text:
- Research article
- Open Access
Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease
© Koprich et al; licensee BioMed Central Ltd. 2010
- Received: 11 July 2010
- Accepted: 28 October 2010
- Published: 28 October 2010
The pathological hallmarks of Parkinson's disease (PD) include the presence of alpha-synuclein (α-syn) rich Lewy bodies and neurites and the loss of dopaminergic (DA) neurons of the substantia nigra (SN). Animal models of PD based on viral vector-mediated over-expression of α-syn have been developed and show evidence of DA toxicity to varying degrees depending on the type of virus used, its concentration, and the serotype of vector employed. To date these models have been variable, difficult to reproduce, and slow in their evolution to achieve a desired phenotype, hindering their use as a model for testing novel therapeutics. To address these issues we have taken a novel vector in this context, that can be prepared in high titer and which possesses an ability to produce neuronally-directed expression, with expression dynamics optimised to provide a rapid rise in gene product expression. Thus, in the current study, we have used a high titer chimeric AAV1/2 vector, to express human A53T α-syn, an empty vector control (EV), or green fluorescent protein (GFP), the latter to control for the possibility that high levels of protein in themselves might contribute to damage.
We show that following a single 2 μl injection into the rat SN there is near complete coverage of the structure and expression of A53T α-syn or GFP appears throughout the striatum. Within 3 weeks of SN delivery of their respective vectors, aggregations of insoluble α-syn were observed in SN DA neurons. The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01). At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition.
In the current implementation of the model, we recapitulate the primary pathological hallmarks of PD, although a proportion of the SN damage may relate to general protein overload and may not be specific for A53T α-syn. Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model. The dynamics of the evolution of the pathology however, provide advantages over current models with respect to providing an initial screen to assess efficacy of novel treatments that might prevent/reverse α-syn aggregation.
- Green Fluorescent Protein
- Tyrosine Hydroxylase
- Substantia Nigra
- Multiple System Atrophy
- Lewy Body
A pathological feature of Parkinson's disease (PD), irrespective of etiology, is the presence of intraneuronal Lewy bodies and Lewy neurites. The appearance of Lewy bodies in different brain regions parallels the development of symptom severity [1–3]. Lewy bodies are composed of at least 45 identified proteins and lipids, although the most significant contribution is made by α-synuclein (α-syn) [4–8]. Loss of dopamine (DA) neurons and the presence of Lewy bodies in the substantia nigra (SN) together provide the definitive diagnosis of PD [9]. The precise role for α-syn in the CNS, however, has yet to be determined. α-syn binds readily to lipids [10] and appears to be associated with vesicles in the presynaptic terminal [11–13] suggesting that the physiological role of α-syn might be related to vesicular release at the lipid membrane.
Human familial and sporadic cases of PD and animal models demonstrate that, in some form, α-syn contributes to the development of PD. Thus, familial forms of PD can be caused by mutations of α-syn (A53T, A30P, and E46K)[14–16] as well as by duplication and triplication of the wildtype (Wt) allele, SNCA [17–19]. Post-mortem analyses of sporadic forms of PD reveal Lewy bodies heavily stained for α-syn, although the mechanism of their production is unclear. Less convincing, perhaps, though still supportive of a role for α-syn in dopaminergic dysfunction in PD, are transgenic mice over-expressing α-syn. These animals show motor abnormalities and impairment in striatal DA release, though no overt loss of DA neurons [13, 20, 21]. Over-expression of α-syn (whether Wt or mutated), through the use of viral vectors, in the rodent [22–25] and primate [26, 27] SN, has provided a more clear indication of toxicity, with DA neuron loss in the SN, dystrophic neurites, reductions in striatal DA, and motor behaviour impairments. Taken together, these data all support the hypothesis that α-syn is toxic to DA neurons.
