Incubation System Research Articles

591. Positivity of Blood Culture on Automated Incubation System After 72 Hours and Clinical Significance from the Large United States Tertiary Care Center

Abstract Background Hospitals commonly use automated blood culture incubation systems that provide continuous monitoring for routine blood cultures. Detecting microorganisms early on can help optimize the utilization of antibiotics (ABx) and enable clinicians to de-escalate or discontinue ABx as necessary, making it an important aspect of antimicrobial stewardship. The BD BACTEC Fx, an automated system for blood culture incubation, holds blood culture bottles until growth is demonstrated or for five days unless an extended incubation period is requested by the infectious disease physician. We conducted a clinical evaluation of the significance of positive blood cultures after 72 hours of incubation time. Methods After receiving approval from the institutional review board (IRB), we obtained positive blood cultures from July 2017 to June 2020, which we then stratified based on positivity from day one to day five. To evaluate clinical significance, we retrospectively reviewed charts for positive blood culture results on day four and day five. An Infectious Diseases subspecialty team determined the clinical significance of each case. Results During the time frame of July 2017 to June 2020, there were 120,320 blood cultures taken. Out of these, 7,558 were determined to be positive, with 76 being positive on day 4 and 22 being positive on day 5. Upon further inspection of patients with positive blood cultures, it was discovered that repeat blood cultures continued to be positive due to either untreated sources or a high burden of bacteremia. It was also found that bottles containing fungus, particularly candida glabrata and candida albicans, tested positive on day 4 requiring treatment. Table 1 and Table 2 contain more detailed results. Blood cultures positive on day 4 Blood cultures positive on day 4 and clinical significance Blood cultures positive on day 5 Blood cultures positive on day 5 and clinical significance Conclusion In our study we have found that not all positive blood cultures at 72 hours translate into clinical scenarios requiring antibiotic therapy. In fact, prolonged antimicrobial treatment in such cases may lead to unnecessary exposure to antibiotics and increased risk of antibiotic resistance. It is important to consider the full context of clinical and laboratory findings when interpreting blood culture results, as well as any potential sources of contamination. Disclosures All Authors: No reported disclosures

The variability in CYP3A4 activity determines the metabolic kinetic characteristics of ketamine

Ketamine is a psychotropic drug that can cause significant neurological symptoms and is closely linked to the activity of the CYP3A4 enzyme. This study aimed to examine the diversity of CYP3A4 activity affects the metabolism of ketamine, focusing on genetic variation and drug-induced inhibition. We used a baculovirus-insect cell expression system to prepare recombinant human CYP3A4 microsomes. Then, in vitro enzyme incubation systems were established and used UPLC-MS/MS to detect ketamine metabolite. In rats, we investigated the metabolism of ketamine and its metabolite in the presence of the CYP3A4 inhibitor voriconazole. Molecular docking was used to explore the molecular mechanism of inhibition. The results showed that the catalytic activity of CYP3A4.5, .17, .23, .28, and .29 significantly decreased compared to CYP3A4.1, with a minimum decrease of 3.13%. Meanwhile, the clearance rate of CYP3A4.2, .32, and .34 enhanced remarkably, ranging from 40.63% to 87.50%. Additionally, hepatic microsome incubation experiments revealed that the half-maximal inhibitory concentration (IC50) of voriconazole for ketamine in rat and human liver microsomes were 18.01 ± 1.20 µM and 14.34 ± 1.70 µM, respectively. When voriconazole and ketamine were co-administered, the blood exposure of ketamine and norketamine significantly increased in rats, as indicated by the area under the concentration-time curve (AUC) and maximum concentration (Cmax). The elimination half-life (t1/2Z) of these substances was also prolonged. Moreover, the clearance (CLz/F) of ketamine decreased, while the apparent volume of distribution (Vz/F) increased significantly. This might be attributed to the competition between voriconazole and ketamine for binding sites on the CYP3A4 enzyme. In conclusion, variations in CYP3A4 activity would result in the stratification of ketamine blood exposure.

Conflicting Views About Interactions Between Pancreatic α-Cells and β-Cells.

