24-h Culture Research Articles

Comparative proteomic analysis reveals the regulatory network of the veA gene during asexual and sexual spore development of Aspergillus cristatus.

Aspergillus cristatus is the predominant fungal population during fermentation of Chinese Fuzhuan brick tea, and belongs to the homothallic fungal group that undergoes a sexual stage without asexual conidiation under hypotonic conditions, while hypertonic medium induces initiation of the asexual stage and completely blocks sexual development. However, the veA deletion mutant only produces conidia in hypotonic medium after a 24-h culture, but both asexual and sexual spores are observed after 72 h. The veA gene is one of the key genes that positively regulates sexual and negatively regulates asexual development in A. cristatus. To elucidate the molecular mechanism of how VeA regulates asexual and sexual spore development in A. cristatus, 2D electrophoresis (2-DE) combined with MALDI-tandem ToF MS analysis were applied to identify 173 differentially expressed proteins (DEPs) by comparing the agamotype (24 h) and teleomorph (72 h) with wild-type (WT) A. cristatus strains. Further analysis revealed that the changed expression pattern of Pmk1-MAPK and Ser/Thr phosphatase signaling, heat shock protein (Hsp) 90 (HSP90), protein degradation associated, sulphur-containing amino acid biosynthesis associated, valine, leucine, isoleucine, and arginine biosynthesis involved, CYP450 and cytoskeletal formation associated proteins were involved in the production of conidia in agamotype of A. cristatus. Furthermore, the deletion of veA in A. cristatus resulted in disturbed process of transcription, translation, protein folding, amino acid metabolism, and secondary metabolism. The carbohydrate and energy metabolism were also greatly changed, which lied in the suppression of anabolism through pentose phosphate pathway (PPP) but promotion of catabolism through glycolysis and tricarboxylic acid (TCA) cycle. The energy compounds produced in the agamotype were mainly ATP and NADH, whereas they were NADPH and FAD in the teleomorph. These results will contribute to the existing knowledge on the complex role of VeA in the regulation of spore development in Aspergillus and provide a framework for functional investigations on the identified proteins.

A colonized millet grain method for Agrobacterium-mediated transformation of the button mushroom Agaricus bisporus

Lack of an efficient transformation system has hampered the molecular breeding of Agaricus bisporus. Here, we describe a highly efficient Agrobacterium-mediated transformation system for A. bisporus using foxtail millet (Setaria italica L. Beauv) grains as cultivation and infection media. Mycelium-millet complexes were prepared for co-culture and treated with ultrasonication for 10 s to improve infection efficiency. After a 72-h culture period, the newly grown mycelium-surrounded millet grain was transferred to selection medium supplemented with 200 μg/mL cefotaxime and 15 μg/mL hygromycin B (hyg) to screen positive transformants. Putative transformants were analyzed for the presence of the hyg gene by polymerase chain reaction and Southern blotting. Expression of eGFP in A. bisporus transformants was detected by fluorescence imaging, and the β-glucuronidase (GUS) protein was detected by histochemical staining. Our protocol resulted in an average 53.85% transformation frequency, and over 85% of the transformants tested remained mitotically stable, even after five successive rounds of subculturing. A feasible method for A. bisporus mushroom transformation using foxtail millet as an innovative culture medium was developed, which will benefit future functional genetic studies of this mushroom.

Effects of insulin-like growth factor-1 on endoplasmic reticulum stress and autophagy in rat gastric smooth muscle cells cultured at different glucose concentrations in vitro.

