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Conceived and designed the experiments: TJM JKB. Performed the experiments: TJM SMP RMB EAL JKB. Analyzed the data: TJM SMP EAL JKB. Wrote the paper: TJM JKB.
The development of an effective therapy for Alzheimer's disease (AD) is a major challenge to biomedical sciences. Because much of early AD pathophysiology includes hippocampal abnormalities, a viable treatment strategy might be to use trophic factors that support hippocampal integrity and function. IGF2 is an attractive candidate as it acts in the hippocampus to enhance memory consolidation, stimulate adult neurogenesis and upregulate cholinergic marker expression and acetylcholine (ACh) release. We performed a seven-day intracerebroventricular infusion of IGF2 in transgenic APPswe.PS1dE9 AD model mice that express green fluorescent protein in cholinergic neurons (APP.PS1/CHGFP) and in wild type WT/CHGFP littermates at 6 months of age representing early AD-like disease. IGF2 reduced the number of hippocampal Aβ40- and Aβ42-positive amyloid plaques in APP.PS1/CHGFP mice. Moreover, IGF2 increased hippocampal protein levels of the ACh-synthesizing enzyme, choline acetyltransferase in both WT/CHGFP and APP.PS1/CHGFP mice. The latter effect was likely mediated by increased protein expression of the cholinergic differentiating factor, BMP9, observed in IGF2-treated mice as compared to controls. IGF2 also increased the protein levels of hippocampal NGF, BDNF, NT3 and IGF1 and of doublecortin, a marker of neurogenesis. These data show that IGF2 administration is effective in reversing and preventing several pathophysiologic processes associated with AD and suggest that IGF2 may constitute a therapeutic target for AD.
Alzheimer's disease (AD) pathology is characterized by cerebral accumulation of extracellular amyloid, intraneuronal neurofibrillary tangles, neurotransmitter abnormalities and, ultimately, loss of synapses and neuronal death. The amyloid is composed of Aβ peptides that are toxic to neurons [1]. Among the numerous neurotransmitter abnormalities in brains of AD patients, the cholinergic defect – thought to contribute to the amnesia that is so prominent in this illness [2]–[4] – is the result of the degeneration and/or malfunction of the basal forebrain cholinergic neurons (BFCN) [3]. BFCN are vulnerable to Aβ toxicity and we [5] and others [6]–[8] showed that Aβ impairs BFCN function in the absence of cell death. Thus, reduction of Aβ accumulation and generation of a trophic environment for BFCN are rational objectives in designing an AD therapy. We tested this idea using insulin-like growth factor 2 (IGF2) as a therapeutic agent based on studies showing that IGF2 mRNA levels decline in the frontal cortex of AD patients at relatively early stages of neuropathology (Braak and Braak 2–3) [9] and on a growing body of data showing beneficial effects of IGF2 on neural function. Intrahippocampal injections of IGF2 in rats [10], [11] and mice [12], [13] enhanced memory function, while antagonizing the action of endogenous IGF2 impaired memory [10], [12], [14] indicating the role of the locally-produced IGF2 in this process. IGF2 is expressed in the subgranular zone of the dentate gyrus, and endogenous IGF2 upregulates the proliferation of neural stem cells in this brain region [15] and, moreover, intrahippocampal injections of IGF2 promote the survival of adult-born neurons in the dentate granule cell layer [12], [13]. IGF2 increases the release of acetylcholine (ACh) from BFCN [16]–[18] and prevents the Aβ-evoked neurotoxicity in cultured septal neurons [19] and in hippocampal cultures [20].
We used the APPswe/PS1deltaE9 (APP.PS1) transgenic mice as a model of AD [21], [22]. To facilitate studies of cholinergic neurons, we crossed these mice with a transgenic strain that expresses the enhanced green fluorescent protein (eGFP) specifically in cholinergic cells [23], [24]. We infused these cholinergic neuron eGFP-expressing AD model mice intracerebroventriculary (icv) with IGF2 for 7 days. This treatment reduced the hippocampal amyloid plaque number, increased protein expression of the ACh–synthesizing enzyme, choline acetyltransferase (CHAT), and the levels of a cholinergic differentiating factor, BMP9, and of trophic factors NGF, BDNF, NT3 and IGF1, as well as a marker of neurogenesis, doublecortin (DCX), indicating that IGF2 exhibits efficacy as an AD treatment in this model.
All animal procedures were performed in accordance with the Animal Welfare Act (Animal Welfare Assurance Number A-3316-01) and the principles of the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Boston University. All surgical procedures were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.
We used the APPswe/PS1deltaE9 (APP.PS1) mice that express murine amyloid precursor protein (APP) with the human Aβ amino acid sequence harboring mutations that cause a familial form of AD (the Swedish mutation APP(K595N/M596L; APPswe) and a mutated form of presenilin 1 (PS1 with exon 9 deleted; PS1dE9) [21]. Although no model of AD fully recapitulates the human disease [25], APP.PS1 mice are well suited for our studies because they exhibit: 1) high production of Aβ peptides in brain and accumulation of amyloid plaques by 4–6 months of age [22], 2) cholinergic defects [26]–[31], and 3) cognitive impairments [31]–[36]. We crossed these mice with a transgenic CHGFP strain that expresses the enhanced green fluorescent protein (eGFP) specifically in cholinergic cells [23], [24]. Thus, our studies were performed on mice that either did not express the AD-related transgenes, designated WT/CHGFP, or their AD model littermates designated APP.PS1/CHGFP. We studied animals at 6 months of age when the APP.PS1 mice exhibit rapid rate of amyloid accumulation [37] and have normal CHAT activity, although they show some impairment of the BFCN projections [26]. This time point represents early, rapidly progressing, stage of pathogenesis. Homozygous CHGFP (B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J) [23], [24] females were crossed to hemizygous APP.PS1 (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax) [22], [26] males to generate WT/CHGFP and APP.PS1/CHGFP experimental subjects.
We performed intraventricular infusion of vehicle or 50 ng/h of human recombinant IGF2 (Peprotech) for 7 days. An equal number of male and female mice at 6 months of age (n=6 per group, 4 groups: PBS WT/CHGFP, PBS APP.PS1/CHGFP, IGF2 WT/CHGFP, IGF2 APP.PS1/CHGFP) were anesthetized using 5% isoflurane in oxygen delivered at a rate of 2.5 L/min. Following induction, isoflurane was given at 2.5% to maintain anesthesia. During the induction phase, mice received 0.04 mg/kg of buprenex analgesic. All surgical steps were performed aseptically on a sterile field. A midsagittal incision was made on the scalp and a subcutaneous tunnel was opened between the shoulder blades, where the Alzet osmotic pumps (model 1002; pumping rate 0.25 µl/h) were implanted. The stereotactic apparatus was used to position the cannula connected to the pump in the lateral ventricle through a small hole in the skull at the following coordinates relative to bregma: posterior: −0.6 mm, lateral: −1.2 mm, depth: −2.0 mm. The cannula was fixed in place with Loctite adhesive (Alzet) and dental cement. The mice received prophylactic antibiotic (ampicillin 35 mg/kg, s.c.) and 1Qml of 0.9% sterile saline s.c. for hydration purposes. The incision was closed with silk sutures and dabbed with Vetbond (3 M). The mice recovered from anesthesia breathing pure oxygen at a rate of 2.5 L/min for approximately 1 min. Following surgery, the mice were placed on a heating pad and subsequently back in their home cages. Animals received buprenex (0.04 mg/kg, s.c.) every 12 h post-operatively for 2 days. They were inspected daily for signs of distress and wound healing was monitored. After 7 days, the mice were killed by CO2 inhalation and their brains rapidly dissected on ice.