In type 1 diabetes, the reduced glucagon response to insulin-induced hypoglycemia has been used to argue that β-cell secretion of insulin is required for the full glucagon counterregulatory response. For years, the concept has been that insulin from the β-cell core flows downstream to suppress glucagon secretion from the α-cells in the islet mantle. This core-mantle relationship has been supported by perfused pancreas studies that show marked increases in glucagon secretion when insulin was neutralized with antisera. Additional support comes from a growing number of studies focused on vascular anatomy and blood flow. However, in recent years this core-mantle view has generated less interest than the argument that optimal insulin secretion is due to paracrine release of glucagon from α-cells stimulating adjacent β-cells. This mechanism has been evaluated by knockout of β-cell receptors and impairment of α-cell function by inhibition of Gi designer receptors exclusively activated by designer drugs. Other studies that support this mechanism have been obtained by pharmacological blocking of glucagon-like peptide 1 receptor in humans. While glucagon has potent effects on β-cells, there are concerns with the suggested paracrine mechanism, since some of the supporting data are from isolated islets. The study of islets in static incubation or perifusion systems can be informative, but the normal paracrine relationships are disrupted by the isolation process. While this complicates interpretation of data, arguments supporting paracrine interactions between α-cells and β-cells have growing appeal. We discuss these conflicting views of the relationship between pancreatic α-cells and β-cells and seek to understand how communication depends on blood flow and/or paracrine mechanisms.

The impacts of CYP3A4 genetic polymorphism and drug interactions on the metabolism of lurasidone

The aim of this study was to investigate the impacts of 24 variants of recombinant human CYP3A4 and drug interactions on the metabolism of lurasidone. In vitro, enzymatic reaction incubation system of CYP3A4 was established to determine the kinetic parameters of lurasidone catalyzed by 24 CYP3A4 variants. Then, we constructed rat liver microsomes (RLM) and human liver microsomes (HLM) incubation system to screen potential anti–tumor drugs that could interact with lurasidone and studied its inhibitory mechanism. In vivo, Sprague–Dawley (SD) rats were applied to study the interaction between lurasidone and olmutinib. The concentrations of the analytes were detected by ultra–performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). As the results, we found that compared with the wild-type CYP3A4, the relative intrinsic clearances vary from 355.77 % in CYP3A4.15 to 14.11 % in CYP3A4.12. A series of drugs were screened based on the incubation system, and compared to without olmutinib, the amount of ID–14283 (the metabolite of lurasidone) in RLM and HLM were reduced to 7.22 % and 7.59 %, and its IC50 were 18.83 ± 1.06 μM and 16.15 ± 0.81 μM, respectively. At the same time, it exerted inhibitory effects both through a mixed mechanism. When co-administration of lurasidone with olmutinib in rats, the AUC(0−t) and AUC(0-∞) of lurasidone were significantly increased by 73.52 % and 69.68 %, respectively, while CLz/F was observably decreased by 43.83 %. In conclusion, CYP3A4 genetic polymorphism and olmutinib can remarkably affect the metabolism of lurasidone.

Evaluation of antifungal activities of woody plant barks using an incubation system with Trametes versicolor

Evaluation of antifungal activities of woody plant barks using an incubation system with Trametes versicolor

Impact of Desulfovibrio ferrophilus IS5 biocorrosion time on X80 carbon steel mechanical property degradation

Three different incubation times (7, 14 and 21 d) were used to immerse X80 carbon steel dogbone coupons in deoxygenated enriched artificial seawater at 28 °C inoculated with Desulfovibrio ferrophilus (strain IS5), a very corrosive sulfate reducing bacterium to cause microbiologically influenced corrosion (MIC). It was found that 21 d immersion had the largest weight loss (24.8 mg cm−2 equivalent to 0.55 mm/year corrosion rate), smallest sessile cell count (6.8 × 108 cell/cm2), deepest pit depth (30.6 μm), and most severe mechanical degradation (9 % loss in ultimate strength and 18 % loss in ultimate strain). Due to nutrient depletion in the static incubation system, corrosion rate and sessile cell count were the highest at 0.82 mm/year and 1.1 × 109 cell cm−2, respectively at 7 d. The results from scanning electron microscopy, X-ray photoelectron spectroscopy and confocal laser scanning microscopy analyses and electrochemical corrosion measurements supported the sessile cell counts and biocorrosion weight loss data.

