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Adrenergic hormones are essential for early heart development. To gain insight into understanding how these hormones influence heart development, we evaluated genomic expression changes in embryonic hearts from adrenergic-deficient and wild-type control mice. To perform this study, we used a mouse model with targeted disruption of the Dopamine β-hydroxylase (Dbh) gene, whose product is responsible for enzymatic conversion of dopamine into norepinephrine. Embryos homozygous for the null allele (Dbh−/−) die from heart failure beginning as early as embryonic day 10.5 (E10.5). To assess underlying causes of heart failure, we isolated hearts from Dbh−/− and Dbh+/+ embryos prior to manifestation of the phenotype and examined gene expression changes using genomic Affymetrix 430A 2.0 arrays, which enabled simultaneous evaluation of >22,000 genes. We found that only 22 expressed genes showed a significant twofold or greater change, representing ∼0.1% of the total genes analyzed. More than half of these genes are associated with either metabolism (31%) or signal transduction (22%). Remarkably, several of the altered genes encode for proteins that are directly involved in retinoic acid (RA) biosynthesis and transport. Subsequent evaluation showed that RA concentrations were significantly elevated by an average of ∼3-fold in adrenergic-deficient (Dbh−/−) embryos compared with controls, thereby suggesting that RA may be an important downstream mediator of adrenergic action during embryonic heart development.
- hormone
- mouse
- heart
- catecholamine
- microarray
- ultrasound
adrenergic hormones play an essential yet unexplained role in the mammalian heart during embryonic/fetal development (17, 43, 44, 56, 99, 109). Norepinephrine (NE) and epinephrine (EPI) are the main adrenergic hormones/neurotransmitters in mammals. In adults, EPI and NE are primarily produced in the chromaffin cells of the adrenal medulla as well as in neurons in the sympathetic and central nervous systems. Numerous other sources of adrenergic hormones have also been found in a diverse array of tissues and organs including the organ of Zuckerkandl (78), retina (30), kidney (28, 52), spleen (28, 52, 77), testes (11), lungs (28, 52, 77), and heart (1, 14, 16, 18, 19, 44, 46, 47, 52, 74–76). In the mid-1990s, it was discovered that NE and/or EPI are vital for embryonic survival in utero since disruption of the genes encoding for the catecholamine biosynthetic enzymes, tyrosine hydroxylase (Th) or dopamine β-hydroxylase (Dbh), resulted in fetal and perinatal lethality due to apparent heart failure (55, 99, 109). In the absence of NE and EPI, embryos survive and appear relatively "normal" until approximately embryonic day 10.5 (E10.5) but then begin to die over the next several days with ∼90% of the Dbh−/− mice not surviving to birth. We showed that disruption of the EPI-specific biosynthetic enzyme, phenylethanolamine n-methyltransferase (Pnmt), results in a loss of EPI but does not display any obvious developmental phenotype (16). Since Pnmt−/− mice still produce NE, this hormone likely compensates, in part, for the loss of EPI. NE and EPI can both stimulate adrenergic receptors.
It is known that while both α- and β-adrenergic receptors are expressed and functional in the developing heart (21, 32, 42, 62, 66, 67, 70, 84, 102), it is primarily the β-receptors that are important for survival through birth and the early postnatal period (25, 49, 70, 79, 98, 100). Drug rescue experiments, for example, showed that the β-agonist isoproterenol could rescue Dbh−/− embryos when supplied in the maternal drinking water (98, 100). In contrast, the α-agonist l-phenylephrine had little effect when administered alone but appeared to slightly improve survival when used in combination with isoproterenol (79, 98, 100). Furthermore, targeted deletion of the β-adrenergic receptor kinase 1 (βARK-1) gene in mice resulted in embryonic lethality due to heart failure with many phenotypic characteristics similar to those described for Dbh−/− embryos (49). In addition, genetic knockout of β1- and β2-adrenergic receptors led to cardiovascular and metabolic defects (8, 85, 86). These studies in total indicate that NE stimulation of β-adrenergic receptors is crucial for heart development and embryonic survival. The downstream targets and molecular signaling mechanisms secondary to the stimulation of adrenergic receptors in the developing heart are not well-defined.
Recent reports suggest that hypoxia may be an important incoming signal that stimulates NE release, which, in turn, stimulates heart rate during hypoxic stress (79, 81). A hypothesis drawn from these studies is that adrenergic-deficient embryos are unable to respond to hypoxic stress by releasing NE and thus cannot properly compensate for lower O2 concentrations by increasing cardiac output. A prediction from this hypothesis is that adrenergic-deficient embryos would become increasingly hypoxic over time and ultimately perish as a result of inadequate blood flow. While there is good evidence supporting the hypoxia hypothesis, it does not appear to provide a full explanation for adrenergic action in the developing heart. Notably, the hypoxia experiments were primarily performed with isolated fetal hearts at E12.5 and later stages, whereas Dbh−/− embryos tend to begin dying a couple of days earlier. Furthermore, most of the supportive evidence stems from ex vivo cultures where conditions were carefully controlled and thus not completely reflective of the in vivo environment. It therefore remains unclear if hypoxia is a causative stimulus for adrenergic action in vivo, particularly at early embryonic stages.
To investigate underlying mechanisms that mediate adrenergic actions in early heart development, we utilized a Dbh−/− mouse model to evaluate physiological and genetic changes associated with the loss of NE and EPI during the early embryonic period extending from E9.5 to E12.5 (17). We employed a combination of noninvasive high-resolution ultrasound imaging and genome-wide microarray analyses to evaluate cardiac changes that occurred due to the absence of endogenous adrenergic hormones. These results suggest that the major categories of genes affected by the absence of adrenergic hormones are those involved in metabolism and signal transduction. In particular, we show that multiple retinoic acid (RA) synthesis and signaling genes display altered expression leading to significant increases in embryonic RA concentrations during a critical phase of early heart development.
All procedures and care of the mice were conducted in accordance with National Institutes of Health guidelines for use of vertebrate animals in research. All animal procedures were reviewed and approved by the appropriate Institutional Animal Care and Use Committees. Breeding and maintenance of the Dbh knockout mouse model were performed as previously described (99). Timed matings were performed with E0.5 defined as noon of the day of vaginal plug verification. All-trans RA (Sigma-Aldrich, St. Louis, MO) was diluted in peanut oil and administered on E10 via oral gavage (50 mg/kg) as previously described (9, 83). Vehicle-only controls received peanut oil only in the same volume as used for RA administration (0.1 ml per mouse).
Embryonic heart rates were evaluated noninvasively in vivo using a Vevo 660 high-resolution ultrasound instrument (Visualsonics, Toronto, Canada). Briefly, pregnant mice were anesthetized with isoflurane and placed onto a heated stage. Maternal body temperature and heart rate were monitored via rectal thermometer and limb ECG leads, respectively. Abdominal hair was removed with Nair hair removal product, and ultrasound data were collected for each embryo using 40 or 55 MHz transducers. Embryonic heart rates were measured using instrument software and confirmed by visual inspection of the recordings. The dams were returned to their cages with heating pads to facilitate recovery. Recordings were performed daily beginning at E9.5.
Embryonic tissue collection.
The dams were killed and the embryos were collected for genotyping and gene expression analyses immediately after the last ultrasound recording session. In most cases, we could readily correlate the position of the embryo with the ultrasound recording. In some cases, however, it was not always clear, especially if the litter was large. Only those embryos that were clearly identified on ultrasound recordings at the time of isolation (i.e., prior to genotype determination) were used for further analysis. For microarray studies, Dbh+/− sire and dam were mated generating Dbh+/+, Dbh+/−, and Dbh−/− offspring collected at E10.5. Furthermore, only embryos that were viable with clearly beating hearts, bright red blood, and no signs of stunted or abnormal morphological development apparent upon visual inspection under a dissecting microscope were used for the microarray, RT-PCR, and RA bioassay assessments. The head was rapidly removed for DNA extraction and genotyping by PCR methods and primers as previously described (99). For the microarray and RT-PCR analyses, the heart was isolated and rapidly frozen for subsequent RNA extraction as described below. For the RA bioassay, the entire embryonic trunk, including the heart, was rapidly frozen following removal of the head for DNA extraction. Tissues were further processed for each assay as described below.
RNA isolation.
Hearts collected for mRNA expression analysis were rapidly frozen in prechilled microfuge tubes on dry ice and stored at −80°C for subsequent use. Frozen embryonic hearts were homogenized in TRIzol (Sigma Aldrich, St. Louis, MO), and total RNA was extracted by established methods as previously described (15). Total RNA quality and yield were assessed with NanoDrop 1000. Only total RNA samples showing an OD260/OD280 ratio between 1.8 and 2.1 were used for microarray and real-time PCR.
Microarray data analysis.
Mouse Genome 430A 2.0 Arrays (AffyMetrix, Santa Clara, CA) were used to analyze gene expression differences in Dbh+/+ and Dbh−/− hearts at E10.5. Purified total RNA was prepared from frozen embryonic hearts and delivered to Gene Logic (Gaithersburg, MD) to perform the microarray analysis. RNA quality-control results and basic statistical analysis of gene expression results were provided by Gene Logic. Access to these files as well as the raw microarray data files from this experiment is provided at the following web link: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=rfghpyusacumoho&acc=GSE33906, These files include individual chip data, MAS 5 signal intensities, detection calls, and detection P values. Probe set annotations included in these files provide the gene name, ontology, pathway membership, and Unigene ID for each gene on the array. Additional information about the basic statistical analysis can be found below under Statistical methods. The networks and canonical pathways were generated through the use of Ingenuity Pathway Analysis (IPA) algorithms (Ingenuity Systems, http://www.ingenuity.com).
Real-time PCR analysis.
Genomic DNA was removed from the isolated RNA by digestion with RNase-free DNase (Promega, Madison, WI). The cDNA was prepared using Affinity Script QPCR cDNA synthesis kit (600559; Stratagene, Cedar Creek, TX). We used 0.4 μg total RNA for each cDNA synthesis reaction. Real-time PCR reactions were performed using IQ SYBR Green super mix (Bio-Rad Laboratories, Hercules, CA) and run in the Bio-Rad ICycler. Oligonucleotide sequences and annealing temperatures are shown in Table 1.