In normal human, α-syn is evenly distributed throughout the neuropil [28]. In PD, however, staining in surviving dopamine neurons is largely in clumps, or aggregates, that are seen throughout the soma. A large proportion of these α-syn aggregates are resistant to protein digestion techniques and considered to be insoluble [29–32]. It is tempting to speculate that aggregation of α-syn is a toxic process in PD and thus, that solubilising, or de-aggregating would be an effective approach to disease modification. However, using post-mortem tissue, it is difficult to discern whether the neurons that have already died in PD had α-syn aggregates similar to those that have survived. Animal models of synucleinopathy are thus important to critically assess the relevance of targeting α-syn aggregation as a therapy for PD. While the gene delivery based animal models of synucleinopathy, have proved better than transgenic models, in so far as reproducing overt nigrostriatal damage, limitations exist. Thus, to date, none of the viral vector models, whether based upon adeno-associated viral vectors (AAV) or lentiviruses have proved optimal for addressing these questions or providing a reliable platform upon which drug discovery programmes might usefully build. Thus, in some cases the timeframe for development of damage is long (8-16 weeks) [22, 23, 25], and in others there is high variability within studies [24, 27]. We propose that these limitations are purely technical, relating to the specifics of the virus and that a delivery vector, with a strong promoter, and selective neuronal expression, if produced in high titer could produce a model of PD synucleinopathy that would have even greater utility. Our preferred model would have the following properties: (1) high levels of expression of α-syn in SN and striatum, after SN delivery (2) pathology that developed over a time period that allowed investigation of effects of manipulations initiated before and after α-syn damage had occurred (3) relatively rapid evolution of the model, 3-6 wks, to minimise logistical challenges in running studies with the model (4) an evolving behavioural deficit (5) pathology that had characteristics similar to Lewy bodies and neurites e.g. intraneuronal inclusions of α-syn in the SN and dystrophic neurites in the striatum and (6) damage that was specific for α-syn and not purely a result of general over-expression of a protein.
As a first step towards achieving these goals we have developed a novel AVV model of PD alpha synucleinopathy. This vector is based on combining the distinct advantages of serotype 2 (neuronal tropism, ability to produce high titers, and to purify on a heparin sulphate column) with that of serotype 1 (excellent ability to penetrate brain tissue), using a chimeric approach to its construction. Thus, AAV1/2 is a vector that expresses both AAV serotype 1 and 2 on the viral capsid in a one to one ratio (details in methods section).
In the current study we have used this AAV1/2 vector to produce a rat model of PD based on the targeted expression of human A53T α-syn in the SN and using GFP and an empty vector as controls. The principle aim of this study was to provide an initial assessment of whether these vectors can drive expression in neurons of the substantia nigra, whether that protein is transported to terminals in the striatum and whether such expression is associated with aggregate-like pathology and loss of dopaminergic phenotype. To this end we report that high titer AAV1/2 vectors produce a relatively rapid course (3 weeks) of dopaminergic nigrostriatal pathology in the presence of α-syn aggregates and dystrophic axonal morphology and that GFP also shows evidence of toxicity.
Expression of GFP and human A53T alpha-synuclein along the nigrostriatal path
Expression of GFP following delivery of AAV1/2-GFP to the SN was also shown to co-localize with TH-immunoreactive neurons (confirmed with z-stacks) throughout the SN (Figure 1C, D). GFP aggregates were observed in the majority of cells within the SN that expressed TH (Figure 1D).
Nigral delivery of AAV1/2-A53T alpha-synuclein and AAV1/2-GFP produce neuronal loss in the SN
Further analysis was conducted to determine whether viral vector mediated reductions in TH-immunoreactive neurons of the SN were due to a loss in phenotypic expression or neuronal death. To address this we performed stereological cell counts in the SN that was immunohistochemically labelled with a universal neuron marker, NeuN. The numbers of NeuN-immunoreactive cells were significantly different across treatment groups [F(2,12) = 33.29, P < 0.0001] (Figure 4E-H). Delivery with AAV1/2-A53T alpha-synuclein produced a 42% and a 29% decrease in NeuN-immunoreactive cells compared to AAV1/2-empty vector (P < 0.01) and AAV1/2-GFP (P < 0.01) treated groups respectively. Animals receiving AAV1/2-GFP showed a significant reduction in NeuN-immunoreactive cells compared to AAV1/2-EV controls (19% reduction, P < 0.01).