Arsenic methylation and microbial communities in paddy soils under alternating anoxic and oxic conditions

Arsenic (As) methylation in soils affects the environmental behavior of As, excessive accumulation of dimethylarsenate (DMA) in rice plants leads to straighthead disease and a serious drop in crop yield. Understanding the mobility and transformation of methylated arsenic in redox-changing paddy fields is crucial for food security. Here, soils including un-arsenic contaminated (N-As), low-arsenic (L-As), medium-arsenic (M-As), and high-arsenic (H-As) soils were incubated under continuous anoxic, continuous oxic, and consecutive anoxic/oxic treatments respectively, to profile arsenic methylating process and microbial species involved in the As cycle. Under anoxic-oxic (A-O) treatment, methylated arsenic was significantly increased once oxygen was introduced into the incubation system. The methylated arsenic concentrations were up to 2–24 times higher than those in anoxic (A), oxic (O), and oxic-anoxic (O-A) treatments, under which arsenic was methylated slightly and then decreased in all four As concentration soils. In fact, the most plentiful arsenite S-adenosylmethionine methyltransferase genes (arsM) contributed to the increase in As methylation. Proteobacteria (40.8%–62.4%), Firmicutes (3.5%–15.7%), and Desulfobacterota (5.3%–13.3%) were the major microorganisms related to this process. These microbial increased markedly and played more important roles after oxygen was introduced, indicating that they were potential keystone microbial groups for As methylation in the alternating anoxic (flooding) and oxic (drainage) environment. The novel findings provided new insights into the reoxidation-driven arsenic methylation processes and the model could be used for further risk estimation in periodically flooded paddy fields.

No major differences in perinatal and maternal outcomes between uninterrupted embryo culture in time-lapse system and conventional embryo culture.

Is embryo culture in a closed time-lapse system associated with any differences in perinatal and maternal outcomes in comparison to conventional culture and spontaneous conception? There were no significant differences between time-lapse and conventional embryo culture in preterm birth (PTB, <37 weeks), low birth weight (LBW, >2500 g) and hypertensive disorders of pregnancy for singleton deliveries, the primary outcomes of this study. Evidence from prospective trials evaluating the safety of time-lapse incubation for clinical use show similar embryo development rates, implantation rates, and ongoing pregnancy and live birth rates when compared to conventional incubation. Few studies have investigated if uninterrupted culture can alter risks of adverse perinatal outcomes presently associated with IVF when compared to conventional culture and spontaneous conceptions. This study is a Swedish population-based retrospective registry study, including 7379 singleton deliveries after fresh embryo transfer between 2013 and 2018 from selected IVF clinics. Perinatal outcomes of singletons born from time-lapse-cultured embryos were compared to singletons from embryos cultured in conventional incubators and 71 300 singletons from spontaneous conceptions. Main perinatal outcomes included PTB and LBW. Main maternal outcomes included hypertensive disorders of pregnancy (pregnancy hypertension and preeclampsia). From nine IVF clinics, 2683 singletons born after fresh embryo transfer in a time-lapse system were compared to 4696 singletons born after culture in a conventional incubator and 71 300 singletons born after spontaneous conception matched for year of birth, parity, and maternal age. Patient and treatment characteristics from IVF deliveries were cross-linked with the Swedish Medical Birth Register, Register of Birth Defects, National Patient Register and Statistics Sweden. Children born after sperm and oocyte donation cycles and after Preimplantation Genetic testing cycles were excluded. Odds ratio (OR) and adjusted OR were calculated, adjusting for relevant confounders. In the adjusted analyses, no significant differences were found for risk of PTB (adjusted OR 1.11, 95% CI 0.87-1.41) and LBW (adjusted OR 0.86, 95% CI 0.66-1.14) or hypertensive disorders of pregnancy; preeclampsia and hypertension (adjusted OR 0.99, 95% CI 0.67-1.45 and adjusted OR 0.98, 95% CI 0.62-1.53, respectively) between time-lapse and conventional incubation systems. A significantly increased risk of PTB (adjusted OR 1.31, 95% CI 1.08-1.60) and LBW (adjusted OR 1.36, 95% CI 1.08-1.72) was found for singletons born after time-lapse incubation compared to singletons born after spontaneous conceptions. In addition, a lower risk for pregnancy hypertension (adjusted OR 0.72 95% CI 0.53-0.99) but no significant difference for preeclampsia (adjusted OR 0.87, 95% CI 0.68-1.12) was found compared to spontaneous conceptions. Subgroup analyses showed that some risks were related to the day of embryo transfer, with more adverse outcomes after blastocyst transfer in comparison to cleavage stage transfer. This study is retrospective in design and different clinical strategies may have been used to select specific patient groups for time-lapse versus conventional incubation. The number of patients is limited and larger datasets are required to obtain more precise estimates and adjust for possible effect of additional embryo culture variables. Embryo culture in time-lapse systems is not associated with major differences in perinatal and maternal outcomes, compared to conventional embryo culture, suggesting that this technology is an acceptable alternative for embryo incubation. The study was financed by a research grant from Gedeon Richter. There are no conflicts of interest for all authors to declare. N/A.