RA bioassay.
F9-RARE-LacZ cells were kindly provided by Drs. Michael Wagner and Alexander Moise (SUNY Downstate Medical Center, Brooklyn, NY) (104). These cells were maintained in L-15 Leibovitz media containing 20% heat inactivated, charcoal-stripped fetal bovine serum and 1% PenStrep (vol/vol) as previously described (104). We used a modification of previous assay methods for quantification of β-galactosidase activity (91, 104, 108) as described below. The cells were grown to confluence (80–90%) on 100 mm gelatin-coated dishes then plated at 60% confluence on 24-well dishes and allowed to settle overnight. Immediately before the assay individual embryonic trunks were homogenized via 5 s sonication twice in F9-RARE-LacZ cell culture assay media (passage media plus 1.6 g/l d-glucose) under dim lighting. The homogenate was centrifuged at 8,000 g for 5 min to remove cell debris before transfer to the F9-RARE-LacZ cells. Immediately prior to adding the homogenate, we removed the normal passage media from the F9-RARE-LacZ cells and added 0.9 ml of assay media followed by 100 μl of the embryonic trunk homogenate. The cells were then incubated overnight at 37°C and 5% CO2. For the detection of β-galactosidase activity using the fluorescein di-β-d-galactopyranoside (FDG) substrate, cocultured F9-RARE-lacZ cells were lysed in 100μl of Z-buffer (0.8 g Na2HPO4, 0.28 g NaH2PO4, 0.5 ml 1 M KCL, 0.05 ml 1 M MgSO4, and 0.135 ml β-mercaptoethanol, all dissolved in pure water at a final volume of 50 ml) via 5 s sonication, and added to a 96-well plate with the addition of 2 μl of 1 mM FDG. Light production was measured with an EnVision Multilabel plate reader (PerkinElmer).
Statistical methods.
Data are presented as means ± SE, except where specified otherwise. Student's t-test (two-sample comparisons) or one-way analysis of variance (three or more sample comparisons) was performed with P < 0.05 required to reject the null hypothesis. For post hoc testing, the Bonferroni transformation was used to assess statistical significance of differences between specific groups. The basic statistical analysis of gene expression data from the microarray provides the mean, standard deviation, and percent present (P) values for each gene. For pairwise statistical comparison between Dbh+/+ and Dbh−/− E10.5 gene expression data (n = 4 each), fold-change and t-test P values are indicated for each gene on the array. For canonical pathway analysis, Fisher's exact test was used to determine significance (P < 0.05) (Ingenuity Systems, http://www.ingenuity.com).
To investigate how adrenergic deficiency leads to embryonic heart failure and death in utero, we used high-resolution ultrasound imaging to evaluate cardiac performance in adrenergic-deficient (Dbh−/−) and adrenergic-competent (Dbh+/+ and Dbh+/−) mouse hearts in vivo. Representative images from this analysis are shown in Fig. 1, A and B, where sagittal views of an E10.5 mouse illustrate the ability to detect cardiac contractions and ability to measure crown-rump length as an estimation of embryonic age (51). Heart rates were assessed on viable E9.5, E10.5, and E11.5 embryos. Embryos were subsequently collected for genotyping and mRNA expression profiling as described in materials and methods. No significant differences were observed in mean heart rates from adrenergic-deficient mouse embryos compared with controls at any of these ages (Fig. 1C).
Overall attrition rates were similar between genotypes at E9.5 and E10.5 but increased dramatically in the Dbh−/− group at E11.5, with roughly half of the adrenergic-deficient population failing to survive to this stage of development (Fig. 1D). These findings are consistent with the original reports that most Dbh−/− mice survive through E10.5 but then steadily decline in numbers over the next few days with >80% dead before birth in heterozygous (Dbh+/−) dams (99). The most notable differences were observed in the heart rate and rhythm of the Dbh−/− embryos shortly before they died after E10.5. Having recorded heart rates from the same sets of embryos on successive developmental days, we were able to witness this phenomenon directly. Heart rates would appear essentially normal for all genotypes at E9.5, E10.5, and E11.5, but occasionally a few Dbh−/− hearts that were beating well at E9.5, E10.5, or even E11.5 began to develop slow, labored contractions sometimes with associated asynchronous and arrhythmic activity. An example of one such recording of a Dbh−/− heart at E10.5 is shown in Supplemental Video 1.1 The heart rate of this Dbh−/− embryo had been ∼60 beats/min (bpm) at E9.5, similar to that of the adrenergic-competent embryos (Fig. 1C), but had declined to ∼34 bpm by E10.5, and stopped sometime between E10.5 and E11.5, as no heart beat was detected on the final round of ultrasound measurements for this particular litter. Other Dbh−/− embryos within the same litter survived with little or no observable difference in heart rates through E11.5. In other cases, the slow labored irregular contractile activity first appeared at E11.5 or E12.5, typically followed by death within 24 h. Consequently, it appears that beating rates develop normally during the early phase of heart development but then start to decline beginning around E10.5 in some Dbh−/− hearts but not in others. There appears to be a window that extends for a few days where the heart is vulnerable to the absence of adrenergic hormones, but this does not always begin at E10.5.
We took advantage of our echocardiographic assessments to identify viable hearts of various genotypes (Dbh+/+, Dbh+/−, and Dbh−/−) that were beating at normal physiological rates. These hearts were immediately collected for analysis of gene expression changes at E10.5, near the beginning of the developmental period where embryos show greatest vulnerability to adrenergic deficiency (99, 109). The objective of this experiment was to determine if there were underlying alterations in gene expression profiles that precede the physiological decline and ultimate death of Dbh−/− embryos in utero. To minimize confounding variables related to downstream gene expression changes incurred as a consequence of heart failure in general, we sought to obtain early specimens that were asymptomatic. The criteria that we used included assessment of heart rates, crown-rump lengths, and general morphological/structural appearance at time of dissection. The embryos collected for this analysis were indistinguishable from E10.5 wild-type controls in terms of these criteria. Notably, all appeared to be in relatively good health with adequate cardiac function at the time of isolation at E10.5.
We identified four viable Dbh−/− (experimental) and four Dbh+/+ (control) age- and size-matched E10.5 mouse hearts for microarray analysis. To assess changes in gene expression between these two groups, we extracted RNA and analyzed the samples using Mouse Genome 430A 2.0 Arrays (Affymetrix, Santa Clara, CA). These arrays permit simultaneous evaluation of >22,000 annotated genes in the mouse genome. The microarray data were analyzed using the Affymetrix MAS5.0 software to compare the two groups according to the following criteria: 1) P value < 0.05, 2) ≥2-fold change, and 3) detection call and signal must be present (P) in at least half of the samples from either group. This yielded a list of 22 genes (23 transcripts) that were significantly (P < 0.05) altered by a factor of two or more between E10.5 Dbh−/− and Dbh+/+ hearts (Table 2). Nine of these genes were downregulated in Dbh−/− hearts relative to Dbh+/+ hearts, with the RA biosynthetic enzyme, β-carotene 15,15′-monooxygenase (Bcmo1), and phospholipase A2, group IID (Pla2g2d) genes displaying the greatest decreases (-2.7-fold). Twelve genes showed marked increase in expression, with fibroblast growth factor 20 (Fgf20) commanding the greatest increase (+5.3-fold). One gene, fibromodulin (Fmod), was identified twice (from two different target sequences on the array) as a gene displaying increased expression (+2.2-fold and +2.7-fold) in the adrenergic-deficient hearts. An analysis of general ontology categories of the altered genes identified from the microarray data shows that most changes were found in metabolic (31%) or signal transduction (22%) genes (Fig. 2). Other categories showing significant expression changes included those encoding for extracellular matrix (ECM) and structural proteins (17%), transport proteins (17%), transcription factors (4%), and proteins of unknown function (9%).
To gain insight about how these various alterations in gene expression may be connected, we performed pathway analysis using the Ingenuity Systems Knowledge Database (http://www.ingenuity.com). The top networks identified were: amino acid metabolism, molecular transport, and small molecule biochemistry (Fig. 3A) and cellular development, nervous system development, and developmental disorders (Fig. 3B). The top biological functions and canonical pathways associated with the altered genes are highlighted in Table 3.
Of particular note, two of the most significantly altered genes are directly involved in RA metabolism (Bcmo1 and Rdh12: see boldfaced items in Table 2 and the top canonical pathways in Table 3). A representative comparison of changes observed from the microarray data with independent assessments of gene expression by RT-PCR is shown for the Bcmo1 gene in Fig. 4, where it can be seen that similar data were obtained by either method. The RT-PCR results showed a slightly larger 3.7-fold decrease in Bcmo1 gene expression compared with the 2.7-fold decrease obtained from the microarray data (Fig. 4A). Verification of the accuracy of the RT-PCR amplification with the Bcmo1 primers is shown by agarose gel electrophoresis in Fig. 4B at the termination of the RT-PCR reactions. These data support the finding that Bcmo1 mRNA expression is decreased in adrenergic-deficient E10.5 mouse hearts relative to controls.
We independently evaluated a sample of additional genes from the microarray analysis using RT-PCR methods. These genes include key RA indicator genes such as Aldehyde dehydrogenase family 1, subfamily a2 (Aldh1a2, also known as Retinaldehyde dehydrogenase 2 or Raldh2) and Retinol-binding protein 1 (Rbp1). The results are summarized in Table 4. The Aldh1a2 gene encodes for the principal rate-limiting enzyme for RA biosynthesis and is essential for heart development and embryonic survival (72). Changes in Rbp1 expression generally parallel changes in RA concentration, and thus expression of this gene is often used as an indicator of increased or decreased RA concentrations (22, 45, 105). We also measured Retinol dehydrogenase 12 (Rdh12) expression using RT-PCR but could not precisely determine fold-difference for this gene because expression was undetectable in 75% of the wild-type control heart samples (data not shown). Rdh12 expression was detected in 50% of the Dbh−/− hearts both by RT-PCR and microarray analyses, suggesting that its expression was likely induced in adrenergic-deficient E10.5 mouse hearts relative to wild-type controls.