The purpose of the study was to observe changes in endoplasmic reticulum stress (ERS)- and autophagy-related proteins in gastric smooth muscle tissues of diabetic rats with gastroparesis, investigate the effect of insulin-like growth factor 1 (IGF-1) on ERS and autophagy in rat gastric smooth muscle cells cultured under different glucose concentrations, and explore the influence of IGF-1 on development of diabetic gastroparesis (DGP). After establishing a rat model of DGP, rats were divided into normal control (NC) and 6-week diabetic model (DM6W) groups. Expression of ERS-related and autophagy-related proteins was detected by western blot analysis and immunofluorescence assay in rat gastric smooth muscle tissue and in vitro-cultured rat gastric smooth muscle cells exposed to different glucose concentrations and treatment with IGF-1 for 24 or 48h. Changes in glucose-regulated-protein-78 (GRP78), growth arrest and DNA damage-inducible gene 153 (CHOP), and microtubule-associated protein 1A/1B light chain 3B (LC3) expression levels were detected by western blot analysis, and GRP78 and LC3 expression were examined by confocal laser-scanning microscopy. In vivo expression levels of GRP78, CHOP, and LC3 were significantly higher in the DM6W group compared with the NC group (p < 0.001). Twenty-four hours after cells were cultured at different glucose concentrations in vitro, expression of GRP78, CHOP, and LC3II/Iwas significantly higher in the high glucose-treated group compared with the normal glucose group (p < 0.05). After IGF-1 intervention, CHOP and GRP78 expression were significantly higher in the normal glucose + IGF-1 group compared with the normal glucose group (p < 0.01), while no significant difference was found between high glucose and high glucose + IGF-1 groups. LC3II/Iexpression was significantly lower in the normal glucose + IGF-1 group compared with the normal glucose group, and was significantly lower in the high glucose and high glucose + IGF-1 groups (p < 0.05). After 48h of culture, CHOP expression was significantly higher and LC3II/I expression was significantly lower in the high glucose group compared with the normal glucose group (p < 0.05), but no significant change in GRP78 expression was observed between these two groups. After IGF-1 intervention, there was no difference in CHOP or GRP78 expression between normal glucose + IGF-1 and normal glucose groups. However, CHOP and GRP78 expression were significantly lower in the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). There was no significant difference in LC3II/I expression between normal glucose + IGF-1 and normal glucose groups, or high glucose + IGF-1 and high glucose groups. Results of confocal laser-scanning microscopy showed significantly lower expression of LC3II/Iin the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). ERS and autophagy were involved in the occurrence of DGP. IGF-1 exerted an inhibitory effect on ERS in rat gastric smooth muscle cells cultured under high glucose conditions, and this inhibitory effect increased with time. IGF-1 inhibited the level of autophagy in rat gastric smooth muscle cells cultured under high glucose conditions at early stages, which may be achieved through inhibition of ERS.

Abstract O-44: ORGANIZATIONAL CHARACTERISTICS AND RESOURCES IN LATIN-AMERICAN PEDIATRIC INTENSIVE CARE UNITS. PRELIMINARY REPORT OF REAL-CIP (REALIDAD EN AMERICA LATINA DE CUIDADOS INTENSIVOS PEDIÁTRICOS) STUDY.

Aims & Objectives: To describe organizational characteristics and resources of PICUs in Latin-America. Methods Web-based survey submitted to Latin-American PICUs. Results 50 out 98 centers responded the survey from 10 countries (38% Chile, 20% Uruguay, 20% Argentina). Centers characteristics: 66% public, 74% mixed [intermediate (IMCU) and intensive care (PICU)], 76% academic, 24% have critical care training program. PICU beds median 7(6,12) and IMCU 6(5,10). Physician/patient ratio was 0.3(0.2,0.5) and 0.25(0.16,0.4) for PICU and IMCU (p<0.05 for difference between centers). 24h intensivist presence was full in 56%, but <50% in 32% centers. Nurse/patient ratio was 0.5 (0.33,0.5) and 0.33 (0.2,0.5) for PICU and IMCU; nurse technician(clinical-assistant)/patient ratio 0.33 (0.25,0.5) (p<0.05). 37% centers had respiratory therapist dedicated to ICU, 54% available during night shift. Clinical protocols use for cardiac arrest 94%, Sepsis 80%, MV 78%, weaning 66%. All centers have invasive and non-invasive MV, HFNC was available in 78%. Scores use for pain56%, sedation62%, withdrawal42% and delirium12%. Severity scores use: PIM2/3 70%, PRISM 26%, PELOD 24%. Basic monitoring was available in all centers; advanced monitoring availability: cardiac output 26%, PAC 6%, FHM 32%, NIRS 22% and volumetric capnography 50%. Rescue therapies available: iNO50%, surfactant60%, TRRC60%, ECMO16%, TTM48%. 24h blood chemistry, Blood cultures and CRP were available in all centers, but lactate only in 94% and procalcitonin 64%. Conclusions This survey highlights substantial variation in critical care organization and resources in Latin-American PICUs. These disparities must be considered for guidelines and collaborative interventional studies. On future research, is the impact of these differences on clinical outcomes will be studied.