Effect of Stainless Steel Tank Incubation System on Development of Ovine Embryos

Effect of Stainless Steel Tank Incubation System on Development of Ovine Embryos

Removal of perfluorooctanoic acid (PFOA) in the liquid culture of Phanerochaete chrysosporium

Perfluorooctanoic acid (PFOA) is becoming a concern due to its persistence, bioaccumulation, and potential harmful effects on humans and the environment. In this study, the fungus Phanerochaete chrysosporium (P. chrysosporium) was used to remove the PFOA in liquid culture system. The results showed that the average activities of laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP) enzymes secreted by P. chrysosporium were 0.0003 U/mL, 0.013 U/mL, and 0.0059 U/mL, respectively, during the incubation times of 0–75 days. The pH of 3 and incubation time of 45–55 days were the optimum parameters for the three enzymes activities. The enzyme activities in P. chrysosporium incubation system were firstly inhibited by adding PFOA and then they were enhanced after 14 days. The maximum removal efficiency of PFOA (69.23%) was achieved after 35 days in P. chrysosporium incubation system with an initial PFOA concentration of 0.002 mM and no veratryl alcohol (VA). Adsorption was not a main pathway for PFOA removal and the PFOA adsorbed in fungi mycelial mat accounted for merely 1.91%. The possible products of PFOA contained partially fluorinated aldehyde, alcohol, and aromatic ring. These partially fluorinated compounds might result from PFOA degradation via a combination of cross-coupling and rearrangement of free radicals.

Mechanism of extracellular electron transport and reactive oxygen mediated Sb(III) oxidation by Klebsiella aerogenes HC10

Microbial oxidation and the mechanism of Sb(III) are key governing elements in biogeochemical cycling. A novel Sb oxidizing bacterium, Klebsiella aerogenes HC10, was attracted early and revealed that extracellular metabolites were the main fractions driving Sb oxidation. However, linkages between the extracellular metabolite driven Sb oxidation process and mechanism remain elusive. Here, model phenolic and quinone compounds, i.e., anthraquinone-2,6-disulfonate (AQDS) and hydroquinone (HYD), representing extracellular oxidants secreted by K. aerogenes HC10, were chosen to further study the Sb(III) oxidation mechanism. N2 purging and free radical quenching showed that oxygen-induced oxidation accounted for 36.78% of Sb(III) in the metabolite reaction system, while hydroxyl free radicals (·OH) accounted for 15.52%. ·OH and H2O2 are the main driving factors for Sb oxidation. Radical quenching, methanol purification and electron paramagnetic resonance (EPR) analysis revealed that ·OH, superoxide radical (O2•−) and semiquinone (SQ−•) were reactive intermediates of the phenolic induced oxidation process. Phenolic-induced ROS are one of the main oxidants in metabolites. Cyclic voltammetry (CV) showed that electron transfer of quinone also mediated Sb(III) oxidation. Part of Sb(V) was scavenged by the formation of the secondary Sb(V)-bearing mineral mopungite [NaSb(OH)6] in the incubation system. Our study demonstrates the microbial role of oxidation detoxification and mineralization of Sb and provides scientific references for the biochemical remediation of Sb-contaminated soil.

