoinduced reductive Reformatsky reaction by cooperative dual-metal catalysis is described. This methodology enables the implementation of this venerable reaction in environmentally friendly conditions, obviating the need for a stoichiometric amount of metals. A broad range of synthetically useful ß-hydroxy esters can be efficiently prepared in moderate to high yields using this protocol.

bla KPC-2 overexpression and bla GES-5 carriage as major imipenem/relebactam resistance mechanisms in Pseudomonas aeruginosa high-risk clones ST463 and ST235, respectively, in China.

Pseudomonas aeruginosa high-risk clones pose severe threats to public health. Here, we characterize the imipenem/relebactam (IR) resistance mechanisms in P. aeruginosa high-risk clones sequence type 235 (ST235) and ST463 in China. Minimum inhibitory concentrations (MICs) were determined, and Illumina short-read sequencing was performed for 1,168 clinical carbapenem-resistant P. aeruginosa (CRPA) isolates. The gene copy number and expression level were analyzed by Illumina sequencing depth and reverse transcription-quantitative PCR, respectively. Resistance conferred by bla GES-5 was evaluated by cloning experiments. ST463 and ST235 accounted for 9.8% (115/1,168) and 4.5% (53/1,168) of total isolates, respectively, and showed high frequencies of extensively drug-resistant and difficult-to-treat resistant phenotypes. The overall IR-resistant rate in CRPA was 21.0% (245/1,168). However, the IR resistance rate was 81.7% (94/115) in ST463-PA and 52.8% (28/53) in ST235-PA. Of the ST463 isolates, 92.2% (106/115) were Klebsiella pneumoniae carbapenemase-producing P. aeruginosa (KPC-PA), and all 94 IR-resistant ST463-PA produced KPC-2. Compared to IR-susceptible ST463 KPC-2-PA, IR-resistant ST463 KPC-2-PA exhibited significantly higher bla KPC-2 copy numbers and expression levels. In ST463 KPC-2-PA, 16 mg/L relebactam resulted in additional fourfold reductions in imipenem MIC50/90 values compared to 4 mg/L relebactam. In ST235, 1.9% (1/53) carried bla IMP carbapenemase and 54.7% (29/53) carried bla GES carbapenemase. Other than the IMP producer, all 27 IR-resistant ST235-PA produced GES-5. Cloning experiments revealed that imipenem resistance in bla GES-5-carrying PAO1 transformants was generally unaffected by relebactam. In conclusion, IR-resistant CRPA isolates in China were mainly distributed in P. aeruginosa high-risk clones ST463 and ST235. The major underlying IR resistance mechanisms were bla KPC-2 overexpression and bla GES-5 carriage.

Unraveling the functional consequences of a novel germline missense mutation (R38C) in the yeast model of succinate dehydrogenase subunit B: insights into neurodegenerative disorders.

This study explores the implications of a novel germline missense mutation (R38C) in the succinate dehydrogenase (SDH) subunit B, which has been linked to neurodegenerative diseases. The mutation was identified from the SDH mutation database and corresponds to the SDH2R32C allele, mirroring the human SDHBR38C mutation. By subjecting the mutant yeast model to hydrogen peroxide (H2O2) stress, simulating oxidative stress, we observed heightened sensitivity to oxidative conditions. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed significant regulation (p < 0.05) of genes associated with antioxidant systems and energy metabolism. Through gas chromatography-mass spectrometry (GC-MS) analysis, we examined yeast cell metabolites under oxidative stress, uncovering insights into the potential protective role of o-vanillin. This study elucidates the biological mechanisms underlying cellular oxidative stress responses, offering valuable insights into its repercussions. These findings shed light on innovative avenues for addressing neurodegenerative diseases, potentially revolutionizing therapeutic strategies.

Characterization of two novel VIM-type metallo-ß-lactamases, VIM-84 and VIM-85, associated with the spread of IncP-2 megaplasmids in Pseudomonas aeruginosa.