The hippocampus was homogenized in a lysis buffer containing 0.05 M Tris-HCl pH 7.5, 0.15 M NaCl, 1% NP-40, 1 mM Na-orthovanadate, 0.001% sodium fluoride, 1% protease inhibitor cocktail (Sigma). BDNF, NT3, and NGF were assayed using the Emax® immunoassay system (Promega) and FGF2 and IGF1 were assayed using the Quantikine® sandwich ELISA kit (R&D Systems) according to manufacturer's instructions and as described in our previous publications [38]–[41]. Aβ40 and Aβ42 levels were assayed using Aβ40 Human ELISA Kit and Aβ42 Human ELISA Kit according to the manufacturer's instructions (Invitrogen).
Forty µg of hippocampal protein per sample was subjected to PAGE electrophoresis using 4–12% Bis-Tris Midi Gel (Invitrogen) and transferred to a blotting membrane with the iBlot system (Invitrogen). The membrane probed with Goat anti-CHAT (11000, Millipore) was blocked with Western Blocker Solution (Sigma). All other membranes were blocked with 5% milk in TBS/1.5% Tween (TBS-T), washed with TBS-T, and probed overnight with either rabbit anti-p75NTR (13000, Advanced Targeting Systems), mouse anti-GFAP (11000, Cell Signaling Technology), rabbit anti-TrkA (11000, Millipore), rabbit anti-DCX (11000, Cell Signaling Technology), rat anti-ALK-1 (11000, R & D Systems), rabbit anti-BMP9 (11000, Abcam), or mouse anti-β-actin (15000, Sigma). Following incubation with the primary antibody, blots were incubated in species-specific anti-IgG-HRP: anti-Rabbit-HRP (14000, Bio-Rad), anti-Goat/Sheep-HRP (12000; Sigma), or anti-mouse-HRP (12000, Bio-Rad).
Reactive bands were detected with SuperSignal West Femto chemiluminescent substrate (Pierce, Rockford IL). Chemiluminescence was captured with a Kodak ImageStation 440CF and the band intensities were quantified with Kodak 1D Image Analysis software.
Following the dissection of the basal forebrain, tissues were homogenized in buffer RLT (Qiagen) and frozen at −70°C. Total RNA was extracted from homogenized samples using an RNAeasy kit (Qiagen) according to manufacturer's instructions. RNAs were used for reverse transcriptase PCR using Superscript™ One-Step RT-PCR with Platinum® Taq (Invitrogen Life Technologies). First strand cDNA synthesis was performed using the extracted total RNA (10 ng for β-actin, 25 ng for Chat, and 50 ng of RNA for Bmp9), oligo dT primer and reverse transcriptase at 48°C (45 min). Primers used for PCR include β-actin (Forward: CACAGCTGAGAGGGAAATC, Reverse: TCAGCAATGCCTGGGTAC), Chat (Forward: CGGGATCCTGCCTCATCTCTGGTGT, Reverse: GGCGGAATTCAATCACAACAT), and Bmp9 (Forward: TAAACCTCAGCGGCATTCC, Reverse: AAACGACCATGCTTCCTTCC). PCR was performed using Platinum Taq DNA polymerase with a denaturing step for 2 min at 94°C, followed by 32–40 cycles of 1 min at 94°C, 1 min at 58°C and 2 min at 72°C (32 cycles for β-actin, 36 for Chat, and 40 for Bmp9) and terminated by an elongation step at 72°C for 7 min. PCR products were displayed on a 10% polyacrylamide gel and stained with ethidium bromide. PCR products were visualized with Kodak Image Station 440 and product intensities were quantified using Kodak software.
Brains were dissected and immediately fixed in 5 volumes of PLP fixative (4% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate; pH 7.4) at 4°C overnight and cryoprotected using 10% and 20% glycerol/2% dimethylsulfoxide, in 0.1 M PBS, pH 7.3 (24 h each). Serial, frozen sections (40 µm, coronal) were cut with a sliding microtome from the anterior frontal pole of to the caudal occipital region. All sections intended to be subjects of comparative analyses were processed together and incubated for the same time periods in all of the reagents. For Aβ42 immunohistochemistry, sections were treated with>95% formic acid (Sigma) for 2 min with gentle agitation, washed with PBS, and then transferred to a solution of PBS/10% Goat Serum (Gibco) for 1 h at room temperature (RT). Sections were probed with rabbit anti-Aβ42 (12500, Invitrogen) overnight at RT in 0.3% triton-X 100, 2% goat serum (Gibco), 0.008% sodium azide PBS. Following washing, sections were probed with goat anti-Rabbit-HRP (Millipore, 11000) in a solution of 2% goat serum/PBS for 3 h at RT. After incubation in a developing solution containing diaminobenzidine (DAB), sodium imidazole, and hydrogen peroxide, sections were mounted on subbed slides. Photomicrographs were taken with 2X and 4X magnification objectives and images of the hippocampus were analyzed with ImageJ software (NIH).
BFCN were imaged using fluorescence microscopy of GFP-expressing cells and by immunofluorescence following anti-75 antibody staining. For p75 immunofluorescence, free floating sections were incubated for 3 h in a blocking buffer consisting of 10% normal donkey serum and 0.3% Triton X-100 in PBS and subsequently overnight in 1% BSA, 0.3% Triton X-100 in PBS containing the rabbit anti-p75 polyclonal antibody (Cell Signaling Technology, 13200). The sections were rinsed with PBS, blocked in the aforementioned blocking buffer for 3 h and incubated in the dark for 6 h with secondary Alexa Fluor-594 donkey anti-rabbit IgG antibody (Life Technologies, 11000). After the final PBS rinse, the sections were mounted on on SuperfrostPlus slides (Fisher) allowed to dry at RT in the dark, coverslipped and stored at −20°C. Each PBS rinse step consisted of 3×10 min washes and all incubations were performed at RT on a rotating shaker. BFCN were imaged with Olympus IX81/DSU spinning disc confocal microscope. Exposure settings were adjusted using image acquisition software (IPLab v.4.0; BD Biosciences). These settings, including the exposure time, were kept constant in each detection channel for all sections imaged and no pixels read saturation. Subsequently these images were used to estimate average BFCN GFP fluorescence intensity and cell area with the NIH ImageJ software. All animals were used for this analysis (n=6 per group) and all GFP-positive cells in each image were included.
Hippocampal sections were used to image immunofluorescence staining of DCX within the dentate gyrus. The protocol described above was followed except goat anti-DCX (1250, Santa Cruz) and secondary Alexa Fluor-594 donkey anti-goat IgG antibody (Life Technologies,11000) were used instead.
Data for all experiments, presented as means ± SEM (n=6), were analyzed by t-test or a one- or two-way ANOVA, as appropriate. Post hoc analyses were performed with a Fisher's LSD test.
We assessed the amyloid plaque deposition in APP.PS1/CHGFP mice using Aβ40 and Aβ42 immunohistochemical staining and measuring plaque number per unit of tissue area in the anterior (bregma approximately −1.5 mm), intermediate (bregma approximately −2.4 mm) and posterior (bregma approximately −3 mm) hippocampus (Figure 1). The data were analyzed by a one-way ANOVA using the hippocampal sub-regions as a repeated measure. A 7-day infusion of IGF2 caused a significant reduction in the Aβ40- and Aβ42-positive plaque number (by 50–60%) (Figure1). We also measured the hippocampal levels of the solubilized Aβ40 and Aβ42 peptides using ELISA. There were no significant effects of IGF2 infusion on Aβ40 and Aβ42 levels by this method (data not shown).