Mitigation of soil N2O emissions by decomposed straw based on changes in dissolved organic matter and denitrifying bacteria

The return of decomposed straw represents a less explored potential option for reducing N2O emissions. However, the mechanisms underlying the effects of decomposed straw return on soil N2O mitigation are still not fully clear. Therefore, we used a helium atmosphere robotized continuous flow incubation system to compare the soil N2O and N2 emissions from four treatments: CK (control: no straw), WS (wheat straw), IWS (wheat straw decomposed with Irpex lacteus), and PWS (wheat straw decomposed with Phanerochaete chrysosporium). All the treatments have been fertilized with the same amount of KNO3. Furthermore, we also analyzed i) the chemodiversity of soil dissolved organic matter (DOM), ii) the nirS, nirK, and nosZ gene copies and relative abundances of denitrifying bacterial communities (DBCs), and iii) the specific linkages between N2O emissions and DOM and DBC. The results showed that the WS, IWS and PWS treatments increased N2O emissions compared to the CK treatment. However, applying decomposed straw to soil, especially straw treated with P. chrysosporium, effectively decreased the soil N2O and increased N2 emissions compared to WS and IWS. Moreover, the IWS and PWS treatments increased the CHO composition, but they decreased the CHON and CHOS compositions of heteroatomic compounds of DOM compared with the WS and CK treatments. Furthermore, the WS, IWS and PWS treatments all significantly increased the nirS and nosZ gene copies compared with the CK treatment. Additionally, compared with the other treatments, the PWS treatment significantly shaped the DBC and led to a higher relative abundance of Pseudomonas with nirS and nosZ genes. Meanwhile, Network analysis showed that the mitigation of N2O was closely related to particular DOM molecules, and specific DBC taxa. These results highlight the potential for decomposed straw amendments to mitigate of soil N2O emissions not only by changing soil DOM but also mediating the soil DBC.

Inhibitory mechanism of vortioxetine on CYP450 enzymes in human and rat liver microsomes.

Vortioxetine is a novel anti-major depression disorder drug with a high safety profile compared with other similar drugs. However, little research has been done on drug-drug interactions (DDI) about vortioxetine. In this paper, the inhibitory effect of vortioxetine on cytochrome P450 (CYP450) and the type of inhibitory mechanism were investigated in human and rat liver microsomes. We set up an in vitro incubation system of 200μL to measure the metabolism of probe substrates at the present of vortioxetine at 37°C. The concentrations of the metabolites of probe substrates were all measured by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. It was found no time-dependent inhibition (TDI) of vortioxetine through determination of half-maximal inhibitory concentration (IC50) shift values. The enzymes and metabolites involved in this experiment in human and rats were as follows: CYP3A4/CYP3A (midazolam); CYP2B6/CYP2B (bupropion); CYP2D6/CYP2D (dextromethorphan); CYP2C8/CYP2C-1 (amodiaquine); CYP2C9/CYP2C-2 (losartan); and CYP2C19/CYP2C-3 (mephenytoin). We found that vortioxetine competitively inhibited CYP2C19 and CYP2D6 in human liver microsomes (HLMs) with inhibition constant (Ki) values of 2.17μM and 9.37μM, respectively. It was noncompetitive inhibition for CYP3A4 and CYP2C8, and its Ki values were 7.26μM and 6.96μM, respectively. For CYP2B6 and CYP2C9, vortioxetine exhibited the mixed inhibition with Ki values were 8.55μM and 4.17μM, respectively. In RLMs, the type of vortioxetine inhibition was uncompetitive for CYP3A and CYP2D (Ki = 4.41 and 100.9μM). The inhibition type was competitive inhibition, including CYP2B and CYP2C-2 (Ki = 2.87 and 0.12μM). The inhibition types of CYP2C-1 and CYP2C-3 (Ki = 39.91 and 4.23μM) were mixed inhibition and noncompetitive inhibition, respectively. The study of the above mechanism will provide guidance for the safe clinical use of vortioxetine so that the occurrence of DDI can be avoided.