To facilitate comparison of the microarray and RT-PCR results, we performed correlation analysis with linear regression and 95% confidence intervals for each of the target genes shown in Table 4. While the RT-PCR and microarray results showed similar degree and direction of change in most cases, some exceptions were also observed. For example, we did not observe good correlation for Neurofibromin 1 (Nf1). The microarray results suggested 2.2-fold decreased expression for Nf1 in Dbh−/− hearts, but the RT-PCR results indicated a 1.2-fold increase. To investigate this discrepancy, we tried an alternative RT-PCR primer set for Nf1 as there are several splice variants of this gene; however, this still did not reproduce the decreased expression of Nf1 observed in the microarray dataset. With the exception of Nf1, all other targets examined showed similarity between the microarray and RT-PCR results, both in terms of the magnitude and direction of the change. From this subset of genes, all lie within the 95% confidence intervals for linear regression analysis (data not shown). In general, these results validate the findings of the microarray dataset.
Since previous studies had suggested that hypoxia may be an important physiological inducer of adrenergic action in the developing heart (79–81, 101), we evaluated potential expression changes in well-known hypoxia-sensitive genes such as hypoxia-inducible factor 1α (Hif1α) and the vascular endothelial growth factor A (VegfA), but no significant differences were observed (see following web link for access to full microarray dataset and basic statistical analysis: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=rfghpyusacumoho&acc=GSE33906). To examine this further, we performed RT-PCR analysis on VegfA expression in adrenergic-deficient and control mouse hearts across multiple ages including E9.5, E10.5, and E11.5, but VegfA expression was remarkably consistent in both groups during this period (Fig. 5). These data suggest that adrenergic-deficient embryos are not in hypoxic distress during this early period of embryonic development.
As indicated above, there were a number of RA metabolic and other genes whose expression was significantly altered on the microarray (Table 2), and several of these were verified by independent RT-PCR analysis (Table 4). These results prompted us to probe the microarray data further to determine if other genes involved with RA synthesis and signaling were also altered in adrenergic-deficient hearts. Closer examination of the microarray data for RA-related genes revealed that many of them were altered in adrenergic-deficient mouse hearts. For example, a notably altered gene involved in RA synthesis was Aldh1a2 (1.5-fold increase, P = 0.089), and a gene involved in RA storage was lecithin retinol acetyltransferase (Lrat) (2.2-fold increase, P = 0.078) (33, 54, 73). Although these genes did not meet the arbitrary conventional threshold for significance (P < 0.05), they nevertheless displayed consistent changes in a relatively small sample size (n = 4 for each genotype, Dbh+/+ and Dbh−/−). Similarly, several other genes involved in RA metabolism also displayed increased expression in Dbh−/− E10.5 mouse hearts relative to control Dbh+/+ hearts (Fig. 6A). Although some of these gene expression changes were not found to be "significant," they appeared to be indicative of a trend toward increased production of RA in adrenergic-deficient hearts. We have included a schematic diagram to show how each of the products of these genes is involved in RA metabolism (Fig. 6B).
To determine if RA concentrations were actually altered in concert with the observed gene expression changes in RA metabolic genes in adrenergic-deficient mouse embryos, we employed a sensitive bioassay (91, 104, 108) to assess RA concentrations in both adrenergic-deficient (Dbh−/−) and adrenergic-competent (Dbh+/−) embryos. Using this approach, we could accurately measure RA concentrations between 30 and 300 pM as shown by the nearly linear (r2 = 0.97) standard curve over this range of concentrations (Fig. 7A). Embryonic RA concentrations fell within this range. When normalized to the total amount of protein per sample, the average RA concentration in wild-type embryos was 14.6 ± 4.8 pM/mg total protein. Quantitative comparison with Dbh−/− embryos showed that RA concentrations were significantly elevated (P < 0.05) by nearly threefold (43.4 ± 10.1 pM/mg total protein) compared with Dbh+/− controls (Fig. 7B). These results demonstrate that RA concentrations were sharply increased in adrenergic-deficient E10.5 mouse embryos during a critical period of development when the heart is exquisitely sensitive to hormonal fluctuations (13, 29, 51, 68, 84, 88, 94, 99, 106, 109).
If the gene expression changes observed on the microarray were due to increases in embryonic RA concentrations resulting from adrenergic deficiency, then one might predict that administration of exogenous RA should produce similar effects. To test this idea, we administered RA (50 mg/kg) to the pregnant dams and then measured embryonic RA concentrations at 1, 2, and 4 h from samples collected at E10.5. Little change was observed at 1 h, but RA concentrations increased >3-fold above control (vehicle-only gavage) by 2 h and remained at similar though slightly declining levels through 4 h (Fig. 8A). In parallel, we also measured gene expression changes for a few representative RA-associated genes in isolated E10.5 hearts from these embryos. As shown in Fig. 8B, both Bcmo1 and Fgf20 were highly responsive to RA administration, with peak expression levels observed at 4 h. In contrast, Rbp1 expression was mildly increased similar to that observed in the microarray analysis (Fig. 6) and did not appear to fluctuate much over this time course. These results demonstrate that cardiac Rbp1, Bcmo1, and Fgf20 were responsive to increases in embryonic RA concentrations at E10.5.
In this study, we have examined in vivo heart rates and cardiac gene expression changes associated with adrenergic deficiency in a mouse model (Dbh−/−) of embryonic heart failure. Our results show that embryonic heart rates were remarkably similar in adrenergic-deficient and adrenergic-competent hearts until shortly before the onset of heart failure and death. Physiological signs of heart failure included bradycardia with associated arrhythmia and asynchrony. When these signs appeared, the embryos were typically found dead within 24 h. Surprisingly little change was observed in hypoxia-sensitive genes, suggesting that adrenergic-deficient embryos may not necessarily experience abnormal or excessive hypoxic stress during this critical early period of heart development (E9.5–E11.5) in mouse embryos. Our results thus suggest that adrenergic deficiency may lead to heart failure and death due to mechanisms other than those induced by hypoxia.
The use of noninvasive high-resolution ultrasound imaging provided a glimpse into the physiological characteristics of cardiac performance in utero, thereby enabling us to assess the effects of adrenergic deficiency on embryonic heart rates compared with control embryos vivo. This extends earlier work that evaluated heart rates in explanted E12.5 Th−/− embryos at room temperature, where a slight but significant decrease of ∼15% was observed in heart rates from the adrenergic-deficient group (109). More recent studies showed similar heart rates for adrenergic-deficient (Th−/−) and control hearts in ex vivo cultures at physiological temperature under normoxic conditions but significantly slower rates for the adrenergic-deficient hearts under hypoxic conditions (79). An additional study also showed similar heart rates for Th−/− and Th+/+ embryos in vivo following acutely administered oxygen with the animals under anesthesia (81). We did not specifically examine hypoxia in the present study, but our results are consistent with the previous findings, indicating that there is little or no difference in heart rates in adrenergic-deficient embryos compared with controls under normal conditions. Our results are in alignment with previous findings of in vivo studies performed with Th−/− embryos by specifically showing that Dbh−/− embryos exhibit heart rates indistinguishable from Dbh+/+ and Dbh+/− controls until shortly before death when physiological signs of heart failure become evident.
Measurement of genome-wide changes in gene expression in asymptomatic hearts prior to the onset of heart failure revealed a limited population of 22 genes from a total of >22,000 total genes that showed significant expression changes of twofold or greater magnitude. More than half of the 22 altered genes are involved in either metabolism or signal transduction pathways, including two genes that directly participate in RA synthesis, Bcmo1 and Rdh12. Interestingly, both Bcmo1 and Rdh12 encode for enzymes that produce retinal; however, they utilize different substrates. Retinol is the substrate for Rdh12, and β-carotene is the substrate for Bcmo1. In the case of Rdh12, there are multiple isoforms that comprise the retinol dehydrogenase family of enzymes, and based on our microarray data it is clear that several of these are expressed much more robustly than Rdh12 in the embryonic heart (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=rfghpyusacumoho&acc=GSE33906). No significant differences in expression of the other isoforms of the Rdh gene were observed from the microarray analysis of adrenergic-deficient and control embryonic mouse hearts. Despite the apparently minor role for Rdh12 under these circumstances, induction of its expression in adrenergic-deficient hearts during embryonic development appears to serve as an indicator of perturbations in RA metabolism.
In contrast, the Bcmo1 gene product is the primary enzyme and only isoform characterized to generate two molecules of retinal for each molecule of β-carotene (4, 27, 82). Although the Bcmo1 gene does not appear to be essential for mouse development when vitamin A is sufficient in the diet (38), its role becomes much more important when vitamin A intake is limited (53, 61). Bcmo1 is a highly conserved gene found in animals as diverse as fruit flies, chickens, and mammals (3). Moreover, Bcmo1 is critical for embryogenesis in zebrafish (59). Expression of Bcmo1 is known to be robust in early mammalian embryogenesis (82), consistent with our observation that it is expressed in the embryonic heart. These results suggest that appropriate Bcmo1 expression and regulation are important for animal development. In our study, Bcmo1 expression was significantly decreased roughly threefold in Dbh−/− hearts at E10.5, coincident with the observed elevations in RA concentrations measured in Dbh−/− embryos at this stage of development. Interestingly, however, its expression increased dramatically in response to maternal RA administration. These data imply that the decreased expression of Bcmo1 in Dbh−/− hearts may not solely be due to the observed increase in RA concentrations but likely involve other regulatory components. Indeed, previous examination of the Bcmo1 promoter identified a RA-responsive element (RARE) and a putative cAMP-responsive element immediately adjacent to the RARE, as well as a number of other transcriptional regulatory elements (27). The fact that Bcmo1 expression is responsive to RA in mouse embryos is consistent with the RARE in its promoter and earlier studies showing that this gene is sensitive to transcriptional regulation in the presence of RA in other systems (4, 97). An intriguing hypothesis generated from our results is that the cAMP-responsive element in the Bcmo1 promoter plays an important role in stimulating Bcmo1 expression in the embryonic heart during this formative period of early heart development. Future experiments aim to test this and other hypotheses that will explain how Bcmo1 gene expression is influenced by developmental hormones. It is nonetheless clear that Bcmo1 expression is responsive to hormonal influences in the embryonic heart, thus providing one of the first tangible pieces of evidence indicating that there may be functional cross talk between adrenergic and RA signaling pathways in the embryonic heart.
