Utility of IMP3, p53, and S100P immunohistochemical stains in distinguishing reactive atypia from dysplasia in cholecystectomy specimens.

BACKGROUND: Distinguishing reactive atypia from dysplasia in cholecystectomy specimens can be histologically challenging. The aim of this study was to evaluate the utility of IMP3, p53, and S100P immunostains in differentiating reactive atypia from dysplasia in cholecystectomies. METHODS: Fifty-four cholecystectomies were reviewed and characterized into 5 groups: 2 normal, 29 reactive atypia, 16 low-grade dysplasia, 2 high-grade dysplasia, and 5 adenocarcinoma. IMP3, p53, and S100P immunostains were performed and evaluated. IMP3 (nuclear) and S100P (nuclear or nuclear/cytoplasmic) were categorized into negative or positive expression, and p53 was categorized into wild-type and aberrant/mutant expression. Chi-square test was used for statistical analysis. RESULTS: The patients were mostly middle-aged women (mean 44, range 19-87 years, 81% female), with predominantly Hispanic White ethnicity (80%). The majority of the normal and reactive atypia cases showed negative IMP3 (100% and 75.9%, respectively) and wild-type p53 (100% and 89.7%, respectively) staining. Over half (56.3%) of the low-grade dysplasia and all the high-grade dysplasia cases showed IMP3 positivity. Aberrant p53 staining pattern was seen in half of both low and high-grade dysplasia cases. Adenocarcinoma showed IMP3 positivity in 80% and p53 aberrancy in all cases. S100P showed no statistical significance among the diagnostic categories. Significant differences in staining patterns were found between reactive atypia vs. low-grade dysplasia, and reactive atypia vs. low-grade + high-grade dysplasia using a combination of IMP3 and p53 stains (all p < 0.05). CONCLUSIONS: In challenging cholecystectomies, IMP3 positivity or aberrant p53 expression may serve as a useful adjunct to support a diagnosis of dysplasia over reactive atypia.

Association of insulin-like growth factor II mrna-binding protein 3 (IMP3) expression with prognostic and morphological factors in endometrial cancer.

Objective: Endometrial cancer (EC) is a heterogeneous disease with recurrence rates ranging from 15 to 20%. The discrimination of cases with a worse prognosis aims, in part, to reduce the length of surgical staging in cases with a better prognosis. This study aimed to evaluate the association between Insulin-like growth factor II mRNA-binding protein 3 (IMP3) expression and prognostic and morphological factors in EC. Methods: This retrospective, cross-sectional, analytical study included 79 EC patients - 70 endometrioid carcinoma (EEC) and 9 serous carcinoma (SC) - and 74 benign endometrium controls. IMP3 expression was evaluated by immunohistochemistry-based TMA (Tissue Microarray), and the results were associated with morphological and prognostic factors, including claudins 3 and 4, estrogen and progesterone receptors, TP53, and KI67. Results: IMP3 expression was significantly higher in SC compared to EEC in both extent (p<0.001) and intensity (p=0.044). It was also significantly associated with worse prognostic factors, including degree of differentiation (p=0.024, p<0.001), staging (p<0.001; p<0.001) and metastasis (p=0.002; p<0.001). IMP3 expression was also significant in extent (p=0.002) in endometrial tumors compared with controls. In addition, protein TP53 and KI67 showed significant associations in extent and intensity, respectively. Conclusion: IMP3 expression was associated with worse prognostic factors studied. These findings suggest that IMP3 may be a potential biomarker for EC poorer prognosis.

[Clinical and neuropsychological features and changes to regional cerebral blood flow in |patients with Parkinson's disease dementia].