This study aimed to characterize two novel VIM-type metallo-ß-lactamases, VIM-84 and VIM-85, and reveal the important role of the IncP-2 type megaplasmids in the spread of antimicrobial resistance (AMR) genes. VIM-84 and VIM-85 were encoded by two novel genes bla VIM-84 and bla VIM-85 which showed similarity to bla VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. The bla VIM-85 gene was identified in a megaplasmid, which was related to 17 megaplasmid sequences with sizes larger than 430 kb, deposited previously in Genbank. A comparative analysis of complete plasmid sequences showed highly similar backbone regions and various AMR genes. A phylogenetic tree revealed that these megaplasmids, which were widely distributed globally, were vehicles for the spread of AMR genes. The bla VIM-24, bla VIM-84, and bla VIM-85 genes were cloned into pGK1900, and the recombinant vectors were further transformed into Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. The antimicrobial susceptibility test of the cloning strains showed high levels of resistance to ß-lactams while they remained susceptible to aztreonam. Enzymatic tests revealed that both, VIM-84 and VIM-85, exhibited higher activity in hydrolyzing ß-lactams compared to VIM-24. A D117N mutation found in VIM-24 affected binding to the antibiotics. IMPORTANCE The metallo-ß-lactamases-producing Pseudomonas aeruginosa strains play an important role in hospital outbreaks and the VIM-type enzyme is the most prevalent in European countries. Two novel VIM-type enzymes in our study, VIM-84 and VIM-85, have higher levels of resistance to ß-lactams and greater hydrolytic activities for most ß-lactams compared with VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. Notably, the genes bla VIM-85 are carried by three different IncP-2-type megaplasmids which are distributed locally and appear responsible for the spread of antimicrobial resistance genes in hospital settings.

Chinese expert consensus on the construction of the fluoroless cardiac electrophysiology laboratory and related techniques.

Transcatheter radiofrequency ablation has been widely introduced for the treatment of tachyarrhythmias. The demand for catheter ablation continues to grow rapidly as the level of recommendation for catheter ablation. Traditional catheter ablation is performed under the guidance of X-rays. X-rays can help display the heart contour and catheter position, but the radiobiological effects caused by ionizing radiation and the occupational injuries worn caused by medical staff wearing heavy protective equipment cannot be ignored. Three-dimensional mapping system and intracardiac echocardiography can provide detailed anatomical and electrical information during cardiac electrophysiological study and ablation procedure, and can also greatly reduce or avoid the use of X-rays. In recent years, fluoroless catheter ablation technique has been well demonstrated for most arrhythmic diseases. Several centers have reported performing procedures in a purposefully designed fluoroless electrophysiology catheterization laboratory (EP Lab) without fixed digital subtraction angiography equipment. In view of the lack of relevant standardized configurations and operating procedures, this expert task force has written this consensus statement in combination with relevant research and experience from China and abroad, with the aim of providing guidance for hospitals (institutions) and physicians intending to build a fluoroless cardiac EP Lab, implement relevant technologies, promote the standardized construction of the fluoroless cardiac EP Lab.

Tail engagement of arrestin at the glucagon receptor.

Arrestins have pivotal roles in regulating G protein-coupled receptor (GPCR) signalling by desensitizing G protein activation and mediating receptor internalization1,2. It has been proposed that the arrestin binds to the receptor in two different conformations, 'tail' and 'core', which were suggested to govern distinct processes of receptor signalling and trafficking3,4. However, little structural information is available for the tail engagement of the arrestins. Here we report two structures of the glucagon receptor (GCGR) bound to ß-arrestin 1 (ßarr1) in glucagon-bound and ligand-free states. These structures reveal a receptor tail-engaged binding mode of ßarr1 with many unique features, to our knowledge, not previously observed. Helix VIII, instead of the receptor core, has a major role in accommodating ßarr1 by forming extensive interactions with the central crest of ßarr1. The tail-binding pose is further defined by a close proximity between the ßarr1 C-edge and the receptor helical bundle, and stabilized by a phosphoinositide derivative that bridges ßarr1 with helices I and VIII of GCGR. Lacking any contact with the arrestin, the receptor core is in an inactive state and loosely binds to glucagon. Further functional studies suggest that the tail conformation of GCGR-ßarr governs ßarr recruitment at the plasma membrane and endocytosis of GCGR, and provides a molecular basis for the receptor forming a super-complex simultaneously with G protein and ßarr to promote sustained signalling within endosomes. These findings extend our knowledge about the arrestin-mediated modulation of GPCR functionalities.

Gut dysbiosis-derived ß-glucuronidase promotes the development of endometriosis.

