In Situ Formed Microalgae-Integrated Living Hydrogel for Enhanced Tumor Starvation Therapy and Immunotherapy through Photosynthetic Oxygenation.

The effectiveness of various cancer therapies for solid tumors is substantially limited by the highly hypoxic tumor microenvironment (TME). Here, a microalgae-integrated living hydrogel (ACG gel) is developed to concurrently enhance hypoxia-constrained tumor starvation therapy and immunotherapy. The ACG gel is formed in situ following intratumoral injection of a biohybrid fluid composed of alginate, Chlorella sorokiniana, and glucose oxidase, facilitated by the crossing-linking between divalent ions within tumors and alginate. The microalgae Chlorella sorokiniana embedded in ACG gel generate abundant oxygen through photosynthesis, enhancing glucose oxidase-catalyzed glucose consumption and shifting the TME from immunosuppressive to immunopermissive status, thus reducing the tumor cell energy supply and boosting antitumor immunity. In murine 4T1 tumor models, the ACG gel significantly suppresses tumor growth and effectively prevents postoperative tumor recurrence. This study, leveraging microalgae as natural oxygenerators, provides a versatile and universal strategy for the development of oxygen-dependent tumor therapies.

Individualized Delivery of Vancomycin by Model-Informed Bayesian Dosing Approach to Maintain an AUC24 Target in Critically Ill Patients.

INTRODUCTION: Monitoring of AUC24 was updated recommendation in the guideline for the therapeutic drug monitoring (TDM) of vancomycin in Chinese pharmacological society published in 2020. Vancomycin pharmacokinetic profiles are diverse and unique in critically ill patients because of the drastic variability of the patients' physiological parameters, while the study for population pharmacokinetic (PPK) models in Chinese critically ill patients has been rarely reported. The objectives of this study were to construct a PPK model to describe the pharmacokinetic characteristics of vancomycin in critically ill patients and to individualize vancomycin dosing by model-informed Bayesian estimation for maintenance of AUC24 target at 400-650 mg h/L recommended by the 2020 guideline. METHODS: Vancomycin with different dosing was administered intravenously over 1 h for critically ill patients, TDM was started at 48 h or 72 h since initiation of vancomycin therapy for patients. Blood samples were collected from patients for trough concentrations or Cmax. Vancomycin concentrations were determined by high-performance liquid chromatography method with ultraviolet detection. PPK model was performed using the nonlinear mixed-effect model (NONMEM®). Individual PK parameters for critically ill patients treated with vancomycin were estimated using a post hoc empirical Bayesian method based on the final PPK model. AUC24 was calculated as the total daily dose divided by the clearance (L/h). RESULTS: The PPK of vancomycin was determined by a one-compartment model with creatinine clearance as fixed effects. The PK estimates in the final model generally agreed with the median estimates and were contained within the 95% CI generated from the bootstrap results, indicating good precision and stability in the final model. The visual predictive check plots showed the adequate predictive performance of the final PK model and supported a good model fit. The model-informed Bayesian estimation was used to predict the AUC24 of critically ill patient by the acquired TDM results, and the dosing adjustment by maintenance of AUC24 at 400-650 mg h/L had made a great therapeutic effect for the case. CONCLUSION: This study established a PPK model of vancomycin in Chinese critically ill patients, and individualized dosing of vancomycin by model-informed Bayesian estimation to maintain an AUC24 target at 400-650 mg h/L has been successfully applied in clinic. This result supports the continued use of model-informed Bayesian estimation to vancomycin treatment in critically ill patients.

Geometric Remodeling of Nitrilase Active Pocket Based on ALF-Scanning Strategy To Enhance Aromatic Nitrile Substrate Preference and Catalytic Efficiency.

Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency. In this study, we developed an active pocket remodeling (ALF-scanning) based on modulating the geometry of the nitrilase active pocket to alter substrate preference and improve catalytic efficiency. Using this strategy, combined with site-directed saturation mutagenesis, we successfully obtained 4 mutants with strong aromatic nitrile preference and high catalytic activity, W170G, V198L, M197F, and F202M, respectively. To explore the synergistic relationship of these 4 mutations, we constructed 6 double-combination mutants and 4 triple-combination mutants. By combining mutations, we obtained the synergistically enhanced mutant V198L/W170G, which has a significant preference for aromatic nitrile substrates. Compared with the wild type, its specific activities for 4 aromatic nitrile substrates are increased to 11.10-, 12.10-, 26.25-, and 2.55-fold, respectively. By mechanistic dissection, we found that V198L/W170G introduced a stronger substrate-residue π-alkyl interaction in the active pocket and obtained a larger substrate cavity (225.66 Å3 to 307.58 Å3), making aromatic nitrile substrates more accessible to be catalyzed by the active center. Finally, we conducted experiments to rationally design the substrate preference of 3 other nitrilases based on the substrate preference mechanism and also obtained the corresponding aromatic nitrile substrate preference mutants of these three nitrilases and these mutants with greatly improved catalytic efficiency. Notably, the substrate range of SmNit is widened. IMPORTANCE In this study, the active pocket was largely remodeled based on the ALF-scanning strategy we developed. It is believed that ALF-scanning not only could be employed for substrate preference modification but might also play a role in protein engineering of other enzymatic properties, such as substrate region selectivity and substrate spectrum. In addition, the mechanism of aromatic nitrile substrate adaptation we found is widely applicable to other nitrilases in nature. To a large extent, it could provide a theoretical basis for the rational design of other industrial enzymes.

Fusidic Acid and Its Major Active Metabolite Penetration into Cerebrospinal Fluid for Assessing Treatment of Intracranial Infections.

Fusidic acid (FA) had excellent antimicrobial effects due to its unique mechanism of action. Since 1962, FA has been widely used in the systemic and topical treatment of staphylococcal infections and exhibits a well-characterized potency against methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and methicillin-resistant coagulase-negative Staphylococci. In view of the spectrum of activity, no cross-resistance with other clinically used antibiotics, and potential penetration into brain tissue, FA was used to treat possible gra-positive bacteria in 3 patients with intracranial infections in the present report. FA and its active metabolite (3-keto FA) were measured in plasma and cerebrospinal fluid (CSF) to assess the treatment of FA, and the results indicated that 1,500 mg per day of FA was sufficient to achieve therapeutic concentrations in both plasma and CSF in intracranial infection patients, while the dosage did not experience unexpected regimen-related toxicity.

Study on the Thermal Stabilizing Process of Layered Double Hydroxides in PVC Resin.

Poly(vinyl chloride) (PVC) is widely used in various fields and requires the use of thermal stabilizers to enhance its thermal stability during processing because of its poor thermal stability. Layered double hydroxides (LDHs) are widely considered to be one kind of highly efficient and environmentally friendly PVC thermal stabilizer. To investigate the thermal stabilizing process of layered double hydroxides (LDHs) in PVC resin, PVC and MgAl-LDHs powders with different interlayer anions (CO32-, Cl-, and NO3-) were physically mixed and aged at 180 °C. The structure of LDHs at different aging times was studied using XRD, SEM, and FT-IR. The results show that the thermal stabilizing process of LDHs on PVC mainly has three stages. In the first stage, the layers of LDHs undergo a reaction with HCl, which is released during the thermal decomposition of PVC. Subsequently, the ion exchange process occurs between Cl- and interlayer CO32-, resulting in the formation of MgAl-Cl-LDHs. Finally, the layers of MgAl-Cl-LDHs react with HCl slowly. During the thermal stabilizing process of MgAl-Cl-LDHs, the peak intensity of XRD reduces slightly, and no new XRD peak emerges. It indicates that only the first step happens for MgAl-Cl-LDHs. The TG-DTA analysis of LDHs indicates that the interaction of LDHs with different interlayer anions has the following order: NO3- < CO32- < Cl-, according to the early coloring in the thermal aging test of PVC composites. The results of the thermal aging tests suggest that LDHs with a weak interaction between interlayer anions and layers can enhance the early stability of PVC significantly. Furthermore, the thermal aging test demonstrates that LDHs with high HCl absorption capacities exhibit superior long-term stabilizing effects on PVC resin. This finding provides a valuable hint for designing an LDHs/PVC resin with a novel structure and excellent thermal stability.

[Experimental study of cardioprotective effects of Cinnamomi Ramulus and Cinnamomi Cortex formula granules on myocardial ischemia/reperfusion injury in rats based on efficacy of "warming and coordinating heart Yang"].

