Enantiopure Corral[4]BINOLs as Ultrastrong Receptors for Recognition and Differential Sensing of Steroids.

The precise recognition and sensing of steroids, a type of vital biomolecules, hold immense practical value across various domains. In this study, we introduced corral[4]BINOLs (C[4]BINOLs), a pair of enantiomeric conjugated deep-cavity hosts, as novel synthetic receptors for binding steroids. Due to the strong hydrophobic effect of their deep nonpolar, chiral cavities, the two enantiomers of C[4]BINOLs demonstrated exceptionally high recognition affinities (up to 1012 M-1) for 16 important steroidal compounds as well as good enantioselectiviy (up to 15.5) in aqueous solutions, establishing them as the most potent known steroid receptors. Harnessing their ultrahigh affinity, remarkable enantioselectivity, and fluorescence emission properties, the two C[4]BINOL enantiomers were employed to compose a fluorescent sensor array which achieved discrimination and sensing of 16 structurally similar steroids at low concentrations.

Catalytic Dehydrogenation of Formic Acid Promoted by Triphos-Co Complexes: Two Competing Pathways for H2 Production.

In this study, we reported the synthesis and structural characterization of a triphos-CoII complex [(κ3-triphos)CoII(CH3CN)2]2+ (1) and a triphos-CoI-H complex [(κ2-triphos)HCoI(CO)2] (4). The facile synthetic pathways from 1 to [(κ3-triphos)CoII(κ2-O2CH)]+ (1') and [(κ3-triphos)CoI(CH3CN)]+ (2), respectively, as well as the interconversion between [(κ3-triphos)CoI(CO)2]+ (3) and 4 have been established. The activation energy barrier, associated with the dehydrogenation of a coordinated formate fragment in 1' yielding the corresponding 2 accompanied by the formation of H2 and CO2, was experimentally determined as 23.9 kcal/mol. With 0.01 mol % loading of 1, a maximum TON ∼ 1735 within 18 h and TOF ∼ 483 h-1 for the first 3 h could be achieved. Kinetic isotope effect (KIE) values of 2.25 (kHCOOH/kDCOOH) and 1.36 (kHCOOH/kHCOOD) for the dehydrogenation of formic acid and its deuterated derivatives, respectively, implicate that the H-COOH bond cleavage is likely the rate-determining step. The catalytic mechanism proposed by density functional theory (DFT) calculations coupled with experimental 1H NMR and gas chromatography-mass spectrometry (GC-MS) analysis unveils two competing pathways for H2 production; specifically, deprotonating a HCOO-H bond by a proposed Co-H intermediate C and homolytic cleavage of the CoII-H moiety of C, presumably via a dimeric Co intermediate D containing a [Co2(µ-H)2]2+ core, to yield the corresponding 2 and H2.

Ethnically unique disease burden and limitations of current expanded carrier screening panels.

OBJECTIVES: The purpose of the study is to identify the recessive diseases currently affecting real-world pediatric patients in Taiwan, and whether current extended carrier screening panels have the coverage and detective power to identify the pathogenic variants in the carrier parents. METHODS: A total of 132 trio-samples were collected from May 2017 to March 2022. The participants were parents of pediatric intensive care unit patients who were critically ill or infants with abnormal newborn screening results. A retrospective carrier screening scheme was applied to analyze only the carrier status of pathogenic or likely pathogenic recessive variants resulting in diseases in their children. The recessive disorders diagnosed in our cohort were compared with the gene content in commercial panels. RESULTS: Mutations in COQ4, PEX1, OTC, and IKBKG were the most frequently identified. In the parents of 44 children with confirmed diagnoses of recessive diseases, 47 (53.40%) screened positive for being the carriers of the same recessive disorders diagnosed in their children. The commercial panels covered 35.13% to 54.05% of the disorders diagnosed in this cohort. CONCLUSION: Clinicians and genetic counselors should be aware of the limitations of current extended carrier screening and interpret negative screening results with caution. Future panels should also consider genes with ethnically unique mutations such as pathogenic variants of the COQ4 gene in the East Asian population.