Expression of A53T alpha synuclein, but not GFP, reduces tyrosine hydroxylase expression in the striatum
To see whether the observed reduction in TH expression was maintained, we analyzed tissues that had been exposed to AAV1/2-A53T α-syn for an additional 3 weeks. Thus, 6 weeks following injection of AAV1/2-A53T α-syn we found that striatal TH remained significantly reduced (t[8] = 2.57, P < 0.05; 167 ± 21 vs. 223 ± 4 [mean optical density ± SEM]) compared to its respective non-injected side. Further analysis of dopaminergic fibres was conducted using dopamine transporter (DAT) as a marker to support the apparent loss of nigrostriatal projections at this timepoint. We found that striatal DAT levels (nCi/g) were also significantly reduced (t[8] = 2.81, P < 0.05; 287 ± 65 vs. 483 ± 24 [mean optical density ± SEM]) compared to its respective non-injected side.
Dystrophic axonal morphology is present in the striatum of AAV1/2 A53T alpha-synuclein injected rats
The objective of the current study was to develop a rat model of Parkinson's disease based on the targeted over expression of human A53T α-syn that produced pathology in a time frame amenable to initial in vivo evaluation of potential therapeutics. We show that within 3 weeks of injection of high titer AAV1/2 viral vectors (5.1 × 1012 gp/ml) into the SN there are significant signs of dopaminergic toxicity in the nigrostriatal pathway.
Delivery of a single 2 μl injection of AAV1/2 A53T α-syn to the rat SN produced excellent anatomical coverage of the target region, the SN. Thus, α-syn expression was observed in the vast majority of TH-immunoreactive neurons spanning the anterior-posterior and dorso-ventral boundaries of the SN. Transport of α-syn and GFP by DA neurons of the SN was determined by examining fibre staining in the primary target structure, the striatum. Indeed, the entire striatum was filled with human α-syn or GFP stained fibres, indicating that A35T α-syn and GFP are able to travel along the nigrostriatal path and confirms that the majority of SN TH-immunoreactive neurons remaining are producing the vector mediated proteins. Thus, with respect to coverage of SN DA neurons and their axons projecting to the striatum, the AAV1/2 construct utilized in this experiment was highly effective. It is likely that the properties of the serotype 1 component of our construct and the high titer that could be purified afforded the AAV1/2 its ability to penetrate brain tissue with such efficacy [33, 34]. In a study by McFarland et al (2009), several AAV serotypes of similar concentration were compared, and serotype 1 had superior coverage of expression at the level of the SN compared to others (serotypes 2, 5, and 8) [33]. Use of the AAV2/5 construct with titers greater than those used here (6.2 - 6.7 × 1013 gc/ml) has also produced coverage of the SN in similar magnitudes using only a single 2 μl injection into the SN [35] and may provide an alternative means of producing models with excellent anatomical coverage, though the impact of such high titers on non-specific damage remains to be evaluated (see below).
Three weeks after delivery of AAV1/2-A53T α-syn, aggregates of human α-syn were observed in nigral TH-immunoreactive cells. Aggregates of α-syn were determined to be insoluble as indicated by being resistant to proteinase K digestion, a cardinal feature observed in PD [7, 28, 29, 32]. There was a reduced number of nigral TH-immunoreactive neurons, as well as reduction in total neuronal (NeuN) number, and, within the striatum, a loss of TH-immunoreactive fibre density and the presence of α-syn/TH-positive dystrophic neurites. Furthermore, this reduction in TH fibre density was present through 6 weeks with a concomitant reduction in striatal DAT demonstrating a persistence of effect. Thus, the approach of AAV1/2 delivery of A53T α-syn was able to replicate, in only three weeks, the major pathological hallmarks of PD. The model used here is not the first AAV-based delivery of α-syn to replicate α-syn aggregation, dystrophic neurites, and similar patterns of nigral cell loss. This has been previously demonstrated in other AAV α-syn rat models [22, 23, 25, 35], although with longer durations to achieve endpoints (8-16 weeks) and variable degrees of cell loss (32-50%). However, the magnitude of the losses (53% reduction in SN DA neurons), the low variability (n = 6/group) and the short time course of development (3 weeks) of the pathologies seen in the current study, highlight some of the potential advantages of using a high titer AAV1/2 vector when considering a model for assessing potential therapeutics targeting the toxicity of α-syn in PD.