This study aimed to parallelly investigate the cardioprotective activity of Cinnamomi Ramulus formula granules(CRFG) and Cinnamomi Cortex formula granules(CCFG) against acute myocardial ischemia/reperfusion injury(MI/RI) and the underlying mechanism based on the efficacy of "warming and coordinating the heart Yang". Ninety male SD rats were randomly divided into a sham group, a model group, CRFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, and CCFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, with 15 rats in each group. The sham group and the model group were given equal volumes of normal saline by gavage. Before modeling, the drug was given by gavage once a day for 7 consecutive days. One hour after the last administration, the MI/RI rat model was established by ligating the left anterior descending artery(LAD) for 30 min ischemia followed by 2 h reperfusion except the sham group. The sham group underwent the same procedures without LAD ligation. Heart function, cardiac infarct size, cardiac patho-logy, cardiomyocyte apoptosis, cardiac injury enzymes, and inflammatory cytokines were determined to assess the protective effects of CRFG and CCFG against MI/RI. The gene expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain protein 3(NLRP3) inflammasome, apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate specific proteinase-1(caspase-1), Gasdermin-D(GSDMD), interleukin-1ß(IL-1ß), and interleukin-18(IL-18) were determined by real-time quantitative polymerase chain reaction(RT-PCR). The protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD were determined by Western blot. The results showed that both CRFG and CCFG pretreatments significantly improved cardiac function, decreased the cardiac infarct size, inhibited cardiomyocyte apoptosis, and reduced the content of lactic dehydrogenase(LDH), creatine kinase MB isoenzyme(CK-MB), aspartate transaminase(AST), and cardiac troponin Ⅰ(cTnⅠ). In addition, CRFG and CCFG pretreatments significantly decreased the levels of IL-1ß, IL-6, and tumor necrosis factor-α(TNF-α) in serum. RT-PCR results showed that CRFG and CCFG pretreatment down-regulated the mRNA expression levels of NLRP3, caspase-1, ASC, and downstream pyroptosis-related effector substances including GSDMD, IL-18, and IL-1ß in cardiac tissues. Western blot revealed that CRFG and CCFG pretreatments significantly decreased the protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD in cardiac tissues. In conclusion, CRFG and CCFG pretreatments have obvious cardioprotective effects on MI/RI in rats, and the under-lying mechanism may be related to the inhibition of NLRP3/caspase-1/GSDMD signaling pathway to reduce the cardiac inflammatory response.

Pathway engineering facilitates efficient protein expression in Pichia pastoris.

Pichia pastoris has been recognized as an important platform for the production of various heterologous proteins in recent years. The strong promoter AOX1, induced by methanol, with the help of the α-pre-pro signal sequence, can lead to a high expression level of extracellular protein. However, this combination was not always efficient, as protein secretion in P. pastoris involves numerous procedures mediated by several cellular proteins, including folding assisted by endoplasmic reticulum (ER) molecular chaperones, degradation through ubiquitination, and an efficient vesicular transport system. Efficient protein expression requires the cooperation of various intracellular pathways. This article summarizes the process of protein secretion, modification, and transportation in P. pastoris. In addition, the roles played by the key proteins in these processes and the corresponding co-expression effects are also listed. It is expected to lay the foundation for the industrial protein production of P. pastoris. KEY POINTS: • Mechanisms of chaperones in protein folding and their co-expression effects are summarized. • Protein glycosylation modifications are comprehensively reviewed. • Current dilemmas in the overall protein secretion pathway of Pichia pastoris and corresponding solutions are demonstrated.

Individualized model for predicting COVID-19 deterioration in patients with cancer: A multicenter retrospective study.