Identification of necroptosis-related lncRNAs for prognosis prediction and screening of potential drugs in patients with colorectal cancer.

BACKGROUND: Tumor recurrence and metastasis lead to a poor prognosis in colorectal cancer (CRC). Necroptosis is closely related to the tumor microenvironment (TME) and affects tumor recurrence and metastasis. We aimed to stratify CRC patients according to necroptosis-related long noncoding RNAs (lncRNAs), which can be used to not only evaluate prognosis and improve precision medicine in clinical practice but also screen potential immunotherapy drugs. AIM: To stratify CRC patients according to necroptosis-related lncRNAs (NRLs), which can be used to not only evaluate prognosis and improve precision medicine in clinical practice but also screen potential immunotherapy drugs. METHODS: LncRNA expression profiles were collected from The Cancer Genome Atlas. NRLs were identified by coexpression analysis. Cox regression analysis identified a NRL signature. Then, the value of this signature was comprehensively and multidimensionally evaluated, and its reliability for CRC prognosis prediction was assessed with clinical CRC data and compared with that of six other lncRNA signatures. Gene set enrichment analysis, TME analysis and half-maximal inhibitory concentration (IC50) prediction were also performed according to the risk score (RS) of the signature. RESULTS: An 8-lncRNA signature significantly associated with overall survival (OS) was constructed, and its reliability was validated with clinical CRC data. Most of the areas under the receiver operating characteristic curves (AUCs) values for 1-, 3- and 5-year OS for this signature were higher than those for the other six lncRNA signatures. OS, disease-specific survival and the progression-free interval were all significantly poorer in the high-risk group. The RS of the signature showed good concordance with the predicted prognosis, with AUCs for 1-, 3- and 5-year OS of 0.79, 0.81 and 0.77, respectively. Additionally, the calibration plots for this signature combined with clinical factors showed that this combination could effectively improve the ability to predict OS. The RS was correlated with tumor stage, lymph node metastasis and distant metastasis. Most of the enriched Kyoto Encyclopedia of Genes and Genomes and Gene Ontology terms were tumor metastasis-related pathways in the high-risk group; these patients showed greater infiltration of immunosuppressive cells, such as cancer-associated fibroblasts, hematopoietic stem cells and M2 macrophages, but less infiltration of infiltrating antitumor effector immune cells, such as cluster of differentiation 8+ T cells and regulatory T cells (Tregs). We explored additional potential immune checkpoint genes and potential immunotherapeutic and chemotherapeutic drugs with relatively low IC50 values. CONCLUSION: We identified an NRL signature with strong fidelity that could stably predict prognosis and might be an indicator of the TME of CRC. Furthermore, additional potential immunotherapeutic and chemotherapeutic drugs were explored.

Curated incidence of lysosomal storage diseases from the Taiwan Biobank.

Lysosomal storage diseases (LSDs) are a group of metabolic disorders resulting from a deficiency in one of the lysosomal hydrolases. Most LSDs are inherited in an autosomal or X-linked recessive manner. As LSDs are rare, their true incidence in Taiwan remains unknown. In this study, we used high-coverage whole-genome sequencing data from 1,495 Taiwanese individuals obtained from the Taiwan Biobank. We found 3826 variants in 71 genes responsible for autosomal recessive LSDs. We first excluded benign variants by allele frequency and other criteria. As a result, 270 variants were considered disease-causing. We curated these variants using published guidelines from the American College of Medical Genetics and Genomics (ACMG). Our results revealed a combined incidence rate of 13 per 100,000 (conservative estimation by pathologic and likely pathogenic variants; 95% CI 6.92-22.23) to 94 per 100,000 (extended estimation by the inclusion of variants of unknown significance; 95% CI 75.96-115.03) among 71 autosomal recessive disease-associated genes. The conservative estimations were similar to those in published clinical data. No disease-causing mutations were found for 18 other diseases; thus, these diseases are likely extremely rare in Taiwan. The study results are important for designing screening and treatment methods for LSDs in Taiwan and demonstrate the importance of mutation curation to avoid overestimating disease incidences from genomic data.