This study aimed to clarify associations of clinical and neuropsychological features and change in regional cerebral blood flow (rCBF) on 123I-IMP-SPECT in patients with Parkinson's disease (PD) who developed dementia. Sixty-one PD patients (mean age, 65.9 ± 8.6 years; mean disease duration, 11.0 ± 11.0 years) were recruited and followed-up for two years. Clinical and neuropsychological characteristics, and rCBF from SPECT were compared between PD patients who developed dementia (PDD+) and those who remained undemented (PDD-). Thirty-eight PD patients (62.3%) were diagnosed with PD-MCI at baseline. During follow-up, 22 PD patients (36%) developed dementia (PDD+). Univariate logistic regression models showed that Hoehn and Yahr scale 4 (odds ratio [OR] 5.85; 95% confidence interval [CI] 1.35-30.75]), visual hallucination (OR 5.95; 95%CI 1.67-25.4]), and PD-MCI (OR 6.47; 95%CI 1.57-39.63]) represented a significant risk factor for PDD+. Among neuropsychological parameters, WAIS (Wechsler Adult Intelligence Scale)-III block design (OR 6.55; 95%CI 1.66-29.84), letter number sequencing (OR 7.01; 95%CI 1.65-36.64), digit-symbol coding (OR 3.90; 95%CI 1.13-14.2), Wechsler Memory Scale, revised (WMS-R) visual paired associates II (delayed recall) (OR 4.68; 95%CI 1.36-17.36), Logical memory I (immediate recall) (OR 8.30; 95%CI 1.37-90.89), Logical memory II (delayed recall) (OR 6.61; 95%CI 1.35-44.33), Visual reproduction I (immediate recall) (OR 7.67; 95%CI 2.11-31.40), and Visual reproduction II (delayed recall) (OR 5.64; 95%CI 1.62-21.47) were significant risk factors. Decreased rCBF assessed using the general linear model (two-sample t-test) by SPM8 was observed in the left precuneus (0, -66, 16), right cuneus (6, -76, 30), and left angular gyrus (-46, -74, 32) in PDD+ compared with PDD- patients. Collectively, we have here shown that clinical and neuropsychological characteristics as well as changes to rCBF in PD patients who converted to PDD+. These features should be carefully monitored to detect the development of dementia in PD patients.

Long noncoding RNA H19 knockdown promotes angiogenesis via IMP2 after ischemic stroke.

AIMS: This study aimed to explore the effects of long noncoding RNA (lncRNA) H19 knockdown on angiogenesis and blood-brain barrier (BBB) integrity following cerebral ischemia/reperfusion (I/R) and elucidate their underlying regulatory mechanisms. METHODS: A middle cerebral artery occlusion/reperfusion model was used to induce cerebral I/R injury. The cerebral infarct volume and neurological impairment were assessed using 2,3,5-triphenyl-tetrazolium chloride staining and neurobehavioral tests, respectively. Relevant proteins were evaluated using western blotting and immunofluorescence staining. Additionally, a bioinformatics website was used to predict the potential target genes of lncRNA H19. Finally, a rescue experiment was conducted to confirm the potential mechanism. RESULTS: Silencing of H19 significantly decreased the cerebral infarct volume, enhanced the recovery of neurological function, mitigated BBB damage, and stimulated endothelial cell proliferation following ischemic stroke. Insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) is predicted to be a potential target gene for lncRNA H19. H19 knockdown increased IMP2 protein expression and IMP2 inhibition reversed the protective effects of H19 inhibition. CONCLUSION: Downregulation of H19 enhances angiogenesis and mitigates BBB damage by regulating IMP2, thereby alleviating cerebral I/R injury.

Characterization of a novel multi-resistant Pseudomonas juntendi strain from China with chromosomal blaVIM-2 and a megaplasmid coharboring blaIMP-1-like and blaOXA-1.

BACKGROUND: Pseudomonas juntendi is a newly identified opportunistic pathogen, of which we have limited understanding. P. juntendi strains are often multidrug resistant, which complicates clinical management of infection. METHODS: A strain of Pseudomonas juntendi (strain L4326) isolated from feces was characterized by MALDI-TOF-MS and Average Nucleotide Identity BLAST. This strain was further subject to whole-genome sequencing and Maximum Likelihood phylogenetic analysis. The strain was phenotypically characterized by antimicrobial susceptibility testing and conjugation assays. RESULTS: We have isolated the novel P. juntendi strain L4236, which was multidrug resistant, but retained sensitivity to amikacin. L4236 harbored a megaplasmid that encoded blaOXA-1 and a novel blaIMP-1 resistance gene variant. P. juntendi strain L4236 was phylogenetically related to P. juntendi strain SAMN30525517. CONCLUSION: A rare P. juntendi strain was isolated from human feces in southern China with a megaplasmid coharboring blaIMP-1-like and blaOXA-1. Antimicrobial selection pressures may have driven acquisition of drug-resistance gene mutations and carriage of the megaplasmid.