AAV1/2 delivery of GFP was employed to control for the effects of high expression of any exogenous protein. This control demonstrated that the high titer of AAV1/2-GFP employed here caused significant nigral dopaminergic toxicity and thus should be considered when interpreting the toxic effects produced by delivery of AAV1/2 α-syn or other high titer vectors. The toxicity of AAV1/2-GFP however, could not account for all the A53T α-syn induced damage in the nigra, being significantly less, and being limited to the SN (no loss in striatal TH-immunoreactive or signs of dystrophic neurites). Thus, three weeks following the delivery of AAVs, aggregates of both human α-syn and GFP were observed in TH-immunoreactive neurons of the SN. Aggregates were seen in the majority of cells expressing both TH and α-syn or GFP. Unbiased stereology demonstrated that AAV1/2-A53T α-syn and, to a lesser extent, AAV1/2-GFP resulted in significant reductions in TH-immunoreactive neurons compared to AAV1/2-EV injected rats. In a recent study by Sanchez-Guajardo et al. (2010) AAV2/5 was used to deliver human α-syn (6.2 × 1012 gp/ml) or GFP (7.9 × 1012 gp/ml) as a control. The results of that study showed that GFP and α-syn expression produced ~20% and 50% SN DA neuron loss, respectively. These data support those shown here, in that expression of a control protein can be toxic and needs to be considered in the interpreation of the results.
To determine whether the TH-immunoreactive cell loss in the SN of the current study was indicative of a down-regulation of TH or frank neuronal cell loss, stereological assessment of NeuN-positive cells was performed [22, 36, 37]. NeuN cell counts were also significantly reduced in AAV1/2-A53T α-syn and AAV1/2-GFP injected animals when compared to AAV1/2-EV controls. In both cases (TH and NeuN cell number), the rats that received AAV1/2-A53T α-syn had significantly greater cell loss than AAV1/2-GFP administered animals. The toxicity of AAV1/2-GFP is unlikely to result from toxicity due to the vector itself, as, in so far as the dependent measures used in this study, injection of AAV1/2-EV produced no evidence of degeneration, in either the SN or the striatum. This suggests that overloading of the control protein might, in itself, be the cause of some of the SN damage seen with AAV1/2-GFP used at this titer. Indeed, a similar effect of GFP toxicity to SN DA neurons has been reported with the use of AAV8 (4 μl of 1 × 1010 vector genomes/ml) four weeks following injection [38]. Burdening the DA neurons of the SN with excessive protein likely reduces their fitness and leads to their demise. Cellular systems, lysosomal and ubiquitin proteasome, designed to deal with protein turnover have been shown to be significantly more active in DA neurons following viral vector mediated expression of α-syn and also in PD patients [29], although to no avail, indicating that insufficient clearance of unwanted or misfolded proteins may pose a significant threat. These findings highlight the importance of not only EV controls, which are often used, but also control protein vectors to define whether generalised protein overload underlies damage attributed to purported toxic proteins such as α-syn.
However, while there may be a non-α-syn-specific component to SN damage caused by delivery of AAV1/2-A53T α-syn, the striatal damage, loss of TH and dystrophic neurite pathology, appears to be specific to the expression of A53T α-syn. Thus, if this high titer AAV1/2 model were to be used, for instance to evaluate novel therapeutic strategies targeting α-syn, it might be appropriate to concentrate on striatal endpoints. There currently exist several classes of drugs that could be used in this initial screen that have been shown to reduce α-syn levels in vivo. For example, one of the statin drugs, lovastatin, was reported to reduce α-syn levels in the cortex of transgenic mice that overexpress α-syn [39]; rifampicin (a bacterialcidal antibiotic) has been shown to reduce established α-syn aggregate load and associated insoluble oligomers in a mouse model of multiple system atrophy that resulted in a decrease in neurodegeneration [40]; and finally rapamycin, a macrolytic lactone, that has been shown to reduce α-syn levels ostensibly via activation of autophagy [41, 42]. However, in future implementations of this model, it may be useful to evaluate lower titers of AAV1/2 to define if such a dose can be identified which will produce SN toxicity that can be completely ascribed to A53T α-syn delivery and have no component that might be caused by generalised protein overload.