Utility of whole-exome sequencing for patients with multiple congenital anomalies with or without intellectual disability/developmental delay in East Asia population.

BACKGROUND: Congenital anomalies (CAs) with or without intellectual disability (ID)/developmental delay (DD) comprise a heterogeneous spectrum of diseases that affect approximately 3% of live births worldwide. Recently, whole-exome sequencing (WES) demonstrated the highly heterogeneous genetic causes of CAs. The purpose of this study was to evaluate a referral system to increase the yield of WES for CAs. METHODS: From August 2018 to July 2019, patients with CAs, with or without ID/DD, after excluding gross chromosomal aberrations, were referred to geneticists in two medical centers. Variant prioritization was conducted with an AI-assisted tool for whole exomes or a CA-related gene panel. RESULTS: Forty patients (27 males and 13 females) with CAs were enrolled in the study with a mean age of 4.71 years (range, 0.01-18.2). Pathogenic variants in 14 genes were discovered in 16 patients (three patients with CHD7 and 13 patients with one gene each of ATP6V1B2, TAF6, COL4A3BP, ANKH, BMP2, SMARCA4, CUL4B, PGAP3, SOX11, FBN2, PTPN11, SOS1, or PROKR2), with a positive diagnostic rate of 40%. Among the 16 positive cases, 13 (81%) also had ID/DD. The inheritance was autosomal dominant in 13 (81%), autosomal recessive in two (13%), and X-linked in one (6%). Only five patients received a correct clinical diagnosis before WES. The analyses of patients with a negative genetic diagnosis revealed a phenotype and gene mutation load similar to those of the positive-finding patients but with a lower percentage of ID/DD. CONCLUSIONS: The careful selection of patients by experienced geneticists and the exclusion of chromosomal aberrations raises the positive rate of the molecular diagnosis for CAs to 40%. However, more than half of the patients with CAs still do not have a genetic diagnosis by current technologies.

K3V2O3F4(IO3)3: a high-performance SHG crystal containing both five and six-coordinated V5+ cations.

The combination of d0 transition metal oxofluorides with iodate anions helps to synthesize polar crystals. Herein, a novel polar crystal, K3V2O3F4(IO3)3, which is the first metal vanadium iodate with two types of V5+-centered polyhedra (VO4F2 octahedron and VO3F2 trigonal bipyramid), has been prepared hydrothermally. It crystallizes in the polar space group of Cmc21 and its structure displays an unprecedented 0D [V2O3F4(IO3)3]3- anion, which is composed of Λ-shaped cis-[VO2F2(IO3)2]3- and [VO2F2(IO3)]2- anions interconnected via the corner-sharing of one oxo anion. The synergy gained from the VO4F2, VO3F2 and IO3 groups resulted in K3V2O3F4(IO3)3 exhibiting both a strong second-harmonic generation (SHG) response (1.3 × KTiOPO4) under 2050 nm laser irradiation and a large birefringence (0.158 @ 2050 nm). This study provides a facile route for designing SHG materials by assembling various vanadium oxide-fluoride motifs and iodate anions into one compound.

Comparison of GATK and DeepVariant by trio sequencing.