OBJECTIVE: To explore the role of gut dysbiosis-derived ß-glucuronidase (GUSB) in the development of endometriosis (EMs). DESIGN: 16S rRNA sequencing of stool samples from women with (n = 35) or without (n = 30) endometriosis and from a mouse model was conducted to assess gut microbiome changes and identify molecular factors influencing the development of endometriosis. Experiments in vivo in an endometriosis C57BL6 mouse model and in vitro verified the level of GUSB and its role in the development of EMs. SETTING: Department of Obstetrics and Gynecology, The First Affiliated Hospital of Sun Yat-sen University; Guangdong Provincial Clinical Research Center for Obstetrical and Gynecological Diseases. PATIENT(S): Women of reproductive age with a histological diagnosis of endometriosis were enrolled in the endometriosis group (n = 35) and infertile or healthy age-matched women who had undergone a gynecological or radiological examination in the control group (n = 30). Fecal and blood samples were taken the day before surgery. Paraffin-embedded sections from 50 bowel endometriotic lesions, 50 uterosacral lesions, 50 samples without lesions, and 50 normal endometria were collected. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Changes in the gut microbiome of patients with EMs and mice and the effect of ß-glucuronidase on the proliferation and invasion of endometrial stromal cells and the development of endometriotic lesions were assessed. RESULT(S): No difference in α and ß diversity was found between patients with EMs and controls. Immunohistochemistry analysis showed higher ß-glucuronidase expression in bowel lesions and uterosacral ligament lesions than in the normal endometrium (p<0.01). ß-Glucuronidase promoted the proliferation and migration of endometrial stromal cells during cell counting kit-8, Transwell, and wound-healing assays. Macrophage levels, especially M2, were higher in bowel lesions and uterosacral ligament lesions than in controls, and ß-glucuronidase promoted the M0 to M2 transition. Medium conditioned by ß-glucuronidase-treated macrophages promoted endometrial stromal cell proliferation and migration. ß-Glucuronidase increased the number and volume of endometriotic lesions and number of macrophages present in lesions in the mouse EMs model. CONCLUSION(S): This ß-Glucuronidase promoted EMs development directly or indirectly by causing macrophage dysfunction. The characterization of the pathogenic role of ß-glucuronidase in EMs has potential therapeutic implications.

Overexpression of blaGES-1 due to a strong promoter in the class 1 integron contributes to decreased ceftazidime-avibactam susceptibility in carbapenem-resistant Pseudomonas aeruginosa ST235.

Sequence type 235 (ST235) Pseudomonas aeruginosa, harboring so-called international, high-risk, or widespread clones, is associated with relatively high morbidity and mortality, partly due to multiantibiotic and high-level antibiotic resistance. Treatment of infections caused by such strains with ceftazidime-avibactam (CZA) is often successful. However, CZA resistance in carbapenem-resistant P. aeruginosa (CRPA) strains has been consistently reported with the increasing use of this drug. Likewise, we identified thirty-seven CZA-resistant ST235 P. aeruginosa strains from among 872 CRPA isolates. A total of 10.8% of the ST235 CRPA strains were resistant to CZA. Site-directed mutagenesis, cloning, expression, and whole-genome sequencing analysis revealed that overexpression of blaGES-1, which was carried in a class 1 integron of the complex transposon Tn6584, occurred due to a strong promoter, contributing to CZA resistance. Moreover, such overexpression of blaGES-1 combined with an efflux pump resulted in high-level resistance to CZA, considerably reducing the therapeutic options available for treating infections caused by ST235 CRPA. Considering the widespread presence of ST235 P. aeruginosa strains, clinicians should be aware of the risk of CZA resistance development in high-risk ST235 P. aeruginosa. Surveillance initiatives for preventing further dissemination of high-risk ST235 CRPA isolates with CZA resistance are essential.

An XDR Pseudomonas aeruginosa ST463 Strain with an IncP-2 Plasmid Containing a Novel Transposon Tn6485f Encoding blaIMP-45 and blaAFM-1 and a Second Plasmid with Two Copies of blaKPC-2.

The increased carbapenem resistance among Pseudomonas aeruginosa has become a serious health issue worldwide. We reported an extensively drug-resistant (XDR) P. aeruginosa PA30 isolate which belonged to sequence type ST463 and contained an IncP-2 plasmid (pPA30_1) carrying two genes, namely, blaIMP-45 and blaAFM-1, which encoded the metallo-ß-lactamases AFM-1 and IMP-45, respectively. Additionally, the strain had a plasmid (pPA30_2) with two copies of the blaKPC-2 genes embedded. The plasmid pPA30_1 was highly similar to the previously reported plasmid pHS17-127, which has the same genetic architecture. This plasmid contained blaIMP-45, located in a second gene cassette of the integron In786, carried by a Tn1403-derivative transposon acquiring an ISCR27n3-blaAFM-1 structure. Interestingly, the transposon in pPA30_1 acquired an extra ISCR1-qnrVC6 module and formed a novel transposon, which was subsequently annotated as Tn6485f. The blaKPC-2 genes in pPA30_2 underwent duplication due to the inversion of the IS26-blaKPC-2-IS26 element, which resulted in two copies of blaKPC-2. IMPORTANCE The ST463 clone is an emerging high-risk sequence type that is spreading with blaKPC-2-containing plasmids. The core blaKPC-2 genetic platform is ISKpn27-blaKPC-2-ISKpn6 in almost all samples, and the adjacent region beyond the core platform varies by IS26-mediated inversion or duplication events, amplifying the blaKPC-2 gene copies. The ST463 P. aeruginosa strain PA30 in our study contains another two metallo-ß-lactamase genes, namely, blaIMP-45 and blaAFM-1, in a novel transposon Tn6485f that is harbored by the IncP-2 megaplasmid. The pPA30_1 carrying blaIMP-45 and blaAFM-1 is highly related to pHS17-127 from the ST369 P. aeruginosa strain, indicating the putative dissemination of the megaplasmid between different clones.