Changes in gene expression such as those noted above provide strong circumstantial evidence that RA synthesis and signaling are awry in adrenergic-deficient hearts, but perhaps the most convincing direct evidence comes from our demonstration that RA concentrations themselves were significantly elevated in adrenergic-deficient mouse embryos. This finding is consistent with the observed increased expression of RA biosynthesis genes such as Aldh1a2 and Rdh12, as well as retinol-binding proteins, Rbp1 and Rbp7, and RA-binding proteins, Crabp1 and Crabp2 (69, 87, 92). Increased expression of the gene encoding the RA storage enzyme, Lrat, is also consistent with a previous report showing that Lrat is transcriptionally stimulated by RA (110). Interestingly, published reports have shown that Lrat and Bcmo1 appear to be reciprocally expressed during early mouse development (53), as well as in obese adult rats (96), in a manner that appears not entirely dissimilar to the inverse expression profiles for Lrat and Bcmo1 observed in Dbh−/− embryonic mouse hearts.
The hypothesis that RA synthesis was perturbed in adrenergic-deficient hearts is further supported by closer examination of other genes involved in RA metabolism, many of which displayed altered expression patterns. Notable among these were Aldh1a2 and Lrat, whose products play critical roles in RA synthesis (72) and storage (33, 54, 73), respectively. Additional supporting evidence is provided by the microarray results showing that several known RA-responsive genes also showed significantly altered gene expression in adrenergic-deficient embryonic mouse hearts compared with controls. These include the Pla2g2d (20), Nf 1 (41, 64), Mgp (7), and Col9a3 (107) genes (Table 2). This hypothesis is also supported by the pathway analysis shown in Fig. 3B, where tretinoin, all-trans RA, is seen to be at the center of several connected pathways and thus may serve to coordinate and integrate expression of many of the associated genes shown.
One of these genes, Fgf20, appears to merit special attention because it was the most strongly and significantly induced (5.3-fold increase, P < 0.001, n = 4/group) gene in Dbh−/− hearts from the entire microarray analysis (see Table 2). Moreover, it is clear that various Fgf isoforms serve as important mediators of RA actions during development (12, 71). Of particular relevance is a study by Lavine et al. (60) that showed Fgf20 expression in E10.5 mouse hearts where it was primarily found in ventricular (left and right) epicardium and endocardium, as well as in the outflow tract region. Fgf20 is closely related to Fgf9 and Fgf16 in terms of sequence homology, cardiac spatio-temporal expression patterns, and Fgf receptor selectivity (31, 48, 60). Although RA regulation of Fgf20 has not been previously examined directly, Lavine et al. (60) showed that RA treatment of embryonic mouse hearts significantly induced Fgf9. These findings are consistent with the results from the present study showing that Fgf20 expression was strongly induced when embryonic RA concentrations were elevated following maternal administration of RA and in Dbh−/− mouse embryos at E10.5. Taken together, these findings suggest that Fgf20 expression is stimulated by RA in E10.5 mouse hearts and that the increased expression of Fgf20 in adrenergic-deficient hearts may be a direct result of the elevated RA concentrations observed in these embryos. Further testing will be required to confirm this hypothesis and determine what consequence(s) may result from transient increases in Fgf20 expression in the developing heart.
Several additional genes and pathways from the microarray analysis described here appear likely to play a role in the observed cardiac phenotypes found in adrenergic-deficient mouse embryos. For example, two of the 22 significantly altered genes from Table 2 are ion channel subunits. One encodes for a calcium channel subunit (Cacna2d2), and the other for a potassium channel (Kcnj6). The Cacna2d2 subunit has previously been shown to be expressed in the heart, and it appears to increase the size of both L- and T-type calcium currents when expressed in a heterologous system (23). Other studies have shown that pace-making and atrial tissue express higher levels of this subunit than the ventricles (37, 65). In contrast, the Kcnj6 gene encodes for an ATP-sensitive inward-rectifying K+ channel (89). We did not perform electrophysiological characterization in the present study but recently reported slowed atrial-ventricular conduction, decreased connexin 43 expression, and increased propensity for cardiac arrhythmias in adrenergic-deficient embryonic mouse hearts (2). It is certainly conceivable that altered expression of Kcnj6 and/or Cacna2d2 could contribute to the observed electrophysiological impairments in these animals, though further experimental evaluation is needed to determine if this is true. At present, there is no indication that either of these ion channel genes are regulated by RA, but several independent reports suggest that elevated RA can lead to delayed ventricular activation (103), decreased connexin 43 expression (103), diminished growth of atrioventricular tissue (5), and accelerated expression of atrial calcium and potassium currents in differentiating cardiomyocytes (24). Thus, the elevated RA concentrations in adrenergic-deficient embryos may indeed contribute to the observed electrophysiological phenotypes, thereby providing additional fodder for future experimentation in this area.
One of the more curious findings stemming from the microarray analysis is that at least two of the genes identified have been shown to be involved in bone formation. The Mgp gene is involved in regulation of bone mineralization and has previously been shown to be regulated by RA (7). The Ebf2 gene, on the other hand, has not been shown to be sensitive to RA, but it apparently "regulates osteoblast-dependent differentiation of osteoclasts" (41). While it is entirely unclear why expression of these genes is altered in adrenergic-deficient hearts, it is intriguing to note that adrenergic-deficient mice display impaired leptin-dependent bone formation (95). Consequently, Mgp and/or Ebf2 could represent novel molecular intermediaries for adrenergic influence on bone formation, thereby opening the door for another new area of investigation.
Study limitations.
The present evidence shows that RA concentrations are increased in adrenergic-deficient embryos, but further study is required to determine if the observed RA fluctuations contribute to embryonic/fetal heart failure in adrenergic-deficient mice. We also recognize that significant gene expression changes observed in our analysis of the microarray data represent genes that appear to be unrelated to RA synthesis or signaling. We were initially excited about Nf1 as a potential target since earlier studies had shown that it is also essential for embryonic heart development (6, 26, 57), but we were unable to confirm altered Nf1 expression with independent RT-PCR assays. We tried two different primer sets targeted to different known splice variants of the Nf1 gene, but there are many other variants that would need to be evaluated before a definitive conclusion can be reached about whether or not this gene is truly influenced by adrenergic hormones in the developing heart. We have not yet systematically verified all the significant gene alterations observed from the microarray data with independent RT-PCR and other methods, but this dataset should prove useful for subsequent investigations into the underlying mechanisms of adrenergic action in the developing heart.
The elevations in embryonic RA concentrations likely contribute to the heart failure phenotype observed in Dbh−/− mice, though this is probably not the whole story since earlier studies have shown that maternal administration of excess RA or vitamin A does not necessarily produce heart failure and embryonic death (83, 87, 90). Nevertheless, overexpression of RA receptors has been reported to cause dilated cardiomyopathy and lead to congestive heart failure in adult mice (10, 93). On the other hand, it is well-established that too much or too little RA can lead to congenital cardiac malformations similar to those seen in human patients (34, 35, 58). In this context, it is potentially interesting to note that excess β-adrenergic stimulation has also been shown to produce similar types of congenital cardiac defects when applied during an analogous window of development in chick embryos (39, 40). It is therefore possible that teratogenic influences of adrenergic stimulation may be mediated, in part, through RA-dependent mechanisms. Additional experiments are required to test this hypothesis.
Study conclusions.
The major conclusion from this report is that adrenergic deficiency results in significant alterations in RA synthesis in the developing mouse embryo near midgestation (E10.5). Although it has been known for many years that adrenergic and RA hormones are each essential for heart development, the present study is the first report to show that disruption of adrenergic signaling significantly influences RA metabolism in the embryonic mouse heart. Interestingly, recent studies have shown that the Th gene is regulated by RA and expressed early in the developing heart (50, 63). The Th gene encodes for the enzyme that catalyzes conversion of L-tyrosine to L-DOPA, which is generally considered the rate-limiting step in adrenergic hormone production. Furthermore, the adrenal gland appears to be a major site of RA production during mouse fetal development (36). Thus, there is mounting evidence suggesting that adrenergic and RA signaling pathways are functionally interconnected during a critical phase of heart development, and perhaps beyond.
This work was supported by National Heart, Lung, and Blood Institute Grant R01HL-78716 to S. N. Ebert.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: K.O., D.W., and S.N.E. conception and design of research; K.O., C.N.B., H.-L.N., C.M., D.W., and S.N.E. performed experiments; K.O., C.N.B., H.-L.N., C.M., and S.N.E. analyzed data; K.O., H.-L.N., D.W., and S.N.E. interpreted results of experiments; K.O., C.N.B., C.M., and S.N.E. prepared figures; K.O. drafted manuscript; K.O., C.N.B., H.-L.N., C.M., D.W., and S.N.E. edited and revised manuscript; K.O., C.N.B., H.-L.N., C.M., D.W., and S.N.E. approved final version of manuscript.
Current address for K. Osuala: Wayne State Univ., School of Medicine, 540 E. Canfield, Detroit, MI 48201 (e-mail: kosuala{at}med.wayne.edu).
↵1 The online version of this article contains supplemental material.
- Copyright © 2012 the American Physiological Society
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text:
Brand Label: Ag
Available for Purchase Online at NORDSTROM for $228.00Pin It
Take your casual look to a luxe new level with this dreamy-soft cashmere sweater cut with a long, easy fit and extra-wide cuffs that add a playful finish. Color(s): deep currant, heather grey, true black. Brand: AG. Style Name: Ag Uma V-Neck Cashmere Sweater. Style Number: 5465760. Available in stores.