While next-generation sequencing (NGS) has transformed genetic testing, it generates large quantities of noisy data that require a significant amount of bioinformatics to generate useful interpretation. The accuracy of variant calling is therefore critical. Although GATK HaplotypeCaller is a widely used tool for this purpose, newer methods such as DeepVariant have shown higher accuracy in assessments of gold-standard samples for whole-genome sequencing (WGS) and whole-exome sequencing (WES), but a side-by-side comparison on clinical samples has not been performed. Trio WES was used to compare GATK (4.1.2.0) HaplotypeCaller and DeepVariant (v0.8.0). The performance of the two pipelines was evaluated according to the Mendelian error rate, transition-to-transversion (Ti/Tv) ratio, concordance rate, and pathological variant detection rate. Data from 80 trios were analyzed. The Mendelian error rate of the 77 biological trios calculated from the data by DeepVariant (3.09 ± 0.83%) was lower than that calculated from the data by GATK (5.25 ± 0.91%) (p < 0.001). DeepVariant also yielded a higher Ti/Tv ratio (2.38 ± 0.02) than GATK (2.04 ± 0.07) (p < 0.001), suggesting that DeepVariant proportionally called more true positives. The concordance rate between the 2 pipelines was 88.73%. Sixty-three disease-causing variants were detected in the 80 trios. Among them, DeepVariant detected 62 variants, and GATK detected 61 variants. The one variant called by DeepVariant but not GATK HaplotypeCaller might have been missed by GATK HaplotypeCaller due to low coverage. OTC exon 2 (139 bp) deletion was not detected by either method. Mendelian error rate calculation is an effective way to evaluate variant callers. By this method, DeepVariant outperformed GATK, while the two pipelines performed equally in other parameters.

Diagnosis of a Single-Nucleotide Variant in Whole-Exome Sequencing Data for Patients With Inherited Diseases: Machine Learning Study Using Artificial Intelligence Variant Prioritization.

BACKGROUND: In recent years, thanks to the rapid development of next-generation sequencing (NGS) technology, an entire human genome can be sequenced in a short period. As a result, NGS technology is now being widely introduced into clinical diagnosis practice, especially for diagnosis of hereditary disorders. Although the exome data of single-nucleotide variant (SNV) can be generated using these approaches, processing the DNA sequence data of a patient requires multiple tools and complex bioinformatics pipelines. OBJECTIVE: This study aims to assist physicians to automatically interpret the genetic variation information generated by NGS in a short period. To determine the true causal variants of a patient with genetic disease, currently, physicians often need to view numerous features on every variant manually and search for literature in different databases to understand the effect of genetic variation. METHODS: We constructed a machine learning model for predicting disease-causing variants in exome data. We collected sequencing data from whole-exome sequencing (WES) and gene panel as training set, and then integrated variant annotations from multiple genetic databases for model training. The model built ranked SNVs and output the most possible disease-causing candidates. For model testing, we collected WES data from 108 patients with rare genetic disorders in National Taiwan University Hospital. We applied sequencing data and phenotypic information automatically extracted by a keyword extraction tool from patient's electronic medical records into our machine learning model. RESULTS: We succeeded in locating 92.5% (124/134) of the causative variant in the top 10 ranking list among an average of 741 candidate variants per person after filtering. AI Variant Prioritizer was able to assign the target gene to the top rank for around 61.1% (66/108) of the patients, followed by Variant Prioritizer, which assigned it for 44.4% (48/108) of the patients. The cumulative rank result revealed that our AI Variant Prioritizer has the highest accuracy at ranks 1, 5, 10, and 20. It also shows that AI Variant Prioritizer presents better performance than other tools. After adopting the Human Phenotype Ontology (HPO) terms by looking up the databases, the top 10 ranking list can be increased to 93.5% (101/108). CONCLUSIONS: We successfully applied sequencing data from WES and free-text phenotypic information of patient's disease automatically extracted by the keyword extraction tool for model training and testing. By interpreting our model, we identified which features of variants are important. Besides, we achieved a satisfactory result on finding the target variant in our testing data set. After adopting the HPO terms by looking up the databases, the top 10 ranking list can be increased to 93.5% (101/108). The performance of the model is similar to that of manual analysis, and it has been used to help National Taiwan University Hospital with a genetic diagnosis.

A novel deep intronic variant strongly associates with Alkaptonuria.

