Screening and Assessment of Selected Chilli (<i>Capsicum annuum</i> L.) Genotypes for Drought Tolerance at Seedling Stage

This study was undertaken to investigate oxidative stress tolerant mechanisms in chilli (Capsicum annuum L.) under drought genotypes through evaluating morphological, physiological, biochemical and stomatal parameters. Twenty genotypes were evaluated for their genetic potential to drought stress tolerant at seedling stage. Thirty days old seedlings were exposed to drought stress induced by stop watering for the following 10 days and rewatering for the following one week as recovery. Based on their survival performance, two tolerant genotypes viz. BD-10906 and BD-109012 and two susceptible genotypes viz. BD-10902 and RT-20 were selected for studying the oxidative stress tolerance mechanism. Drought reduced root and shoot length, dry weight, ratio, petiole weight and leaf area in both tolerant and susceptible genotypes, and a higher reduction was observed in susceptible genotypes. Lower reduction of leaf area and photosynthetic pigments were also found in tolerant genotypes. Moreover, tolerant genotypes showed higher recovery than susceptible genotypes after the removal of stress. A higher reduction of relative water content (RWC) may cause an imbalance between absorbed and transpirated water in susceptible genotypes. Higher accumulation of proline in tolerant genotypes might be helpful to for better osmotic maintenance than that in susceptible genotypes. Tolerant genotypes showed higher antioxidant activity as they showed DPPH radical scavenging percentage than the susceptible genotypes. Moreover, closer stomata in tolerant genotypes than susceptible ones helped to avoid dehydration in tolerant genotypes. Thus, the above morphological, physiological, biochemical and stomatal parameters helped to show better tolerance in chilli under drought stress.     

Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa

A reduction in plasma membrane cholesterol is one of the early events that either triggers or is closely associated with capacitation of mammalian spermatozoa. In this investigation, we have examined the effects of cholesterol efflux on tyrosine phosphorylation, lipid diffusion, and raft organization in boar spermatozoa. Results show that a low level of cholesterol efflux, mediated by 5 mM methyl-beta-cyclodextrin (MBCD), enhances capacitation and induces phosphorylation of two proteins at 26 and 15 kDa without affecting sperm viability. Lipid diffusion rates under these conditions are largely unaffected except when cholesterol efflux is excessive. Low-density Triton X100-insoluble complexes (lipid rafts) were isolated from spermatozoa and found to have a restricted profile of proteins. Capacitation-associated cholesterol efflux has no effect on raft composition, but cholesterol depletion destabilizes them completely and phosphorylation is suppressed. During MBCD-mediated capacitation, the distribution of GM1 gangliosides on spermatozoa changes in a sequential manner from overlying the sperm tail to clustering on the sperm head. It is concluded that there is a safe window for removal of plasma membrane cholesterol from spermatozoa within which protein phosphorylation and polarized migration of lipid rafts take place. A preferential loss of cholesterol from the nonraft pool may be the stimulus that promotes raft clustering over the anterior sperm head.     

Concurrent inhibition of NF‐κB,cyclooxygenase‐2, and epidermal growth factor receptor leads to greater anti‐tumor activity in pancreatic cancer

Inactivation of survival pathways such as NF‐κB, cyclooxygenase (COX‐2), or epidermal growth factor receptor (EGFR) signaling individually may not be sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by recent clinical trials. 3,3′‐Diindolylmethane (B‐DIM) is an inhibitor of NF‐κB and COX‐2 and is a well‐known chemopreventive agent. We hypothesized that the inhibition of NF‐κB and COX‐2 by B‐DIM concurrently with the inhibition of EGFR by erlotinib will potentiate the anti‐tumor effects of cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo. Inhibition of viable cells in seven PC cell lines treated with B‐DIM, erlotinib, or gemcitabine alone or their combinations was evaluated using 3‐(4,5‐dimetylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Significant inhibition in cell viability was observed in PC cells expressing high levels of COX‐2, EGFR, and NF‐κB proteins. The observed inhibition was associated with an increase in apoptosis as assessed by ELISA. A significant down‐regulation in the expression of COX‐2, NF‐κB, and EGFR in BxPC‐3, COLO‐357, and HPAC cells was observed, suggesting that simultaneous targeting of EGFR, NF‐κB, and COX‐2 is more effective than targeting either signaling pathway separately. Our in vitro results were further supported by in vivo studies showing that B‐DIM in combination with erlotinib and gemcitabine was significantly more effective than individual agents. Based on our preclinical in vitro and in vivo results, we conclude that this multi‐targeted combination could be developed for the treatment of PC patients whose tumors express high levels of COX‐2, EGFR, and NF‐κB. J. Cell. Biochem. 110: 171–181, 2010. © 2010 Wiley‐Liss, Inc.     

Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K-Ras transgenic mouse model of pancreatic cancer

Human pancreatic cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre (KC), and K-Ras;Pdx1-Cre;INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT-PCR. We found over-expression of miR-21, miR-221, miR-27a, miR-27b, and miR-155, and down-regulation of miR-216a, miR-216b, miR-217, and miR-146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI-derived RInk-1 cells. Mechanistic investigations revealed a significant induction of EGFR, K-Ras, and MT1-MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk-1 cells compared to human MIAPaCa-2 cells. Furthermore, miR-155 knock-down in RInk-1 cells resulted in the inhibition of cell growth and colony formation consistent with down-regulation of EGFR, MT1-MMP, and K-Ras expression. In addition, miR-216b which target Ras, and forced re-expression of miR-216b in RInk-1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR, and MT1-MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA-targeted agents for designing personalized therapy for PC.     

Co-culture of stromal and erythroleukemia cells in a perfused hollow fiber bioreactor system as an in vitro bone marrow model for myeloid leukemia

We have developed a hematopoietic co-culture system using the hollow fiber bioreactor (HFBR) as a potential in vitro bone marrow model for evaluating leukemia. Supporting stroma using HS-5 cells was established in HFBR system and the current bioprocess configuration yielded an average glucose consumption of 640 mg/day and an average protein concentration of 6.40 mg/mL in the extracapillary space over 28 days. Co-culture with erythroleukemia K562 cells was used as a model for myelo-leukemic cell proliferation and differentiation. Two distinct localizations of K562 cells (loosely adhered and adherent cells) were identified and characterized after 2 weeks. The HFBR co-culture resulted in greater leukemic cell expansion (3,130 fold vs. 43 fold) compared to a standard tissue culture polystyrene (TCP) culture. Majority of expanded cells (68%) in HFBR culture were the adherent population, highlighting the importance of cell-cell contact for myelo-leukemic proliferation. Differentiation tendencies in TCP favored maturation toward monocyte and erythrocyte lineages but maintained a pool of myeloid progenitors. In contrast, HFBR co-culture exhibited greater lineage diversity, stimulating monocytic and megakaryocytic differentiation while inhibiting erythroid maturation. With the extensive stromal expansion capacity on hollow fiber surfaces, the HFBR system is able to achieve high cell densities and 3D cell-cell contacts mimicking the bone marrow microenvironment. The proposed in vitro system represents a dynamic and highly scalable 3D co-culture platform for the study of cell-stroma dependent hematopoietic/leukemic cell functions and ex vivo expansion.     

Investigating factors associated with success of breastfeeding in first-time mothers undergoing epidural analgesia: a prospective cohort study

Background

We investigated the possible risk factors that could influence the likelihood of breastfeeding at 5 to 9 weeks postpartum with our primary aim being to analyse the associations between psychological vulnerabilities, such as peripartum depression and anxiety, and continued breastfeeding. Our secondary aim was to investigate other non-psychological factors’ influence on continued breastfeeding.

Methods

A prospective cohort study was conducted in KK Women’s and Children’s Hospital in Singapore. Healthy nulliparous parturients at ≥36 weeks gestation with a singleton fetus who received epidural analgesia were recruited. Demographic and anaesthetic data were obtained. Self-reported psychological and pain determinants such as anxiety (State-Trait Anxiety Inventory), depression (Edinburgh Postnatal Depression Scale), stress (Perceived Stress Scale), pain susceptibility (Pain Catastrophizing Scale) and pain perception (McGill Pain Questionnaire) were also recorded at baseline. A phone interview was then performed at 5 to 9 weeks postpartum to obtain information on breastfeeding status.