Response of human normal and leukemia cells to factors released by amnion fragments in vitro.

Amnion is a membrane surrounding the embryo/fetus which determine growth factors and interleukins with angiogenic, immunogenic, and anti-inflammatory properties. The aim of the present study was to investigate the effects of conditioned culture medium from 24-h cultures of human amnion (hAM CCM) on migration and proliferation of human umbilical vein endothelial primary cells (HUVECs), freshly isolated bone marrow mononuclear cells (BM MNCs), and Jurkat leukemia cell line. Amnion membrane was freshly isolated from healthy placenta and its fragments cultured in vitro to produce hAM CCM. Members of the IGFBP protein family made up one third of all assayed proteins present in the hAM medium. The hAM CCM did not affect the proliferation rate of HUVECs or MNCs, but we observed more intensive migration of those cells, and lower expression of CD31 surface antigen on HUVECs as compared to control cultures. In contrast, Jurkat cells did not respond to hAM CCM treatment by proliferation or mobility change. The conditioned medium from 24-h cultures of human amnion is easy to obtain and is a convenient source of various growth and other factors that may be useful in practical medicine.

Modulation of large dense core vesicle insulin content mediates rhythmic hormone release from pancreatic beta cells over the 24h cycle.

The rhythmic nature of insulin secretion over the 24h cycle in pancreatic islets has been mostly investigated using transcriptomics studies showing that modulation of insulin secretion over this cycle is achieved via distal stages of insulin secretion. We set out to measure β-cell exocytosis using in depth cell physiology techniques at several time points. In agreement with the activity and feeding pattern of nocturnal rodents, we find that C57/Bl6J islets in culture for 24h exhibit higher insulin secretion during the corresponding dark phase than in the light phase (Zeitgeber Time ZT20 and ZT8, respectively, in vivo). Glucose-induced insulin secretion is increased by 21% despite normal intracellular Ca2+ transients and depolarization-evoked exocytosis, as measured by whole-cell capacitance measurements. This paradox is explained by a 1.37-fold increase in beta cell insulin content. Ultramorphological analyses show that vesicle size and density are unaltered, demonstrating that intravesicular insulin content per granule is modulated over the 24h cycle. Proinsulin levels did not change between ZT8 and ZT20. Islet glucagon content was inversely proportional to insulin content indicating that this unique feature is likely to support a physiological role. Microarray data identified the differential expression of 301 transcripts, of which 26 are miRNAs and 54 are known genes (including C2cd4b, a gene previously involved in insulin processing, and clock genes such as Bmal1 and Rev-erbα). Mouse β-cell secretion over the full course of the 24h cycle may rely on several distinct cellular functions but late night increase in insulin secretion depends solely on granule insulin content.

Induction of PIR-A/B+ DCs in the in vitro inflammatory condition and their immunoregulatory function.

Dendritic cells (DCs), primary antigen-presenting cells, are now well known as an immunoregulator of many aspects of immune responses including inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis. We have reported that PIR-A/Bhigh cDCs (conventional DCs) appeared in dextran sodium sulfate (DSS)-induced colitis and serve as a negative immunoregulator in an animal model of IBD. The immunoregulatory role of PIR-A/B+ cDCs was confirmed in both an in vitro culture system and an in vivo transfer experiment. Here, we have investigated the differentiation process of PIR-A/B+ cDCs in an in vitro inflammatory environment and examined their functions. cDCs were isolated from the large intestinal lamina propria from C57BL/6 mice and cultured in an inflammatory environment (IL-1, IL-6, TNFα, and LPS). The appearance of PIR-A/B+ cDCs was determined after 24h, and the in vitro-induced PIR-A/B+ cDCs were functionally and genetically examined. PIR-A/B+ cDCs were detected after a 24-h culture only in the inflammatory environment, and the cells acted as a negative immunoregulator when examined in an allogenic mixed leukocyte reaction (MLR). The message level of IL-27 was highly upregulated in PIR-A/B+ cDCs, while that of high mobility group box 1 protein (HMGB1) was downregulated in these cells. This was well in accordance with the fact that PIR-A/B+ cDCs showed a suppressive function against activated T cells. We found that PIR-A/B+ cDCs produced IL-27, as verified by an ELISA assay, and that the inhibitory effect by PIR-A/B+ cDCs was, at least partially, due to IL-27. Furthermore, CD85d+ cells, a human counterpart of mouse PIR-A/B+ cDCs, were found in the lamina propria of the colon of the patients with ulcerative colitis, but not in the similar part of the non-inflammatory area of colon specimens from patients with colon cancer. PIR-A/B+ cDCs induced in an in vitro inflammatory environment model showed a suppressive function against activated T cells by producing an inhibitory cytokine.