Alkaptonuria is a rare autosomal recessive inherited disorder of tyrosine metabolism, which causes ochronosis, arthropathy, cardiac valvular calcification, and urolithiasis. The epidemiology of alkaptonuria in East Asia is not clear. In this study, patients diagnosed with alkaptonuria from January 2010 to June 2020 were reviewed. Their clinical and molecular features were further compared with those of patients from other countries. Three patients were found to have alkaptonuria. Mutation analyses of the homogentisate 1,2-dioxygenase gene (HGD) showed four novel variants c.16-2063 A > C, p.(Thr196Ile), p.(Gly344AspfsTer25), and p.(Gly362Arg) in six mutated alleles (83.3%). RNA sequencing revealed that c.16-2063 A > C activates a cryptic exon, causing protein truncation p.(Tyr5_Ile6insValTer17). A literature search identified another 6 patients with alkaptonuria in East Asia; including our cases, 13 of the 18 mutated alleles have not been reported elsewhere in the world. Alkaptonuria is rare in Taiwan and East Asia, with HGD variants being mostly novel and private.

N6-methyladenosine Regulator-Mediated Immune Genes Identify Breast Cancer Immune Subtypes and Predict Immunotherapy Efficacy.

Breast cancer (BRCA) is a heterogeneous malignancy closely related to the tumor microenvironment (TME) cell infiltration. N6-methyladenosine (m6A) modification of mRNA plays a crucial regulator in regulating the immune microenvironment of BRCA. Immunotherapy represents a paradigm shift in BRCA treatment; however, lack of an appropriate approach for treatment evaluation is a significant issue in this field. In this study, we attempted to establish a prognostic signature of BRCA based on m6A-related immune genes and to investigate the potential association between prognosis and immunotherapy. We comprehensively evaluated the m6A modification patterns of BRCA tissues and non-tumor tissues from The Cancer Genome Atlas and the modification patterns with TME cell-infiltrating characteristics. Overall, 1,977 TME-related genes were identified in the literature. Based on LASSO and Cox regression analyses, the m6A-related immune score (m6A-IS) was established to characterize the TME of BRCA and predict prognosis and efficacy associated with immunotherapy. We developed an m6A-IS to effectively predict immune infiltration and the prognosis of patients with BRCA. The prognostic score model represented robust predictive performance in both the training and validation cohorts. The low-m6A-IS group was characterized by enhanced antigen presentation and improved immune checkpoint expression, further indicating sensitivity to immunotherapy. Compared with the patients in the high-score group, the overall survival rate after treatment in the low-score group was significantly higher in the testing and validation cohorts. We constructed an m6A-IS system to examine the ability of the m6A signature to predict the infiltration of immune cells of the TME in BRCA, and the m6A-IS system acted as an independent prognostic biomarker that predicts the response of patients with BRCA in immunotherapy.

Andrographolide mitigates cardiac apoptosis to provide cardio-protection in high-fat-diet-induced obese mice.

Excessive intake of high fat diet (HFD) and associated obese conditions are critical contributors of cardiac diseases. In this study, an active metabolite andrographolide from Andrographis paniculata was found to ameliorate HFD-induced cardiac apoptosis. C57/BL6 mouse were grouped as control (n = 9), obese (n = 8), low dose (25 mg/kg/d) andrographolide treatment (n = 9), and high dose (50 mg/kg/d) andrographolide treatment (n = 9). The control group was provided with standard laboratory chow and the other groups were fed with HFD. Andrographolide was administered through oral gavage for 1 week. Histopathological analysis showed increase in apoptotic nuclei and considerable cardiac-damages in the obese group signifying cardiac remodeling effects. Further, Western blot results showed increase in pro-apoptotic proteins and decrease in the proteins of IGF-1R-survival signaling. However, feeding of andrographolide significantly reduced the cardiac effects of HFD. The results strongly suggest that andrographolide supplementation can be used for prevention and treatment of cardiovascular disease in obese patients.

Cellular apoptosis and cardiac dysfunction in STZ-induced diabetic rats attenuated by anthocyanins via activation of IGFI-R/PI3K/Akt survival signaling.