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subset: commoncrawl


text: I never had any trouble with Stellarium v. 0.12.??. After receiving e-mail notification of the availability of v. 0.13.0, I downloaded from SourceForge.net and installed v. 0.13.0. Everything proceeded normally. When I try to launch Stellarium, a Windows message pops up immediately reporting that Stellarium has stopped working. A second Windows message then pops up saying that Windows will close the program and notify me if there is a solution. I have not received a notice from Windows with a solution. I subsequently removed the previous version of Stellarium thinking that might be the problem and tried again. Same result. Then I removed the installed Stellarium v. 0.13.0 and repeated the process several times of downloading v. 0.13.0, installing, launching, experiencing failure of the program and removing it when the program would not run. Every time I got the same result. It will not run. I tried to recapture the previous version so that I could use the older program once more, but my repeated steps of installing and removing apparently left me with only prior installations of v. 0.13.0 and I could not go back to V. 0.12.??. Any suggestions would be appreciated. I am installing on an HP Laptop with Windows 7 (up to date) both Win.32 and Win.64. My security software is Kaspersky Internet Security 2014 (up to date), but I am not getting any warnings from Kaspersky when I download and install v. 0.13.0. Help!


text:
The Hotel Ibis is found on the picturesque shores of Lake Rotorua, providing 145 guestrooms within a 5-minute walk of the town centre.
The main draw for this intriguing city is the marvellous geothermals and the Polynesian Spa, where many spend their day soaking in the naturally warm water, is only fifteen minutes away, standing alongside the Rotorua Museum.
The town's many cafes and restaurants are within easy reach as well, but if guests choose to dine in house, the Ibis Restaurant is superb. Serving European cuisine, the restaurant is open for breakfast and dinner, while the iBar is the perfect place to relax with friends over a drink or two.
The rooms are designed to provide exceptional comfort and include sound proof windows to help guests get a good nights sleep. Each room has an in-room safe, TV, private bathroom with hairdryer, tea and coffee maker and ironing facilities.
Hotel Features:
● 145 guestrooms
● Restaurant & bar
● Laundry & dry cleaning service
● Free parking
● WiFi at an additional charge


text:
Quality, clean and comfortable. Highly recommend.Wayne2017-09-03T00:00:00Z
Absolutely perfect stay. Room was as described, comfy and the garden beautiful. The little extra touches such as freshly baked muffins and continental breakfast was unexpected but so appreciated.Laura2017-08-27T00:00:00Z
Jane's bungalow is exactly how it is described . It was very clean and tidy and well presented with quality fittings and accessories. It's in a great location and had all i needed for a comfortable stay. Would stay here again.ian2017-05-26T00:00:00Z
We didn't get to meet Jane as she was working away but very helpful and warm in her correspondence. Lou her hubby who was the perfect host! We just loved the stunning garden and beautiful guest house which was immaculately clean, tastefully decorated and had a lovely atmosphere and welcome bottle of wine. Chookies popped their head up to say hello in the garden too! It was such and easy and pleasant walk into Albury and Lou gave us great recommendations! People here are very warm and friendly and we enjoyed our trip immensely and can't wait to come back.Mike2016-11-06T00:00:00Z
We LOVED staying here, it's 5 stars from all of us. Jane and her husband were very accomodating, the bungalow was private, had its own patio in a beautiful garden, our pups were allowed to stay too. Wine and cheese on arrival with a continental breakfast and all amenities included. We will definitely come back again.Khris2016-12-24T00:00:00Z
Jane's studio is absolutely gorgeous and extremely comfortable in a naturally, tranquil and peaceful setting. Really nice.Suzanne2017-03-02T00:00:00Z
Lovely bungalow convinent to the centre of town via car or walkingMarian2017-04-09T00:00:00Z
This place was great, we messaged Jane to say we'd be arriving late and the room was heated when we arrived. Breakfast and coffee was lovely, and the bed was super comfy. Definitely recommend to anyone. Lovely quiet street as well.Kelli2016-09-16T00:00:00Z
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East Albury, New South Wales, Australien
I had a fantastic stay at Vivien's beautiful warm home. She was a lovely host, supa friendly & very helpful with local tips & places to eat! A home away form home.maylei2016-05-04T00:00:00Z
My experience was nothing short of amazing. The host is beautifully mannered & all accommodating. Will definitely be coming back :)Nick2016-04-23T00:00:00Z
Amazing!!! Vivien was a very nice host. She was welcoming us even we arrived so late. The beauty and cleanliness house had everything we need and her two cats are very friendly. We really love here. We all had a warm and relaxing stay in her house. Thank you Vivien. Really highly recommend to stay in Vivien's house.Ariel2016-10-31T00:00:00Z
Viviene was very welcoming even though I arrived late at night. We had a lovely chat over breakfast. Would definately stay again!Natalie2016-10-10T00:00:00Z
Vivien is a very nice and host. Her place is so lovely. The two cats are so cute. We are so thankful for vivien's hospitality. Highly recommended!Jing2017-04-11T00:00:00Z
We extended our stay with Vivien because we loved being there. Lovely home, comfortable bed, quiet neighbourhood and great host.Mary2016-09-03T00:00:00Z
Vivien's place is perfect!She is very nice. would like to come back again!Crystal2017-06-01T00:00:00Z
Vivien welcomed us with a cup of tea and cake. Her home is beautiful, the bedroom lovely and the bed super comfortable. Everything we could want was at hand. A very pleasant stay with a very pleasant host.Helen2017-03-01T00:00:00Z
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Albury, New South Wales, Australien
We had the most comfortable stay in this beautifully furnished apartment. Terry and Row provided five star luxury with such great attention to detail. Loved it!Zaheeda2016-12-31T00:00:00Z
Absolutely perfect! Beautifully styled and finished. It's exactly as it appears in the pictures. We were staying with our 2 kids (3 and 8) and there was plenty of space for us all. Lovely, peaceful courtyard and lots of thoughtful touches like a range of T2, tasty treats, fresh bread and a fridge filled with the essentials. We got in late so it was a relief to not have to run out to the shops and Terry and Row had also left lights and heat on for us. They were quick to respond to any questions we had and even allowed us to check out later then planned. Thanks for a perfect stay Terry and Row, we will definitely be back.Lauren2017-06-04T00:00:00Z
The unit was lovely, easy to find, close to town if you wanted to walk into eat. The fresh eggs, milk bread & croissants were lovely as were the pantry bits. The way the unit was set up with the TV room was fantastic. The bed was REALLY comfy as were the pillows (I usually use my own but not this time). Super shower and the "extra" bits were great. Couldn't fault the unit in anyway. When we arrived, after dark, the light inside were all on, the music was playing. Really lovely atmosphere. There are renovations on some of the other units being done at the moment but as we were out and about both days this did not bother us in anyway.Diane2016-05-19T00:00:00Z
Place was more that expected and we loved it.Andrew2017-07-16T00:00:00Z
Very comfortable well equiped, nicely appointed and provisioned apartment, a great base for visiting the city of Albury.Donald2017-07-31T00:00:00Z
Nice neighborhood. Close to shops. Very comfortable and clean. Would be good for a short or a long stay.Katherine2016-07-11T00:00:00Z
Terry and Row are great hosts. Their attention to the smaller details make staying with them a wonderful experience. The panorama cinema room made for a great night in after a long day's drive. The bed was super comfortable and the location is perfect for walking to the main area for something to eat. I would definitely recommend staying here.Tracey2015-12-21T00:00:00Z
Terry and Row provided a wonderful unit for my two week stay. The flat was very comfortable, well positioned and great value for money! Terry and Row were fast to respond and very kind with all communications. Would highly recommend their unit.Matt2017-02-11T00:00:00Z
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Albury, New South Wales, Australien
Just stayed overnight on the way to Melbourne from Sydney. Very clean and comfortable room with ensuite shower room in a quiet area. The host Vivien was nice and kind lady and this place is recommendable for a break during long distance driving.John H.2016-02-17T00:00:00Z
Vivien made us feel at home from the moment we arrived, the room was done well and neat. We had a pleasant stay at Vivien and she was a wonderful host. The added bonus is her pets (2 lovely cats) very friendly. I would recommend Vivien place for a stayMURALIDHARAN2016-04-01T00:00:00Z
Another great repeat stay with Vivien. Great company and wonderful accommodationTony2016-09-09T00:00:00Z
I really enjoyed staying at Vivien's lovely home. Vivien's house was comfortable and homely with everything supplied for a relaxing visit. Vivien was a warm and welcoming host and a pleasure to chat with. I would highly recommend Vivien's place to stay if you need somewhere in Albury.Di2016-06-03T00:00:00Z
Vivien was a very warm & welcoming host, very easy to communicate with & extremely helpful... Our room and ensuite were clean, comfortable & beautifully appointed... We would not hesitate to stay there again or recommend her house to other travellers to Albury...Megan2017-06-03T00:00:00Z
First time airBnB'er. Stressful time made way easier by a lovely clean, comfy room and two gorgeous cats to talk to. Vivien lovely tooLeanne2015-12-14T00:00:00Z
Had a wonderful 3 night stay in this gorgeous home in Albury. Very quiet and peaceful. Host was friendly, helpful and engaging. Large room, comfy bed and private ensuite fantastic. Shared use of kitchen and living space and a few cuddles from the 2 cats made it even better.Diane2015-09-09T00:00:00Z
I had a quick stopover on the way through to Sydney, the room was very clean, bed comfortable and Vivienne was lovely. Slept very well and was in my way. So much better than a hotel. ThanksJustin2016-03-16T00:00:00Z
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Albury, New South Wales, Australien
Well appointed 3 bedroom townhouse with everything you need for a trip to Albury. Highly recommended.Matt2017-09-28T00:00:00Z
Great location, great house!Ginny2017-09-03T00:00:00Z
Great place,great location,great hostBrad2017-10-01T00:00:00Z
Amazing!!! Everything was so well thought of, was perfect. Wouldn't hesitate to stay there again. It was a beautiful place, so clean. The beds were comfortable. Plenty of space for the children.Katherine2017-08-28T00:00:00Z
Great place.Trent2017-09-22T00:00:00Z
Absolutely loved itKatrina2017-09-01T00:00:00Z
Fantastic place to stay in Albury, highly recommend.George2017-08-01T00:00:00Z
Amazing service, beautiful unit and fantastic location. Samara was very welcoming and accomodating with our plans.Amy2017-09-06T00:00:00Z