An in vitro evaluation of kratom (Mitragyna speciosa) on the catalytic activity of carboxylesterase 1 (CES1)

Kratom, (Mitragyna Speciosa Korth.) is a plant indigenous to Southeast Asia whose leaves are cultivated for a variety of medicinal purposes and mostly consumed as powders or tea in the United States. Kratom use has surged in popularity with the lay public and is currently being investigated for possible therapeutic benefits including as a treatment for opioid withdrawal due to the pharmacologic effects of its indole alkaloids. A wide array of psychoactive compounds are found in kratom, with mitragynine being the most abundant alkaloid. The drug-drug interaction (DDI) potential of mitragynine and related alkaloids have been evaluated for effects on the major cytochrome P450s (CYPs) via in vitro assays and limited clinical investigations. However, no thorough assessment of their potential to inhibit the major hepatic hydrolase, carboxylesterase 1 (CES1), exists. The purpose of this study was to evaluate the in vitro inhibitory potential of kratom extracts and its individual major alkaloids using an established CES1 assay and incubation system. Three separate kratom extracts and the major kratom alkaloids mitragynine, speciogynine, speciociliatine, paynantheine, and corynantheidine displayed a concentration-dependent reversible inhibition of CES1. The experimental Ki values were determined as follows for mitragynine, speciociliatine, paynantheine, and corynantheidine: 20.6, 8.6, 26.1, and 12.5 μM respectively. Speciociliatine, paynantheine, and corynantheidine were all determined to be mixed-type reversible inhibitors of CES1, while mitragynine was a purely competitive inhibitor. Based on available pharmacokinetic data, determined Ki values, and a physiologically based inhibition screen mimicking alkaloid exposures in humans, a DDI mediated via CES1 inhibition appears unlikely across a spectrum of doses (i.e., 2–20g per dose). However, further clinical studies need to be conducted to exclude the possibility of a DDI at higher and extreme doses of kratom and those who are chronic users.

Contrasting effects of dissimilatory Fe(III)/As(V) reduction on arsenic mobilization of Al coprecipitated ferrihydrite in simulated groundwater

Arsenic pollution caused by dissimilatory As(V)/Fe(III)-reducing microorganisms in oxygen-starved sediments has aroused widespread concern. During the alternate redox cycles, precipitation and dissolution of iron (oxyhydr)-oxides often introduce various impurities, among which aluminum (Al) is the most prevalent one. Therefore, it is of great significance to devote some of our attention to the arsenic contamination induced by the bio-reduction of aluminum‑iron (oxyhydr)-oxides. In this study, we synthesized aluminum-ferrihydrite (AlFh) with Al/(Al + Fe) mol% of 0, 10, 20 and 30 successfully. After discussing the adsorption mechanism of arsenate by these materials, the biotic experiment with the presence of Shewanella oneidensis MR–1 (MR-1) or As(V) reducing bacteria Citrobacter sp. JH012–1 (JH012–1) were conducted. Our results suggested that the coprecipitated of Al inhibited the bio-reduction of Fe(III). In MR–1 incubation systems, due to the impaired Fe(III) reduction, As(V) release in the initial stage of Al coprecipitate Fh was inhibited. The released As(V) was then immobilized into the secondary mineral phases. In JH012–1 incubation systems the incorporated aluminum result in a higher degree of As dissolution and As(III) accounts for a relatively high proportion in the liquid phase. Spectroscopic analysis demonstrated that with the increase of Al content in AlFh, the relative proportions of surface As(III) were 63.53%, 58.60%, 54.91% and 50.35% respectively. The enhanced release of As in JH012–1 experiment was mainly ascribed to the poor adsorption capacity of As(III) on AlFh. These findings will help to complement the bio-migration behavior of As in natural aluminum‑iron (oxyhydr)-oxides.

