Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration

Human embryonic stem cells (hESCs) can be induced and differentiated to form a relatively homogeneous population of neuronal precursors in vitro. We have used this system to screen for genes necessary for neural lineage development by using a pooled human short hairpin RNA (shRNA) library screen and massively parallel sequencing. We confirmed known genes and identified several unpredicted genes with interrelated functions that were specifically required for the formation or survival of neuronal progenitor cells without interfering with the self-renewal capacity of undifferentiated hESCs. Among these are several genes that have been implicated in various neurodevelopmental disorders (i.e., brain malformations, mental retardation, and autism). Unexpectedly, a set of genes mutated in late-onset neurodegenerative disorders and with roles in the formation of RNA granules were also found to interfere with neuronal progenitor cell formation, suggesting their functional relevance in early neurogenesis. This study advances the feasibility and utility of using pooled shRNA libraries in combination with next-generation sequencing for a high-throughput, unbiased functional genomic screen. Our approach can also be used with patient-specific human-induced pluripotent stem cell-derived neural models to obtain unparalleled insights into developmental and degenerative processes in neurological or neuropsychiatric disorders with monogenic or complex inheritance.     

Effect of coenzyme Q10 supplementation on mitochondrial function after myocardial ischemia reperfusion.

BACKGROUND: Coenzyme Q10 (CoQ10) protects myocardium from ischemia-reperfusion (IR) injury as evidenced by improved recovery of mechanical function, ATP, and phosphocreatine during reperfusion. This protection may result from CoQ10's bioenergetic effects on the mitochondria, from its antioxidant properties, or both. The purpose of this study was to elucidate the effects of CoQ10 supplementation on mitochondrial function during myocardial ischemia-reperfusion using an isolated mitochondrial preparation. METHODS: Isolated hearts (n = 6/group) from rats pretreated with liposomal CoQ10 (10 mg/kg iv, CoQ10), vehicle (liposomal only, Vehicle), or saline (Saline) 30 min before the experiments were subjected to 15 min of equilibration (EQ), 25 min of ischemia (I), and 40 min of reperfusion (RP). Left ventricular-developed pressure (DP) was measured. Mitochondria were isolated at end-equilibration (end-EQ), at end-ischemia (end-I), and at end-reperfusion (end-RP). Mitochondrial respiratory function (State 2, 3, and 4, respiratory control index (RCI, ratio of State 3 to 4), and ADP:O ratio) was measured by polarography using NADH (alpha-ketoglutarate, alpha-KG)- or FADH (succinate, SA)-dependent substrates. RESULTS: CoQ10 improved recovery of DP at end-RP (67 +/- 11% in CoQ10 vs 47 +/- 5% in Vehicle and 50 +/- 11% in Saline, P < 0.05 vs Vehicle and Saline). CoQ10 did not change preischemic mitochondrial function. IR decreased State 3 and RCI in all groups using either substrate. CoQ10 had no effect in the mitochondrial oxidation of alpha-KG at end-I. CoQ10 improved State 3 at end-I when SA was used (167 +/- 21 in CoQ10 vs 120 +/- 10 in Saline and 111 +/- 10 ng-atoms O/min/mg protein in Vehicle, P < 0.05). Using alpha-KG as a substrate, CoQ10 improved RCI at end-RP (4.2 +/- 0.2 in CoQ10 vs 3.2 +/- 0.2 in Saline and 3.0 +/- 0.3 in Vehicle, P < 0.05). Using SA, CoQ10 improved State 3 (181 +/- 10 in CoQ10 vs 142 +/- 9 in Saline and 140 +/- 12 ng-atoms O/min/mg protein in Vehicle, P < 0.05) and RCI (2.21 +/- 0.06 in CoQ10 vs 1.85 +/- 0.11 in Saline and 1.72 +/- 0.08 in Vehicle, P < 0.05) at end-RP. CONCLUSIONS: The cardioprotective effects of CoQ10 can be attributed to the preservation of mitochondrial function during reperfusion as evidenced by improved FADH-dependent oxidation.     

Psychobiological modulation in anxious and depressed patients after a mindfulness meditation programme: a pilot study

Mindfulness meditation is an ancient and simple form of meditation that has been said to induce several important physical and psychological benefits. The present study was designed with the aim of investigating the psychobiological effects of mindfulness meditation practice in a clinical population. Sixteen patients with depression and anxiety symptoms were submitted to a mindfulness meditation programme for the period of 2 months. Psychological well‐being [5‐item Mental Health (MH‐5)] and several endocrine parameters [adrenocorticotropic hormone (ACTH), cortisol, dehydroepiandrosterone‐sulphate (DHEAS), thyroid‐stimulating hormone, triiodothyronine, thyroxine and parathyroid hormone (PTH)] were assessed before and after the meditation programme. At post‐test, the MH‐5 score improved, while the levels of DHEAS and ACTH were raised, and those of PTH decreased. Our results suggest that mindfulness meditation can exert a significant psychobiological modulation by enhancing psychological well‐being, as well as by regulating the levels of several hormonal parameters on different axes. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of cell death and vertebral end plate bone mineral density in the annulus of the aging sand rat.