To determine the effects of IGF2 on septal BFCN, we imaged these GFP-expressing cells in brain sections using fluorescence microscopy. To further verify the identity of these neurons we also stained the brain sections with an antibody to the BFCN protein marker, the low-affinity neurotrophin receptor, p75NGFR. As expected, septal BFCN expressed this protein (Figure 2A). We quantified the intensity of GFP fluorescence of BFCN as well as their average cell size. The intensity of BFCN fluorescence was reduced in untreated APP.PS1/CHGFP mice by 22% as compared to the WT/CHGFP mice (Figure 2B). IGF2 increased BFCN fluorescence intensity by 23% in the WT/CHGFP mice and by 35% in the APP.PS1/CHGFP mice (Figure 2B). Similarly, the average size of septal BFCN was reduced by 21% in APP.PS1/CHGFP mice as compared to WT/CHGFP mice and IGF2 increased the size of BFCN by approximately 30–34% in both the wild type and AD model mice (Figure 2C). In addition we found a small increase in the septal Chat mRNA levels in IGF2 infused mice of both genotypes as compared to untreated controls (Figure 2D). Hippocampal CHAT protein levels were similar in WT/CHGFP and APP.PS1/CHGFP mice and were increased by IGF2 infusion by approximately 50% in both WT/CHGFP and APP.PS1/CHGFP animals (Figure 2E).
The acquisition of the cholinergic phenotype of BFCN during development [42]–[44] and its maintenance in adulthood [41] is controlled by bone morphogenetic protein 9 (BMP9, also known as growth/differentiation factor 2, GDF2). Therefore we measured the levels of BMP9 protein in the hippocampus in the control- and IGF2-infused mice. APP.PS1/CHGFP mice were characterized by a 40% increase in the basal levels of BMP9 protein relative to the WT/CHGFP animals and IGF2 infusion increased those levels in both mouse lines leading to similarly high BMP9 protein amounts in WT/CHGFP (2-fold vs controls) and APP.PS1/CHGFP mice (45% increase vs controls) (Figure 3A, B). BMP9 signals via its specific type I receptor, ALK1 [45]–[48] and we showed that ALK1 is expressed by BFCN [24]. We observed a reduction of ALK1 protein levels in the hippocampus of the WT/CHGFP mice infused with IGF2 (70% of controls). This effect of IGF2 was absent in APP.PS1/CHGFP mice (Figure 3A, C). Our previous studies showed that Bmp9 mRNA is expressed in mouse septum [42]. We found that IGF2 increased Bmp9 transcript expression in the septum of WT/CHGFP mice (by 47%) but this effect was not observed in APP.PS1/CHGFP mice (Figure 3D, E).
IGF2 infusion also modulated the expression of other proteins that are trophic to BFCN (Figure 4). We found a statistically significant overall increases in the levels of NGF, NT3, BDNF, and IGF1 in the hippocampus of IGF2-treated mice. In contrast IGF2 infusion significantly reduced hippocampal FGF2 levels (to 70% of controls in APP.PS1/CHGFP mice) (Figure 4E).
Previous studies showed that IGF2 stimulates adult hippocampal neurogenesis. We measured the levels of doublecortin (DCX, a marker of neuronal precursor cells and immature neurons) as an index of this process [49], [50]. Using confocal microscopy of hippocampal sections stained with an anti-DCX antibody, an increase in DCX-positive staining by IGF2 infusion was readily apparent (Figure 5A). We also found a 60% increase in the levels of DCX in the hippocampus of IGF2-infused wild type and AD-model mice as compared to controls using immunoblot analysis (Figure 5B).
APP.PS1 mice are reportedly characterized by hippocampal gliosis that increases with age as determined using GFAP immunostaining and qPCR assays [51], [52]. Using immunoblots, we found early signs of increased GFAP expression the hippocampus of APP.PS1/CHGFP mice (Figure 6). IGF2 infusion had no effect on GFAP levels.
The results show that administration of IGF2 to the brain of the AD model APP.PS1/CHGFP mice ameliorates the central pathophysiologic feature of AD, the accumulation of amyloid plaques in the hippocampus. This action of IGF2 was observed after only a one week infusion, consistent with the notion that plaque turnover can be rapid. Previous studies showed that plaque formation is exceptionally fast occurring within one day in APP.PS1 mice [53]. Thus, it is conceivable that IGF2 could inhibit the generation of new plaques, an effect that would manifest as fewer plaques in the IGF2-treated mice as compared to controls. Similarly, plaque burden may be reduced within days in APP.PS1 mice by certain drugs, e.g. PPARγ- agonists [54], indicating that if IGF2 acted by accelerating plaque clearance, this too could be observed within the time period of IGF2 administration.
We present novel observations that IGF2 administration increases hippocampal CHAT protein levels and Chat mRNA expression in the septum, and moreover, that the IGF2-treated mice are characterized by increased size of septal BFCN. The latter observation points to novel trophic actions of IGF2 on cholinergic neuronal morphology in vivo. Our findings related to modulation of CHAT levels by IGF2 are consistent with in vitro studies showing that IGF2 is a trophic factor for cultured BFCN as assessed by cell viability and CHAT expression [55]–[57]. Moreover, fetal IGF2-treated BFCN transplanted into the hippocampus exhibited higher long-term survival than control neurons [58]. IGF2 was equally as effective in elevating CHAT expression in WT/CHGFP- as in APP.PS1/CHGFP mice. These data indicate that the AD-model mice analyzed at 6 months of age, i.e. relatively early during the progression of the AD-like pathology, exhibit similar responsiveness to IGF2 as their wild type siblings.
The antiamyloidogenic action of IGF2 observed in APP.PS1/CHGFP mice may be the consequence of the increased cholinergic tone evoked by IGF2 because cholinergic neurotransmission activates α-secretase which hydrolyzes APP within the Aβ sequence precluding amyloid formation [59]. Indeed, in APP.PS1 mice subjected to a specific BFCN lesion, produced by the immunotoxin anti-p75NGFR-saporin, there is a rapid acceleration of amyloid plaque deposition in the hippocampus that can be appreciated within days [60], [61] indicating that normal cholinergic innervation slows down the generation of new plaques. Similarly, in AD model mice with deleted m1 muscarinic receptor, Aβ levels and amyloid plaque numbers are increased [62]. However, in contrast to the reduction of Aβ42 plaque density in the hippocampus of IGF2-infused mice, we found no effect of IGF2 on total Aβ40 and Aβ42 levels measured by ELISA in hippocampal extracts. Interestingly other reports also indicated relatively more robust effects of various treatments on plaque burden as compared to Aβ peptide levels in mouse models of AD [63]–[65]. One possible mechanism that could explain our observations might be that IGF2 slows down the aggregation and deposition of the Aβ peptides into plaques or, alternatively, that IGF2 promotes plaque clearance (e.g. by glial cells), involving transfer of the peptide from plaques to an intracellular compartment where it may be degraded. If this were the case, higher amounts of Aβ peptides would be present in non-plaque form in brains of IGF2-treated mice as compared to the vehicle-infused animals. Our ELISA method – which includes amyloid solubilization in guanidine hydrochloride – was designed to detect all Aβ42 (i.e., present in plaques, in diffuse extracellular amyloid and the intracellular peptide).
