The impact of CYP3A4 genetic polymorphism on crizotinib metabolism and drug-drug interactions.

To elucidate the impact of CYP3A4 activity inhibition and genetic polymorphism on the metabolism of crizotinib. Enzymatic incubation systems for crizotinib were established, and Sprague-Dawley rats were utilized for in vivo experiments. Analytes were quantified using LC-MS/MS. Upon screening 122 drugs and natural compounds, proanthocyanidins emerged as inhibitor of crizotinib metabolism, exhibiting a relative inhibition rate of 93.7%. The IC50 values were 24.53 ± 0.32 µM in rat liver microsomes and 18.24 ± 0.12 µM in human liver microsomes. In vivo studies revealed that proanthocyanidins markedly affected the pharmacokinetic parameters of crizotinib. Co-administration led to a significant reduction in the AUC(0-t), Cmax of PF-06260182 (the primary metabolite of crizotinib), and the urinary metabolic ratio. This interaction is attributed to the mixed-type inhibition of liver microsome activity by proanthocyanidins. CYP3A4, being the principal metabolic enzyme for crizotinib, has its genetic polymorphisms significantly influencing crizotinib's pharmacokinetics. Kinetic data showed that the relative metabolic rates of crizotinib across 26 CYP3A4 variants ranged from 13.14% (CYP3A4.12, 13) to 188.57% (CYP3A4.33) when compared to the wild-type CYP3A4.1. Additionally, the inhibitory effects of proanthocyanidins varied between CYP3A4.12 and CYP3A4.33, when compared to the wild type. Our findings indicate that proanthocyanidins coadministration and CYP3A4 genetic polymorphism can significantly influence crizotinib metabolism.

A modified nucleoside O6-methyl-2'-deoxyguanosine-5'-triphosphate exhibits anti-glioblastoma activity in a caspase-independent manner.

Resistance to temozolomide (TMZ), the frontline chemotherapeutic agent for glioblastoma (GBM), has emerged as a formidable obstacle, underscoring the imperative to identify alternative therapeutic strategies to improve patient outcomes. In this study, we comprehensively evaluated a novel agent, O6-methyl-2'-deoxyguanosine-5'-triphosphate (O6-methyl-dGTP) for its anti-GBM activity both in vitro and in vivo. Notably, O6-methyl-dGTP exhibited pronounced cytotoxicity against GBM cells, including those resistant to TMZ and overexpressing O6-methylguanine-DNA methyltransferase (MGMT). Mechanistic investigations revealed that O6-methyl-dGTP could be incorporated into genomic DNA, disrupting nucleotide pools balance, and inducing replication stress, resulting in S-phase arrest and DNA damage. The compound exerted its anti-tumor properties through the activation of AIF-mediated apoptosis and the parthanatos pathway. In vivo studies using U251 and Ln229 cell xenografts supported the robust tumor-inhibitory capacity of O6-methyl-dGTP. In an orthotopic transplantation model with U87MG cells, O6-methyl-dGTP showcased marginally superior tumor-suppressive activity compared to TMZ. In summary, our research, for the first time, underscores the potential of O6-methyl-dGTP as an effective candidate against GBM, laying a robust scientific groundwork for its potential clinical adoption in GBM treatment regimens.

Using new indices to predict metabolism dysfunction-associated fatty liver disease (MAFLD): analysis of the national health and nutrition examination survey database.