The 2019 novel coronavirus has spread rapidly around the world. Cancer patients seem to be more susceptible to infection and disease deterioration, but the factors affecting the deterioration remain unclear. We aimed to develop an individualized model for prediction of coronavirus disease (COVID-19) deterioration in cancer patients. The clinical data of 276 cancer patients diagnosed with COVID-19 in 33 designated hospitals of Hubei, China from December 21, 2019 to March 18, 2020, were collected and randomly divided into a training and a validation cohort by a ratio of 2:1. Cox stepwise regression analysis was carried out to select prognostic factors. The prediction model was developed in the training cohort. The predictive accuracy of the model was quantified by C-index and time-dependent area under the receiver operating characteristic curve (t-AUC). Internal validation was assessed by the validation cohort. Risk stratification based on the model was carried out. Decision curve analysis (DCA) were used to evaluate the clinical usefulness of the model. We found age, cancer type, computed tomography baseline image features (ground glass opacity and consolidation), laboratory findings (lymphocyte count, serum levels of C-reactive protein, aspartate aminotransferase, direct bilirubin, urea, and d-dimer) were significantly associated with symptomatic deterioration. The C-index of the model was 0.755 in the training cohort and 0.779 in the validation cohort. The t-AUC values were above 0.7 within 8 weeks both in the training and validation cohorts. Patients were divided into two risk groups based on the nomogram: low-risk (total points ≤ 9.98) and high-risk (total points > 9.98) group. The Kaplan-Meier deterioration-free survival of COVID-19 curves presented significant discrimination between the two risk groups in both training and validation cohorts. The model indicated good clinical applicability by DCA curves. This study presents an individualized nomogram model to individually predict the possibility of symptomatic deterioration of COVID-19 in patients with cancer.

L-valine production in Corynebacterium glutamicum based on systematic metabolic engineering: progress and prospects.

L-valine is an essential branched-chain amino acid that cannot be synthesized by the human body and has a wide range of applications in food, medicine and feed. Market demand has stimulated people's interest in the industrial production of L-valine. At present, the mutagenized or engineered Corynebacterium glutamicum is an effective microbial cell factory for producing L-valine. Because the biosynthetic pathway and metabolic network of L-valine are intricate and strictly regulated by a variety of key enzymes and genes, highly targeted metabolic engineering can no longer meet the demand for efficient biosynthesis of L-valine. In recent years, the development of omics technology has promoted the upgrading of traditional metabolic engineering to systematic metabolic engineering. This whole-cell-scale transformation strategy has become a productive method for developing L-valine producing strains. This review provides an overview of the biosynthesis and regulation mechanism of L-valine, and summarizes the current metabolic engineering techniques and strategies for constructing L-valine high-producing strains. Finally, the opinion of constructing a cell factory for efficiently biosynthesizing L-valine was proposed.

Enhancing ß-alanine production from glucose in genetically modified Corynebacterium glutamicum by metabolic pathway engineering.

To directly produce ß-alanine from glucose by microbial fermentation, a recombinant Corynebacterium glutamicum strain with high efficiency of ß-alanine production was constructed in this study. To do this, the biosynthetic pathway of ß-alanine in an L-lysine-producing strain XQ-5 was modified by enhancing carbon flux in biosynthetic pathway and limiting carbon flux in competitive pathway. This study showed that replacement of L-aspartate kinase (AK) with wild-type AK and disruption of lactate dehydrogenase and alanine/valine aminotransferases increase ß-alanine production because of decreasing the by-products accumulation. Moreover, L-aspartate-α-decarboxylase (ADC) from Bacillus subtilis was designed as the best enzyme for increasing ß-alanine production, and its variant (BsADCE56S/I88M) showed the highest activity for catalyzing L-aspartate to generate ß-alanine. To further increase ß-alanine production, expression level of BsADCE56S/I88M was controlled by optimizing promoter and RBS, indicating that Pgro plus ThirRBS is the best combination for BsADCE56S/I88M expression and ß-alanine production. The resultant strain XQ-5.5 produced 30.7 ± 2.3 g/L of ß-alanine with a low accumulation of lactate (from 5.2 ± 0.14 to 0.2 ± 0.09 g/L) and L-alanine (from 7.6 ± 0.22 to 3.8 ± 0. 32 g/L) in shake-flask fermentation and produced 56.5 ± 3.2 g/L of ß-alanine with a productivity of 0.79 g/(L·h) and the glucose conversion efficiency (α) of 39.5% in feed-batch fermentation. This is the first report of genetically modifying the biosynthetic pathway of ß-alanine that improves the efficiency of ß-alanine production in an L-lysine-producing strain, and these results give us a new insight for constructing the other valuable biochemical. KEY POINTS: • Optimization and overexpression of the key enzyme BsADC increased the accumulation of ß-alanine. • The AK was replaced with wild-type AK to increase the conversion of aspartic acid to ß-alanine. • A 56.5-g/L ß-alanine production in fed-batch fermentation was achieved.