Integrated Analysis of Patient Networks and Plasmid Genomes to Investigate a Regional, Multispecies Outbreak of Carbapenemase-Producing Enterobacterales Carrying Both blaIMP and mcr-9 Genes.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of imipenemase (IMP)-encoding CPE among diverse Enterobacterales species between 2016 and 2019 across a London regional network. METHODS: We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE-positive patients. Genomes of IMP-encoding CPE isolates were overlaid with patient contacts to imply potential transmission events. RESULTS: Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, and Escherichia coli); 86% (72 of 84) harbored an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68 of 72). Phylogenetic analysis of IncHI2 plasmids identified 3 lineages showing significant association with patient contacts and movements between 4 hospital sites and across medical specialties, which was missed in initial investigations. CONCLUSIONS: Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multimodal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks.SummaryThis was an investigation, using integrated pathway networks and genomics methods, of the emergence of imipenemase-encoding carbapenemase-producing Enterobacterales among diverse Enterobacterales species between 2016 and 2019 in patients across a London regional hospital network, which was missed on routine investigations.

Biochemical communication between filament-forming enzymes: Potential Regulatory Roles of Metabolites in Enzyme Co-assemblies with CTP Synthase.

A host of metabolic enzymes reversibly self-assemble to form membrane-less, intracellular filaments under normal physiological conditions and in response to stress. Often, these enzymes reside at metabolic control points, suggesting that filament formation affords an additional regulatory mechanism. Examples include cytidine-5'-triphosphate (CTP) synthase (CTPS), which catalyzes the rate-limiting step for the de novo biosynthesis of CTP; inosine-5'-monophosphate dehydrogenase (IMPDH), which controls biosynthetic access to guanosine-5'-triphosphate (GTP); and ∆1-pyrroline-5-carboxylate (P5C) synthase (P5CS) that catalyzes the formation of P5C, which links the Krebs cycle, urea cycle, and proline metabolism. Intriguingly, CTPS can exist in co-assemblies with IMPDH or P5CS. Since GTP is an allosteric activator of CTPS, the association of CTPS and IMPDH filaments accords with the need to coordinate pyrimidine and purine biosynthesis. Herein, a hypothesis is presented furnishing a biochemical connection underlying co-assembly of CTPS and P5CS filaments - potent inhibition of CTPS by glutamate γ-semialdehyde, the open-chain form of P5C.

The interaction of the azetidine thiazole side chain with the active site loop (ASL) 3 drives the evolution of IMP metallo-ß-lactamase against tebipenem.

Imipenemase (IMP) metallo-ß-lactamases (MBLs) hydrolyze almost all available ß-lactams including carbapenems and are not inhibited by any commercially available ß-lactamase inhibitor. Tebipenem (TP) pivoxil is the first orally available carbapenem and possesses a unique bicyclic azetidine thiazole moiety located at the R2 position. TP has potent in vitro activity against Enterobacterales producing extended-spectrum and/or AmpC ß-lactamases. Thus far, the activity of TP against IMP-producing strains is understudied. To address this knowledge gap, we explored the structure activity relationships of IMP MBLs by investigating whether IMP-6, IMP-10, IMP-25, and IMP-78 [MBLs with expanded hydrolytic activity against meropenem (MEM)] would demonstrate enhanced activity against TP. Most of the Escherichia coli DH10B strains expressing IMP-1 variants displayed a ≥twofold MIC difference between TP and MEM, while those expressing VIM or NDM variants demonstrated comparable MICs. Catalytic efficiency (kcat/KM) values for the TP hydrolysis by IMP-1, IMP-6, IMP-10, IMP-25, and IMP-78 were significantly lower than those obtained for MEM. Molecular dynamic simulations reveal that V67F and S262G substitutions (found in IMP-78) reposition active site loop 3, ASL-3, to better accommodate the bicyclic azetidine thiazole side chain, allowing microbiological/catalytic activity to approach that of comparison MBLs used in this study. These findings suggest that modifying the R2 side chain of carbapenems can significantly impact hydrolytic stability. Furthermore, changes in conformational dynamics due to single amino acid substitutions should be used to inform drug design of novel carbapenems.

Development of an automated region-of-interest-setting method based on a deep neural network for brain perfusion single photon emission computed tomography quantification methods.