Modulation of gingival cell response towards dental composites

ObjectiveThis aim of this study was to investigate the cell behavioural response on clinically applied dental composites in exposition-relevant target cells, i.e. human gingival fibroblasts (HGF) and epithelial keratinocytes (HGK). MethodsHGF and HGK were exposed to eluates of Ceram X™, Filtek™ Silorane, Filtek™ Supreme XTE, Fusio™ Liquid Dentin and Vertise™ Flow. Eluates were created by storing material samples in respective cell culture medium, for 24h and 72h (n=17), according to ISO 10993-12:2012. Cell response was evaluated at eluate exposure periods of 24h and 72h by (i) impedance analysis-based real-time monitoring of adhesion and proliferation, (ii) semi-quantitative indirect immunofluorescence (sq-IIF) detection of tissue-specific biomarkers, and (iii) ELISA-detection of pro-inflammatory interleukin (IL)-6. ResultsGenerally, cell behavioural response towards the eluates was gradual in HGK and HGF, the latter exhibiting a less pronounced modulation per se. In HGK, ERK 1/2 was mainly activated after 24h by Fusio™ Liquid Dentin and Vertise™ Flow, while an increase in biomarker expression occurred time-delayed. A 72h exposure of HGK to eluates of Filtek™ Supreme XTE, Fusio™ Liquid Dentin and Vertise™ Flow significantly decreased secreted IL-6 amounts. In HGK, the impedance analysis revealed less proliferation and/or adhesion in case of Fusio™ Liquid Dentin and Vertise™ Flow with matched other composites. SignificanceIn detail, protein expression and secretion is modulated particularly in terms of signal transduction, differentiation and inflammation. On cell biological level, all tested materials modulated the analysed features of cell behaviour with emphasis on the self-adhering composites.

IMPROVEMENT OF BUFFALO EMBRYO DEVELOPMENT IN VITRO; EFFECT OF DIFFERENT CULTURE MEDIA

The aim of the present study to compare 4 different types of media, SOF, TCM-119, CR1aa and KSOM, for in vitro culture of buffalo embryo. This study was under taken in the Department of Artificial insemination and embryo transfer, Animal Reproduction Research Institute (ARRI), Al-Haram, Giza .The selec&#116;ed cumulus oocyte complexes(COCs) with a compact cumulus cells and evenly granulated ooplasm were in vitro matured at 38.5°C in atmosphere of 5% CO2 with high humidity .After 24h of culture, oocyte were used for in vitro fertilization . Following 18h of insemination, oocyte were randomly assigned to investigate the effect of different types of media from the zygote to day 7 post-insemination . there were no significant difference in cleavage rate among 4 media However, the morula and blastocyst stage increased significantly(P<0.05) in using SOF (26.03, 17.88% respectively) and also by using CR1aa (25.37, 16.42% respectively) compared with TCM (11.86, 5.08% respectively) and KSOM (9.62, 3.85 %respectively).There is no significant difference between SOF and CR1aa.In conclusion, the current data reveled that SOF is the best media used in embryo development in buffalo. Key words: SOF. In vitro culture media. Buffalo

Water-Based Suspensions of Iron Oxide Nanoparticles with Electrostatic or Steric Stabilization by Chitosan: Fabrication, Characterization and Biocompatibility.