Developmental toxicity in early chicken embryos on exposure to an organophosphorus flame retardant, tris(2-chloroisopropyl) phosphate

Tris(2-chloroisopropyl) phosphate (TCIPP) is an organophosphate flame retardant detected in the environment and eggs, feathers, and livers. Early-developmental-stage avian embryos are vulnerable to the toxic effects of chemicals. However, studies on the specific effects of TCIPP on avian embryonic development are limited. We aimed to investigate the toxicity of TCIPP in early chicken embryos using a previously developed shell-less incubation system. Fertilized chicken (Gallus gallus domesticus) eggs (n = 220) were exposed to 50 or 500 nmol TCIPP/(g egg) or dimethyl sulfoxide (DMSO) as a vehicle control on Day 0 of incubation. Development of 198 embryos was monitored from Days 3–9 of incubation, and 22 embryos on Day 4 and 74 embryos on Day 9 were dissected. Messenger RNA expression levels for several genes were measured in embryos on Day 4. Both TCIPP-exposed groups showed a significant reduction in survival rate. Imaging analyses revealed significant decreases in body length, head and bill length, eye diameter, and forelimb and hindlimb length in both TCIPP-treated groups. TCIPP exposure significantly impaired the development of extraembryonic blood vessels and the production of red blood cells. A TCIPP-dose-dependent decreasing trend in heart rate was observed on Days 4–7. The somitic angle increased significantly on Days 4–6, and embryos with curved somites showed cleavage in the back and gaps between somites, resulting in asymmetrical somite formation. A significant correlation was found between the somitic angle and FGF8 expression levels, suggesting that TCIPP exposure affects somite formation through an altered FGF-signaling pathway. Embryos with somitic deformities in TCIPP-exposed groups had significantly reduced survival rates, indicating that abnormal segment formation directly increased mortality. Finally, eye weight and ocular luminosity values were significantly reduced, suggesting that TCIPP may also affect eye development. Overall, these findings highlight severe toxic effects of TCIPP on avian embryonic development, including in vascularization, cardiac function, and somite and ocular development.

Promotion effects of salt-alkali on ammonia volatilization in a coastal soil

Coastal ecosystems are highly susceptible to salt-related problems due to their formation process and geographical location. As such ecosystems are the most accessible land resources on Earth, clarifying and quantifying the effects of salt-alkali conditions on N concentration and ammonia (NH3) volatilization are pivotal for promoting coastal agricultural productivity. The challenge in establishing this effect is to determine how salt-alkali conditions impact NH3 volatilization through direct or indirect interactions. An incubation experiment using a coastal soil from a paddy farmland, combined with the structural equation modeling (SEM) method, was conducted to reveal the net effects of salt-alkali on NH3 volatilization and the role of environmental and microbial factors in mutual interaction networks. The specific experimental design consisted of four salt treatments (S1, S2, S3, and S4: 1‰, 3‰, 8‰, and 15‰ NaCl by mass of soil, respectively), four alkaline treatments (A1, A2, A3, and A4: 0.5‰, 1‰, 3‰, and 8‰ NaHCO3 by mass of soil, respectively) and a control without NaCl or NaHCO3 addition (CK), and each treatment had three urea concentrations (N1, N2, and N3: 0.05, 0.10, and 0.15 g N kg-1 soil, respectively) and three replicates. At the N1, N2, and N3 levels, NH3 volatilization increased by 9.31%–34.98%, 3.07%–26.92%, and 2.99%–43.61% as the NaCl concentration increased from 1‰ to 15‰, respectively, compared with CK. With an increase in the NaHCO3 concentration from 0.5‰ to 8‰, NH3 volatilization increased by 8.36%–56.46%, 5.49%–30.10%, and 30.72%–73.18% at the N1, N2, and N3 levels, respectively, compared with CK. According to the SEM method, salinity and alkalinity had positive direct effects on NH3 volatilization, with standardized path coefficients of 0.40 and 0.19, respectively. Considering the total effects (net positive and negative effects) in the SEM results, alkalinity had a greater influence than salinity (total standardized coefficient 0.104 > 0.086). Nitrogen concentrations in the incubation system showed a direct positive effect on NH3 volatilization (standardized path coefficient = 0.78), with an obvious decrease under elevated salinity and alkalinity levels. Additionally, gene abundances of nitrogen-transforming microbes indirectly increased NH3 volatilization (total indirect standardized coefficient = 0.31). Our results indicated that potential NH3 emissions from coastal saline areas could be enhanced more by soil alkalization than by salinization.