Our studies show that IGF2 infusion generates a trophic environment for BFCN by increasing the levels of several proteins that are neuroprotective for these neurons, indicating that IGF2 may not only act directly on BFCN but also, by creating a trophic milieu for these cells, supports their function via additional paracrine mechanisms. In particular, IGF2 dramatically increased hippocampal BMP9 protein levels in both the wild type and AD model mice. We have previously shown that BMP9 infused icv to mice with experimental injury to these neurons, prevents BFCN loss [41] and a 7-day icv infusion of BMP9 in APP.PS1/CHGFP mice reduces hippocampal and cortical amyloidosis and counteracts the cholinergic defect [66]. In contrast, IGF2 lowered the levels of the BMP9 receptor, ALK1, specifically in WT/CHGFP mice, possibly leading to desensitization/tolerance to the IGF2-induced BMP9 in these animals. In APP.PS1/CHGFP mice no such downregulation of ALK1 was observed suggesting that the BMP9 induced by IGF2 would be expected to signal productively in these mice. Moreover, hippocampal BMP9 levels were higher in APP.PS1/CHGFP mice than in WT/CHGFP controls, suggesting that the induction of BMP9 synthesis may be part of an adaptive response to the AD-like pathophysiologic process that occurs in APP.PS1/CHGFP mice. While there are no data on the levels of BMP9 in AD brain, the levels of a related protein, BMP6 (but not BMP2 and BMP7), are increased in the hippocampus of AD patients and in a mouse AD model [67]. Similarly the levels of BMP4 mRNA [68] and the number of BMP4-positive cells [69] are reportedly increased in the hippocampus of APP.PS1 mice.
IGF2 increased the hippocampal levels of NGF, BDNF, and NT3 to varying degrees in WT/CHGFP- and APP.PS1/CHGFP mice. These neurotrophins support the viability, the cholinergic phenotype, axonal growth, synaptogenesis, and function of BFCN. NGF, whose levels rose slightly in IGF2-treated mice, is a prototypic trophic factor for septal cholinergic neurons [70] whose therapeutic utility for AD has been explored [71] (http://clinicaltrials.gov/ct2/show/NCT00876863). In contrast to the small response of NGF levels to the IGF2 treatment, hippocampal levels of BDNF increased considerably (by 60%) in IGF2-infused APP.PS1/CHGFP mice, suggesting that some of the actions of IGF2 on BFCN could be mediated by the rise in BDNF. BDNF supports BFCN survival and elevates CHAT expression in cell culture [72]–[76]. Moreover, BDNF is necessary for postnatal maturation of BFCN in vivo [77]. In rats, treatment with BDNF prevents axotomy-induced degeneration and loss of CHAT expression in BFCN [78], [79]. NT3 levels in the hippocampus were increased by 30% in IGF2-treated mice as compared to controls. This neurotrophin promotes the extension of cholinergic axons towards their hippocampal and cortical target neurons and facilitates cholinergic synapse formation on these cells [80]. Furthermore, NT3 is neuroprotective for cortical neurons cultured in the presence of Aβ and attenuates Aβ-mediated apoptosis of these cells [81].
Interestingly, purified BFCN from AD patients are characterized by reduced expression of mRNAs encoding the NGF receptor, TRKA, the BDNF receptor, TRKB, and the NT3 receptor, TRKC, as compared to matched control subjects [82], [83], suggesting that increased levels of the neurotrophin ligands for these receptors could functionally offset reductions in their levels and/or activity. Indeed, recent reports indicate that administration of a small molecule TRKB agonist [84] or BDNF delivery via a viral vector [85] reversed memory impairments seen transgenic AD model mice.
Previous studies showed that the levels of IGF1 tend to be reduced in plasma of AD patients [86] and that APP.PS1 mice, crossed with a strain engineered to have reduced circulating IGF1 levels, are characterized by high brain amyloid burden [87]. In contrast, administration of IGF1 reduces brain amyloidosis in AD model mice [88]. There is also evidence that IGF1 protects neurons against Aβ-induced toxicity in culture [89]. Thus, the modestly increased levels of IGF1 by IGF2 seen in APP.PS1/CHGFP mice may potentially help to slow down the AD-like pathophysiologic process in these animals.
IGF2 infusion decreased the levels of hippocampal FGF2 both in the wild type- and APP.PS1/CHGFP mice. These data are consistent with our previous studies showing that icv administration of BMP9 in mice reduces the hippocampal levels of FGF2 [41]. Perhaps, the increased expression of BMP9 evoked by IGF2 administration (see above) was responsible for the reductions of FGF2 levels. However, previous studies showed that in mouse models of AD, hippocampal expression of FGF2, mediated by a viral vector, improves memory function, enhances long-term potentiation in the CA1 region and reduces Aβ levels [90], indicating a potential for the use of FGF2 as a therapeutic agent for AD.
AD model mice, including the APP.PS1 mice, exhibit impaired adult hippocampal neurogenesis as they age [91]–[94]. In this study, the 6-month-old APP.PS1/CHGFP mice had similar expression of DCX as the wild type mice. Our data showing enhanced DCX expression evoked by IGF2 are consistent with previous reports that this growth factor upregulates neurogenesis [12], [13], [15].
IGF2 signals by activating several classes of receptors including the insulin receptor (IR), insulin-like growth factor receptor 1 (IGF1R) and IGF2R [95]. These proteins are expressed in brain in cell-specific manner [95] and mediate the IGF2 signal either via a tyrosine kinase cascade (IR, IGFR1) or via a G-protein (IGF2R) [96]. As noted above, IGF2 facilitates memory function. Depending on the type of memory examined these effects are mediated by IGF2R (inhibitory avoidance) [10], [11] or IGF1R (fear extinction) [12]. It will be interesting to determine which receptors mediate the multiple IGF2 actions reported here.
In conclusion we report that IGF2 is effective in reducing the amyloid burden, enhancing cholinergic function, and generating a neurotrophic milieu for cholinergic neurons in a model of AD, thus providing evidence that the IGF2 signaling pathway may constitute a novel therapeutic target in AD.
We would like to thank Olivia Huleatt for her assistance with the immunohistochemical work performed in this study.
This work was supported by an NIH grant AG032709 to JKB and by a Boston University Alzheimer's Disease Center Pilot- and an Alzheimer's Association New Investigator grants to TJM. RMB was supported by an NIH T32 AG00015 training grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Slow Cooked Mexican Beef
Slow cooked Mexican beef 101
Cook in the oven, on the stovetop, or in the slow cooker, it's a great dish to make ahead of time and will taste even better the next day.
- Cut the chuck into 3cm cubes. Trim away any fat.
- Brown it in small batches, this will give the meat a good colour and a rich, deep flavour.
- Saute onions and other vegies like carrots and celery until they just soften and begin to colour. The natural sugars in the onion and garlic will begin to caramelize giving the dish subtle undertones.
- Add your Mexican flavourings - ground cumin, coriander, dried oregano and sweet paprika. Stir for a minute or two to let the flavours develop.
- Add stock, or a mix or stock/tomato puree. As a guide, 1kg of chuck will need about 3-4 cups of liquid. The liquid should cover the meat by about 2-3cm in the casserole dish or slow cooker. A little more liquid may be needed if you cook it on the stovetop.
- A fresh bay leaf or two will flavour the casserole as it cooks. Keep chopped coriander for adding just before serving.
- Cook the chuck slowly, over a low heat on the stovetop or in a moderately-slow oven. The mixture mustn't boil only simmer. Check the heat and regulate the heat up or down as needed as it cooks.
- Make it in advance if you like, it will keep for 2-3 days in the fridge. Take care to reheat it well, the meat and the sauce must both be very hot before serving.
Serve with all your favourite Mexican sides... warm tortillas, shredded lettuce, sour cream, guacamole and jalapenos.

