Two Point Mutations in CYP4CE1 Promoter Contributed to the Differential Regulation of CYP4CE1 Expression by FoxO between Susceptible and Nitenpyram-Resistant Nilaparvata lugens.

Four P450s were reported to be important for imidacloprid resistance in Nilaparvata lugens, a major insect pest on rice, which was confirmed in this study in an imidacloprid-resistant strain (ImiR). Here we found that only two (CYP4CE1 and CYP6ER1) from these four P450 genes were overexpressed in a nitenpyram-resistant strain (NitR) when compared to a susceptible strain (SUS). CYP4CE1 RNAi reduced nitenpyram and imidacloprid resistance in NitR and ImiR strains, with a greater reduction in nitenpyram resistance. The transcription factor FoxO mediated nitenpyram resistance in NitR and ImiR strains, but it was not differentially expressed among strains. The potential reason for the differential regulation of FoxO on CYP4CE1 expression was mainly from sequence differences in the CYP4CE1 promoter between susceptible and resistant insects. In six FoxO response elements predicted in the CYP4CE1 promoter, the single-nucleotide polymorphisms were frequently detected in over 50% of NitR and ImiR individuals. The luciferase reporter assays showed that two mutations, -650T/G and -2205T/A in two response elements at the positions of -648 and -2200 bp, mainly contributed to the enhanced regulation on CYP4CE1 expression by FoxO in resistant insects. The frequency was over 69% for both -650T/G and -2205T/A detected in NitR and ImiR individuals but less than 20% in SUS insects. In conclusion, CYP4CE1 overexpression importantly contributed to nitenpyram resistance in N. lugens, and two mutations in the CYP4CE1 promoter of resistant insects led to an enhanced regulation on CYP4CE1 expression by FoxO.

Clinical Outcomes and Risk Factors for Death in Critically Ill Patients with Carbapenem-Resistant Klebsiella pneumoniae Treated with Ceftazidime-Avibactam: A Retrospective Study.

Purpose: Carbapenem-Resistant Klebsiella pneumoniae (CRKP) is a significant public health threat, because it is associated with substantial morbidity and mortality. However, the risk factors associated with treatment failure of ceftazidime-avibactam (CAZ-AVI) and the need for CAZ-AVI-based combination remain unclear. Methods: We conducted a retrospective study of critically ill patients (age: > 18 years) diagnosed with CRKP infections and treated with CAZ-AVI for at least 24 h between June 2020 and December 2022 at Henan Provincial People's Hospital. Results: This study included a total of 103 patients who received CAZ-AVI. Of these, 91 (88.3%) patients received the standard dosage of 2.5 g every q8h, while only 20 (19.4%) received monotherapy. The Kaplan-Meier curves showed that the all-cause 30-day mortality was significantly higher among patients who experienced septic shock than those who did not. There was no significant difference in mortality between monotherapy and combination therapy. Dose reduction of CAZ-AVI was associated with a significantly increased mortality rate. Independent risk factors for the 30-day mortality included higher APACHE II score (HR: 1.084, 95% CI: 1.024-1.147, p = 0.005) and lower lymphocyte count (HR: 0.247, 95% CI: 0.093-0.655, p = 0.005). Conversely, a combination therapy regimen containing carbapenems was associated with lower mortality (HR: 0.273, 95% CI: 0.086-0.869, p = 0.028). Conclusion: Our study suggests that CAZ-AVI provides clinical benefits in terms of survival and clinical response in critically ill patients with CRKP infection. A higher APACHE II score and lower lymphocyte count were associated with 30-day mortality, while the combination therapy regimen containing carbapenems was the only protective factor. CAZ-AVI dose reduction was associated with an increased mortality rate. Futher large-scale studies are needed to validate these findings.

Insecticidal toxicity of ω-Atypitoxin-Cs1a and its inhibitory effects on insect voltage-gated calcium channels.