Functional evaluation of CYP2C19 and CYP3A4 gene polymorphism on ibuprofen metabolism

AimIbuprofen is the most commonly used analgesic. CYP polymorphisms are mainly responsible for the differences in drug metabolism among individuals. Variations in the ability of populations to metabolize ibuprofen can lead to drug exposure events. The aim of this study was to evaluate the effects of CYP2C19 and CYP3A4 polymorphisms on ibuprofen metabolism in a Chinese population. MethodsFirst, 31 CYP2C19 and 12 CYP3A4 microsomal enzymes were identified using an insect expression system. Then, variants were evaluated using a mature incubation system. Moreover, ibuprofen metabolite content was determined via ultra-performance liquid chromatography-tandem mass spectrometry analysis. Finally, kinetic parameters of CYP2C19 and CYP3A4 genotypes were determined via Michaelis-Menten curve fitting. ResultsMost variants exhibited significantly altered intrinsic clearance compared to the wild type. In the CYP2C19 metabolic pathway, seven variants exhibited no significant alterations in intrinsic clearance (CLint), six variants exhibited significantly high CLint (121–291%), and the remaining 15 variants exhibited substantially reduced CLint (1–71%). In the CYP3A4 metabolic pathway, CYP3A4*30 was not detected in the metabolite content due to the absence of activity, and 10 variants exhibited significantly reduced CLint. ConclusionTo the best of our knowledge, this is the first study to assess the kinetic characteristics of 31 CYP2C19 and 12 CYP3A4 genotypes on ibuprofen metabolism. However, further studies are needed on poor metabolizers as they are more susceptible to drug exposure. Our findings suggest that the kinetic characteristics in combination with artificial intelligence to predict the toxicity of ibuprofen and reduce any adverse drug reactions.

Advanced in vitro hemocompatibility assessment of biomaterials using a new flow incubation system

Physiologically relevant in vitro hemocompatibility assessment of biomaterials remains challenging. We present a new setup that enables standardized whole blood incubation of biomedical materials under flow. A blood volume of 2 mL is recirculated over test surfaces in a custom-made parallel plate incubation system to determine the activation of hemostasis and inflammation. Controlled physiological shear rates between 125 s−1 and 1250 s−1 and minimized contact to air are combined with a natural-like pumping process. A unique feature of this setup allows tracing adhesion of blood cells to test surfaces microscopically in situ. Validation testing was performed in comparison to previously applied whole blood incubation methodologies. Experiments with the newly developed setup showed that even small obstacles to blood flow activate blood (independent of materials-induced blood activation levels); that adhesion of blood cells to biomaterials equilibrates within 5 to 10 min; that high shear rates (1250 compared to 375 s−1) induce platelet activation; and that hemolysis, platelet factor 4 (PF4) release and platelet loss - but not thrombin formation – depend on shear rate (within the range investigated, 125 to 1250 s−1).

Elucidation of the relationship between evodiamine-induced liver injury and CYP3A4-mediated metabolic activation by UPLC-MS/MS analysis.

Evodiamine (EVD), which has been reported to cause liver damage, is the main constituent of Evodia rutaecarpa (Juss.) Benth and may be bioactivated into reactive metabolites mediated by cytochrome P450. However, the relationships between bioactivation and EVD-induced hepatotoxicity remain unknown. In this study, comprehensive hepatotoxicity evaluation was explored, which demonstrated that EVD caused hepatotoxicity in both time- and dose-dependent manners in mice. By application of UPLC-Q/TOF-MS/MS, two GSH conjugates (GM1 and GM2) derived from reactive metabolites of EVD were identified, in microsomal incubation systems exposed to EVD with glutathione (GSH) as trapping agents. CYP3A4 was proved to be the main metabolic enzyme. Correspondingly, the N-acetyl-L-cysteine conjugate derived from the degradation of GM2 was detected in the urine of mice after exposure to EVD. For the first time, the iminoquinone intermediate was found in EVD-pretreated rat bile by the high-resolution MS platform. Pretreatment with ketoconazole protected the animals from hepatotoxicity, decreased the protein expression of cleaved caspase-1 and -3, but increased the area under the serum-concentration-time curve of EVD in blood determined by UPLC-QQQ-MS/MS. Depletion of GSH by buthionine sulfoximine exacerbated EVD-induced hepatotoxicity. These results implicated that the CYP3A4-mediated metabolic activation was responsible for the observed hepatotoxicity induced by EVD.