Objectives: A simple noninvasive microsphere (SIMS) method using 123I-IMP and an improved brain uptake ratio (IBUR) method using 99mTc-ECD for the quantitative measurement of regional cerebral blood flow have been recently reported. The input functions of these methods were determined using the administered dose, which was obtained by analyzing the time activity curve of the pulmonary artery (PA) for SIMS and the ascending aorta (AAo) for the IBUR methods for dynamic chest images. If the PA and AAo regions of interest (ROIs) can be determined using deep convolutional neural networks (DCNN) for segmentation, the accuracy of these ROI-setting methods can be improved through simple analytical operations to ensure repeatability and reproducibility. The purpose of this study was to develop new PA and AAo-ROI setting methods using a DCNN (DCNN-ROI method). Methods: A U-Net architecture based on convolutional neural networks was used to determine the PA and AAo candidate regions. Images of 290 patients who underwent 123I-IMP RI-angiography and 108 patients who underwent 99mTc-ECD RI-angiography were used. The PA and AAo-ROI results for the DCNN-ROI method were compared to those obtained using manual methods. The counts for the input function on the PA and AAo-ROI were determined by integrating the area under the curve (AUC) counts of the time-activity curve of PA and AAo-ROI, respectively. The effectiveness of the DCNN-ROI method was elucidated through a comparison with the integrated AUC counts of the DCNN-ROI and the manual ROI. Results: The coincidence ratio for the locations of the PA and AAo-ROI obtained using the DCNN method and that for the manual method was 100%. Strong correlations were observed between the AUC counts using the DCNN and manual methods. Conclusion: New ROI- setting programs were developed using a deep convolution neural network DCNN to determine the input functions for the SIMS and IBUR methods. The accuracy of these methods is comparable to that of the manual method.

Clinical and Microbiological Characteristics of Bacteremia Caused by Carbapenemase-producing Enterobacterales in Minami Ibaraki Area, Japan.

Although recent propagation of carbapenemase-producing Enterobacterales (CPE) has become a problem worldwide, the picture of CPE infection in Japan has not fully been elucidated. In this study, we examined clinical and microbiological characteristics of invasive CPE infection occurring at 8 hospitals in Minami Ibaraki Area between July 2001 to June 2017. Of 7294 Enterobacterales strains isolated from independent cases of bacteremia and/or meningitis, 10 (0.14%) were CPE (8 Enterobacter cloacae-complex, 1 Escherichia coli, and 1 Edwardsiella tarda）, all of which had the blaIMP-1 gene and susceptible to gentamicin and trimethoprim/sulfamethoxazole. These strains were isolated from 7 adult and 2 infant bacteremia (1 infant patient developed CPE bacteremia twice) after 2007. The most common portal of entry was intravenous catheters. All of the adult patients were recovered, while the infant patients eventually died. Genomic analyses showed that the 8 E. cloacae-complex strains were classified into 5 groups, each of which was exclusively detected in specific facilities at intervals of up to 3 years, suggesting persistent colonization in the facilities. This study showed that invasive CPE infection in the area was rare, caused by IMP-1-type CPE having susceptibility to various antibiotics, and nonfatal among adult patients.

Understanding the processes underpinning IMPlementing IMProved Asthma self-management as RouTine (IMP2ART) in primary care: study protocol for a process evaluation within a cluster randomised controlled implementation trial.

BACKGROUND: Providing supported self-management for people with asthma can reduce the burden on patients, health services and wider society. Implementation, however, remains poor in routine clinical practice. IMPlementing IMProved Asthma self-management as RouTine (IMP2ART) is a UK-wide cluster randomised implementation trial that aims to test the impact of a whole-systems implementation strategy, embedding supported asthma self-management in primary care compared with usual care. To maximise opportunities for sustainable implementation beyond the trial, it is necessary to understand how and why the IMP2ART trial achieved its clinical and implementation outcomes. METHODS: A mixed-methods process evaluation nested within the IMP2ART trial will be undertaken to understand how supported self-management was implemented (or not) by primary care practices, to aid interpretation of trial findings and to inform scaling up and sustainability. Data and analysis strategies have been informed by mid-range and programme-level theory. Quantitative data will be collected across all practices to describe practice context, IMP2ART delivery (including fidelity and adaption) and practice response. Case studies undertaken in three to six sites, supplemented by additional interviews with practice staff and stakeholders, will be undertaken to gain an in-depth understanding of the interaction of practice context, delivery, and response. Synthesis, informed by theory, will combine analyses of both qualitative and quantitative data. Finally, implications for the scale up of asthma self-management implementation strategies to other practices in the UK will be explored through workshops with stakeholders. DISCUSSION: This mixed-methods, theoretically informed, process evaluation seeks to provide insights into the delivery and response to a whole-systems approach to the implementation of supported self-management in asthma care in primary care. It is underway at a time of significant change in primary care in the UK. The methods have, therefore, been developed to be adaptable to this changing context and to capture the impact of these changes on the delivery and response to research and implementation processes.