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Albury, New South Wales, Australien
Cute little apartment! Perfect for an overnight stay on your way up or down the Hume. Breakfast options were great! There are a lot of extra little touches that make this a lovely place to stay.Emma2017-09-09T00:00:00Z
Ronelle's place was absolutely perfect for our stopover between Sydney and Melbourne. Everything was provided which meant we only had to change our clothes! Breakfast was fantastic! Spotlessly clean. We'll be back!Claire2017-09-25T00:00:00Z
Perfect in every way!Emily 2017-09-13T00:00:00Z
Great place to stay for family. Very clean as its brand new place. Everything we needed was provided for us to stay overnight. Was last minute booking traveling from Melbourne to Syd but host was kind and quick to answer :) Thank you! Surely we will be back for next trip to Melbourne!Jane2017-09-15T00:00:00Z
Great accommodation, beautifully presented and clean with everything you could possibly need. Warm and very comfortable for a family of 4.Michelle2017-08-20T00:00:00Z
Incredible! Ronelle's place was so impeccably presented you cannot fault it! Everything we could have needed was catered for! Will definitely stay again! For us it was the perfect stop over on our way from Sydney to Melbourne.Emma2017-09-29T00:00:00Z
Lovely, clean, stylish townhouse Ronelle was a great hostAnna2017-09-22T00:00:00Z
The place was even better than the photos. Very impressive interior decor. Ronelle was very nice and fast with communication. The place was very close to the town and the airport which was great for us as my husband flew into Albury that night. For anyone staying in Albury, I would highly recommend this place!Nao2017-09-28T00:00:00Z
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Springdale Heights, New South Wales, Australien
This was a lovely space to stay whilst travelling to Albury.Jordan2016-09-26T00:00:00Z
Was a pleasure staying here. The house is lovely and very thoughtfully set up for guests to find everything they need. All the facilities and bedrooms were lovely and clean and you wouldn't even know that it is still being renovated! Plenty of space and storage that accommodated 4 adults and 2 children very easily. It is comfortable to stay in as the evaporative cooling worked wonderfully. The host went above and beyond with a wonderful selection of breakfast items, fresh milk, tea and coffee. Also wanted to say thank you for being so thoughtful and setting up the port-a-cot for our baby with linen and blankets as well. She slept like a log in it!Ben2016-03-29T00:00:00Z
Daniel's house was beautifully furnished, very comfortable and with every comfort. The beds were very luxurious and the kitchen stunning. Arrangements for our arrival were organised well and Daniel very friendly. The suburb in which the house is situated very amenable. Thanks for such a pleasant stay.Richard2015-11-03T00:00:00Z
We had a very comfortable stay in Daniel's lovely place. It was spacious, very comfortable and spotlessly clean. Great location nearby to town and cafes. Super nice touch to leave cool drinks and snacks too. Great to deal with thank you.Justine2015-12-18T00:00:00Z
It was a great accommodation for our short one night stay. Everything we needed was provided. I'd highly recommend it.Ievgen2016-03-23T00:00:00Z
Very spacious and lovely quiet area. Close to the main area and very accessible by car. Daniel was a great host, he was very accommodating and let us check in early.Ebony2017-07-30T00:00:00Z
It was a lovely stay at Daniel's place. The house was impeccably clean and neat and we love the complimentary tea, coffee and snacks provided. We had a good night's rest after a long drive from Sydney.Nadiah2015-12-16T00:00:00Z
Nice place exactly as described. Daniel gave clear instruction for checking in, the house was clean & tidy, beds were comfortable, and the location is great. There are parks nearby and it's only a short walk into town.Clarissa2016-02-18T00:00:00Z
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South Albury, New South Wales, Australien
Clean and great value with some nice thoughtful touches that made us feel welcomeMyf2016-08-29T00:00:00Z
Place as expected and good communication with hostNiamh2017-09-29T00:00:00Z
Immaculate. Spotlessly clean. Comfy overnight stay between syd n melb. The personal touches, restaurant recommendations, tourist info , nice wine and chocolate makes it special. Very easy check in and out. No need to coordinate times, most convenient.Christina2016-04-11T00:00:00Z
Fantastic stay. Bianca and rebecca were super helpful throughout the booking process! Highly recommend the stay. Apartment was close to the Main Street, very clean and spacious and exactly what we wanted!Emma2015-07-20T00:00:00Z
- Very narrow parking facility. If you have a 4WD, SUV, MPV then your passengers needed to get off the car first or they can't get out. I had to knock the neighbour next door to move her car as she wasn't parking straight so I even can't park my car in very narrow space. Off street parking is not safe. - The neighbour put out loud music which you can hear from the first bedroom. - Towels and linens were already turning yellowish (rather than white) which was due to be replaced. - Squeaky spring mattresses or bed frames when turning the sleeping position which was annoying as you need quietness to sleep. - Carpet and rugs were due for steam cleaned. Not recommended if you have crawling son. - Before you left, you were 'obligated' to wash the dishes which nothing stated in the house rules. - Before you left, you were also 'obligated' to put furniture back in place. In example: we moved coffee table less than a metre away beside the TV as it was a hazard for my crawling son, but then you were 'expected' to move it back. - Not convenient to move stuffs from your car as unit was on 3rd floor.San2017-07-16T00:00:00Z
Lovely unit with heaps of space and great facilities. Some nearby eateries are within easy walking distance for reasonably fit people. Very helpful communication with Bianca. Really liked the place.Ren2017-01-28T00:00:00Z
Very clean warm and comfortable.Marni2017-07-01T00:00:00Z
Great place to stay. Would recommend to others.Leigh2017-06-10T00:00:00Z
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Med plads til
Albury, New South Wales, Australien
Hidden Gem on Dean Street. Awesome location and great hospitality... and there's a bubble bath!Lindsay2017-07-03T00:00:00Z
Lovely flat right on the main street which was perfect for work and getting round, with plenty of spots for breakfast, lunch and dinner nearby. Carolyn was a great host and was happy to either have a chat or allow privacy for me to work on my laptop. Would stay again next time I'm in Albury.Luke2017-07-12T00:00:00Z
Very modern, clean bedroom with private bathroom. Very spacious. It is located in the center of CBD with easy access to shops. It also has a parking space in a private car park. The host is very nice , she stayed up late waiting for me to arrive. Very good value for money, I enjoyed my stay very much.Eric2017-08-07T00:00:00Z
Carolyn's place is in a great location in the Main Street of Albury, it's very clean and modern and also very homely. Carolyn was a lovely host and made me feel very welcomeJames2017-08-05T00:00:00Z
Fantastic accommodation. Great host, really easy to communicate with. Place was exactly as described. I would highly recommend.adam and Alex2017-07-19T00:00:00Z
Great location, very nice place - highly recommendShannon2017-09-22T00:00:00Z
Very easy to find spot in centre of Albury, great for business or a night out.Lindsay2017-07-29T00:00:00Z
The apartment was close to everything and lovely and clean it was a god sendGayle 2017-09-25T00:00:00Z
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Med plads til
Albury, New South Wales, Australien
Everything about this experience was fantastic. Michelle was very prompt in her responses and was very accommodating. She made sure she was home to great us when we arrived and she instantly made us feel at home. The space was modern and cozy and the bed and pillows were very comfortable. There was a variety of breakfast provisions on offer and some chocolates on arrival which were greatly appreciated. She was also very accommodating in terms if us bringing our dog. After a long car ride she was very happy to have a big yard to run around in and a couple of mates to play with. Michelle even left some treats for our dog. This was an ideal place to stop for a night between Sydney and Melbourne and I would definitely stay here again.Amy2016-05-18T00:00:00Z
My husband and his little dog stayed here on the way to Melbourne. He raved to me about the size and amenity (everything you could possibly need) of the accommodation as well as the friendliness of the hosts. He's now insisting we stay here every time we drive to Melbourne. Highly recommended.Sonia2017-02-07T00:00:00Z
This apartment was a little haven - I was only in Albury for a short while, but once I arrived I wished I was staying longer - so I could hang out in the apartment and enjoy it even more. Have already recommended it to people! Michelle was very friendly, helpful and relaxed.Cathy2016-07-23T00:00:00Z
We really enjoyed our stay. Michelle and Charles (and their dogs) were wonderful hosts and made us feel very welcome. The room had everything we needed; nice furnishings, a comfy big bed and a good area to make a snack or a cup of tea. Within about 10mins we could also walk into the town centre and also come back without disturbing anyone as we had our own entrance. Definitely recommend.Daniel2016-02-06T00:00:00Z
A great stop over. The unit is a garage conversion - but so comfortable and it has everything you need at your fingertips, including wifi. The host is welcoming and very accommodating. Definitely a place to return to in future travels.Cat2016-07-22T00:00:00Z
Our hosts Michelle and Charles were very friendly and welcoming. The room was immaculate and had a great array of added extras like fruit, sourdough bread, cereals and bathroom items. The bed was comfortable and overall we were very happy with our stay. Thank you Michelle and Charles ☺Loretta2016-09-25T00:00:00Z
It really was a pleasure to stay in Michelle's meticulously clean, warm and comfortable studio. The bed was very comfortable, furnishings great and handy location. Michelle was excellent with her communication, welcoming on arrival and helpful throughout the stay. She had gone to a great deal of trouble with lots of special touches and surprises. She thought of everything to make the stay enjoyable. We felt so at home and we wanted to stay longer. We'll be back, for sure.Christine2016-06-07T00:00:00Z
Absolutely beautifulAshley2017-06-22T00:00:00Z
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Med plads til
East Albury, New South Wales, Australien
We loved staying at Gwyneth's. Super comfy bed, warm & cosey room. Definately worth a stay in Albury. Made us feel at home.Michele2017-08-08T00:00:00Z
Very comfortable and friendly place to stay. All of the amenities were spotless and the bedroom was spacious. We had a pleasant stay and would recommend to everyone.Ryan2017-07-02T00:00:00Z
A 5 star hotel room would be like this one.Jeff2017-07-09T00:00:00Z
This was a perfect stopover on our drive from Sydney to Melbourne. A beautiful home and our room was enormous, a very comfortable stay and offers superb value!Thomas2017-07-13T00:00:00Z