PLN: Parasitic-Like Network for Barely Supervised Medical Image Segmentation.

It is known that annotations for 3D medical image segmentation tasks are laborious, time-consuming and expensive. Considering the similarities existing in inter-slice and inter-volume, we believe that the delineation way and the model architecture should be tightly coupled. In this paper, by introducing an extremely sparse annotation way of labeling only one slice per 3D image, we investigate a novel barely-supervised segmentation setting with only a few sparsely-labeled images along with a large amount of unlabeled images. To achieve this goal, we present a new parasitic-like network including a registration module (as host) and a semi-supervised segmentation module (as parasite) to deal with inter-slice label propagation and inter-volume segmentation prediction, respectively. Specifically, our parasitism mechanism effectively achieves the collaboration of these two modules through three stages of infection, development and eclosion, providing accurate pseudo-labels for training. Extensive results demonstrate that our framework is capable of achieving high performance on extremely sparse annotation tasks, e.g., we achieve Dice of 84.83% on LA dataset with only 16 labeled slices. The code is available athttps://github.com/ShumengLI/PLN.

A novel KPC-113 variant conferring carbapenem and ceftazidime-avibactam resistance in a multidrug-resistant Pseudomonas aeruginosa isolate.

OBJECTIVES: This study aimed to characterize a novel KPC-113 variant from a clinical Pseudomonas aeruginosa isolate R20-14. METHODS: Genomic DNA of R20-14 was subjected to Illumina and Oxford Nanopore sequencing. The horizontal transmission of plasmid was evaluated with conjugation experiments. Minimum inhibitory concentrations of bacterial strains were obtained using broth microdilution methods. KPC-113 detectability of different carbapenemase detection methods was tested. The kinetic parameters of KPC-113 were compared with those of KPC-2 by a spectrophotometer. Structure modelling and molecular docking of KPC-2 and KPC-113 were performed using Schrödinger. RESULTS: R20-14, a sequence type 3903 multidrug-resistant strain, was resistant to carbapenems and ceftazidime-avibactam (CZA) concurrently. S1-nuclease pulsed-field gel electrophoresis and genomic analysis revealed a blaKPC-113-carrying plasmid pR20-14, which resembled the previously reported type I KPC-encoding P. aeruginosa plasmids and exhibited a high conjugation frequency. KPC-113, with a glycine residue insertion between Ambler positions 266 and 267 in KPC-2, conferred both carbapenem and CZA resistance in DH5α and PAO1 transformants. Diagnostic tests showed that KPC-113 acted in a similar manner to KPC-2. Compared with KPC-2, KPC-113 presented reduced catalytic ability to carbapenems and ceftazidime, meanwhile responding poorly to avibactam inhibition. Modelling structure revealed that KPC-113 possibly had a more flattened binding pocket than KPC-2, leading to the change of ligand binding modes. CONCLUSIONS: KPC-113 is a novel KPC variant mediating both CZA resistance and carbapenem resistance. It is of great concern that blaKPC-113 could transfer horizontally with great efficiency and inactivate carbapenems and CZA simultaneously. Great efforts should be made to prevent its spread in clinical settings.

Ischemic accumulation of succinate induces Cdc42 succinylation and inhibits neural stem cell proliferation after cerebral ischemia/reperfusion.