Reviving resilience: MEcPP-mediated ASK1-IMPα-9-TRP2 stress-responsive module.

Chloroplasts are not only critical photosynthesis sites in plants, but they also participate in plastidial retrograde signaling in response to developmental and environmental signals. MEcPP (2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate) is an intermediary in the methylerythritol phosphate (MEP) pathway in chloroplasts. It is a critical precursor for the synthesis of isoprenoids and terpenoid derivatives, which play crucial roles in plant growth and development, photosynthesis, reproduction, and defense against environmental constraints. Accumulation of MEcPP under stressful conditions triggers the expression of IMPα-9 and TPR2, contributing to the activation of abiotic stress-responsive genes. In this correspondence, we discuss plastidial retrograde signaling in support of a recently published paper in Molecular Plant (Zeng et al. 2024). We hope that it can shed more insight on the retrograde signaling cascade.

Occurrence, Impact, and Multilocus Sequence Analysis of Alder Yellows Phytoplasma Infecting Common Alder and Italian Alder in Southern Italy.

Alder yellows (ALY) phytoplasma (16SrV-C) is associated with ALY, a disease of several Alnus (alder) species in Europe and A. rubra in North America. In all affected species, the symptoms are similar. However, latent infections are common. ALY phytoplasma includes different strains which may be occasionally transmitted to grapevines leading to some grapevine yellows diseases. In the current study, visual symptom assessment and PCR-based methods using universal and group-specific phytoplasma primers were used to update and extend knowledge on the occurrence, impact, and genetic diversity of ALY phytoplasma in declining and non-symptomatic A. glutinosa and A. cordata trees in the Basilicata and Campania regions of southern Italy. ALY phytoplasma was detected in 80% of alder trees examined. In symptomatic trees, no other cause of disease was observed. More than half of alder trees that tested phytoplasma-positive proved to be latently infected. A considerable genetic variability was observed among the newly recorded ALY phytoplasma strains in southern Italy in almost of the genes examined. These included 16S rRNA, 16S/23S rDNA spacer region, ribosomal protein rpsV (rpl22) and rpsC (rps3), map, imp, and groEL genes. Eleven new genotypes were identified at map gene sequence level. However, the genetic differences observed were not related to plant host species, geographical origin, and symptoms shown by infected alder trees. Also, this study indicates that ALY phytoplasma is more widespread than previously thought.

Activation of stress-response genes by retrograde signaling-mediated destabilization of nuclear importin IMPα-9 and its interactor TPR2.

Stress-induced retrograde signal transmission from the plastids to the nucleus has long puzzled plant biologists. To address this, we performed a suppressor screen of the ceh1 mutant, which contains elevated 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP) levels, and identified the gain-of-function mutant impα-9, which shows reversed dwarfism and suppressed expression of stress-response genes in the ceh1 background despite heightened MEcPP. Subsequent genetic and biochemical analyses established that the accumulation of MEcPP initiates an upsurge in Arabidopsis SKP1-like 1 (ASK1) abundance, a pivotal component in the proteasome degradation pathway. This increase in ASK1 prompts the degradation of IMPα-9. Moreover, we uncovered a protein-protein interaction between IMPα-9 and TPR2, a transcriptional co-suppressor and found that a reduction in IMPα-9 levels coincides with a decrease in TPR2 abundance. Significantly, the interaction between IMPα-9 and TPR2 was disrupted in impα-9 mutants, highlighting the critical role of a single amino acid alteration in maintaining their association. Disruption of their interaction results in the reversal of MEcPP-associated phenotypes. Chromatin immunoprecipitation coupled with sequencing analyses revealed that TPR2 binds globally to stress-response genes and suggested that IMPα-9 associates with the chromatin. They function together to suppress the expression of stress-response genes under normal conditions, but this suppression is alleviated in response to stress through the degradation of the suppressing machinery. The biological relevance of our discoveries was validated under high light stress, marked by MEcPP accumulation, elevated ASK1 levels, IMPα-9 degredation, reduced TPR2 abundance, and subsequent activation of a network of stress-response genes. In summary, our study collectively unveils fresh insights into plant adaptive mechanisms, highlighting intricate interactions among retrograde signaling, the proteasome, and nuclear transport machinery.