Reconstruction of the Diaminopimelic Acid Pathway to Promote L-lysine Production in Corynebacterium glutamicum.

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.

[Anti-inflammatory effect and mechanism of ethanol extract from Saposhnikoviae Radix in LPS-induced inflammation mouse model].

This paper aimed to explore the anti-inflammatory effect of ethanol extract from Saposhnikoviae Radix in a lipopolysaccharide(LPS)-induced inflammation mouse model and its regulation of TLR4/NF-κB signaling pathway. The ethanol extract from Saposhnikoviae Radix was separated and purified on the macroporous adsorption resin and its main chemical components were identified by UPLC-QE/MS. The identification results showed that the top ten components of ethanol extract from Saposhnikoviae Radix were mainly chromones and coumarins. A mouse model of inflammation induced by intraperitoneal injection of LPS was used to investigate the anti-inflammatory effects of ethanol extract from Saposhnikoviae Radix after intragastric administration for seven successive days. Mice in all groups except for the control group were treated with intraperitoneal injection of LPS(0.015 g·kg~(-1)) one hour after the last administration, and twelve hours later, the blood was sampled and separated and the broncoalveolar lavage fluid(BALF) was collected. The levels of nitric oxide(NO), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1ß(IL-1ß) in mouse serum and BALF were detected by ELISA. The harvested lung tissue was stained with hematoxylin-eosin(HE) for observing the pathological changes, followed by the detection of protein expression levels of related molecules in TLR4/NF-κB signaling pathway by Western blotting. The results showed that the ethanol extract from Saposhnikoviae Radix significantly ameliorated the pathological conditions in lung tissue of model mice, reversed the increase in NO, TNF-α, IL-6, and IL-1ß levels of mouse serum and BALF, down-regulated the protein expression levels of Toll-like receptor 4(TLR4), myeloid differentiation factor(MyD88), and phosphorylated nuclear transcription factor κB-p65/nuclear transcription factor κB-p65(P-NF-κB p65/NF-κB p65), and up-regulated the NF-κB inhibitory protein α(IκBα). The ethanol extract from Saposhnikoviae Radix exhibited a good anti-inflammatory effect in the LPS-induced acute inflammation muse model, which might be related to the inhibition of the activation of TLR4/NF-κB inflammatory signaling pathway. Chromones and coumarins have been proved to be the active components for its anti-inflammatory effects.

[Mechanism of Jingfang Granules in relieving alcohol and protecting liver based on bioinformatics technology].

The present study explored the potential mechanism of Jingfang Granules in relieving alcohol and protecting liver by network pharmacology and molecular docking and verified the effects and related pathways by animal experiments. The active components of Jingfang Granules were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Targets of drugs and diseases were obtained from PubChem, Swiss Target Prediction and CTD. The common targets were uploaded to STRING to plot the protein-protein interaction(PPI) network. The core targets were screened out and the target organs were identified by Bio GPS and Metascape, followed by Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of common targets. The acute drunk mouse model was established and the effects of Jingfang Granules on serum ethanol level and the expression of proteins related to the phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt) signaling pathway in the liver tissue of mice were observed. A total of 187 active components of Jingfang Granules were obtained, including 47 common targets with alcoholic liver injury. GO enrichment analysis and KEGG pathway analysis showed that Jingfang Granules might play the role of relieving alcohol and protecting liver through the PI3 K-Akt signaling pathway. The drug-component-target and component-target-pathway networks revealed that the important active components of Jingfang Granules in relieving alcohol and protecting liver included quercetin, 5-O-methylvisamminol, glyasperin M, glyasperin B and hederagenin. Molecular docking showed that the active components had a good affinity with AKT1, EGFR, ESR1 and PTGS2. Experimental results showed that Jingfang Granules(15 and 10. 5 g·kg-1) could significantly reduce the content of serum ethanol in mice and up-regulate the protein expression ratios of p-PI3 K/PI3 K and p-Akt/Akt in the liver tissue. Jingfang Granules could relieve alcohol and protect liver through multi-component and multitarget, and the mechanism may be related to the activation of the PI3 K-Akt signaling pathway.

Pyridoxal isonicotinoyl hydrazone inhibition of FXR is involved in the pathogenesis of isoniazid-induced liver injury.