Clinical and experimental evidence points towards the presence of α-syn aggregates in catecholaminergic neurons in PD as a contributing factor to reduced neuronal health and ultimately cell death. Irrespective of etiology, the vast majority of PD cases receiving post-mortem evaluation show Lewy bodies and Lewy neurites which stain heavily for α-syn [5]. This suggests that of the multiple upstream mechanisms, whether of genetic and/or non-genetic origin, responsible for PD, there appears to be convergence that leads to synucleinopathy. Such convergence defines α-syn as a key intervention point at which therapeutics could broadly target PD. It remains unclear whether the end product of α-syn accumulation, the aggregated form in Lewy bodies, is toxic or whether species leading up to this form should be the focus of therapeutic strategies. In vivo models such as that described here will prove useful in understanding which types of therapies will be effective by providing a relatively rapid screening platform upon which to test drugs capable of preventing aggregate formation or by reversing/de-aggregating inclusions. Care must be taken however, in interpreting actions where the damage cannot be completely ascribed to the α-syn per se, where there might be some role of generalised increased demand on protein handling systems. Indeed, more work is needed to refine models that do not generate toxicity in the control protein condition. This will likely require lower vector concentrations and a somewhat longer time course. There may always be a balance between non-specific toxicity and duration of evolution of pathology. However, we could envisage an ideal model based around the current AAV1/2 technology where pathology and behavioural deficits, selective for α-syn over generalised over-expression of protein, evolved over a relatively short period of time, perhaps 6 weeks, but where there was an intermediate stage, perhaps at 3 weeks, where there was functional disruption of DA systems though in the absence of overt cell loss. Treatments that were deemed efficacious in the rapid onset model, such as that described here, could then be further evaluated in a more protracted or progressive model to define in greater detail their therapeutic potential.
Animals and vector delivery
24 female Sprague Dawley rats (~280 g, Charles River) were stereotaxically injected with either AAV1/2 empty vector, GFP, or human A53T alpha-synuclein under isoflurane/oxygen anesthesia. In each case, a single 2 μl injection of viral vector was delivered to the SN at a rate of 0.2 μl/min according to the following coordinates: AP, -5.2 mm; ML, -2.0 mm; DV, -7.5 mm (skull) using a microinjector (Stoelting, Kiel, WI) and according to the atlas of Paxinos and Watson (1997). Animals were housed in pairs in a temperature controlled environment, kept on regular 12 hr light/dark cycle and allowed food and water ad libitum. All procedures were conducted under a permit that had received local IACUC approval (University Health Network).
Adeno associated vectors were custom ordered from GeneDetect Ltd. (Auckland, New Zealand). Each vector was driven by a chicken beta actin (CBA) promoter hybridized with the cytomegalovirus (CMV) immediate early enhancer sequence. In addition, a woodchuck post-transcriptional regulatory element (WPRE) and the presence of a bovine growth hormone (BGH) polyadenylation sequence for high transcription following transduction was incorporated. Viral titers were 5.1 × 1012 genomic particles (gp)/ml for each vector used and were determined by quantitative PCR (Applied Biosystems 7900 QPCR) with primers directed to the WPRE region incorporated within the AAV expression cassette, thus representing the number of functional physical particles of AAV in the solution containing the genome to be delivered. WPRE primers used produced a 113 bp product and were as follows (5' to 3'): FWD GGCTGTTGGGCACTGACAAT; REV CCGAAGGGACGTAGCAGAAG. AAV1/2 is a chimeric vector where the capsid expresses AAV1 and AAV2 serotype proteins in a 1:1 ratio and use the inverted terminal repeats (ITR's) from AAV2 according to the following scheme: ITR (from AAV2)----CMV/CBA promoter----human A53T alpha-synuclein or GFP----WPRE-bGH-polyA----ITR (from AAV2) [43]. Viral vectors were affinity purified against immobilized heparin sulphate proteoglycan and concentrated by modified Iodixanol/cation exchange/Q-Sepharose. Purity was verified by SDS-PAGE. Three vectors were produced using this design, in the first, the gene for mutated human alpha-synuclein was inserted, this vector is termed AAV1/2-A53T α-syn, the second vector expressed green fluorescent protein (GFP), AAV1/2-GFP, and in the third vector the space for the gene was left empty, AAV1/2-empty vector (EV). Similar AAV1/2 chimeric vectors have previously been used for other purposes i.e. expressing proteins in experimental models of Huntington's disease and epilepsy [34, 44].