Emergence of carbapenem-resistant Pseudomonas aeruginosa ST179 producing both IMP-16 and KPC-2: a case study of introduction from Peru to Spain.

We describe four cases of a novel carbapenem-resistant Pseudomonas aeruginosa ST179 clone carrying the blaKPC-2 or blaKPC-35 gene together with blaIMP-16, imported from Peru to Spain and isolated from leukemia patients. All isolates were multidrug-resistant but remained susceptible to fosfomycin, cefiderocol, and colistin. Whole-genome sequencing revealed that blaKPC-2 and blaKPC-35 were located in an IncP6 plasmid, whereas blaIMP-16 was in a chromosomal type 1 integron. This study highlights the global threat of multidrug-resistant P. aeruginosa clones and underscores the importance of monitoring and early detection of emerging resistance mechanisms to guide appropriate treatment strategies. The importation and spread of such clones emphasize the urgent need to implement strict infection control measures to prevent the dissemination of carbapenem-resistant bacteria. IMPORTANCE: This is the first documented case of a Pseudomonas aeruginosa ST179 strain carrying the blaKPC-35 gene, and it represents the first report of a P. aeruginosa co-harboring blaIMP-16 and either blaKPC-2 or blaKPC-35, which wre imported from Peru to Spain, highlighting a threat due to the capacity of spreading carbapenem-resistance via plasmid conjugation.

Boron-Salphen Conjugate based Molecular Probe Exhibiting Fluorescence On-Off-On Response in Detection of Cu2+ and ATP through Displacement Approach.

Synthesis and photophysical properties of a fluorescent probe HBD is described. Probe upon interaction with metal ions, anions and nucleoside pyrophosphates (NPPs) showed fluorescence quenching with Cu2+ due to chelation enhanced quenching effect (CHEQ). Moreover, interaction of ensemble HBD.Cu2+ with anions and NPPs showed fluorescence "turn-On" response with ATP selectively. "On-Off-On" responses observed with Cu2+ and ATP is attributed to an interplay between ESIPT and TICT processes. Cyclic voltammogram of probe exhibited quasi-reversible redox behaviour with three oxidation and two reduction potentials and the change in band gaps of probe suggested the interaction with Cu2+ and ATP. The 2 : 1 and 1 : 1 binding stoichiometry for an interaction between probe and Cu2+ (LOD, 62 nM) and ensemble, HBD.Cu2+ with ATP (LOD, 0.4 µM) respectively are realised by Job's plot and HRMS data. Cell imaging studies carried out to detect Cu2+ and ATP in HeLa cells. Also, the output emission observed with Cu2+ and ATP is utilized to construct an implication (IMP) logic gate. Test paper strips showed naked-eye visible color responses to detect Cu2+ and ATP. In real water samples probe successfully detected copper (0.03 µM) between 5-6.5 ppb level (ICP-MS method).

Clonal transmission of blaIMP-4-carrying ST196 Klebsiella pneumoniae isolates mediated by the IncN plasmid in China.