BACKGROUND: Metabolism dysfunction-associated fatty liver disease (MAFLD), is the most common chronic liver disease. Few MAFLD predictions are simple and accurate. We examined the predictive performance of the albumin-to-glutamyl transpeptidase ratio (AGTR), plasma atherogenicity index (AIP), and serum uric acid to high-density lipoprotein cholesterol ratio (UHR) for MAFLD to design practical, inexpensive, and reliable models. METHODS: The National Health and Nutrition Examination Survey (NHANES) 2007-2016 cycle dataset, which contained 12,654 participants, was filtered and randomly separated into internal validation and training sets. This study examined the relationships of the AGTR and AIP with MAFLD using binary multifactor logistic regression. We then created a MAFLD predictive model using the training dataset and validated the predictive model performance with the 2017-2018 NHANES and internal datasets. RESULTS: In the total population, the predictive ability (AUC) of the AIP, AGTR, UHR, and the combination of all three for MAFLD showed in the following order: 0.749, 0.773, 0.728 and 0.824. Further subgroup analysis showed that the AGTR (AUC1 = 0.796; AUC2 = 0.690) and the combination of the three measures (AUC1 = 0.863; AUC2 = 0.766) better predicted MAFLD in nondiabetic patients. Joint prediction outperformed the individual measures in predicting MAFLD in the subgroups. Additionally, the model better predicted female MAFLD. Adding waist circumference and or BMI to this model improves predictive performance. CONCLUSION: Our study showed that the AGTR, AIP, and UHR had strong MAFLD predictive value, and their combination can increase MAFLD predictive performance. They also performed better in females.

Functional evaluation of CYP2C19 and CYP3A4 gene polymorphism on ibuprofen metabolism.

AIM: Ibuprofen 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. METHODS: First, 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. RESULTS: Most 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. CONCLUSION: To 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.

Dectin-1 plays a deleterious role in high fat diet-induced NAFLD of mice through enhancing macrophage activation.

The innate immune response and inflammation contribute to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). Dectin-1 is a pathogen recognition receptor in innate immunity. In this study, we investigated the role of Dectin-1 in the pathogenesis of NAFLD. We first showed that Dectin-1 expression was significantly elevated in liver tissues of patients with NASH. NAFLD was induced in mice by feeding high fat diet (HFD) for 24 weeks. At the end of treatment, mice were sacrificed, and their blood and liver tissues were collected for analyses. We showed HFD feeding also increased liver Dectin-1 levels in mice, associated with macrophage infiltration. Either gene knockout or co-administration of a Dectin-1 antagonist laminarin (150 mg/kg twice a day, ip, from 16th week to 24th week) largely protected the livers from HFD-induced lipid accumulation, fibrosis, and elaboration of inflammatory responses. In primary mouse peritoneal macrophages (MPMs), challenge with palmitate (PA, 200 µM), an abundant saturated fatty acid found in NAFLD, significantly activated Dectin-1 signaling pathway, followed by transcriptionally regulated production of pro-inflammatory cytokines. Dectin-1 was required for hepatic macrophage activation and inflammatory factor induction. Condition media generated from Dectin-1 deficient macrophages failed to cause hepatocyte lipid accumulation and hepatic stellate activation. In conclusion, this study provides the primary evidence supporting a deleterious role for Dectin-1 in NAFLD through enhancing macrophage pro-inflammatory responses and suggests that it can be targeted to prevent inflammatory NAFLD.

Development of a prediction model for the risk of 30-day unplanned readmission in older patients with heart failure: A multicenter retrospective study.

BACKGROUND AND AIM: Heart failure (HF) imposes significant global health costs due to its high incidence, readmission, and mortality rate. Accurate assessment of readmission risk and precise interventions have become important measures to improve health for patients with HF. Therefore, this study aimed to develop a machine learning (ML) model to predict 30-day unplanned readmissions in older patients with HF. METHODS AND RESULTS: This study collected data on hospitalized older patients with HF from the medical data platform of Chongqing Medical University from January 1, 2012, to December 31, 2021. A total of 5 candidate algorithms were selected from 15 ML algorithms with excellent performance, which was evaluated by area under the operating characteristic curve (AUC) and accuracy. Then, the 5 candidate algorithms were hyperparameter tuned by 5-fold cross-validation grid search, and performance was evaluated by AUC, accuracy, sensitivity, specificity, and recall. Finally, an optimal ML model was constructed, and the predictive results were explained using the SHapley Additive exPlanations (SHAP) framework. A total of 14,843 older patients with HF were consecutively enrolled. CatBoost model was selected as the best prediction model, and AUC was 0.732, with 0.712 accuracy, 0.619 sensitivity, and 0.722 specificity. NT.proBNP, length of stay (LOS), triglycerides, blood phosphorus, blood potassium, and lactate dehydrogenase had the greatest effect on 30-day unplanned readmission in older patients with HF, according to SHAP results. CONCLUSIONS: The study developed a CatBoost model to predict the risk of unplanned 30-day special-cause readmission in older patients with HF, which showed more significant performance compared with the traditional logistic regression model.

