Development Of A High-Throughput Assay For Screening Plant Extracts That Can Extend The Chronological Life Span Of Yeast Saccharomyces Cerevisiae

Bioactive compounds are produced in different parts of a plant, and the compounds can be extracted as plant extracts (PEs). Some bioactive compounds have pharmacological properties such as antioxidant and lifespan-extending activities on organisms in vitro and in vivo. The budding yeast Saccharomyces cerevisiae is a model organism for studying the mechanisms of ageing because yeast shares conserved pathways to higher eukaryotes. The chronological life span (CLS) of yeast is defined as the capacity of cells to remain viable in a non-dividing state. In this study, leaf extracts of of Manihot esculenta, Wodyetia bifurcata and Tabernaemontana divaricata were found to extend yeast CLS. A high-throughput 96-well based CLS assay was developed to enable quantitative screening of many PEs simultaneously to identify plant extracts with yeast CLS-extending activity

Childhood obesity management shifting from health care system to school system: Intervention study of school-based weight management programme

© 2014 Lee et al.; licensee BioMed Central Ltd.Background: Home and school environments conducive for unhealthy eating and physical inactivity are precursors of obesity. The aim of this study is evaluation of the effectiveness of a multi-component school-based weight management programme for overweight and obese primary school children via a home-school joint venture. Methods: This study made use of variety of behavioural modification strategies integrating into the Health Promoting School approach to promote healthy lifestyles. The participants were overweight and obese students aged between 8 and 12 from six participating schools. The interventions involved students attending ten 75 minutes after-school sessions and one 3-hour week-end session of practical interactive and fun activities on healthy eating and exercise, and meal plan together with parents and printed tailor-made management advices. Parents received an introductory seminar with 2 sets of specially designed exercise for their overweight children. The tools to measure bodyweight and fat percentage and standing height were bio-impedance body fat scale and a portable stadiometer. Self-administered questionnaire was used to measure knowledge, attitudes and behaviours. McNemar test was utilized to compare the proportions of behaviour changes within the same group to assess for the trends of changes. BMI z-score and body fat percentage of intervention participants at baseline, 4 month and 8 month were compared pair-wisely using tests of within subject contrasts in repeated measures ANOVA to assess for programme sustainability. Results: Those students in the intervention group reduced their BMI z-score (-0.21, 95% CI -0.34 to -0.07, P = 0.003) and body fat (-2.67%, 95% CI -5.12 to -0.22, P = 0.033) compared to wait list control group with statistical significant, and the intervention group also had a significant reduction in BMI z-score (-0.06, 95% CI -0.11, -0.007, P = 0.028) and body fat (-1.71%, 95% CI, -3.44 to 0.02, P = 0.052) after a 4 month maintenance period. Improvement of dietary habits and positive attitudes towards exercise were observed among the intervention group. Conclusion: School based weight management programme integrated into a Health Promoting School approach with improved school policies and environment in supporting individual skills of obese students and their parents appears to be a promising practice for sustaining weight control.Link_to_subscribed_fulltex

Generation of NSE-MerCreMer Transgenic Mice with Tamoxifen Inducible Cre Activity in Neurons

To establish a genetic tool for conditional deletion or expression of gene in neurons in a temporally controlled manner, we generated a transgenic mouse (NSE-MerCreMer), which expressed a tamoxifen inducible type of Cre recombinase specifically in neurons. The tamoxifen inducible Cre recombinase (MerCreMer) is a fusion protein containing Cre recombinase with two modified estrogen receptor ligand binding domains at both ends, and is driven by the neural-specific rat neural specific enolase (NSE) promoter. A total of two transgenic lines were established, and expression of MerCreMer in neurons of the central and enteric nervous systems was confirmed. Transcript of MerCreMer was detected in several non-neural tissues such as heart, liver, and kidney in these lines. In the background of the Cre reporter mouse strain Rosa26R, Cre recombinase activity was inducible in neurons of adult NSE-MerCreMer mice treated with tamoxifen by intragastric gavage, but not in those fed with corn oil only. We conclude that NSE-MerCreMer lines will be useful for studying gene functions in neurons for the conditions that Cre-mediated recombination resulting in embryonic lethality, which precludes investigation of gene functions in neurons through later stages of development and in adult

HOXB5 binds to multi-species conserved sequence (MCS+9.7) of RET gene and regulates RET expression