Post-mortem measures
Three weeks after AAV1/2 injection, animals were administered an overdose of pentobarbital and killed by exsanguination with saline followed by 4% paraformaldehyde, brains removed and processed for immuno-labeling. Brains were sectioned frozen in the coronal plane at a thickness of 40 μm on a sliding microtome (Leica Microsystems Inc., Richmond Hill, ON) and 6 series of sections were stored in cryoprotectant (30% glycerol, 30% ethoxyethanol, 40% PBS). A single series of sections were processed for visualization of tyrosine hydroxylase (TH) and NeuN via the biotin-labelled antibody procedure. Briefly, following several washes in a PBS solution containing 0.2% Triton X-100 (PBS-T), endogenous peroxidase was quenched in a 3% hydrogen peroxide solution and background staining was then inhibited in a 10% normal goat serum/2% bovine serum albumin solution. Tissue was then incubated with primary antibodies overnight: rabbit anti-TH antibody (1:1000, Pel-Freez, Rogers, AR), mouse anti-NeuN antibody (1:200, Millipore, Billerica, MA). After three washes in PBS-T, sections were sequentially incubated in biotinylated goat anti-rabbit or mouse IgG (1:300; Vector, Burlingame, CA) for 1 h and the Elite avidin-biotin complex (ABC Kits; Vector, Burlingame, CA) for 1 h separated by three washes in PBS. Immunostaining was visualized following a reaction with 3,3-diaminobenzidine (Vector, Burlingame, CA). Sections were then mounted on glass slides, allowed to dry, dipped into dH20, dehydrated through graded alcohols (70%, 95%, 100%), cleared in xylenes, and coverslipped with DPX mounting medium (Electron Microscopy Sciences, Hatfield, PA).
Triple label immunofluorescence (Confocal microscopy, Zeiss Axioplan with LSM510META) to reveal TH (1:1000, Pel-Freez, Rogers, AR) and human alpha-synuclein (1:500, Zymed, San Fransico, CA) or GFP (1:2000, Abcam, Cambridge, MA), and Hoechst (nuclear stain) simultaneously to provide detail regarding co-localization and whether expression was nuclear and/or cytoplasmic. Images were taken throughout the Z-axis to confirm co-localization of α-syn and GFP within individual TH neurons.
TH and NeuN stained sections of the SN were used for stereological estimation of dopamine neuron numbers using optical fractionator from the Stereo Investigator software package (v. 7, MBF Biosciences, Williston, VT). The user was blinded to group assignment by coded slides. Nine sections spanning the entire anterior/posterior extent of the SN, separated by 240 μm (1/6 series), were used for counting. All TH-immunoreactive neurons of the SN (pars compacta and pars reticulata) were included within each contour, of each section. NeuN contours were closely matched with cases used for TH [22]. Parameters used for TH stereological counting were grid size, 300 μm × 300 μm; counting frame, 80 μm × 80 μm, and 2 μm guard zones. Parameters used for NeuN stereological counting were grid size, 480 μm × 480 μm; counting frame, 80 μm × 80 μm, and 2 μm guard zones. Tissue thickness was determined by the user at each counting site. All final values represent estimated total by number weighted section thickness and were only included if their Gunderson coefficient of error (m = 1) was less than 0.09.