Ketamine exerts dual effects on the apoptosis of primary cultured hippocampal neurons from fetal rats in vitro.

Ketamine, a noncompetitive N-methyl D-aspartate (NMDA) receptor antagonist, is widely used in pediatric clinical practice. The neuroprotective and neurotoxic effects of ketamine on brain neurons during development remain controversial. The reason may be related to the different concentrations of ketamine used in practice and the small range of concentrations used in previous studies. In this study, cultured hippocampal neurons were treated with ketamine in a wide range of concentrations to comprehensively observe the effects of different concentrations of ketamine on neurons. We demonstrated that low concentrations of ketamine (10 µM, 100 µM and 1000 µM) promoted neuronal survival (p < 0.05) and reduced neuronal apoptosis (p < 0.05) compared with those of the control group. High concentrations of ketamine (2000 µM, 2500 µM and 3000 µM) reduced neuronal survival (p < 0.05) and promoted neuronal apoptosis (p < 0.05). The p38 MAPK inhibitor SB203580 reduced neuronal apoptosis induced by high concentrations of ketamine (2500 µM) (p < 0.05). Our findings indicate that ketamine exerts a dual effect on the apoptosis of primary cultured fetal rat hippocampal neurons in vitro and that the neurotoxic effects of ketamine are related to activation of the p38 MAPK signaling pathway.

Predicting delayed methotrexate elimination in pediatric acute lymphoblastic leukemia patients: an innovative web-based machine learning tool developed through a multicenter, retrospective analysis.

BACKGROUND: High-dose methotrexate (HD-MTX) is a potent chemotherapeutic agent used to treat pediatric acute lymphoblastic leukemia (ALL). HD-MTX is known for cause delayed elimination and drug-related adverse events. Therefore, close monitoring of delayed MTX elimination in ALL patients is essential. OBJECTIVE: This study aimed to identify the risk factors associated with delayed MTX elimination and to develop a predictive tool for its occurrence. METHODS: Patients who received MTX chemotherapy during hospitalization were selected for inclusion in our study. Univariate and least absolute shrinkage and selection operator (LASSO) methods were used to screen for relevant features. Then four machine learning (ML) algorithms were used to construct prediction model in different sampling method. Furthermore, the performance of the model was evaluated using several indicators. Finally, the optimal model was deployed on a web page to create a visual prediction tool. RESULTS: The study included 329 patients with delayed MTX elimination and 1400 patients without delayed MTX elimination who met the inclusion criteria. Univariate and LASSO regression analysis identified eleven predictors, including age, weight, creatinine, uric acid, total bilirubin, albumin, white blood cell count, hemoglobin, prothrombin time, immunological classification, and co-medication with omeprazole. The XGBoost algorithm with SMOTE exhibited AUROC of 0.897, AUPR of 0.729, sensitivity of 0.808, specificity of 0.847, outperforming the other models. And had AUROC of 0.788 in external validation. CONCLUSION: The XGBoost algorithm provides superior performance in predicting the delayed elimination of MTX. We have created a prediction tool to assist medical professionals in predicting MTX metabolic delay.

Pharmacological inhibition of MyD88 suppresses inflammation in tubular epithelial cells and prevents diabetic nephropathy in experimental mice.