RET gene is crucial for the development of enteric nervous system, and dys-regulation of RET expression causes Hirschsprung disease. HOXB5 regulates RET transcription, and perturbations in transcriptional regulation by HOXB5 caused reduced RET expression and defective enteric nervous system development in mice. The mechanisms by which HOXB5 regulate RET transcription are unclear. Thus, unraveling the regulatory mechanisms of HOXB5 on RET transcription could lead to a better understanding of the etiology of Hirschsprung disease. In this study, we identified and confirmed HOXB5 binding to the multi-species conserved sequence (MCS+9.7) in the first intron of the RET gene. We developed a RET mini-gene reporter system, and showed that MCS+9.7 enhanced HOXB5 trans-activation from RET promoter in human neuroblastoma SK-N-SH cells and in chick embryos. The deletion of HOXB5 binding site interfered with HOXB5 trans-activation. Furthermore, transfection of HOXB5 induced endogenous RET transcription, enhanced the co-precipitation of TATA-box binding protein with the transcription start site of RET, and induced histone H3K4 trimethylation in chromatin regions upstream and downstream of RET transcription start site. In conclusion, (i) HOXB5 physically interacted with MCS+9.7 and enhanced RET transcription, (ii) HOXB5 altered chromatin conformation and histone modification of RET locus, which could facilitate the formation of transcription complex, and enhance RET transcription, (iii) expression of RET was mediated by a complex regulatory network of transcription factors functioning in a synergistic, additive and/or independent manners. Hence, dys-regulation of RET expression by HOXB5 could result in insufficient RET expression and Hirschsprung disease. © 2014 Elsevier Ltd.Link_to_subscribed_fulltex

Identification of Tropical Plant Extracts That Extend Yeast Chronological Life Span

Certain plant extracts (PEs) contain bioactive compounds that have antioxidant and lifespan-extending activities on organisms. These PEs play different roles in cellular processes, such as enhancing stress resistance and modulating longevity-defined signaling pathways that contribute to longevity. Here, we report the discovery of PEs that extended chronological life span (CLS) in budding yeast from a screen of 222 PEs. We identified two PEs, the leaf extracts of Manihot esculenta and Wodyetia bifurcata that extended CLS in a dose-dependent manner. The CLS-extending PEs also conferred oxidative stress tolerance, suggesting that these PEs might extend yeast CLS through the upregulation of stress response pathways

Statistics of the generation of <i>NSE-MerCreMer</i> transgenic mice.

a<p>, Number of transgenic lines showing expression of transgene in brain and intestine as detected by RT-PCR or Western blot (WB).</p

Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable

<div><p>Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth <i>in vivo</i>. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.</p></div

Generation of <i>NSE-MerCreMer</i> transgenic lines.

<p>A, Transgenic construct <i>NSE-MerCreMer</i> consists of a 1.8 kb rat <i>NSE</i> gene promoter and the cDNA encoding the tamoxifen inducible Cre recombinase (MerCreMer). X-gal staining of <i>NSE-MerCreMer</i> and <i>pCAG-CAT-LacZ</i> co-transfected HeLa cells with (+OHT) or without (−OHT) the addition of synthetic ligand 4-OHT. B, PCR amplification of <i>NSE-MerCreMer</i> transgene using <i>CreR1</i> and <i>CreF1</i> primer pair generated a 374 bp DNA fragment from genomic DNA of <i>NSE-MerCreMer</i> transgenic mice (Tg) but not from non-transgenic mice (NTg). ‘+ve’ denotes PCR amplification using the transgenic construct as template DNA. C, RT-PCR analysis showed expression of the transgene (<i>MerCreMer</i>) in the brain (Br, +RT) and intestine (I, +RT) of transgenic mouse. RT-PCR for mouse <i>β</i>-actin (<i>β</i>-<i>actin</i>) was included to check the integrity of the RNA. Reverse transcriptase was omitted in the first strand cDNA synthesis which served as a negative control (−RT) to ascertain the PCR product was not amplified from genomic DNA. D, Western blot analysis using anti-body against ligand binding domain of estrogen receptor α (ERα) detected a protein band of MerCreMer (113 kDa) in the intestine (I), and a protein band of ERα (67 kDa) in the uterus (U) of transgenic mouse. Abbreviation: M, DNA size marker.</p

Tamoxifen induction of Cre activity in neurons of the enteric nervous system.

<p>Intestine of <i>NSE-MerCreMer</i>/<i>R26R</i> double transgenic mice of lines #778 and #805 fed with tamoxifen (A, C) or corn oil (B, D) were analyzed by immuno-fluorescence for <i>β</i>-galactosidase (green). Dotted regions were magnified and shown as insets. Abbreviations: m, mucosa; cm, circular muscle; lm, longitudinal muscle.</p