BACKGROUND: Excessive use of chemical insecticides raises concerns about insecticide resistance, urging the development of novel insecticides. Peptide neurotoxins from spider venom are an incredibly rich source of ion channel modulators with potent insecticidal activity. A neurotoxin U1-Atypitoxin-Cs1a from the spider Calommata signata was annotated previously. It was of interest to investigate its insecticidal activity and potential molecular targets. RESULTS: Cs1a was heterologously expressed, purified and pharmacologically characterized here. The recombinant neurotoxin inhibited high-voltage-activated calcium channel currents with an median inhibitory concentration (IC50 ) value of 0.182 ± 0.026 µm on cockroach DUM neurons and thus was designated as ω-Atypitoxin-Cs1a. The recombinant Cs1a was toxic to three insect pests of agricultural importance, Nilaparvata lugens, Spodoptera frugiperda and Plutella xylostella with median lethal concentration (LD50 ) values of 0.121, 0.172 and 0.356 nmol g-1 , respectively, at 24 h postinjection. Cs1a was equivalently toxic to both insecticide-susceptible and -resistant insects. Cs1a exhibited low toxicity to Danio rerio with an LD50 of 2.316 nmol g-1 . CONCLUSION: Our results suggest that ω-Atypitoxin-Cs1a is a potent CaV channel inhibitor and an attractive candidate reagent for pest control and resistance management. © 2023 Society of Chemical Industry.

Multiple acetylcholinesterases in Pardosa pseudoannulata brain worked collaboratively to provide protection from organophosphorus insecticides.

Acetylcholinesterase (AChE) is an essential neurotransmitter hydrolase in nervous systems of animals and its number varies among species. So far, five AChEs have been identified in the natural enemy Pardosa pseudoannulata. Here we found that Ppace1, Ppace2 and Ppace5 were highly expressed in the spider brain, among which the mRNA level of Ppace5, but not Ppace1 and Ppace2, could be up-regulated by organophosphorus insecticides at their sublethal concentrations. In spider brain, the treatment by organophosphorus insecticides at the sublethal concentrations could increase total AChE activity, although high concentrations inhibited the activity. The activity that increased from the sublethal concentration pretreatment could compensate for the activity inhibition due to subsequent application of organophosphorus insecticides at lethal concentrations, and consequently reduce the mortality of spiders. PpAChE1 and PpAChE2 were highly sensitive to organophosphorus insecticides, and their activities would be strongly inhibited by the insecticides. In contrast, PpAChE5 displayed relative insensitivity towards organophosphorus insecticides, but with the highest catalytic efficiency for ACh. That meant the up-regulation of Ppace5 under insecticide exposure was important for maintaining AChE activity in spider brain, when PpAChE1 and PpAChE2 were inhibited by organophosphorus insecticides. The study demonstrated that multiple AChEs in the spider brain worked collaboratively, with part members for maintaining AChE activity and other members responding to organophosphorus inhibition, to provide protection from organophosphorus insecticides. In fields, high concentration insecticides are often applied when ineffective controls of insect pests occur due to relative-low concentration of insecticides in last round application. This application pattern of organophosphorus insecticides provides more chances for P. pseudoannulata to survive and controlling insect pests as a natural enemy.

Studies on Virulence and Extended-Spectrum ß-Lactamase-Producing Uropathogenic Escherichia coli Isolates and Therapeutic Effect of Fosfomycin in Acute Pyelonephritis Mice.

The understanding about virulence factors (VFs) and the drug resistance of uropathogenic Escherichia coli (UPEC) helps us understand the pathogenesis of urinary tract infections (UTIs) and make better decisions for clinical treatment. This study examined the correlation between the extended-spectrum ß-lactamases (ESBLs) phenotype and VFs in UPEC strains. In addition, we validated the therapeutic potential of fosfomycin in acute pyelonephritis mice. From May 2017 to November 2018, 22 nonduplicate E coli. strains were isolated from UTI patients. PCR was utilized to detect the distribution of virulence genes. We also analyzed the ESBL phenotype in E coli. We further evaluated the therapeutic effect of intravenous fosfomycin treatment in the acute pyelonephritis (APN) model. All 22 UPEC strains expressed the type 1 fimbriae (FimH) gene and more than 50% (12/22) of strains produced ESBLs. The detection rates of the iron acquisition-associated genes ChuT and IutA were 77.3% (n = 17) and 50% (n = 11) and those of P fimbria papA and papC genes were 45% (n = 10) and 50% (n = 11), respectively. Though the VFs were closely related with pathologenicity, the relationship between VFs and ESBLs still needs further investigation. Furthermore, intravenous fosfomycin 800 mg/kg significantly reduced the bacterial load and the inflammatory infiltration in the bladder and kidney, maintaining the structural integrity of the kidney. Intravenous fosfomycin administration can be used for the treatment of acute pyelonephritis caused by highly pathogenic and drug-resistant UPEC strains.

A validated UHPLC-MS/MS method for simultaneous determination of lumiracoxib and its hydroxylation and acyl glucuronidation metabolites in rat plasma: Application to a pharmacokinetic study.