Emerging evidence shows that chronic inflammation mediated by toll-like receptors (TLRs) contributes to diabetic nephropathy. Myeloid differentiation primary-response protein-88 (MyD88) is an essential adapter protein of all TLRs except TLR3 in innate immunity. It is unclear whether MyD88 could be a therapeutic target for diabetic nephropathy. Here, we used a new small-molecule MyD88 inhibitor, LM8, to examine the pharmacological inhibition of MyD88 in protecting kidneys from inflammatory injury in diabetes. We showed that MyD88 was significantly activated in the kidney of STZ-induced type 1 diabetic mice in tubular epithelial cells as well as in high glucose-treated rat tubular epithelial cells NRK-52E. In cultured tubular epithelial cells, we show that LM8 (2.5-10 µM) or MyD88 siRNA attenuated high-concentration glucose-induced inflammatory and fibrogenic responses through inhibition of MyD88-TLR4 interaction and downstream NF-κB activation. Treatment with LM8 (5, 10 mg/kg, i.g.) significantly reduced renal inflammation and fibrosis and preserved renal function in both type 1 and type 2 diabetic mice. These renoprotective effects were associated with reduced MyD88-TLR4 complex formation, suppressed NF-κB signaling, and prevention of inflammatory factor expression. Collectively, our results show that hyperglycemia activates MyD88 signaling cascade to induce renal inflammation, fibrosis, and dysfunction. Pharmacological inhibition of MyD88 may be a therapeutic approach to mitigate diabetic nephropathy and the inhibitor LM8 could be a potential candidate for such therapy.

Introducing electrolysis to enhance anaerobic digestion resistance to acidification.

The phenomenon that the anaerobic system is inhibited by acid has always been a bottleneck hindering the application of anaerobic digestion (AD) technology. We tried to introduce electrolysis into AD to improve the acidification resistance, and eventually the productivity of the energy. In a batch fermentation device, the ability of electrochemical anaerobic digestion (EAD) to resist acidification was evaluated in current intensity, electrode potential, AC impedance, microbial community, pH value, and volatile fatty acids (VFAs). The results showed that the average concentration of VFAs in EAD was 32.9% lower than that in AD, the energy efficiency of EAD is 53.25% higher than AD, indicating that EAD has stronger anti-acidification ability and energy conversion efficiency than AD. When the EAD reaches a steady state, the current intensity fluctuates in the range of 7-12 mA, the electrode potential difference is maintained at 600 ± 5 mV, and the internal resistance decreases from 3333.3 ± 16Ω at startup to 68.9 ± 1.4Ω at the steady state, indicating that the EAD has stronger resistance to acidification may be due to the degradation of some VFAs on the electrode surface. Furthermore, the 16S rRNA sequencing analysis showed that the dominant electricity-producing bacteria on EAD anode surface were Clostridium, Hydrogenophaga and Trichloromonas, with a relative abundance of 40.32%, while the relative abundance of electrogenic bacteria in AD bulk solution and EAD bulk solution were about 1/2 and 1/4 that of EAD anode film, suggesting that the electricity-producing bacteria on the electrode surface play an important role in the degradation of VFAs.

Circulating sFasL Levels Predict the Severity and Outcome of Burn Injury: A Prospective Observational Study.

BACKGROUND: Severe burn injury activates shock, inflammation, and blood cell system, but inappropriate reactions may lead to adverse outcomes. Soluble Fas ligand (sFasL) participates in apoptosis and inflammatory response. The circulating sFasL levels we investigated in association with the burn severity, shock, inflammation, blood cells, and mortality in patients with severe burns. METHODS: A total of 56 patients with severe burns were recruited. The levels of sFasL and the biomarkers reflecting shock, organ damage, inflammation, and blood cells at 48 h postburn were analyzed. We compared the practical situation of patients that stratified by median sFasL levels and investigated the predictive value of sFasL for mortality. RESULTS: High circulating sFasL levels were associated with the higher degrees of burn index, shock index, lactate, N-terminal probrain natriuretic peptide, total bilirubin, blood urea nitrogen, creatinine, tumor necrosis factor-α, interleukin-1ß, interleukin-8, intercellular adhesion molecule 1, and complement 3, and the lower degrees of oxygenation index, lymphocytes, and platelets. Multiple linear regression analysis showed that the higher tumor necrosis factor-α (P < 0.001) and the lower oxygenation index (P = 0.031) and lymphocytes (P = 0.043) were associated with the higher sFasL. High sFasL (a unit is 50 ng/L) (odds ratio [OR] 5.50 [95% CI 1.04-29.20], P = 0.045) was an independent predictor of increased mortality by multivariate logistic regression analysis. CONCLUSIONS: High circulating sFasL at 48 h postburn in patients with severe burns reflect shock, proinflammatory response, organ damage, and lymphocyte reductions and predict 30-day mortality.