BACKGROUND CONTEXT: The relationship between disc degeneration and end plate sclerosis is poorly understood. The sand rat is an excellent, economical small-animal model in which disc degeneration is age related, spontaneous, reliable, and well characterized. This model is used here to evaluate disc degeneration, disc cell viability, and vertebral end plate bone mineral density (BMD) in lumbar sites. PURPOSE: To determine the proportion of live and dead cells and end plate bone mineral density in the aging sand rat annulus. STUDY DESIGN: Young and old sand rats were used in work approved by the Institutional Animal Care and Use Committee. Outcome measures were the percentage of live/dead annulus cells in the disc and the BMD of cranial and caudal end plates of lumbar vertebrae. METHODS: Bone densitometry was used to obtain endplate BMD on lumbar spines of 16 young sand rats aged 2 to 6 months and 26 older animals aged 22 to 46 months. X-ray films were analyzed for wedging, end plate calcification, and disc-space narrowing. Additional discs were also harvested and incubated with fluorochromes, and the percentage of live or dead cells were determined for the outer, inner annulus, and entire annulus. RESULTS: Radiographically old animals had significantly greater incidence of lumbar wedging (p<0.004) and a significantly greater incidence of end plate calcification and disc-space narrowing (p<0.01). In the live-dead study, the mean percentage of dead annulus cells for the three age groups were significantly different for the outer annulus (p<0.001), inner annulus (p=0.005), and total annulus (p<0.0001). The percentages of dead cells for the entire annulus were 46.14%+/-7.99% (age 2-6 months), 48.13%+/-17.32% (age, 13-19 months), and 76.80%+/-7.27% (age 26-38 months). The percentage of dead disc cells correlated significantly with age for outer annulus, inner annulus, and total annulus (p<0.006). The percentage of dead cells in the entire annulus and the inner annulus correlated significantly with end plate BMD (p<0.02). CONCLUSIONS: Data are novel and show that in very aged sand rats, end plate BMD is significantly greater than that of young animals. Live/dead cell analyses showed increasing cell death in both outer and inner annulus, which correlated significantly with age and with end plate BMD.     

Effects of gonadal steroid withdrawal on serum phosphate and FGF-23 levels in men

INTRODUCTION: Fibroblast growth factor (FGF-23) is a novel phosphaturic factor. Current data suggest that serum phosphate, dietary phosphate and 1,25 dihydroxyvitamin D regulate circulating FGF-23 levels in vivo. We examined if hypogonadism-induced increases in serum phosphate are associated with increases in circulating FGF-23 in healthy men in the absence of dietary manipulation. MATERIALS AND METHODS: 25 healthy men were administered goserelin acetate (GnRH analog) 3.6 mg subcutaneously every 4 weeks for 12 weeks to induce acute testosterone and estrogen deficiency. Subjects consumed an ad libitum diet. Morning fasting blood and urine samples were collected to measure serum phosphate, serum intact FGF-23, PTH, and the maximum tubular reabsorption of phosphate (T(m)P/GFR) at baseline, weeks 4 and 12. The changes in serum FGF-23 and phosphate at weeks 4 and 12 were compared to baseline using paired t-tests. RESULTS: Goserelin therapy decreased mean serum testosterone levels from 543+/-160 ng/dL to 33+/-15 ng/dL at week 4 (p<0.001), and to 20+/-10 ng/dL at week 12 (p<0.001). Serum phosphate increased significantly from 3.4+/-0.6 mg/dL to 3.9+/-0.4 mg/dL at week 4 (p=0.002), and to 4.3+/-0.4 mg/dL at week 12 (p<0.001). T(m)P/GFR increased significantly from 3.2+/-0.6 mg/dL to 3.6+/-0.5 mg/dL at week 4 (p<0.004), and to 4.1+/-0.6 mg/dL at week 12 (p<0.001). FGF-23 levels, however, did not change during the 12-week study. CONCLUSIONS: Gonadal steroid deprivation increased serum phosphate levels in men but did not affect serum FGF-23 concentrations. The absence of any change in circulating FGF-23 suggests that supraphysiologic levels of serum phosphate may be required to stimulate circulating FGF-23 or that FGF-23 production is primarily sensitive to changes in dietary phosphate or 1,25 dihydroxyvitamin D within this physiologic serum phosphate range.     

New contribution to the study of ventricular remodeling and valve rings in dilated cardiomyopathy: anatomical and histological evaluation

Introduction

Idiopathic dilated cardiomyopathy causes great impact but many aspects of its pathophysiology remain unknown.

Objective

To evaluate anatomical and histological aspects of hearts with idiopathic dilated cardiomyopathy and compare them to a control group, evaluating the behavior of the perimeters of the atrioventricular rings and ventricles and to compare the percentage of collagen and elastic fibers of the atrioventricular rings.

Methods

Thirteen hearts with cardiomyopathy and 13 normal hearts were analysed. They were dissected keeping the ventricular mass and atrioventricular rings, with lamination of segments 20%, 50% and 80% of the distance between the atrioventricular groove and the ventricular apex. The sections were subjected to photo scanning, with measurement of perimeters. The atrioventricular rings were dissected and measured digitally to evaluate their perimeters, later being sent to the pathology laboratory, and stained by hematoxylin-eosin, picrosirius and oxidized resorcin fuccin.

Results

Regarding to ventricles, dilation occurs in all segments in the pathological group, and the right atrioventricular ring measurement was higher in idiopathic dilated cardiomyopathy group, with no difference in the left side. With respect to collagen, both sides had lower percentage of fibers in the pathological group. With respect to the elastic fibers, there was no difference between the groups.

Conclusion

There is a change in ventricular geometry in cardiomyopathy group. The left atrioventricular ring does not dilate, in spite of the fact that in both ventricles there is lowering of collagen.