Lumiracoxib is a selective cyclooxygenase-2 (COX-2) inhibitor. The aim of this study was to develop a simple and sensitive ultra-high performance liquid chromatography tandem mass spectrometric method (UHPLC-MS/MS) for the simultaneous determination of lumiracoxib and its circulating metabolites 4'-Hydroxyl-lumiracoxib and lumiracoxib-acyl-glucuronide in rat plasma. The analytes and diclofenac (internal standard, IS) were extracted using acetonitrile containing 0.2 % formic acid. Chromatographic separation was executed on ACQUITY BEH C18 column (2.1 × 50 mm, 1.7 µm) with water containing 0.2 % formic acid and acetonitrile as mobile phase. Mass detection was achieved in positive multiple reactions monitoring (MRM) mode, with precursor-to-product transitions at m/z 294.1 > 248.1, m/z 310.1 > 264.1, m/z 470.1 > 276.1 and m/z 296.0 > 250.0 for lumiracoxib, 4'-hydroxyl-lumiracoxib, lumiracoxib-acyl-glucuronide and for IS, respectively. The developed LC-MS/MS method was validated based on the guidance of U.S. Food and Drug Administration. The linearity was evident (r > 0.995) over the concentration ranges of 1-1000 ng/mL for lumiracoxib, 1-500 ng/mL for 4'-hydroxyl-lumiracoxib and 1-200 ng/mL for lumiracoxib-acyl-glucuronide, respectively. The precision (RSD) did not exceed 8.23 % and accuracy (RE) ranged from -7.85 % to 9.50 %. The extraction recovery was more than 80.54 %. All the analytes were demonstrated to be stable under the tested storage and processing conditions. The validated LC-MS/MS method has been successfully applied to the pharmacokinetic study of lumiracoxib and its metabolites in the rats after orally administered with lumiracoxib.

Visible-light-mediated photocatalytic cross-coupling of acetenyl ketones with benzyl trifluoroborate.

In this report, we describe a simple visible light-triggered Barbier-type reaction by employing acetenyl ketones with benzyl trifluoroborates. Through a radical-radical cross-coupling process, this photocatalytic protocol furnished a wide range of tertiary propargyl alcohols. Mechanistic investigation indicated that proton-coupled electron transfer (PCET) might be involved in the photochemical transformations.

Simultaneous determination of Guanfu base G and its active metabolites by UPLC-MS/MS in rat plasma and its application to a pharmacokinetic study.

To establish a rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC-MS/MS) method for the determination of concentration of guanfu base G (GFG) and its active metabolites in rat plasma. The GFG and its active metabolites and the internal standard (phenacetin) were separated on an Acquity UPLC(®) BEH C18 chromatography column (2.1mm×50mm I.D., 1.7µm) using gradient elution with a mobile phase of methanol and ultrapure water at a flow rate of 0.4mL/min. The detection was performed on a Xevo triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 472.26→m/z 310.03 for GFG and m/z 180.00→m/z 109.99 for phenacetin (IS) using a positive electrospray ionization interface. The lower limit of quantification (LLOQ) was 1ng/mL, the limit of detection (LOD) was 0.3ng/mL, and the linear calibration curve was obtained over the concentration range of 1-200ng/mL. The intra-day and inter-day assay variations were measured to be below 10.97%, and the accuracy values (relative error) ranged from 95.4% to 103.6%. After validation, this method was successfully applied to a pharmacokinetic study where rats were intravenous administration of 5mg/kg GFG. A simple, rapid, sensitive, and accurate method for the determination of the concentration of GFG and its metabolites in rat plasma was developed and validated.

A simple and sensitive HPLC-UV method for the determination of ponicidin in rat plasma and its application in a pharmacokinetic study.

A simple, rapid, selective and sensitive HPLC-UV method has been developed and validated for the determination of ponicidin in rat plasma. The analyte was extracted from rat plasma by liquid-liquid extraction with ethyl acetate as the extraction solvent. The LC separation was performed on a Zorbax Eclipse XDB C(18) analytical column (150 × 4.6 mm i.d., 5 µm) with an isocratic mobile phase consisting of methanol-water-phosphoric acid (45:55:0.01, v/v/v) at a flow rate of 1.0 mL/min. There was a good linearity over the range of 0.1-25 µg/mL (r = 0.9995) with a weighted (1/C(2) ) least square method. The lower limit of quantification was proved to be 0.1 µg/mL. The accuracy was within ±10.0% in terms of relative error and the intra- and inter-day precisions were less than 9.1% in terms of relative standard deviation. After validation, the method was successfully applied to characterize the pharmacokinetics of ponicidin in rats.