Recent advances of small molecule fluorescent probes for distinguishing monoamine oxidase-A and monoamine oxidase-B in vitro and in vivo.

Monoamine oxidases (MAO-A and MAO-B) are the two flavin adenine dinucleotide (FAD) enzymes that play an important role in neurotransmitter homeostasis and in protection against biogenic amines. The two MAO enzymes are related to various diseases such as neurological disorders, cancer or other systemic diseases. It is crucial to distinguish these two subtypes in order to explore the pathogenesis and pathophysiology of different diseases. In this review, the relationship between MAOs and related diseases is briefly introduced. Additionally, we summarize the recent advances in small molecule fluorescent probes for specific detection of MAO-A and MAO-B.

Ultra-low NA step-index large mode area Yb-doped fiber with a germanium doped cladding for high power pulse amplification.

High concentration rare earth doped, large mode area (LMA) step-index fibers, which feature a very high cladding absorption per unit length at the pump wavelength, high efficiency, and excellent beam quality, are ideal for high power pulsed fiber lasers/amplifiers where large effective mode areas and short device lengths are crucial in order to reduce detrimental nonlinear effects associated with high peak power operation. In this Letter, we realize low numerical aperture (NA) high absorption fibers, simply by employing a germanium (Ge)-doped cladding rather than a pure silica cladding to offset the high refractive index associated with using a high concentration of ytterbium (Yb) in the core. This approach allows us to separate the two inter-linked fiber design parameters of pump absorption and NA in a step-index fiber. Using a conventional modified chemical vapor deposition process combined with solution doping, a low NA (0.04), LMA (475µm2) silica fiber is fabricated with a cladding absorption value of >20dB/m, which is the highest value among LMA step-index fibers with NA<0.06 so far reported to the best of our knowledge. The fabricated Yb-doped fiber was tested in a high-power picosecond amplifier system and enabled the generation of 190 ps laser pulses with a 101 µJ pulse energy and 0.5 MW peak power at an average power of 150 W.

[Moderated mediation analysis for symptoms of attention deficit/hyperactivity disorder with the symptoms of anxiety in children].

OBJECTIVE: To study the moderated mediation for attention deficit/hyperactivity disorder (ADHD) with the symptoms of anxiety in children. METHODS: A total of 12 271 students were included with an average age of 8.9±1.9 years, including 6 743 male students and 5 508 female students, and 20 students with missing data on gender. Child psychological trauma questionnaires (parents version) and Conners questionnaires (parent version) were completed by the parents of primary school students. The data was studied by univariate analysis, multivariate analysis and moderated mediation analysis. RESULTS: The results of the univariate analysis showed that in all subjects, boys, and girls, the scores of hyperactivity index and childhood trauma were positively correlated with the score of anxiety (P<0.01), and ADHD and childhood trauma positively predicted anxiety disorder (P<0.001). The results of the multivariate analysis showed that in all subjects, boys, and girls, the scores of hyperactivity index (ADHD symptoms) and childhood trauma positively predicted the score of anxiety (P<0.001), and both ADHD and childhood trauma positively predicted anxiety disorder (P<0.001). The results of the moderated mediation analysis showed that childhood trauma was a mediating factor for the relationship between hyperactivity index and anxiety index in boys and girls (P<0.05), and sex moderated the relationship between hyperactivity index and anxiety index (P<0.001). CONCLUSIONS: ADHD symptoms/ADHD are closely associated with anxiety symptoms/anxiety disorder. Childhood trauma exerts a mediating effect on the relationship between ADHD symptoms and anxiety symptoms, and sex moderates the relationship between ADHD symptoms and anxiety symptoms.

Inhibition of miR-148b ameliorates myocardial ischemia/reperfusion injury via regulation of Wnt/ß-catenin signaling pathway.