subset: commoncrawl


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Family and friends are extremely important to me. I am looking for someone who feels the same.
I am quick witted and sarcastic at times, but it is well placed and never mean spirited. Laughter turns me on. If you can make me laugh with quick wit, you're all right in my book! Being able to converse in a witty and intelligent way is a MUST. If you can laugh at yourself and be goofy that's a bonus!
I truly enjoy traveling and hope to find someone who also enjoys traveling. It's nice just to relax with that special person and enjoy a drink and a movie at home as well.
I love seafood and sushi but there's nothing wrong w/a good steak every now & then.


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(Mar. 16, 2010) On March 2, 2010, the General Court of the European Union rejected a challenge, brought by "the world's largest steel producer" Arcelor S.A., to the EU's 2003 Emissions Trading Directive. The Luxembourg-based company argued that several European Community law principles – the right to property and to the pursuit of economic activity, equal treatment, freedom of establishment, legal certainty – were violated by certain Directive provisions. It therefore sought both the annulment of the provisions and damages for the harm it suffered as a result of the Directive's adoption. The action was lodged against the European Parliament and the Council of the European Union on January 15, 2004 (when the General Court was the Court of First Instance of the European Communities). (Gabriela Forbes, EU Court Dismisses Challenge to Emissions Trading Rules, PAPER CHASE NEWSBURST, Mar. 3, 2010, available at http://jurist.law.pitt.edu/paperchase/2010_03_03_indexarch.php
The General Court dismissed as inadmissible the action for annulment of the Directive. It noted that while a company may bring an action against Community acts that concern it directly and individually, "Arcelor is neither individually nor directly concerned by the directive." (Press Release, No. 17/10, The General Court of the European Union, The General Court Dismisses the Action Brought by Arcelor Challenging the Validity of the Emissions Trading Directive (Mar. 2, 2010), available athttp://curia.europa.eu/jcms/upload/docs/application/pdf/2010-03/cp100017
en.pdf.) The Directive applies, the Court stated, "in a general and abstract manner, to all of the operators" covered in its annex, and therefore it did not result in unequal treatment. The Court also rejected Arcelor's application for damages, finding that the company has not shown that, in adopting the directive, "the Community legislature committed a sufficiently serious breach" of the principles mentioned above "to give rise to non-contractual liability on the part of the Community." (Id.)
Aside from dismissing the action, the Court ordered Arcelor to bear its own costs and those incurred by the Parliament and the Council, but ordered the European Commission, which intervened in support of the Parliament and Council, to bear its own costs. (Case T-16/04, Arcelor v Parliament and Council, Court of Justice of the European Union website, Mar. 2, 2010, available at http://curia.europa.eu/jurisp/
cgi-bin/form.pl?lang=EN&Submit=rechercher&numaff=T-16/04.) The company has two months to appeal the decision, limited to points of law only, to the EU Court of Justice (Press Release, supra.)
The 2003 Directive establishes a scheme for greenhouse gas emission allowance trading within the European Community "in order to promote reductions of greenhouse gas emissions in a cost-effective and economically efficient manner" (art. 1). (Directive 2003/87/EC of the European Parliament and of the Council of 13 October 2003 [as amended to Apr. 23, 2009] Establishing a Scheme for Greenhouse Gas Emission Allowance Trading Within the Community and Amending Council Directive 96/61/EC, 2003 OFFICIAL JOURNAL OF THE EUROPEAN UNION (L 275) 32, available at http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:2003L0087:
20090625:EN:HTML.) Under the scheme, which is compatible with the United Nations Framework Convention on Climate Change and the Kyoto Protocol, EU Member States allocate GHG emissions allowances to domestic operators of installations (the types of which are listed in an annex to the Directive), in accordance with national allocation plans. Operators that reduce emissions may sell their surplus allowances to other operators; conversely, operators with emissions in excess of their allowances may purchase the necessary allowances from an operator with a surplus. (Press release, supra.)


text:
BANGOR, Maine — The Bangor-based New England Telehealth Consortium, working to link more than 400 health care facilities in northern New England to a telecommunications network, recently announced its 125th connection.
"Connecting the 125th healthcare site to the NETC network is a testament to years of planning, designing and building," says Jim Rogers, the founder of NETC and president of ProInfoNet, the company managing the implementation of the NETC network, in a press release. "From satellites enabling mobile health vans to serve rural Maine to crystal clear long-distance consultations for surgery, NETC is powering a cost-savings health care revolution."
Health care facilities linking to NETC's network are instantly sharing potentially life saving information and stand to save millions of dollars in annual technology costs.
"In health care, quality and reliability come with a cost," says Brian Thibeau, president of New England Telehealth Consortium. "NETC is affording rural and urban providers quality and reliability at significant savings while enabling health care facilities throughout Northern New England to provide patients access to a higher level care."
FairPoint Communications is the largest vendor partner in NETC.
"FairPoint's network provides flexible and expandable bandwidth and Ethernet-based solutions that NETC needs," says Mike Reed, FairPoint's Maine state president. "Since April 2008, FairPoint has invested more than $193 million in the communications infrastructure and technology in northern New England. This investment positions us to serve the technology needs of NETC through our extensive 'enterprise class' network with more than 15,000 miles of fiber offering IP/Ethernet services across Maine, New Hampshire and Vermont."
NETC has increased access to the most rural regions of New England by furnishing mobile clinics with satellite technology. Vans equipped with video conferencing, prescription dispensing and electronic health records are enabling the Maine Migrant Health Program to care for workers harvesting crops in Maine's rural Washington County.
The New England Telehealth Consortium, a regional group of rural and urban health care members, formed in response to the Federal Communications Commissions' Rural Health Care Pilot Program of the Telecom Act of 1996. The Act dedicated funding for aiding rural hospitals, clinics, and physicians through advanced telecommunications


text:
Melbourne: The tightening of media rights was the reason for the freeze on payments to Australian cricketers, International Cricket Council (ICC) president Malcolm Gray said on Wednesday. The Australian Cricket Board (ACB) and Australian Cricketers Association (ACC) have advised international and state cricketers their payments would not increase as predicted in the next two years. This follows a slowing in the growth rate projected for 2001-05. Gray said the freeze in payments was due to a slow down in the world economy and television rights 'flattening'. "Television deals won't necessarily decrease but that golden era of the 1990s has come and gone and I think it was a bubble," he said. "You've got to remember that cricket was financed almost by the chook raffle up until 20 years ago and then television came along over the last two decades and it was absolutely huge growth to all sports, including cricket." "The television market today is not what it was three years ago and I think that flatter market will continue." The player payment pool for 2002-03 is A$ 24 million ($15.3 million) and this had been projected to increase to A$ 26 million and A$ 28 million in each of the next two financial years. The ACB has revised the player payment pool projections to A$ 25 million and A$ 27 million for the next two years. Copyright AFP 2001
ACB to donate 00 to repossess Don's cap
'Sledging Aussies are not liked around the world'
Aussie cricketers to earn lesser than projected