Present day biomedical applications, including magnetic biosensing, demand better understanding of the interactions between living systems and magnetic nanoparticles (MNPs). In this work spherical MNPs of maghemite were obtained by a highly productive laser target evaporation technique. XRD analysis confirmed the inverse spinel structure of the MNPs (space group Fd-3m). The ensemble obeyed a lognormal size distribution with the median value 26.8 nm and dispersion 0.362. Stabilized water-based suspensions were fabricated using electrostatic or steric stabilization by the natural polymer chitosan. The encapsulation of the MNPs by chitosan makes them resistant to the unfavorable factors for colloidal stability typically present in physiological conditions such as pH and high ionic force. Controlled amounts of suspensions were used for in vitro experiments with human blood mononuclear leukocytes (HBMLs) in order to study their morphofunctional response. For sake of comparison the results obtained in the present study were analyzed together with our previous results of the study of similar suspensions with human mesenchymal stem cells. Suspensions with and without chitosan enhanced the secretion of cytokines by a 24-h culture of HBMLs compared to a control without MNPs. At a dose of 2.3, the MTD of chitosan promotes the stimulating effect of MNPs on cells. In the dose range of MNPs 10–1000 MTD, chitosan “inhibits” cellular secretory activity compared to MNPs without chitosan. Both suspensions did not caused cell death by necrosis, hence, the secretion of cytokines is due to the enhancement of the functional activity of HBMLs. Increased accumulation of MNP with chitosan in the cell fraction at 100 MTD for 24 h exposure, may be due to fixation of chitosan on the outer membrane of HBMLs. The discussed results can be used for an addressed design of cell delivery/removal incorporating multiple activities because of cell capability to avoid phagocytosis by immune cells. They are also promising for the field of biosensor development for the detection of magnetic labels.

Quantifying the Effects of Different Neutral Proteases on Human Islet Integrity

Efficient islet release from the pancreas requires the combination of collagenase, neutral protease (cNP), or thermolysin (TL). Recently, it has been shown that clostripain (CP) may also contribute to efficient islet release from the human pancreas. The aim of this study was to evaluate the impact of these proteases on human islet integrity in a prospective approach. Islets were isolated from the pancreas of 10 brain-dead human organ donors. Purified islets were precultured for 3 to 4 d at 37 °C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 °C in Hank’s balanced salt solution supplemented with cNP, TL, or CP. The protease concentrations were calculated for a pancreas of 100 g trimmed weight utilizing 120 dimethyl-casein units of cNP, 70,000 caseinase units of TL, or 200 benzoyl-l-arginine-ethyl-ester units of CP (1×). These activities were then increased both 5× and 10×. After subsequent 24-h culture in enzyme-free culture medium, treated islets were assessed and normalized to sham-treated controls. Compared with controls and CP, islet yield was significantly reduced by using the 5× activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1× activity of cNP, 5× activity of TL, or 10× activity of CP. Although mitochondrial function was significantly lowered by 1× cNP and 5× TL, CP did not affect mitochondria at any concentration. cNP- and TL-incubated islets significantly lost intracellular insulin already at 1× activity, while the 10× activity of CP had to be added to observe a similar effect. cNP and TL have a similar toxic potency regarding islet integrity. CP also induces adverse effects on islets, but the toxic threshold is generally higher. We hypothesize that CP can serve as supplementary protease to minimize cNP or TL activity for efficient pancreas digestion.

In vitro antagonistic and indifferent activity of combination of 3-deoxyanthocyanidins against Toxoplasma gondii.

Toxoplasma gondii is a ubiquitous intracellular zoonotic parasite estimated to affect about 30-90% of the world's human population. The most affected are immunocompromised individuals such as HIV-AIDS and cancer patients, organ and tissue transplant recipients, and congenitally infected children. No effective and safe drugs and vaccines are available against all forms of the parasite. We report here the antagonistic and indifferent activity of the combination of five different formulations of pure synthetic 3-deoxyanthocyaninidin (3-DA) chloride compounds against T. gondii tachyzoites and the synergistic and additive interaction against a human foreskin fibroblast (HFF) cell line in vitro using fluorescence microscopy, trypan blue assay, and fractional inhibitory concentration index. The individual and the combined pure 3-DA compounds were observed to have effective inhibition against T. gondii parasites with less cytotoxic effect in a ratio of 1:1. The IC50 values for parasite inhibition ranged from 1.88μg/mL (1.51-2.32μg/mL) for luteolinindin plus 7-methoxyapigeninindin (LU/7-MAP) and 2.23μg/mL (1.66-2.97μg/mL) for apigeninindin plus 7-methoxyapigeninindin (AP/7-MAP) combinations at 95% confidence interval (CI) after 48h of culture. We found LU/7-MAP to be antagonistic and AP/7-MAP to be indifferent in interaction against T. gondii growth. Both individual and combination 3-DA compounds not only depicted very strong inhibitory activity against T. gondii, but also had synergistic and additive cytotoxic effects against HFF cells. These synthetic 3-DAs have potential as antiparasitic agents for the treatment of human toxoplasmosis.