Our work aims to elucidate the effect how microRNA-148b (miR-148b) participated in myocardial ischemia/reperfusion (I/R) injury via regulation of Wnt/ß-catenin signaling pathway. The in vivo myocardial I/R models of SD rats and in vitro hypoxia/reoxygenation (H/R) models of H9C2 cells were established. The heart function and infarction area of rats and lactic dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), and superoxide dismutase (SOD) levels were evaluated. Myocardial cell viability was measured using positron emission tomography combined with computer tomography and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick-end labeling method and flow cytometry; quantitative reverse-transcription polymerase chain reaction and western blot were used to detect the related molecules expressions. The myocardial infarction area of rats was significantly increased with reductions in LVSP, + dp/dtmax, - dp/dtmax, LVFS%, LVEF% and standardized uptake value and elevation in left ventricular developed pressure after ischemia/reperfusion (I/R), and the LDH, CK, and MDA levels were enhanced with the decreased SOD. The apoptotic rates were higher in I/R rats and H/R H9C2 cells with upregulated miR-148b and cleaved caspase-3, but decreased Bcl-2/Bax ratio; and meanwhile, the Wnt/ß-catenin pathway was inhibited. Additionally, the H/R-induced H9C2 cells also exhibited decreased cell viability. MiR-148b overexpression further aggravated I/R injury of rats, whereas inhibition of miR-148b reduced I/R and H/R injury through activation of Wnt/ß-catenin pathway. In addition, Wnt-1 small interfering RNA exposure abolished the effect of miR-148b inhibitor on H/R injury of H9C2 cells. Inhibition of miR-148b improved the antioxidative ability and myocardial cell survival to suppress its apoptosis by activating Wnt/ß-catenin signaling pathway, thus ameliorating the myocardial I/R injury.

Mechanisms underlying the regulation of intracellular and luminal pH in vaginal epithelium.

The vagina provides a characteristic low-Na+ and low-pH fluid microenvironment that is considered generally protective. Previous studies have shown that various types of epithelial cells harbor the capacity of intracellular pH (pHi) regulation. However, it remains elusive whether vaginal epithelium could actively regulate pHi by transporting acid-base ions. In this study, we verified that after transient exposure to NH4 Cl, the pHi values could rapidly recover from acidification via Na+ -H+ exchanger (NHE), Na+ -HCO3 - cotransporter (NBC), and carbonic anhydrase in human vaginal epithelial cell line VK2/E6E7. Positive expression of the main acid-base transporters including NHE1-2, NBCe1-2, and NBCn1 mRNA was also detected in VK2/E6E7 cells. Moreover, the in vivo study further showed that interfering with the function of V-type H+ -ATPase, NHE or NBC expressed in vagina impaired vaginal luminal pH homeostasis in rats. Taken together, our study reveals the property of pH regulation in vaginal epithelial cells, which might provide novel insights into the potential role of vaginal epithelium in the formation of the vaginal acidic microenvironment.

Genome-Wide Analysis of LIM Family Genes in Foxtail Millet (Setaria italica L.) and Characterization of the Role of SiWLIM2b in Drought Tolerance.

LIM proteins have been found to play important roles in many life activities, including the regulation of gene expression, construction of the cytoskeleton, signal transduction and metabolic regulation. Because of their important roles in many aspects of plant development, LIM genes have been studied in many plant species. However, the LIM gene family has not yet been characterized in foxtail millet. In this study, we analyzed the whole genome of foxtail millet and identified 10 LIM genes. All LIM gene promoters contain MYB and MYC cis-acting elements that are related to drought stress. Based on the presence of multiple abiotic stress-related cis-elements in the promoter of SiWLIM2b, we chose this gene for further study. We analyzed SiWLIM2b expression under abiotic stress and hormone treatments using qRT-PCR. We found that SiWLIM2b was induced by various abiotic stresses and hormones. Under drought conditions, transgenic rice of SiWLIM2b-overexpression had a higher survival rate, higher relative water content and less cell damage than wild type (WT) rice. These results indicate that overexpression of the foxtail millet SiWLIM2b gene enhances drought tolerance in transgenic rice, and the SiWLIM2b gene can potentially be used for molecular breeding of crops with increased resistance to abiotic stress.

Gambogic Acid Efficiently Kills Stem-Like Colorectal Cancer Cells by Upregulating ZFP36 Expression.