Gwyneth was an amazing host, her place was clean and the room was very comfortable. The bedroom was huge and you had access to everything you would need including wifi and Netflix! The house was very warm and cosy also! Thank you so much for a wonderful stay :)Tien2017-06-17T00:00:00Z
The home was perfect for our stay, and self check in was really easy. Our host was away that weekend so we had the house to ourselves. The bedroom very roomy with a lovely comfortable bed for a sound sleep. The kitchen was terrific, great facilities to prepare and cook. Lovely view out from the kitchen windows to the expansive and green backyard. Beautiful polished wooden floors took our attention as soon as we walked through the front door. Loved the spaciousness and comfort of the house.Aleysha2017-05-07T00:00:00Z
The host canceled this reservation 10 days before arrival. This is an automated posting.Wiruut2017-08-17T00:00:00Z
We really enjoyed our stay at Gwyneth's immaculate and stylish home. She was warm and welcoming and her communication with us was particularly prompt and informative. We explored a city that we previously only paused in and regretted only allowing ourselves two nights to enjoy her hospitality and Albury's relaxed at.roberts2017-03-18T00:00:00Z
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Med plads til
North Albury, New South Wales, Australien
Great service from Donna. Our team members went to work at the Caravan Show so I needed individual beds and rooms. 4 bedrooms worked a treat. Donna left breakfast provisions for the first morning which was very handy as they didn't arrive until late. It was cold in Albury so the electric blankets were great.Bronny2015-08-18T00:00:00Z
We had a great stay, the house was in a great position with a beautiful view. It was quite cold over the weekend so we appreciated being able to cosy up around the heater! The bedrooms and bathroom were clean and tidy. Donna was very accommodating and helpful.Olivia2016-06-13T00:00:00Z
Had the pleasure of meeting Donna upon arrival. She was really nice and accommodating. The place was amazing. Huge, clean and can accommodate many people. Great stay, highly recommend!Ben2016-05-16T00:00:00Z
Once again Donna and her house delivered the goods. Only a short stop over this time but as usual Donna was an outstanding host. Love the new work that you've done out the back Donna. Thanks again.Damien2016-04-04T00:00:00Z
We loved staying at Donna's lovely home. It was clean, spacious, comfortable and very family friendly. Very close to the thurgoona shops and had a reserve out the back. The neighborhood was very quiet and had everything we needed without heading into Albury. When we arrived Donna had arranged a change table, baby towels and a few goodies for the kids which was very thoughtful. Excellent communication and she made us feel very welcomed. We would recommend Donna's home to family and friends and will be staying here again next time we visit Albury.Ashleigh2015-09-26T00:00:00Z
My friends and I done a three months internship at CSU and Donna's house was relatively close to the Uni (20min walk)Clara2017-08-13T00:00:00Z
Great, extended my stayPaul2017-09-18T00:00:00Z
Everything was great, beautiful place in a great peaceful location.Paul2015-03-03T00:00:00Z
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Med plads til
Thurgoona, New South Wales, Australien
we had a lovely stay, the room is great, we loved the breakfast items and the coffee and milk were extremely appreciated.Jay2016-10-13T00:00:00Z
Ronelle's home is just beautiful. The studio was so well prepared it was truly amazing. From the combination lock for easy check in, to the cell phone chargers available for use, and even midnight snacks for those like me who like a little sweet/salty snack. Breakfast was simple, cereal and milk and toast with jam which was adequate for us. The location was perfect for us as well. Anything we needed Ronnelle came to the rescue immediately and was happy to help. Thank you for everything! We hope to see you again in the near future!Victoria2017-09-28T00:00:00Z
Exceptional spot in a very beautiful town that was shrouded in a cold fog. Ronelle was warm and welcoming. The studio is extremely comfortable and a short drive to restaurants etc. Highly recommendedAnne2017-06-22T00:00:00Z
Place is in a lovely and quiet neighborhood about 10 minutes drive out of Albury. The studio is very well equipped, clean and spacious. Most comfortable.Kevin 2016-11-26T00:00:00Z
Great stay, would and will stay again.Lachlan2017-07-14T00:00:00Z
Great place to stay! Very comfortable and clean, Ronelle provided more than need be, highly recommend to all! Thank you again! :-)Jaymii2017-07-19T00:00:00Z
Gorgeous cosy night sleep. Perfect location and had everything we needed. Highly recommend and wouldnt think twice about staying againDavid2016-10-20T00:00:00Z
An excellent place for our overnight stay in Albury. Warm, comfortable, well appointed and in a quiet residential neighbourhood. Communication was excellent and privacy respected. Many thanks, Ronelle!Andrew2016-07-28T00:00:00Z
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Bed & Breakfast
Med plads til
Lavington, New South Wales, Australien
Place was very clean and comfortable. Gail is a great host and helped me find vegetarian food options in Albury. Great place to stay.Ngaire2017-01-04T00:00:00Z
They were not aware we were coming.John2016-10-06T00:00:00Z
She went anove & beyond & made me really feel at home thereJillian2016-11-13T00:00:00Z
Gail's place is bright and colourful and incredibly welcoming, as is Gail. We arrived very late at night but Gail was there to welcome us and had even put the electric blanket on. Everything you could possibly need was provided for. Thank you Gail our stay was short but very sweet!Trish2017-05-23T00:00:00Z
This is the second time that I have stayed there in the last two months, so I think that says it all. Great place to be able to work or relax, quiet and easy access to the Albury CBD as well as surrounding towns and the winery regions.Pamela2017-06-07T00:00:00Z
Great place - friendly hosts, very clean and comfortable.Scott2017-03-31T00:00:00Z
We have a great visit , room was really comfortable , Gail and her husband were both lovely and it was no trouble to get us little things for the room . The golf course across the road was really handy for dinner after arriving lateNicole2016-06-14T00:00:00Z
Gail and John were wonderful hosts, very friendly and helpful. The space was beautiful perfect for our needs. It was a great location with a beautiful view off the back deck hopefully next time we will have time to sit out there and have a drink and chat with the hosts. Thanks for everything, Nicole and Mili.Nicole2016-03-30T00:00:00Z
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Med plads til
Albury, New South Wales, Australien
This was our first experience with AirB&B and an absolutely fabulous find. We stayed at Kade & Carly's beautiful home for one night only on our way from Melbourne to Sydney. It is easy to find on a hill overlooking Albury. There were kookaburras in the trees and a wallaby was spotted in the nearby grass . This ultra modern house is set away from the road providing accommodation for up to 6 adults . There are 3 bedrooms, 2 bathrooms (separate WC's) with an open lounge and well equipped kitchen. The lounge leads onto a spacious decking area with BBQ facilities and far reaching views of Albury and beyond. Our only disappointment was that we did not spend more time in this beautiful accommodation. Highly recommended. Wonderful hosts. If we come back this way again, you will certainly be on our list of places to stay Best wishes Anne & Co United Kingdom February 201(URL HIDDEN)Anne2017-02-16T00:00:00Z
We came to Albury from Melbourne for the Winery. Great big house with an amazing deck with beautiful view's over Albury. The photo doesn't do it justice. The house had everything you need and was very spacious. The cinema room was amazing, we had lots of laughs playing the Xbox sports games too. We did a walk from the house to town via the track going from the monument through the bush which was so lovely, took about 20 minutes. Thanks again and would 100% recommend.Kay2017-06-11T00:00:00Z
Beautiful home. Awesome cinema room!Nick2017-05-03T00:00:00Z
WOW!!!! This home is nothing but AMAZING!!!! Thank you so much Kade and Carly. This home has everything and more. From a beautiful kitchen, dinning and lounge, to the downstairs cinema room and then there's fantastic views. I only had a brief one nights stay and was disappointed I couldn't stay in the area any longer due to my work commitments. I would highly recommend this home to absolutely anyone. Again greatly appreciated and thank you very much Kade and CarleyMalcolm2017-06-20T00:00:00Z
Awesome good home, feel like back to sweet home. House located on the up hill can see the view of the Albury city and airport.Gan2017-06-29T00:00:00Z
The view from this property is breathtaking! Easy to find he property and close to everything! House was immaculate, and well furnished and owners left instructions for all appliances and electronics. The driveway is very steep and heater takes a while to warm up downstairs area but these are minor inconveniencesKylie2017-06-25T00:00:00Z
Highly recommend this place. The house has everything you need. The 2 bedrooms and theatre room downstairs gave some good space, yes the stairs are a bit hard for very young children, but with a safety gate at the top we had no worries with our 2 1/2 y.o. The views are awesome are we really recommend walking to the monument and down the hill to the botanical gardens which has a really fun kids garden area in it, the kids had a ball. While staying we did the run to Fall Creek to play in the snow which is about 1 hour 45min drive. We would happily stay with these great hosts again.Rhys2017-07-28T00:00:00Z
Kade & Carly's place has everything you require for a wonderful weekend away - and more! Great views, beautifully appointed, plus a theatre room and a gym for added extras. Would highly recommend staying at Kade and Carly's place.Felicity2017-09-26T00:00:00Z
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Med plads til
Albury, New South Wales, Australien
We had another night here after a short trip and wouldn't want to go anywhere else!Eric2017-03-01T00:00:00Z
I honestly recommend this perfect place. Clean and very beautiful. Candace and Marc are so helpful and friendly couple.Daniela2016-11-15T00:00:00Z
Nice and clean place with everything you need. ThanksAlina2016-11-04T00:00:00Z
Lovely people, comfortable bed/room, everything is clean, convenient location, cute little bonuses like Wi-Fi, chocolates and a bottle of water, affordable. I can't see how you could go wrong staying here. :)Kirsty2017-01-12T00:00:00Z
I had a very enjoyable stay at Candace's house. Candace is a welcoming and helpful host. Her house is beautifully furnished and very clean. I would definitely recommend staying there!Adelaide2016-11-25T00:00:00Z
Candace is a very welcoming and caring host. She made sure to double check on me as I was feeling a bit under the weather during my stay. The room was toasty, comfy and beautifully decorated. And the house has everything you'll need including a furry friend. I recommend staying at Candace's :)Penny2017-09-04T00:00:00Z
Great peopleMuhammad 2017-05-19T00:00:00Z
Beautiful house, and the way its designed is amazing! Very lovely & welcoming people. Everything was super clean and it was just perfect for what I needed.Jacinda2016-11-02T00:00:00Z
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Med plads til