Polyhydroxyalkanoate accumulation ability and associated microbial community in activated sludge-derived acetate-fed microbial cultures enriched under different temperature and pH conditions

The influence of temperature and pH during enrichment on the polyhydroxyalkanoate (PHA) accumulation ability and composition of PHA-accumulating microorganisms (PHAAMOs) in enrichment cultures was investigated. Enrichment of PHAAMOs from activated sludge was conducted in acetate-fed sequencing batch reactors using a feast-famine regime under different temperature (20°C, 28°C, and 36°C) and pH (controlled at 7.2 or not) conditions. PHA accumulation ability, which was evaluated in nitrogen- and phosphorus-deficient 24-h single-batch cultures, was greatly enhanced by enrichment, irrespective of the temperature and pH. Enrichment at 20°C or 28°C and without pH control seemed most appropriate for strong PHA accumulation. Analyses of the PHAAMO composition by the clone library method targeting phaC genes, which encode the class I and II PHA synthases, revealed that Burkholderiales were the dominant PHAAMOs in the seed sludge, while Rhodocyclales, specifically Azoarcus spp. and Thauera spp., were dominant after enrichment without pH control, showing a strong ability to accumulate PHA. The results indicated that Azoarcus spp. and Thauera spp. are key PHAAMOs in an enrichment culture based on the feast-famine method, with high PHA accumulation ability.

Delayed Activation Kinetics of Th2- and Th17 Cells Compared to Th1 Cells.

During immune responses, different classes of T cells arise: Th1, Th2, and Th17. Mobilizing the right class plays a critical role in successful host defense and therefore defining the ratios of Th1/Th2/Th17 cells within the antigen-specific T cell repertoire is critical for immune monitoring purposes. Antigen-specific Th1, Th2, and Th17 cells can be detected by challenging peripheral blood mononuclear cells (PBMC) with antigen, and establishing the numbers of T cells producing the respective lead cytokine, IFN-γ and IL-2 for Th1 cells, IL-4 and IL-5 for Th2, and IL-17 for Th-17 cells, respectively. Traditionally, these cytokines are measured within 6 h in flow cytometry. We show here that 6 h of stimulation is sufficient to detect peptide-induced production of IFN-γ, but 24 h are required to reveal the full frequency of protein antigen-specific Th1 cells. Also the detection of IL-2 producing Th1 cells requires 24 h stimulation cultures. Measurements of IL-4 producing Th2 cells requires 48-h cultures and 96 h are required for frequency measurements of IL-5 and IL-17 secreting T cells. Therefore, accounting for the differential secretion kinetics of these cytokines is critical for the accurate determination of the frequencies and ratios of antigen-specific Th1, Th2, and Th17 cells.

Nanoceria-mediated delivery of doxorubicin enhances the anti-tumour efficiency in ovarian cancer cells via apoptosis

Nanocarriers are widely used for effective delivery of anticancer drugs to tumours with potential to improve cancer treatment. Here, we developed a nanoceria (CeO2)-based system for delivery of the anti-cancer drug doxorubicin (DOX) to human ovarian cancer cells. Negatively charged nanoceria could conjugate with the cationic DOX via electrostatic interaction under physiological conditions, forming DOX-loaded nanoceria (CeO2/DOX). CeO2/DOX particles displayed nearly spherical shapes, along with superior drug-loading content (22.41%), loading efficiency (99.51%), and higher cellular uptake and drug release behaviours compared to free DOX. Moreover, DOX was released faster from CeO2/DOX under reductive acidic conditions (pH 5.0, 10 mM glutathione) than under physiological conditions (pH 7.4). The initial intracellular DOX concentration was higher in the free DOX groups than in the CeO2/DOX groups, but quickly reduced to 25% of the initial concentration after 24-h culture. By contrast, CeO2/DOX showed sustained DOX release over time and maintained a high intracellular DOX concentration for up to 72 h. In vitro assays showed that CeO2/DOX exhibited higher cell proliferation inhibition and apoptosis compared with free DOX. These results highlight DOX-loaded nanoceria as a promising therapeutic agent for cancer treatment.