BACKGROUND/AIMS: Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been reported to be a potential novel antitumor drug. Whether GA inhibits putative cancer stem cells (CSCs), which are considered to be the major cause of cancer treatment failure, remains largely unknown. This study investigated whether GA inhibits the CSCs of colorectal cancer (CRC) and its possible mechanisms. METHODS: We performed CCK8 and tumor sphere formation assays, percentage analysis of both side population and CD133+CD44+ cells, and the detection of stem cells markers, in order to assess the role of GA in inhibiting the stem celllike features of CRC. An mRNA microarray was performed to identify the downstream gene affected by GA and rescue assays were performed to further clarify whether the downstream gene is involved in the GA induced decrease of the stem cell-like CRC population. CRC cells were engineered with a CSC detector vector encoding GFP and luciferase (Luc) under the control of the Nanog promoter, which were utilized to investigate the effect of GA on putative CSC in human tumor xenograft-bearing mice using in vivo bioluminescence imaging. RESULTS: Our results showed that GA significantly reduced tumor sphere formation and the percentages of side population and CD133+CD44+ cells, while also decreasing the expression of stemness and EMT-associated markers in CRC cells in vitro. GA killed stem-like CRC cells by upregulating the expression of ZFP36, which is dependent on the inactivation of the EGFR/ ERK signaling pathway. GFP+ cells harboring the PNanog-GFP-T2A-Luc transgene exhibited CSC characteristics. The in vivo results showed that GA significantly inhibited tumor growth in nude mice, accompanied by a remarkable reduction in the putative CSC number, based on whole-body bioluminescence imaging. CONCLUSION: These findings suggest that GA significantly inhibits putative CSCs of CRC both in vitro and in vivo by inhibiting the activation of the EGFR/ ERK/ZFP36 signaling pathway and may be an effective drug candidate for anticancer therapies.

PP2A inhibition by LB-100 protects retinal pigment epithelium cells from UV radiation via activation of AMPK signaling.

AMP-activated protein kinase (AMPK) signaling activation can inhibit Ultra-violet (UV) radiation (UVR)-induced retinal pigment epithelium (RPE) cell injuries. LB-100 is a novel inhibitor of protein phosphatase 2A (PP2A), the AMPKα1 phosphatase. Here, our results demonstrated that LB-100 significantly inhibited UVR-induced viability reduction, cell death and apoptosis in established ARPE-19 cells and primary murine RPE cells. LB-100 activated AMPK, nicotinamide adenine dinucleotide phosphate (NADPH) and Nrf2 (NF-E2-related factor 2) signalings, inhibiting UVR-induced oxidative injuries and DNA damage in RPE cells. Conversely, AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A mutation, almost blocked LB-100-induced RPE cytoprotection against UVR. Importantly, CRISPR/Cas9-mediated PP2A knockout mimicked and nullified LB-100-induced anti-UVR activity in RPE cells. Collectively, these results show that PP2A inhibition by LB-100 protects RPE cells from UVR via activation of AMPK signaling.

Validation and implementation of Planova™ BioEX virus filters in the manufacture of a new liquid intravenous immunoglobulin in China.

There is a continuous need to improve the viral safety of plasma products, and we here report the development and optimization of a manufacturing-scale virus removal nanofiltration step for intravenous immunoglobulin (IVIG) using the recently introduced Planova™ BioEX filter. IVIG throughput was examined for various operating parameters: transmembrane pressure, temperature, protein concentration, and prefiltration methods. The developed procedure was based on filtering undiluted process solution (50.0 g/l IVIG) under constant transmembrane pressure filtration at 294 kPa and 25 °C following prefiltration with a 0.1 µm MILLEX VV filter. The recovery of IgG was approximately 98%, and no substantial changes in biochemical characteristics were observed before and after nanofiltration in scaled-up production. A viral clearance validation study with parvovirus under worst-case conditions performed at the National Institutes for Food and Drug Control of China (NIFDC) showed PPV logarithmic reduction value (LRV) > 4. Improved viral safety of IVIG can be assured by implementing a Planova BioEX nanofiltration step to ensure effective parvovirus clearance under conditions providing excellent protein recovery and no detectable impact on product biochemical properties. This plasma-derived IVIG product is the first to be certified for parvovirus safety by the NIFDC in China.