Ischemic accumulation of succinate causes cerebral damage by excess production of reactive oxygen species. However, it is unknown whether ischemic accumulation of succinate affects neural stem cell proliferation. In this study, we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery. We found that succinate levels increased in serum and brain tissue (cortex and hippocampus) after ischemia/reperfusion injury. Oxygen-glucose deprivation and reoxygenation stimulated primary neural stem cells to produce abundant succinate. Succinate can be converted into diethyl succinate in cells. Exogenous diethyl succinate inhibited the proliferation of mouse-derived C17.2 neural stem cells and increased the infarct volume in the rat model of cerebral ischemia/reperfusion injury. Exogenous diethyl succinate also increased the succinylation of the Rho family GTPase Cdc42 but repressed Cdc42 GTPase activity in C17.2 cells. Increasing Cdc42 succinylation by knockdown of the desuccinylase Sirt5 also inhibited Cdc42 GTPase activity in C17.2 cells. Our findings suggest that ischemic accumulation of succinate decreases Cdc42 GTPase activity by induction of Cdc42 succinylation, which inhibits the proliferation of neural stem cells and aggravates cerebral ischemia/reperfusion injury.

iNKT17 cells play a pathogenic role in ethinylestradiol-induced cholestatic hepatotoxicity.

IL-17 is closely associated with inflammation in intrahepatic cholestasis (IHC). Targeting IL-17 ameliorates IHC in mice. Invariant natural killer T (iNKT) cells are predominantly enriched in the liver and they mediate drug-induced liver injury through their secreted cytokines. However, whether iNKT17 cells are involved in ethinylestradiol (EE)-induced IHC remains unclear. In the present study, the administration of EE (10 mg/kg in vivo and 6.25 µM in vitro) promoted the activation and expansion of iNKT17 cells, which contributed to a novel hepatic iNKT17/Treg imbalance. iNKT cell-deficient Jα18-/- mice and the RORγt inhibitor digoxin (20 µg) alleviated EE-induced cholestatic hepatotoxicity and downregulated the IL-17 signalling pathway. In contrast, the co-administration of EE with recombinant IL-17 (1 µg) to Jα18-/- mice induced cholestatic hepatotoxicity and increased the infiltration of hepatic neutrophils and monocytes. Importantly, the administration of IL-17-/- iNKT cells (3.5 × 105) to Jα18-/- mice resulted in the attenuation of hepatotoxicity and the recruitment of fewer hepatic neutrophils and monocytes than the adoptive transfer of wild-type iNKT cells. These results indicated that iNKT17 cells could exert pathogenic effects. The recruitment and activation of iNKT17 cells could be attributed to the high level of CXCR3 expression on their surface. CXCL10 deficiency ameliorated EE-induced cholestatic liver damage, reduced hepatic CXCR3+ iNKT cells and inhibited RORγt expression. These findings suggest that iNKT17 cells play a key role in EE-induced cholestatic liver injury via CXCR3-mediated recruitment and activation. Our study provides new insights and therapeutic targets for cholestatic diseases.

Percutaneous Left Atrial Appendage Closure With a Novel LAA Occluder for Stroke Prevention in Atrial Fibrillation.

Background: More than 90% of thromboses originate from the left atrial appendage (LAA) in patients with nonvalvular atrial fibrillation (NVAF). Objectives: This study was designed to investigate the safety and efficacy of LAA closure with the Leftear device (Pulse Scientific) in NVAF patients. Methods: A prospective, multicenter, registry-based study was conducted in 200 NVAF patients with CHA2DS2-VASc (congestive heart failure, hypertension, age, diabetes, previous stroke/transient ischemic attack, vascular disease, female sex) scores ≥2. The primary safety endpoint was defined as any serious adverse events. Efficacy was assessed by a primary composite endpoint of hemorrhagic or ischemic stroke, systemic embolism, and cardiac or unexplained death at 1 year of follow-up. Results: The device was implanted in 196 patients, with 1-stop LAA closure combined with atrial fibrillation ablation implemented in 133 patients. The immediate success rate was 100%. There were serious adverse events in 9 patients (4.5%; 95% CI: 1.6%-7.4%), which mainly occurred in 1-stop LAA closure. All pericardial tamponades occurred in 6 patients with 1-stop LAA closure. No patient experienced a major bleeding event or acute device-related thrombus. During the 12-month follow-up period, the risk of the primary composite endpoint was 1.6% (95% CI: 0.3%-4.5%), and statistical noninferiority was achieved (the upper bound of 95% CI: 4.5% < the prespecified maximum annual incidence of 8.0%). Ischemic stroke occurred in 1 patient, 3 patients had incomplete LAA sealing, and no delayed device-related thrombus was found. Conclusions: LAA closure with the novel disc-like occluder shows high procedural success, satisfactory safety, and encouraging efficacy for stroke prevention in patients with NVAF. Compared with 1-stop LAA closure, single LAA closure may be more tolerable. (A multicenter, single-arm clinical trial to evaluate the efficacy and safety of left atrial appendage system for left atrial appendage occlusion in patients with non-valvular atrial fibrillation; ChiCTR1900023035).