Isoniazid (INH)-induced liver injury may be associated with inhibition of the liver farnesoid X receptor (FXR). However, the relationship between FXR and INH-induced liver injury remained unclear. The present study was performed to clarify the role of inhibition of FXR in the pathogenesis of INH-induced liver injury and to further identify potential inhibitors of FXR from INH and its metabolites. HepaRG cells were treated with INH (10 mM) plus mixed bile acids (BA) and rats were treated with INH (60-600 mg/kg p.o.) or INH plus obeticholic acid (OCA, 10 mg/kg), a potent FXR agonist, for seven days. INH can cause BA-dependent toxicity and apoptosis with elevated intracellular bile acids in vitro; indeed, in these studies, liver bile acids and mRNA levels for Cyp7a1, an FXR target gene were increased, while mRNA levels for FXR and Shp were significantly decreased, and these changes could be prevented by co-treatment with the FXR agonist OCA. In silico molecular docking studies showed that INH, acetyl isoniazid, isonicotinic acid and PIH may be potential FXR inhibitors, and a TR-FRET FXR-coactivator assay confirmed that PIH is a strong antagonist of FXR (IC50 = 52 nM). To further determine if PIH also inhibits FXR activity in vivo, rats were treated with PIH directly (5 mg/kg). Liver total bile acids were significantly increased while FXR expression was not changed, but Shp mRNA levels were significantly decreased and Cyp7a1 mRNA was significantly increased, consistent with PIH acting as an FXR antagonist. In summary, PIH inhibition of liver FXR function leading to bile acid accumulation in hepatocytes may be an early pathogenesis event in INH-induced liver injury.

Determination of Total and Unbound Meropenem, Imipenem/Cilastatin, and Cefoperazone/Sulbactam in Human Plasma: Application for Therapeutic Drug Monitoring in Critically Ill Patients.

BACKGROUND: Critically ill patients show several pathophysiological alterations that can complicate antibiotic dosing. Hence, there is a strong rationale to individualize anti-infective dosing in these patients by using therapeutic drug monitoring (TDM). The current study aimed to develop and validate a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of total and unbound plasma concentrations of 3 commonly used antibiotics (meropenem, imipenem/cilastatin, and cefoperazone/sulbactam) in the treatment of infections in critically ill patients in China, which could be suitable for routine TDM in hospital laboratories. METHODS: The unbound drug was separated from the bound drug by ultrafiltration. Simple protein precipitation was used for sample preparation. Meropenem, imipenem/cilastatin, cefoperazone/sulbactam, and their corresponding internal standards were then resolved using the Waters CORTECS C18 column. All the compounds were detected using electrospray ionization in the positive/negative ion-switching mode. RESULTS: The calibration curves were linear for all compounds, with correlation coefficients (R) above 0.99 for total concentrations in human plasma and unbound concentrations in the plasma ultrafiltrate. For both total and unbound drugs, the relative errors and intra-assay/interassay relative standard deviations were below 15%. The limit of quantification was 0.05 mcg/mL for both total plasma concentrations and plasma ultrafiltrate concentrations of all compounds. CONCLUSIONS: The method was simple, rapid, and reliable and is currently being used to provide a TDM service to enhance the efficacious use of the 3 antibiotics.

Incidence and Nutritional Management of Chylothorax after Surgery for Congenital Heart Diseases in Children.

BACKGROUND: Chylothorax is caused by thoracic lymphatic system injury that leads to lymph extravasates in the thoracic cavity. Cardiac surgery was the most common cause. Reports comparing therapeutic effects between enteral nutrition (EN) with medium-chain triglycerides (MCT) and total parenteral nutrition (TPN) are few and inconsistent. Our study aimed to analyze the incidence of chylothorax in children in our hospital and optimum nutritional management modalities. METHODS: We retrospectively reviewed the medical records of children admitted to our hospital with a diagnosis of chylothorax from 2014 to 2018. We analyzed the incidence of chylothorax, therapeutic effectiveness, and cost effectiveness of EN with MCT or TPN. RESULTS: 136 patients with chylothorax after surgery for congenital heart disease (CHD) were identified from 172 patients with chylothorax (79.07%); chylothorax occurred in 5.62% of all 2420 congenital heart disease surgeries that were performed during that period. Tetralogy of Fallot (TOF), ventricular septal defect (VSD), and double-outlet right ventricle (DORV) were the most common primary diagnoses. Fontan surgery, TOF repair, and VSD repair were the most common primary procedures. We enrolled 45 patients with cured chylothorax. Nutrition support costs in the EN with MCT group (n = 28) were significantly lower than in the TPN group (n = 17) (P = .000). Time to resolution and time to removal of the drainage tube were shorter in EN with MCT versus TPN (P = .003), and the length of hospital stay was shorter (P = .032). There were no significant differences between the 2 groups in time from admission to surgery, postoperative days before diagnosing chylothorax, or length of PICU stay (P > .05). CONCLUSIONS: The therapeutic effects of EN with MCT were significantly better than those of TPN, with lower costs. Therefore, we suggest that EN with MCT be chosen first to treat chylothorax caused by surgery with mild chest drainage volume when gastrointestinal tract function is allowed.