Environmentally relevant level of aflatoxin B1 elicits toxic pro-inflammatory response in murine CNS-derived cells

Aflatoxin B1 (AFB1) is a well-known member of aflatoxins (AFs) that is considered among highly stable toxic contaminants of food, worldwide. The impact of AFB1 on neural cells and systems has poorly been understood. To assess the cellular effects of AFB1 on brain, we used murine pure primary astrocytes, sub ventricular zone-derived neural precursor cells (NPCs) and microglia cell line (BV2). Cells were exposed separately to environmentally relevant level (20ng/ml) of AFB1 for 1, 2, 3, 6, 12, 24 and 48h in culture. At each time points, total free radicals production measured by luminol-enhanced cellular chemiluminescence (CL) assay; cytokines production of IL-1β, IL-6, TNF-α and IL-10 were analyzed using Bioplex ELISA and a set of genes involved in the immediate response to danger such as TLR2, TLR4 and iNOS etc. were evaluated by multiplex qPCR. Upon AFB1 exposure production, of the total free radicals significantly increased only in microglial cells after 24h and slightly elevated in the other examined cells. AFB1 also induced secretion of pro-inflammatory cytokines (i.e. TNF-α and IL-6) on both microglial cells (more TNF-α) and astrocytes (more IL-6). mRNA expression of TLR2, TLR4, MyD88 and NF-κB were up-regulated with different timing and levels among cells. Immunotoxicologically, microglial cells, and astrocytes, but not NPCs, are capable of sensing a low level of AFB1. Thus, the pro-inflammatory effects of an environmentally relevant dose of AFB1 on CNS-derived cells in vitro could potentially explain the immune dysregulation in neurodegenerative disorders.

Hypoxia enhances the therapeutic potential of superparamagnetic iron oxide-labeled adipose-derived stem cells for myocardial infarction.

Adipose-derived stem cells (ASCs) induce therapeutic angiogenesis due to pro-angiogenic cytokines secretion. Superparamagnetic iron oxide (SPIO) nanoparticles are critical for magnetic resonance (MR) tracking of implanted cells. Hypoxia is a powerful stimulus for angiogenic activity of ASCs. In this study, we investigated whether therapeutic potency could be enhanced by implantation of hypoxia-preconditioned SPIO-labeled ASCs (SPIOASCs) into the infarcted myocardium. ASCs and SPIOASCs were cultured under 2% O2 (hypoxia) or 95% air (normoxia). Cells were intramyocardially injected into the infarcted myocardium after 48-h culture. We found that hypoxia culture increased the mRNA expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in ASCs and SPIOASCs. The VEGF protein in the conditioned medium was significantly higher in hypoxic ASCs and SPIOASCs than in normoxic ASCs and SPIOASCs. The capillary density and left ventricular contractile function in the infarcted myocardium were significantly higher 4 weeks after implantation with hypoxic ASCs and SPIOASCs than with normoxic ASCs and SPIOASCs. Improvement in the capillary density and left ventricle function didn't differ between hypoxic ASCs-transplanted rats and hypoxic SPIOASCs-transplanted rats. Hypoxic culture enhanced the angiogenic efficiency of ASCs. It was concluded that implantation of hypoxic ASCs or SPIOASCs promotes therapeutic angiogenesis and cardiac function recovery in the infarcted myocardium. SPIO labeling does not impact the beneficial effect of hypoxic ASCs.