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Donate to "Wish List"
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WASHINGTON to KEY WEST for $263 R/T via DELTA
Amazing deal: Washington to Key West flight roundtrip as low as $263.
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Colloidal hoary building complex both as a extra and as an bactericide. This resources that it can be exhausted in the bunking off of an actual unwellness or it can be used-up or utilised after the commencement of a learned profession question (in which case, it has an antibacterial drug office). Any specialistsability suggest mixture silver as a daily supplement, particularly to patients who have spot on hitches beside the immune system: they get raw often, they change assorted diseases out of the blue and they existing disunite forms even if they business near eubacterium or uncomplicated viruses. In these cases, the mixture shiny has the intention to shirk the start of viruses in the quality organism, patch reinforcing, at the same time, the tissues, the in shape cells and the total condition arrangement.
As an antibiotic, the mixture hoary has a chief role: to waste the viruses, germs or fungus kingdom. This is conceivable due to the information that the mixture shiny enables the breathing protein of the microorganism cells - as a result, these cells die in a short patch. Besides, the mixture shiny presents the control of anyone engaged severely smartly - this is why the symptoms are ameliorated in a punctual style. Even more, mixture silver itself is at the double eliminated from the living thing.
In totalling to the roles mentions above, mixture hoary has varied personal effects that can be thoughtful slightly desirable: it improves the skin, as all types of infectionsability and allergies are eliminated, it cleans the wounds (in this case, spout mixture shiny is applied on the pompous areas), it helps the biological process regulations to drive powerfully and it has bad personal effects upon visual percept (in the past, mixture grey was in use to get out the unloved microbes from the newborns' thought).Post ads:
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text:
A group discussion is a congregation (conference) control ended a duration (tele), hence the moniker. It is a contraption for businesses to convey ended long-term distances. Using the telecommunications technology, two or more parties come together from two or much locations.
The technologies that come in below for conference view video conferencing, aural conferencing, and information conferencing.
With the amount of investments that businesses are disbursal on these technologies increasing, it is blatant that the stress of teleconferencing in the house business organization global cannot be unnoticed.Post ads:
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Teleconferencing practical application let businesses to have meetings complete lifelong distances, doings company briefings, worker training sessions, conduct interviews, match up workshops and seminars, just give or take a few everything betwixt personnel who cannot easy come with in cooperation.
It is maximum commonly nearly new in the conglomerate situation for formality train meetings, distances basic cognitive process and training, and job interviewing. So, group discussion plainly makes a conglomerate much efficient and helps the company to let go notes.
How you may very well ask. Let us chew over the following points.Post ads:
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NO MORE FOOTLOOSE: With teleconferencing, a business organization can retrieve a considerable magnitude on transfer outlay. Communication will be by mistreatment the telecom methods, which is more timesaving and favorable.
An organization's force don't have to determination and voyage from one place to another acquisition overheads. Staff meetings, learning, habituation can all be conducted over teleconferencing, reaction hand motion to the least.
APPOINTMENT TIME: Businesses can use conference for recruiting and interviewing upcoming force. One of the benefits of teleconference in the achievement act is that the enterprise has the faculty to examination much candidates for the very cost. This reduces the worth of flying the candidates ended for the interview.
In addition, interviewers from more than than one geographic situation in the said business organization can assist in the examination process, in a unit effort, in need the fuss and disbursal of movement to a intermediate parcel of land. Result is enormous expenditure in your favour.
FACE-ON- FACE-OFF: With teleconference providing the facility of discussion calls, enterprise human resources do not have to instinctively join all different facade to face. Conference calls get content switch relating offices, personnel or from one firm position to another enormously simple, near purely the click of a fastening.
No cumbrous meetings to direct or long-winded mails to amass. In fact, group discussion has reduces the individualized meetings relating human resources to the showing least unless genuinely important. This has besides attenuated the holdup of work and case in attending meetings or reading and replying to mails. And definitely, the debt of group discussion is some little that a formal meeting!
THE NEED IS SPEED: Web-conferencing techniques are as well open with teleconference. This allows the simplified conducing of meetings and devising enterprise diplomacy. With the relief of applications same Excess, PowerPoint, businesses can payment by sending similes from one set to different in a entity of records.
Also, web conferencing includes tick messaging mating that can permit businesses to use instantaneous messages to spread or set up explicitly designed chat flat to doings discussions, meetings and preparation of projects.
GROUPIES NO LONGER: One of the most important advantages of conference is its latent to eat up the outlay of type meetings. The best explicit reward of teleconference is that of train meetings of distributed force inwardly the association.
This unremarkably takes the develop of government meetings, wherever force stumble upon from opposing ranked levels of the business, to revaluation organization objectives, performance, goals, or new administration issues. With teleconferencing, this activity gets simplified near less drive of followers and no condition to order the standing on ceremony set up.
Teleconferencing can besides be nearly new as a way of division information, such as as goods design, inside the same gradable flat of the enterprise. This can relief your business rescue funding by abiding juncture required to impart assemblage and info to not like groups inside the company.
THE CUTTING EDGE: More and more businesses are using teleconferencing to out of harm's way a ruthless assistance. This matched plus results from both tangible and intangible benefits. Definitely once group discussion is used in point of old-time concern travel, location will be a physical advantage of outflow nest egg to the business organisation system.
However, at hand are as well reasons for conference that may not yield determinable benefits, yet alter to the contending pre-eminence of the business organization. For example, it may be practical to allow more than predominant associates meetings inside the concern because costly journey is not mandatory.
The nethermost stripe is that next to teleconferencing, businesses can connect near comparatively smaller number disbursal. Teleconferencing saves loved occurrence and money, which are main to the gain of a conglomerate. There absolutely will be first costs involved next to background up the necessary equipment and technology, but it provides large saving in the overnight run.
So, don't shilly-shally and drop in the teleconferencing engineering at the earliest and set your enterprise on top the roadworthy to profit!!