Effect of high uric acid on the disposition of metformin: in vivo and in vitro studies.

Metformin is always used as the baseline antidiabetic therapy for patients with type 2 diabetes mellitus (T2DM) and hyperuricemia. Metformin is excreted into urine through active secretion mediated by rOCTs and rMATE1.The aim of this study was to identify the effects of high uric acid on the disposition and its mechanism. For the in vivo study, a hyperuricemic animal model was induced by intraperitoneal injection of potassium oxonate (250 mg/kg) in rats. Metformin (100 mg/kg) was administered orally to investigate the pharmacokinetics in control and hyperuricemic rats, respectively. For the in vitro study, HEK293 and HepaRG cells were used to investigate the effect of uric acid (15 mg/dl) on the expression of OCT1, OCT2 and MATE1 and the disposition of metformin, respectively. The in vivo study showed that the AUC0 â†’ 600 of metformin was significantly decreased by 33.3%, whereas the cumulative urinary excretion of metformin was increased by 25.4% in hyperuricemic rats compared with that in control rats. The renal rOCT1, rOCT2 and rMATE1 and hepatic rMATE1 levels were increased in hyperuricemic rats compared with those in control rats, respectively. The in vitro study showed that uric acid could upregulate the expression of OCT2 and MATE1 in HEK293 cells and MATE1 in HepaRG cells and increase the intracellular metformin concentration in these two cell lines. These results demonstrated that a high uric acid level promoted urinary metformin excretion and decreased the plasma metformin concentration; the in vivo and in vitro studies provided a possible explanation being that high uric acid could upregulate the expression of renal metformin transporters OCTs and MATE1.

[Effects of Jingfang n-butanol extraction isolated fraction A on LPS-induced inflammation in RAW264.7 cells].

The LPS-induced RAW264. 7 cells inflammation model was used as a carrier to investigate the in vitro anti-inflammation effects of Jingfang n-butanol extraction(JFNE) isolated fraction A and explore its preliminary anti-inflammation mechanism by observing the regulatory effect on PI3 K/AKT signaling pathway and NF-κB pathway. The RAW264. 7 cells inflammation model was established by stimulating with LPS for 12 h. After 3 h pre-treatment with fraction A,the contents of interleukin-6(IL-6),interleukin-1ß(IL-1ß) and tumor necrosis factor(TNF-α) in the supernatant of RAW264. 7 cells inflammation model were determined by ELISA and the contents of NO in supernatant were assayed by Griess. Reverse transcription-polymerase chain reaction(RT-PCR) method was used to determine the expression of IL-6,IL-1ß,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammatory cells,and the expression levels of phosphorylated and total PI3 K/AKT protein,NF-κB p50,p65,p-p65,p105 protein in cells were determined via Western blot. In addition,LC-MS and database were used to identify the possible chemical constituents in fraction A. The results showed that fraction A could significantly reduce the release levels of NO,IL-6,IL-1ß and TNF-α in the supernatant and the expression of IL-6,IL-1ß,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammation model cells(P<0. 05 or P<0. 01) and significantly inhibit the phosphorylation expression levels of PI3 K and AKT protein and mRNA expressions(P<0. 05 or P<0. 01). Moreover,fraction A could significantly reduce the levels of NF-κB p50,p-p65 and i NOS protein,as well as NF-κB1,Rela mRNA expressions in RAW264. 7 cells,and increase the expression of CHUK gene.A total of 196 compounds were identified from fraction A in the composition analysis,and isoobtusilactone,5-O-methyl-vismitol,emebel(embelin) and prim-O-glucosylcimifugin showed high contents. The results all above showed that fraction A had a certain antiinflammatory effect in LPS-induced RAW264. 7 inflammation model cells,and its anti-inflammatory effects may be related to its regulatory effect on the activation of PI3 K/AKT signaling pathway and NF-kappa B signaling pathway. In addition,emblin may be its effective anti-inflammation chemical composition.