Wodonga, Victoria, Australien
Awesome little place,so stylish and had everything you could want and moreSonya2016-01-26T00:00:00Z
Great location, easy to find. Arrangements for accessing apartment clear and flexible. Description of apartment was accurate, and it was comfortable, well equipped, clean and had a lovely personalised feel & touch to it. Far better than paying an equivalent price for a soulless motel/hotel. Great for self catering.Carmel2016-05-18T00:00:00Z
Perfect location for a trip to Albury. Light and bright with a big kitchen and bathroom that makes you feel right at home. Enjoyed using the Netflix!Rachel2016-12-28T00:00:00Z
Had a great stay at this fully renovated apartment, full of nice touches and thoughtful extras. Lovely balcony with great outlook and short walk to everything. Very relaxing stopover between Sydney and Melbourne, just what we needed and even found a bonus farmers market to get fresh supplies before leaving. Could have happily stayed much longer.tim2016-01-23T00:00:00Z
Stayed at Row and Tina's for 1 week. Very handy location with a short walk to Dean Street (CBD Albury). The unit itself is very well presented and extremely clean. Checking in and out was a breeze with good communication from Row. Would definitely stay here again if required.Darren2016-02-11T00:00:00Z
Modern and stylish apartment with thoughtful hosts. Only stayed 1 night but can confirm the description and photos are accurate. Will happily recommend.Ben2017-08-02T00:00:00Z
Lovely place, neat, clean and very well presented. Easy to find and convenient. Great communication as well. Recommended.Alun2016-02-03T00:00:00Z
Place was like a hotel! Maybe better! Great location too, close to main shops and restaurants, and easy to find. Would definitely book again and recommend to my family and friends!Kr2016-02-13T00:00:00Z
Helt hjem/lejlighed
Med plads til
Albury, Australien
Host was lovely, accomodation comfortable and close to town. Doggies so cute.Darren2017-03-27T00:00:00Z
Mia, Luigi and Loretta (her 2 maltese) are the sweetest hosts ever! They were very warm and welcoming. The room was heated up even before we came to welcome us and the room provided was even fitted with electric blanket which were very comfortable and toasty. We thoroughly enjoyed our stay there and would highly recommend her. Her dogs were super friendly and we had such a fun time we didn't want to leave. Although we only did a one night stopover, we enjoyed ourselves so much that we wished we could stay for longer.Isabella 2017-07-11T00:00:00Z
Mia was a fabulous host and her house was too cute, plus her two dogs Luigi and Loreta were just as welcoming as Mia. Located close to downtown, the botanic gardens, the river, and parks it was a wonderful place for a quick stay in Albury. I would stay again and recommend it for anyone!Marissa2017-01-07T00:00:00Z
Thank you Mia. A great place to stay on a long trip to Sydney. Ideal place to brake the trip. Thank youMorris2017-03-08T00:00:00Z
Lovely renovated cottage. Loads of character. Friendly pets and very comfortable stay. Would stay again and recommend to family and friends.Ashling2017-01-08T00:00:00Z
We received a warm welcome from Mia and her georgeous puppies. A short walk from the house and we were in town for dinner. Enjoyed a restful sleep in a comfortable bed. Thanks again for your hospitality Mia.Hannah2017-01-09T00:00:00Z
Mia's place was beautiful, quiet, cosy and central to everything you would need. Mia was very welcoming and accomodating and i would highly recommend.Laura2017-02-02T00:00:00Z
What a quant beautiful cottage, stepping back in time. I couldn't have asked for a more perfect abode to be at ! Mia was so helpful and her fur babies where so gorgeous, nothing like puppy love ❤ they where just gorgeous. Thankyou Mia so much for sharing your home with us. Rizael & ZetaRizael2017-08-27T00:00:00Z
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Albury, New South Wales, Australien


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Fiction by Andrew Reichard: Water March
By Andrew Reichard April 15th—the morning of our protest on Pennsylvania Avenue. The day we risked injury from a government that responded to rage with derision. I hoped then, and privately, that I at least would always remember it, and the strangest thing is that


text:
Journey to Freedom (1957)
Directed by Robert C. DertanoGenres - Action, Drama, Romance, Spy Film | Run Time - 60 min. | Countries - USA |
Share on
Synopsis by Sandra Brennan
In this espionage thriller, Communist agents pursue a fleeing activist for "Voice of Freedom" from Bulgaria to the United States. Along the way he falls in love with a supportive nurse who helps him recuperate from an injury.
accident, activism, agent [representative], America, chase, Cold-War, defector, doctor/nurse, espionage, freedom, fugitive, hunting, injury, journey, love, refugee, Russia, tracking [following]


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Home Listings shortage drives up prices
Real estate sales in the GTA continued at a blistering pace in April. An all-time record for the month was set with 12,085 transactions, up 7.4 per cent from April 2015. However, the demand for detached homes still far exceeds the supply of homes listed for sale. Detached and semi-detached home sales actually fell from last year, in the City of Toronto. Bidding wars among buyers are driving up prices. "As we move into the busiest time of the year in terms of sales volume, strong competition between buyers will continue to push home prices higher,"says Jason Mercer, the Toronto Real Estate Board's director of market analysis. In April, the average selling price for a detached home in the city was $1,257,958, an increase of 18.9 per cent from last April.
In the 905 regions, the average detached home sold for $881,413 up 20.7 per cent. Detached GTA homes spent an average of 13 days on the market and sold for 104 per cent of the listed price in April. Several factors continue to stoke the real estate furnace in the GTA. Active listings were down by 26.9 for all types of property, creating heightened demand.
Low mortgage interest rates should continue well into next year. Immigrants keep flowing into the GTA, as well as people returning from Western Canada after job losses. There has been strong employment growth during the last twelve months. Demographics also favour the housing market as the echo generation – baby boomers' children – are the prime age to buy real estate. Another factor, particularly in the luxury real estate market, is the controversial topic of foreign investment in real estate.
"Toronto is the most popular Canadian city for Chinese buyers, although I think sometimes Vancouver feels like it is the leader," says Charles Pittar, CEO of Juwai.com, a Chinese international property portal. "The biggest motivation for buyers is the education of their children. They often combine the goal of providing a home for their children who are studying in Canada with other investment and lifestyle goals."
The federal government has pledged to improve their tracking of the impact of foreign investment.


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17 Winter Street Milford, MA 01757
508-473 -2000 Fax: 508-473-6907
508-473 -2000 Fax: 508-473-6907
We are all called to be Good Stewards with God's money. All that we have comes from Him. If tithing is not part of your families life, it is never too late to start! If you think you can't afford to tithe, than you definitely can't afford not to. Here are some ways you can Donate:
Click Here for the Electronic Stewardship Authorization Form - Please Fill out and mail or fax back to the secretary.
Every week St. Mary's Parish Tithe's a portion of all dontations each week. For more information go to the News and Events Page


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THE Radio burbled as I staggered out of the wreckage into the foggy night. Looking over my shoulder, the scene painfully repeated itself: I'd hit a tree and rolled down an embankment. Panic started to cross my mind as I realised my daughter was missing.
I ventured into the misty town ahead of me and a small silhouette started to reveal itself in the distance. I shouted for her; whatever it was giggled ghoulishly and ran down an alley faster than my eye could track. What was going on?
I was curious, I was scared, but mostly I was absolutely overjoyed.
You see, this moment of tension and fear on the original PlayStation was a crossing point for me. Fate had given one horror fan a brain for programming and made him the inventor of Konami's horrific masterpiece – Silent Hill. The other (ie: me) was sat in a dark room, drinking a beer and thinking this game would be another Count Crapula. How wrong I was.
And the next few hours of sheer terror that followed inspired me to write about games.
Needless to say, every succeeding Silent Hill episode has been met with the same ritual: Late night, beer for the nerves, and no interruptions. And predictably each one has lapsed over the time limit I've set myself, with me hitting the wee hours fuelled on nothing but adrenaline and fear.
Ten years after this epiphany, Homecoming has arrived, telling the story of ex-soldier Alex Shepherd who has returned to the town of Shepherd's Glen. Plagued by horrific dreams and odd visions, Alex sets out to discover why his hometown seems to be slipping into hell.
Stylistically, the latest part in the Silent Hill series hasn't changed. Shot in the third person and ghoulishly graphic, it is survival horror at its best and riddled with devices to make your spine tingle. In fact there have also been significant improvements to the sluggish control system – meaning this outing wasn't laced with late night curses at a dodgy attack system.
Purists will argue that this added to the tension and Alex's moves are too good this time. But in all fairness the opportunity to have a decent fighting chance against things – whose only purpose is to scare the bejesus out of you – is a no-brainer. The more skilled the better, I say; and the fact that this episode features crowds of hideous creatures lurching towards you does balance the tension.
New weirdness is more challenging though, and doesn't seem to be something Homecoming has succeeded with. That's not to say the blood-drenched industrial "otherworld" doesn't get a little spooky – more than doffing its cap to classic films like Jacob's Ladder and Ju-On – but this is something I'm getting fairly familiar with now.
For my latest venture I allowed an uninitiated horror fan into this evening ritual, and the effect was exactly like my own almost a decade ago.
Homecoming isn't a bad game but its effect on the stalwart fan will be less than someone new to the series.
RATING: 7/10 – The silent treatment
Skate 2 (Electronic Arts, PS3, £44.99)
SKATE has been one of the few challengers to the Tony Hawk's franchise. In fact with its sales debut it quite literally smashed ol' Hawky's waning series to pieces.
Featuring the "flick it" control system, the game allows you to perform stunts with the analogue sticks on the pad. This of course allowed for more error but more control too and led an overall greater mastery of stunts.
Skate 2 brings the magic back with it a host of new tracks. The controls system's bugs have also been slightly ironed out and new stunts like hand plants have also been added.
Definitely one for the skater fans.
RATING: 8/10 Wheeled out


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