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Happy Eid Mubarak guys!!! well it's the first Eid without my grandma :( but we still must celebrate it with cheer right ahaha(?) These are a few photos of....myself and few snacks and my cute little superman:/
well, those 2 photos taken in the night before this
big day and i had exactly no idea wht i'm doing-_-
This banana chips is really really delicious! you must try it.. and i kinda look like a toothpaste-commercial-girl


text:
For rent by Tangent Global Resources Limited VERIFIED 8 Mar 1:29 pmLekki, Lagos
This lovely house has four bedroom and a boys quarter and it is located before Ikota villa estate. It is well finished and in a good environment.
For more enquiries, please contact the agent
Stay safe!
Watch out for:


text:
About the Book
A photography book by Joel Gilgoff showing the country of Vietnam and its beautiful people. Contains 247 color images.
See More
About the Creator
Joel Gilgoff
Sedona, Arizona, USA
I've been a digital photographer for about 10 years and have published over 125 photography books. I teach advanced Adobe Photoshop classes and self publishing classes in Sedona, Arizona. I enjoy photographing people the most - but still practise all sorts of photography. See my web-site www.colornudes.com for my more creative images.
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MATLAB Newsgroup
(joe krystofik)
Hi, I've used Matlab a lot in the past for DSP and Image processing projects however I have an interest in what matlabs capabilities are with position and acceleration acquisition. I'd like to find some gyroscopes and accelerometers that can be used with matlab. Anyone have any experience with this?
I'm basically looking for some hardware (preferably smaller) that I could hook up to some moving test equipment, take data and transmit it to matlab. I suppose that would be done through a usb connection.
If anyone can help me out with what is good for matlab and direct me to some hardware that would be greatly appreciated. thanks guys
I use a Phidgets 3-axis USB accelerometer. There isn't an event-driven Matlab interface, so I call the Java interface from Matlab; alternatively I'm sure it would be fine to call the C interface from Matlab. It works well for both data logging and real-time interaction, given my fairly modest data rate requirements.
覺 have a project and I use nxt gyroscope and accelerometer but 覺 dont know how to see the graphics of the results . 覺f anyone can help me it would be greatly appreciated thanks a lot.
merlin <firstname.lastname@example.com/> wrote in message <email@example.com>...
> ? have a project and I use nxt gyroscope and accelerometer but ? dont know how to see the graphics of the results . ?f anyone can help me it would be greatly appreciated thanks a lot.
> Dear! I have task of interfacing Gyroscope M750 with Matlab for Real Time modeling. If u can help me in any way about this task, i will b very grateful.
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text:
This section contains 184 words
(approx. 1 page at 400 words per page)
Book 3, Chapter 6 Summary
Jenna moved to Panama City with her family when she was four. Her father tried to do the best he could. Her father felt that he had given them the best, but Jenna and Tony felt as if they had moved to the ghetto. Tony was always trying to protect Jenna from the other guys who would try to touch her and got into many fights. His father even told him to take care of one bully or not come home. Of, course Tony did what his father said. Even though Larry and his wife, Marjorie, worked long hours, and Tony and Jenna would stay out late and run the streets, they felt as if they had a normal and happy childhood.
Book 3, Chapter 6 Analysis
The reader sees that Larry wanted Tony to be tough, and Tony lived up to his father's expectations. He protected himself and Jenna. Even though Marjorie was not very nice to them, they were still happy, especially when it was just the two of them, Jenna and Tony taking on the world.
This section contains 184 words
(approx. 1 page at 400 words per page)


text:
We've finished studying the American Revolution, and are moving into a new unit on American Government; quite appropriate, it seemed, with elections only a few weeks off. So today we discussed the three branches of government and the concept of checks and balances. We're making a lapbook, so Brynning put together a mini-book on the three branches:
His concept of the function of the Executive branch may be more accurate than he knows. (For the record, I did explain that "to execute" in this instance is supposed to mean "to carry out". He came up with the firing squad on his own.)
16 hours ago


text:
I have found the bar scene quite difficult to meet people so have decided to try this out and see what happens.
I am up meeting to grab a drink or dinner but I am also willing to do something different and try something new.
I will eat pretty much anything, I wouldn't say I am picky but I am particular. I like a decent meal. I like Italian, Mexican and good seafood. You pick the place and I will likely find something delicious on the menu.


text:
I'm new to this company, didn't build the network, but am cleaning up some problems. I was told about the 2008 DC that would just quit working every couple of weeks, you couldn't even open AD Users and Computers and users couldn't access files. It finally happened. The only error I could find was lucheckupdates.exe would quit working - otherwise the system looked fine. It does have BE12 on it - I'm working on building a backup server so I can move that off of the server. My plan also includes downgrading this to a member server after I finish my other server builds. This server also houses all of the company data, so I don't want the file server on a DC. Has anyone seen this type of issue? I'm leaning towards Symantec BE12 being the culprit, but wanted to see if anyone has any knowledge of this - I did the usual google search to no avail, other than mentions of Symantec BE. thanks!
3 Replies
Jan 16, 2009 at 4:41 UTC
Anything coming up in the Event Logs?
What is the staus of the OS itself?
Are you patched up on Server 2K8?
You indicated that you don't have any replication servers currently...
Run DCDiag and NetDiag, look for errors or fails in the reports.
Jan 19, 2009 at 8:08 UTC
Was this server just bought for 2008? I have a server here now that just stopped working at different times and it turned out to be hardware issue. I also would not put it past symantec's software to screw it up either. Symantec (all products) have done some unexplainable things to my servers in the past. Don't put that up as the only possibility though. Easiest thing to do would be move BE12 to another server if you can.
Jan 19, 2009 at 8:22 UTC
Hey Big Timmy - yea, it's a PE 2900 - supposably came installed with 2008 server. I'm going to remove BE12 in the next week or so, as soon as I get my backup hardware - I'll let you know what happens.
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


text:
Robot Overlords | 2015
Set in a robot-occupied, futuristic Britain a boy and his friends struggle to survive in the search for his father.
Jump to
British Film Company
British Film Company
Near-future Earth has been conquered by robots, but in a seaside town in England, Sean Flynn and his friends have discovered a way to evade their oppressors. Convinced this his father - an RAF pilot who went missing in the war - is still alive, Sean and his crew set out to find his father and launch a rebellion against the robots.