Cholecystokinin responsiveness varies across the population dependent on metabolic phenotype

Background: Cholecystokinin (CCK) is an important satiety factor, acting at type 1 receptors (CCK1Rs) on vagal afferent neurons; however, CCK agonists have failed clinical trials for obesity. We postulated that CCK1R function might be defective in such patients due to abnormal membrane composition, such as that observed in cholesterol gallstone disease.Objective: Due to the challenges in directly studying CCK1Rs relevant to appetite control, our goal was to develop and apply a method to determine the impact of a patient's own cellular environment on CCK stimulus-activity coupling and to determine whether CCK sensitivity correlated with the metabolic phenotype of a high-risk population.Design: Wild-type CCK1Rs were expressed on leukocytes from 112 Hispanic patients by using adenoviral transduction and 24-h culture, with quantitation of cholesterol composition and intracellular calcium responses to CCK. Results were correlated with clinical, biochemical, and morphometric characteristics.Results: Broad ranges of cellular cholesterol and CCK responsiveness were observed, with elevated cholesterol correlated with reduced CCK sensitivity. This was prominent with increasing degrees of obesity and the presence of diabetes, particularly when poorly controlled. No single standard clinical metric correlated directly with CCK responsiveness. Reduced CCK sensitivity best correlated with elevated serum triglycerides in normal-weight participants and with low HDL concentrations and elevated glycated hemoglobin in obese and diabetic patients.Conclusions: CCK responsiveness varies widely across the population, with reduced signaling in patients with obesity and diabetes. This could explain the failure of CCK agonists in previous clinical trials and supports the rationale to develop corrective modulators to reverse this defective servomechanism for appetite control. This trial was registered at www.clinicaltrials.gov as NCT03121755.

Possible Immunomodulating Effect of Retinol on Cytokines Secretion in Patients with Recurrent Furunculosis

Recurrent furunculosis is an infection of hair follicles which results in formation of abscesses. Previous studies showed that the pathogenesis of the disease may include an immune-mediated component as the proliferative response of peripheral blood lymphocytes to staphylococcal antigen is depressed. The aim of our study was to evaluate cytokines concentration in the plasma of patients with recurrent furunculosis and to determine whether retinol affects the secretion of those cytokines in patients with recurrent furunculosis and healthy subjects. Blood samples were taken from 15 patients with recurrent furunculosis and 15 age-matched healthy subjects. A quantitative determination of selected cytokines (IL-17, 13, 2, 10, 4, IFN-γ, TNF-α) was performed in the plasma at baseline and after 72-h culture of peripheral blood mononuclear cells with and without retinol in both groups. In the plasma of patients with recurrent furunculosis, concentration of IL-10, 2, and TNF-α was significantly higher, whereas IL-13 significantly lower when compared with healthy subjects. After retinol stimulation, the concentration of IL-17 and IFN-γ increased significantly in both groups. Secretion of anti-inflammatory cytokines, especially IL-10 (p < 0.002) and 13 (p < 0.01), achieved lower levels in recurrent furunculosis samples than in those of healthy controls. Network of cytokines differs in patients with recurrent furunculosis from healthy subjects. Retinol stimulation affects secretion of both pro-inflammatory and anti-inflammatory cytokines. Further studies are recommended for better understanding the pathomechanism of recurrent furunculosis and potential clinical use of retinol in patients affected by recurrent furunculosis.

5-Bromoindirubin 3′-(O-oxiran-2-ylmethyl)oxime: A long-acting anticancer agent and a suicide inhibitor for epoxide hydrolase

Indirubin 3′-oxime (Indox (1b)) suppresses cancer cell growth (IC50: 15μM towards HepG2 cells) and inhibits cell cycle-related kinases such as cyclin-dependent kinases and glycogen synthase kinase-3β. We have previously reported that the conjugation of 1b with oxirane, a protein-reactive component, enhanced the cytotoxic activity of Indox as determined from the IC50 value (1.7μM) of indirubin 3′-(O-oxiran-2-ylmethyl)oxime (Epox/Ind (1c)). Here we prepared Epox/Ind derivatives with one or two halogen atoms or a methoxy group on the aromatic ring(s) of an Indox moiety and studied the structure-activity relationships of the substituent(s). We found that bromine-substitution at the 5-position on 1c or any Epox/Ind derivative(s) having bromine on the aromatic ring except Epox/6′-Br-Ind was efficient to improving anticancer activity. Of the 22 Epox/Ind derivatives, 5-bromoindirubin 3′-(O-oxiran-2-ylmethyl)oxime (Epox/5-Br-Ind (2c)) was the best anticancer agent in both short- (24h) (IC50: 0.67μM) and extended-duration (72h) cultures. The high anticancer activity of 2c was partly due to it being a poor substrate and a suicide inhibitor for epoxide hydrolase as epoxide hydrolase was identified as the enzyme primarily responsible for the metabolism of 2c.