Anthocyanins are known cyto-protective agents against various stress conditions. In this study cardio-protective effect of anthocyanins from black rice against diabetic mellitus (DM) was evaluated using a streptozotocin (STZ)-induced DM rat model. Five-week-old male Wistar rats were administered with STZ (55 mg kg-1 , IP) to induce DM; rats in the treatment group received 250 mg oral anthocyanin/kg/day during the 4-week treatment period. DM and the control rats received normal saline through oral gavage. The results reveal that STZ-induced DM elevates myocardial apoptosis and associated proapoptotic proteins but down-regulates the proteins of IGF1R mediated survival signaling mechanism. Furthermore, the functional parameters such as the ejection-fraction and fraction-shortening in the DM rat hearts declined considerably. However, the rats treated with anthocyanins significantly reduced apoptosis and the associated proapoptotic proteins and further increased the survival signals to restore the cardiac functions in DM rats. Anthocyanin supplementation enhances cardiomyocyte survival and restores cardiac function.

Andrographis paniculata extract attenuates pathological cardiac hypertrophy and apoptosis in high-fat diet fed mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Andrographis paniculata (Burm. f.) Nees (Acanthaceae) has a considerable medicinal reputation in most parts of Asia as a potent medicine in the treatment of Endocrine disorders, inflammation and hypertension. AIM OF THE STUDY: Water extract of A. paniculata and its active constituent andrographolide are known to possess anti-inflammatory and anti-apoptotic effects. Our aim is to identify whether A. paniculata extract could protect myocardial damage in high-fat diet induced obese mice. MATERIALS AND METHODS: The test mice were divided into three groups fed either with normal chow or with high fat diet (obese) or with high fat diet treated with A. paniculata extract (2g/kg/day, through gavage, for a week). RESULTS: We found that the myocardial inflammation pathway related proteins were increased in the obese mouse which potentially contributes to cardiac hypertrophy and myocardial apoptosis. But feeding with A. paniculata extract showed significant inhibition on the effects of high fat diet. CONCLUSION: Our study strongly suggests that supplementation of A. paniculata extract can be used for prevention and treatment of cardiovascular disease in obese patients.

Purple rice anthocyanin extract protects cardiac function in STZ-induced diabetes rat hearts by inhibiting cardiac hypertrophy and fibrosis.

Diabetes mellitus (DM) often causes chronic inflammation, hypertrophy, apoptosis and fibrosis in the heart and subsequently leads to myocardial remodeling, deteriorated cardiac function and heart failure. Anthocyanins are strong antioxidants that show effective cardioprotective properties. Our aim was to determine whether anthocyanin extracted from purple rice provides protective effects in DM hearts. Five-week-old male Wistar rats were administered with streptozotocin (STZ) to induce type 1 diabetes. Animals were randomly divided into normal group, DM group (induced by 55mg/kg STZ, i.p.) and DM with anthocyanin group (250mg/kg/day, feeding 4 weeks). After treatment, the left ventricular tissues were collected to observe the relevant changes in the heart and the associated molecular events were determined by Western blotting assay. STZ-induced DM increased the proinflammatory signaling proteins in the heart and triggered the development of cardiac hypertrophy and fibrosis. Significant reduction in the heart function index such as left ventricular end-diastolic dimension and left ventricular end-systolic dimension was observed in the STZ-induced DM rat hearts, suggesting myocardial tissue damage and loss of heart function. Treatment with anthocyanin from purple rice extract, however, reduced the effect of DM and showed significant reduction in cardiac hypertrophy and fibrosis. Anthocyanin therefore restores the deteriorating cardiac functions in DM rats as evident from their heart functional parameters.

[A project to reduce the incidence of facial pressure ulcers caused by prolonged surgery with prone positioning].