text:
The Prodigal Wife (2 of 6) by Jerry Vines
This content is part of a series.The Prodigal Wife (2 of 6)
Jerry Vines
Hosea 2:1-13
There's trouble at the preacher's house. His worse
fears have now been confirmed. The Lord had said to
him, "Go take unto thee, Gomer, thy wife. But Hosea, I
want you to know that she's a prostitute."
This is one of those strange mysteries in the dealings
of the Lord. Acts 5:18 is a helpful verse in that
connection because there it says, "Known unto God are
all his ways from the beginning of the world. There
are some things about the ways of God we do not know.
But God knows. God knows what he is doing. God knows
His plans and His purposes. The Bible says his ways
are not our ways, His thoughts are not our thoughts.
So, this is one of those strange perplexing accounts
in the Bible which will await our going into the Lord
in heaven for full explanation. Somehow the Lord, in
His plan and in His providence had said to Hosea, "You
are to marry a woman who is a prostitute." Now Hosea
finds that just exactly what God had said to him is
coming to pass and it hits him like a laser.
This marital tragedy in the life of the preacher Hosea
is used as a backdrop for God to teach the tremendous
lesson of His love for His people. God loves us with
an infinite love. There is no way you and I could
fully understand or comprehend the great love of God
for us. So, God uses pictures and analogies and
illustrations in the bible. of all the illustrations
which God uses for His love, the illustration of
marriage is probably the most meaningful of all of
them. The marriage relationship is so intense and so
intimate that God uses to describe the love that is to
be between Himself and His redeemed people.
God says I have loved you with an everlasting love. I
have loved you with a love that is out of sight. So,
the marriage relationship is very important and an
individual breaks that marriage vow and sins against
the love of a mate, it is a very ...
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FRESNO, Calif. -- Steve Cleveland was hired to be Fresno
State's basketball coach on Saturday, and the former Brigham Young
coach pledged to restore integrity to a program rocked by years of
Cleveland, who went 138-108 in eight seasons at BYU, replaces
Ray Lopes, who resigned March 17 after a school investigation
determined he had broken an NCAA rule about telephone calls to
"My intent is to run this program at the highest level of
integrity," Cleveland said at a news conference at the Save Mart
Center. "To graduate young men, to have them doing those things on
and off the court that would bring great pride to this community.
"It's not going to be easy, and it's not going to be done next
Thursday, (but) I love the potential of this program, and the
potential of this community."
Jerry Tarkanian, Fresno State's previous coach, spent several
years dodging various scandals that undermined public confidence in
the program. In fact, the Bulldogs' entire athletic department has
been in turmoil since director Scott Johnson resigned just before
women's basketball coach Stacy Johnson-Klein was fired on March 2.
Cleveland's squeaky-clean reputation perhaps was even more
important to Fresno State than his coaching abilities. Though BYU
struggled this season, Cleveland's program largely stayed clear of
any scandals at the Mormon Church-owned university.
"He met and exceeded all the criteria that were set in the
university search," interim athletic director Paul Oliaro said.
"This last year has been a tough one for Steve at BYU, but his
overall performance there has been stellar -- and we know about
tough years, Steve."
Lopes went 50-37 in three seasons at Fresno State, winning the
WAC title in 2003. But the Bulldogs struggled near .500 for the
last two years, overshadowing the opening of the Save Mart Center.
"It was critical for us to attract a coach with high integrity
and a commitment to rules compliance," Fresno State president John
D. Welty said. "A coach who demonstrated a high priority for
student-athletes to perform in the classroom."
Cleveland's departure is yet another setback for BYU, which
recently fired football coach Gary Crowton and reorganized its
athletic department in response to years of slumping
Tom Holmoe, the former Cal football coach who became the
Cougars' new athletic director, already has begun to search for a
new basketball coach. Dave Rose, Cleveland's associate head coach,
is in charge of the program for now.


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Pineda de Mar is an attractive resort that appeals to the more mature couples. Families with young children will also enjoy it for its calm and relaxing atmosphere. The transfer time is around 40 minutes so you can take in your surroundings to get an understanding of the location of the resort and look at all the scenery. The water at the beach is very calm and appealing to people. The sands are pale and fine and you will find plenty of space to laze about on. There are a few apartments in the resort, although you are more likely to find 3-star hotels that cater to your needs.
Pineda de Mar is home to many water sports including windsurfing, snorkelling, scuba diving, jet-skiing and water-skiing. There are also glass-bottom boat trips where you can see shipwrecks and the marine life. Horse-riding, cycling and walking are brilliant ways to see the rest of the Costa Brava. If you would prefer to try some excursions then why not head to the water park for all of the rides, slides and swimming pools. If you still are not satisfied then why not head into Barcelona for some shopping, go the zoo, see the dolphins or go and see the Pyrenees Mountains.
Pineda de Mar has plenty of restaurants with a wide range of cuisines on offer. You will find seafood, Spanish, Italian and Chinese food on offer. Most evening entertainment is organised by hotels, however you will find one very popular nightclub along with some bars. Most dances and music are organised in hotels however you will also find music in the bars and a DJ in the club. There are shops in the resort offering food, clothing, jewellery and little crafts. There are several reliable buses that take you in and around the resort as well as a train that takes you into other resorts. All are reliable, regular and reasonably priced.
The average temperature in Pineda de Mar is around 22°C, however it can reach 30°C during the summer. On average, you will find that you get around 12 hours of sunshine a day during the summer, with this amount halving in the winter. July and August tend to be the hottest months so if you want it a bit milder then it is best to visit in May or June. There is barely any rain in Pineda de Mar, and it mainly falls between September and February, with December being the wettest month. During the autumn, the temperatures tend to be around 21-24°C and in winter they drop to 16°C.


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Dr. Benjamin Moorehead MD
- Sports Medicine Morgantown, WV
Patient Reviews
Reviews reflect patients' level of satisfaction with the care they received from this provider. Dr. Benjamin Moorehead xhas an overall rating of 4.8.
Dr. Benjamin Moorehead, MD is a Doctor primarily located in Morgantown, WV, with another office in Morgantown, WV. He has 15 years of experience. His specialties include Sports Medicine and Internal Medicine. Dr. Moorehead is affiliated with West Virginia University Hospitals-Ruby Memorial, West Virginia University Hospitals and Ruby Memorial Hospital. He speaks English.
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Dr. Benjamin Moorehead has the following 1 area of expertise, 2 specialties and 2 board certifications.
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West Virginia University Hospitals-Ruby Memorial
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women's ministry
Our desire as a women's ministry is to nurture the hearts of women to know God's Word and follow faithfully after Christ demonstrating authenticity and love, and serving as we are uniquely made. Like our Facebook Page to keep up to date!
upcoming events
Growing in the Dark, July17, 7pm, Fireside Room
Women's Bible Study Sign Ups Begin, August 1-31
Growing in the Dark, August 21, 7pm Fireside Room - Going to Norm's after
Women's Ministry Kick Off, August 27, 6-8pm, Fireside Room
A ministry for all ladies at Grace! Single? Married? Kids? 20's? 80's? This is where you can belong. Legacy: Women Impacting Our World is a place where you can enjoy intergenerational fellowship--meet old friends and make new ones, be encouraged in your role as a woman, and grow in your faith.
Check out the Upcoming Events listed above to find out the next Legacy meeting date along with speaker/program information and the registration and cancellation forms.
Registration will be required for each event.
Do you have a desire to experience genuine community? Our desire is to encourage women toward personal and spiritual maturity through the teaching and counsel from God's Word. Small group settings provide opportunities to engage in a deep and personal way. A list of available Bible Studies will be available soon!
growing in the dark
Growing in the Dark is a support group for women facing anxiety and depression. It meets monthly and strives to bring women together in community that understands each others' struggles with anxiety and depression. We want to foster a place where women can share freely, be known, and encourage each other to engage in the hard work of "struggling well." This includes techniques that help us to cope day-to-day and retrain our brains long-term. We also desperately want to help each other know Christ more and grow in our relationship with Him, who is the true Healer.
Growing in the Dark meets the 3rd Monday of the month, from 7:00-8:30PM in the Fireside Room at GCC. Registration not required.
missing motherhood
Are you going through the painful experience of infertility? Have you had a miscarriage or child loss? Do you have a child with special needs? We have a team of women that would love to walk with you through this valley as we seek God together in the pain. For more info please contact Julie Friesen at firstname.lastname@example.com.
encouraging hearts
One of our key values in women's ministry is the instructions by Paul for an older woman to teach the younger women. Our mentoring ministry encourages and provides direction for anyone who wants to be involved in this kind of relationship, either as a mentor or as someone being mentored. Our model for mentoring is taken from 1 Thessalonians 2:7-8: "We loved you dearly. Not content to just pass on the Message, we wanted to give you our hearts. And we did...holding your hand, whispering encouragement, showing you step-by-step how to live well before God..." (MSG) For more info please contact Della Loewen at email@example.com.
women's ministry council
Our Ministry Council is dedicated to meeting the needs of women at Grace Community Church and the community. If you have questions or feedback regarding the programs
offered, then please feel free to contact us! Get in touch with Della Loewen at firstname.lastname@example.com. View the Women's Ministry Council brochure.


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