[Protective effect and mechanism of Xiaoyao San on lipopolysaccharide-induced hippocampal neurons injury].

To investigate the relationship between anti-depressant effect and hippocampal nerve growth of Xiaoyao San,the inflammatory model of hippocampal neuron was induced by lipopolysaccharide( LPS). The effect of Xiaoyao San serum( final concentration of4%,8%) on the cell proliferation activity was detected by immunofluorescence,the levels of BDNF and ß-NGF in the supernatant of hippocampal neurons were detected by ELISA,and the expressions of BDNF,NGF,Trk B,Trk A and CREB mRNA in cell lysate of hippocampal neuron were detected by PCR. Western blot was used to detect the expressions of Trk B,CREB,p-CREB and SYP protein in cell lysate of hippocampal neuron,and to reveal the neuroprotective effect and mechanism of Xiaoyao San. The results showed that8% Xiaoyao San serum could significantly increase in Brdu/Neu N ratio( P<0. 01). 4%,8% Xiaoyao San serum could significantly improve the levels of BDNF and ß-NGF in supernatant( P<0. 05 or P<0. 01),up-regulate the expression of BDNF,NGF,Trk B,Trk A,CREB mRNA and Trk B,p-CREB,SYP protein in cell lysate( P< 0. 05 or P< 0. 01). 8% Xiaoyao San serum could significantly increase CREB protein in cell lysate( P<0. 05),and elevate in p-CREB/CREB ratio( P<0. 01). All the above results indicate that Xiaoyao San has a certain protective effect on LPS induced hippocampal neuron injury,which suggests that the protective effect of Xiaoyao San is related to the promotion of hippocampal nerve growth,which is one of its antidepressant mechanisms.

Advantages with prophylactic PEG-rhG-CSF versus rhG-CSF in breast cancer patients receiving multiple cycles of myelosuppressive chemotherapy: an open-label, randomized, multicenter phase III study.

BACKGROUND: PEG-rhG-CSF reduces neutropenia and improves chemotherapy safety. In China's registration trial (CFDA: 2006L01305), we assessed its efficacy and safety against rhG-CSF, and prospectively explored its value over multiple cycles of chemotherapy. METHODS: In this open-label, randomized, multicenter phase 3 study, breast cancer patients (n = 569) were randomized to receive PEG-rhG-CSF 100 µg/kg, PEG-rhG-CSF 6 mg, or rhG-CSF 5 µg/kg/d after chemotherapy. The primary endpoints were the incidence and duration of grade 3/4 neutropenia during cycle 1. Secondary endpoints included the incidence and duration of grade 3/4 neutropenia during cycles 2-4, the incidence of febrile neutropenia, and the safety. RESULTS: A once-per-cycle PEG-rhG-CSF at either 100 µg/kg or 6 mg was not different from daily injections of rhG-CSF for either incidence or duration of grade 3/4 neutropenia. Interestingly, a substantial difference was noted during cycle 2, and the difference became bigger over cycles 3-4, reaching a statistical significance at cycle 4 in either incidence (P = 0.0309) or duration (P = 0.0289) favoring PEG-rhG-CSF. A significant trend toward a lower incidence of all-grade adverse events was noted at 129 (68.98%), 142 (75.53%), and 160 (82.47%) in the PEG-rhG-CSF 100 µg/kg and 6 mg and rhG-CSF groups, respectively (P = 0.0085). The corresponding incidence of grade 3/4 drug-related adverse events was 2/187 (1.07%), 1/188 (0.53%), and 8/194 (4.12%), respectively (P = 0.0477). Additionally, PFS in metastatic patients preferred PEG-rhG-CSF to rhG-CSF despite no significance observed by Kaplan-Meier analysis (n = 49, P = 0.153). CONCLUSIONS: PEG-rhG-CSF is a more convenient and safe formulation and a more effective prophylactic measure in breast cancer patients receiving multiple cycles of chemotherapy.