Results

329 participants were included into this study, of which 263 (79.9%) of them were still breastfeeding at 5 weeks postpartum. Multivariate logistic regression analysis showed that a higher State-Trait Anxiety Inventory score (Adjusted Odds Ratio [AOR] 0.97; 95% Confidence Interval [CI] 0.94, 1.00) at baseline, higher intrapartum blood loss (AOR 0.76; 95% CI 0.61, 0.93), and occurrence of fetal anomalies (AOR 0.15; 95% CI 0.03, 0.72) were associated with reduced likelihood of breastfeeding at 5 to 9 weeks postpartum. Indians (AOR 0.56; 95% CI 0.20, 1.53), Malays (AOR 0.30; 95% CI 0.14, 0.62) and other ethnicities (AOR 0.36; 95% CI 0.16, 0.83) were less likely to continue breastfeeding compared to Chinese participants. On the other hand, receiving any support services on breastfeeding during the participants’ hospital stay was 3.3 times more likely (AOR 3.30; 95% CI 1.21, 9.02) to increase the likelihood of breastfeeding at 5 to 9 weeks postpartum.

Conclusion

We identified 5 independent association factors that could have significant influences on breastfeeding at 5 to 9 weeks postpartum. Healthcare providers could utilize this risk stratification to identify parturients likely to have poorer breastfeeding outcomes and undertake interventions that may help safeguard optimization of breastfeeding outcomes and parturient care.

Trial registration

Clinicaltrials.gov NCT02278601. Registered 26 October 2014.

     

Prolactin induces MFG-E8 production in macrophages via transcription factor C/EBPβ-dependent pathway

The lactogenic hormone prolactin (PRL) regulates milk protein gene expression in mammary glands. To maintain homeostatic balance in the body, milk fat globule epidermal growth factor 8 (MFG-E8) is vital for phagocytic clearance of apoptotic cells. We investigated the effects of PRL on MFG-E8 expression in macrophages by evaluating its promoter function. Macrophages were stimulated with PRL, and the expression of MFG-E8 was determined using real-time PCR and Western blotting. The role of MFG-E8 on phagocytosis of apoptotic cells in PRL-treated macrophages was assessed using microscopy, while the response of PRL to MFG-E8 expression was evaluated using luciferase assay. Following treatment with PRL, significant up-regulations of the PRL receptor and MFG-E8 were observed in macrophages, though PRL-treated macrophages more efficiently engulfed apoptotic cells. The results of MFG-E8 promoter analysis showed considerable up-regulation of promoter activity in macrophages following PRL treatment and results from mutation analysis of the MFG-E8 promoter suggested that the C/EBPβ binding site was responsible for PRL-induced activation of the MFG-E8 promoter. C/EBPβ activity was found to be up-regulated in PRL-treated cells as revealed by an electrophoretic mobility shift assay (EMSA). In conclusion, PRL is a potent inducer of MFG-E8 expression in macrophages, while its effect is mediated by the presence of a responsive element in the MFG-E8 promoter.     

Intracellular free zinc up-regulates IFN-γ and T-bet essential for Th1 differentiation in Con-A stimulated HUT-78 cells

Zinc deficiency impairs cellular immunity. Up-regulation of mRNA levels of IFN-γ, IL-12Rβ2, and T-bet are essential for Th₁ differentiation. We hypothesized that zinc increases Th₁ differentiation via up-regulation of IFN-γ and T-bet expression. To test this hypothesis, we used zinc-deficient and zinc-sufficient HUT-78 cells (a Th₀ cell line) under different condition of stimulation in this study. We also used TPEN, a zinc-specific chelator, to decrease the bioavailability of zinc in the cells. We measured intracellular free zinc, cytokines, and the mRNAs of T-bet, IFN-γ, and IL-12Rβ2. In this study, we show that in zinc-sufficient HUT-78 cells, mRNA levels of IFN-γ, IL-12Rβ2, and T-bet in PMA/PHA-stimulated cells were increased in comparison to zinc-deficient cells. Although intracellular free zinc was increased slightly in PMA/PHA-stimulated cells, Con-A-stimulated cells in 5 μM zinc medium showed a greater sustained increase in intracellular free zinc in comparison to cells incubated in 1 μM zinc. The cells pre-incubated with TPEN showed decreased mRNA levels of IFN-γ and T-bet mRNAs in comparison to cells without TPEN incubation. We conclude that stimulation of cells by Con-A via TCR, release intracellular free zinc which functions as a signal molecule for generation of IFN-γ and T-bet, and IL-12Rβ2 mRNAs required for Th₁ cell differentiation. These results suggest that zinc increase Th₁ cell differentiation by up-regulation of IFN-γ and T-bet, and IL-12Rbβ2 mRNAs.