BACKGROUND & PROBLEMS: We observed in our institute a 13.6% incidence of prolonged surgery (>4 hours) induced facial pressure ulcers that required prone positioning. Causes identified included: (1) customized silicon face pillows used were not suited for every patient; (2) our institute lacked a standard operating procedure for prone positioning; (3) our institute lacked a postoperative evaluation and audit procedure for facial pressure ulcers. PURPOSE: We designed a strategy to reduce post-prolonged surgery facial pressure ulcer incidence requiring prone positioning by 50% (i.e., from 13.6% to 6.8%). RESOLUTIONS: We implemented the following: (1) Created a new water pillow to relieve facial pressure; (2) Implemented continuing education pressure ulcer prevention and evaluation; (3) Established protocols on standard care for prone-position patients and proper facial pressure ulcer identification; (4) Established a face pressure ulcers accident reporting mechanism; and (5) Established an audit mechanism facial pressure ulcer cases. RESULTS: After implementing the resolution measures, 116 patients underwent prolonged surgery in a prone position (mean operating time: 298 mins). None suffered from facial pressure ulcers. The measures effectively reduced the incidence of facial pressure ulcers from 13.6% to 0.0%. CONCLUSIONS: The project used a water pillow to relieve facial pressure and educated staff to recognize and evaluate pressure ulcers. These measures were demonstrated effective in reducing the incidence of facial pressure ulcers caused by prolonged prone positioning.

Interactions between octaarginine and U-937 human macrophages: global gene expression profiling, superoxide anion content, and cytokine production.

Cell penetrating peptides such as octaarginine (R8) have been widely used as intracellular delivery vectors to import biologically active membrane-impermeable molecules. However, before using these peptides clinically, human immune responses to them must be fully understood. Because macrophages are important for immune responses, we evaluated the interactions between R8 and a human U-937 cell line. Cytotoxicity, binding, internalization, genome-wide profiling of gene expression, intracellular superoxide anion content, and cytokine release were assessed after U-937 cells had been incubated with different amounts of R8. Cytotoxicity was limited for up to 40 microM of R8 and 24 h of incubation. Kinetic analysis of the binding and uptake of cells treated with fluorescein-5-isothiocynate-R8 showed time- and concentration-dependent increases. Microarray analysis identified 4386 genes time-dependently regulated when U-937 macrophages were exposed to 10 microM of R8 for 0.5 h and 4 h; the majority of these genes were upregulated for each time point. Thirty-five upregulated genes responded to the stimuli with immune functions, and, using real-time quantitative reverse transcriptase-polymerase chain reaction analysis, five genes - FOS, OSM, C1R, TNF, IL1R1 - were confirmed. R8 induced superoxide anion production after 0.5 h, but not after longer incubations. Incubating U-937 cells with R8 for up to 24 h did not release the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6. In summary, exposing U-937 macrophages to R8 did not induce proinflammatory cytokine release; however, it generated superoxide anion and affected gene expression.

Interactions between U-937 human macrophages and tyloxapol.

Tyloxapol is reported to prevent macrophages from reacting to endotoxin. However, the intracellular responses that tyloxapol induces in macrophages are still not fully explored. Hence, the objective of this study was to evaluate the intracellular events in macrophages treated with tyloxapol and assess the antioxidant properties of tyloxapol in endotoxin-activated macrophages. Using flow cytometry, we examined intracellular responses in macrophages: reactive oxygen species (ROS) content, mitochondria membrane potential, and cell cycle profiles. We also assessed the antioxidant properties of tyloxapol in endotoxin-activated macrophages. Kinetic hydrogen peroxide production tended to decline with increasing doses. Tyloxapol produced a progressive increase followed by a decline in superoxide anion production in macrophages with increasing doses. Tyloxapol also caused unstable fluctuations in mitochondrial membrane potential. Apoptosis had developed at higher doses after 4h of incubation time. After 2h of tyloxapol-pretreatment, tyloxapol acted as an antioxidant only at lower doses. Most tyloxapol-pretreated cells at lower doses fully recovered from the changes in superoxide anion and hydrogen peroxide production. Our findings contribute to a better understanding of the molecular action of tyloxapol in macrophages and how it protects macrophages against endotoxin.