OBJECTIVES: To investigate the clinical and molecular epidemiological characteristics of blaIMP-4-carrying Klebsiella pneumoniae in a tertiary hospital in China. METHODS: Ten carbapenem-resistant K. pneumoniae (CRKP) isolates carrying the blaIMP-4 gene were collected. Molecular characteristics were analysed using whole-genome sequencing. Plasmid conjugation experiments were used to analyse conjugation of the plasmids. We compared and analysed K. pneumoniae-carrying blaIMP-4 genomic datasets obtained from the National Center for Biotechnology Information (NCBI) with the strains in this study. RESULTS: All 10 CRKP isolates carrying blaIMP-4 were collected from 10 adult patients in the respiratory intensive care unit. These strains were only sensitive to polymyxins and tigecycline due to them simultaneously carrying multiple resistance genes, namely blaOKP-A-5, fosA, oqxA, and oqxB. Notably, R29 harboured two carbapenemase genes (blaNDM-1 and blaIMP-4). These strains had similar drug-resistant phenotypes and genes, all belonging to sequence type (ST)196. Additionally, the patients had experienced spatiotemporal intersection during hospitalization, suggesting that these strains underwent clonal transmission, but they belonged to different clonal clusters from the blaIMP-4-positive K. pneumoniae currently published in the NCBI. Among the 10 strains, blaIMP-4 was located on the IncN plasmid, and six strains had successfully transferred the plasmid to the recipient strain EC600 through plasmid conjugation. CONCLUSIONS: The blaIMP-4-positive ST196 CRKP isolate showed clonal distribution in the respiratory intensive care unit, which was mediated by the IncN plasmid. Consequently, there should be increased monitoring of carbapenem-resistant strains in clinical settings to prevent and control its transmission.

Relative inhibitory activities of the broad-spectrum ß-lactamase inhibitor xeruborbactam in comparison with taniborbactam against metallo-ß-lactamases produced in Escherichia coli and Pseudomonas aeruginosa.

Xeruborbactam is a newly developed ß-lactamase inhibitor designed for metallo-ß-lactamases (MBLs). This study assessed the relative inhibitory properties of this novel inhibitor in comparison with another MBL inhibitor, namely taniborbactam (TAN), against a wide range of acquired MBL produced either in Escherichia coli or Pseudomonas aeruginosa. As observed with taniborbactam, the combination of xeruborbactam (XER) with ß-lactams, namely, ceftazidime, cefepime and meropenem, led to significantly decreased MIC values for a wide range of B1-type MBL-producing E. coli, including most recombinant strains producing NDM, VIM, IMP, GIM-1, and DIM-1 enzymes. Noteworthily, while TAN-based combinations significantly reduced MIC values of ß-lactams for MBL-producing P. aeruginosa recombinant strains, those with XER were much less effective. We showed that this latter feature was related to the MexAB-OprM efflux pump significantly impacting MIC values when testing XER-based combinations in P. aeruginosa. The relative inhibitory concentrations (IC50 values) were similar for XER and TAN against NDM and VIM enzymes. Noteworthily, XER was effective against NDM-9, NDM-30, VIM-83, and most of IMP enzymes, although those latter enzymes were considered resistant to TAN. However, no significant inhibition was observed with XER against IMP-10, SPM-1, and SIM-1 as well as the representative subclass B2 and B3 enzymes, PFM-1 and AIM-1. The determination of the constant inhibition (Ki) of XER revealed a much higher value against IMP-10 than against NDM-1, VIM-2, and IMP-1. Hence, IMP-10 that differs from IMP-1 by a single amino-acid substitution (Val67Phe) can, therefore, be considered resistant to XER.

Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) bifunctional enzyme from Candidatus Liberibacer asiaticus.

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.

PKM2 promotes myoblast growth and inosine monophosphate-specific deposition in Jingyuan chicken.

Inosine monophosphate (IMP) is widely regarded as an important indicator for evaluating the flavour of poultry meat. However, little is known about the molecular mechanisms affecting the specific deposition of IMP. In this study, we functionally verified PKM2 (Pyruvate kinase M2), a candidate gene related to IMP synthesis, in order to reveal the important role of PKM2 in meat flavour and muscle development of Jingyuan chickens. The results showed that the IMP content in breast muscle of Jingyuan chickens was negatively correlated with PKM2 mRNA expression (r = -0.1710), while the IMP content in leg muscle was significantly positively correlated with PKM2 mRNA expression (r = 0.7350) (P < 0.05). During myogenesis, PKM2 promoted the proliferation rate of myoblasts and the expression of proliferation marker genes, inhibited the apoptosis rate and the expression of apoptosis marker genes, and decreased the expression of differentiation marker genes. Up-regulation of PKM2 enhanced the expression of key genes in the purine metabolic pathway and the de novo synthesis pathway of IMP, and suppressed the expression of key genes in the salvage pathway. ELISA assays showed that PKM2 decreased IMP and hypoxanthine (HX) contents, while adenosine triphosphate (ATP) and uric acid (UA) contents were clearly elevated. In summary, these studies revealed that PKM2 regulates myogenesis and specific deposition of IMP, which can be used to improve the quality of Jingyuan chicken meat.