Flow cytometric characterization of interactions between U-937 human macrophages and positively charged catanionic vesicles.

Catanionic vesicles are considered a potential alternative to liposomes for drug delivery systems because of their greater stability and lower cost. Before using catanionic vesicles in vivo, their interactions with macrophages must be fully understood because they are primarily removed from circulation by the macrophages of the mononuclear phagocyte system. Using flow cytometry, we examined the intracellular responses-reactive oxygen species (ROS) content, mitochondrial membrane potential, cell size and complexity, and cell cycle profiles-in U-937 human macrophages treated with positively charged catanionic vesicles. Kinetic hydrogen peroxide production initially increased at lower concentrations (4-10nM) but declined at higher concentrations (40 nM and 80 nM) over the entire incubation period. Superoxide content generation, however, increased over the entire concentration range and incubation period. Catanionic vesicles decreased mitochondrial membrane potential for every concentration after 4h of incubation but caused a significant fluctuation in mitochondrial membrane potential at 6h. After 6h of incubation, catanionic vesicles produced more changes in cell size and complexity than after 4h. The increase in the subG1 population of cells treated with catanionic vesicles at higher doses indicated that apoptosis progressed. Positively charged catanionic vesicles induced different activated patterns of ROS generation and changes in mitochondrial membrane potential than did cationic liposomes. The nature of the interactions between macrophages and catanionic vesicles is of great importance for the design of safer and more effective delivery systems for macrophages. Our findings contribute to a better understanding of the molecular action of catanionic vesicles in the cellular system.

Interactions between U-937 human macrophages and poly(propyleneimine) dendrimers.

Interest in using poly(propyleneimine) (PPI) dendrimers for biomedical applications is increasing. Before using PPI dendrimers in vivo, their interactions with macrophages must be fully understood because they are primarily removed from circulation by the macrophages of the mononuclear phagocyte system. However, few investigators have studied in detail the intracellular responses that cationic dendrimers induce in macrophages. Here we examined the intracellular responses-reactive oxygen species (ROS) content, mitochondria membrane potential, cell size and complexity, and cell cycle profiles-in U-937 human macrophages treated with poly(propyleneimine) dendrimers generation 2 (DAB 2.0) and 3 (DAB 3.0). Our study focused on the concentration ranges within which cell viability was greater than 90% after PPI dendrimers had been incubated for 16 h. For spontaneous ROS generation, DAB 2.0 did not consistently generate hydrogen peroxide production with increasing dosages over the entire culture period while it was capable of generating superoxide content except during the 12 h of incubation. In contrast, DAB 3.0 did not induce any hydrogen peroxide and superoxide production except for an abrupt increase of superoxide content at 60 microg/mL after 6 h of incubation. Our results showed that ROS responses in macrophages were strongly influenced by the nature of the dendrimer surface. Except at 3 h, DAB 2.0 increased mitochondrial membrane potential for every dose and culture period. In contrast, DAB 3.0 caused a significant fluctuation in mitochondrial membrane potential only at 6 h, compared with other incubation times. Exposing macrophages to PPI dendrimers caused dramatic and significant changes in macrophage cell size and complexity, and DAB 3.0 caused greater changes than DAB 2.0 did. For incubation times longer than 1 h, propidium iodide staining showed that cells treated with DAB 2.0 and 3.0 had a higher subG1 phase (indicative of apoptosis) than did untreated cells. PPI dendrimers induced different activated patterns in ROS generation and changes of mitochondrial membrane potential than did other carriers such as cationic liposomes and polyalkylcyanoacrylate. The nature of interactions between macrophages and PPI dendrimers is crucial for the design of safer and more effective delivery systems for macrophages. Our findings provide a novel insight into the cytotoxic effects at the molecular level that dendrimers cause in macrophages.