Proteinase K treatment
Tissue sections from AAV1/2-A53T α-syn rats containing the SN were treated with proteinase K to determine whether the α-syn seen here was soluble (non-aggregated) or insoluble (aggregated) according to the method of Chu et al (2007 and 2009) [28, 29]. Briefly, sections were mounted and dried on Permafrost+ glass slides for at least 8 hrs at 55°C. Sections were then briefly hydrated with TBS-T (10 mM Tris-HCl, pH 7.8; 100 mM NaCl; 0.05% Tween20), and digested with 50 μg/ml PK (Invitrogen, Carlsbad, CA) in TBS-T (10 mM Tris-HCl, pH 7.8; 100 mM NaCl; 0.1% Tween20) for a period of 2.5 h at 55°C. Sections were then fixed for 10 min using 4% paraformaldehyde and then processed for α-syn immunohistochemistry as described above.
The levels of striatal DAT binding were assessed by [125I]-RTI-121 binding autoradiography in sections prepared from fresh-frozen tissue. Briefly, thawed slides (one slide per animal) were placed in binding buffer (2 × 15 min, room temperature) containing 50 mM Tris, 120 mM NaCl and 5 mM KCl. Sections were then placed in the same buffer containing 30 pM [125I]-RTI-121 (Perkin-Elmer, specific activity 2200 Ci/μmol) for 120 min at 25°C to determine total binding. Non-specific binding was defined as that observed in the presence of 100 μM GBR 12909 (Tocris Bioscience). All slides were then washed (4 × 15 min) in ice-cold binding buffer, rinsed in ice-cold distilled water and air-dried. Together with [125I]-microscale standards (Amersham) slides were then apposed to autoradiographic film (Kodak) and left for 7 days at RT before developing. Autoradiograms were analysed using MCID software (Image Research Inc, Ontario, Canada). Densiometric analysis of 3 striata from each animal was carried out whereby a reference curve of c.p.m. versus optical density was calculated from β-emitting [14C] micro-scale standards and used to quantify the intensity of signal as nCi/g. Background intensity was subtracted from each reading. Data were then expressed as mean ± s.e.m. signal intensity for each treatment group. Non-specific binding was calculated in the same way and subtracted from the total to give to give specific binding. Non-specific binding was found to account for <1% of total binding.
Statistical Analysis
For all statistical comparisons we first used a 1-way ANOVA, with significance set at P < 0.05. If ANOVA was significant, all post-hoc tests were conducted using Tukey's Multiple Comparison test. Where only two groups were compared, an unpaired t-test was conducted. Software used to conduct statistical analyses and graph all data was Prism v. 5.02 (GraphPad, La Jolla, CA, USA).
This study was supported by The Cure Parkinson's Trust and the Krembil Foundation. We thank Maria Espinosa for her excellent technical skills that contributed to completing the experiments. We thank the laboratory of Dr. Jeffrey Kordower for kindly providing a protocol for us to conduct the proteinase K digestion component of the study.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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It is easy for physicians to run afoul of numerous federal and state laws when operating or selling their medical practice. The purpose of this article is to briefly discuss the False Claims Act (FCA), and the Anti-Kickback Statute (AKS), the Physician Self-Referral Law (Stark law). The Government agencies charged with enforcing these laws include the Department of Justice, the Department of Health & Human Services Office of Inspector General (OIG), and the Centers for Medicare & Medicaid Services (CMS).
The civil FCA shields the Government from being overcharged or sold inferior goods or services. It is illegal to present claims for payment to Medicare or Medicaid you know or should know are false or fraudulent. If a claim results from a kickback, or is made in violation of the Stark law, that alone may render it false or fraudulent creating liability under the civil FCA and the AKS or Stark law. No specific intent to defraud is required. The civil FCA defines "knowing" to include not only actual knowledge but also instances in which the person acted in deliberate ignorance or reckless disregard of the truth or falsity of the information. The penalties are severe; Physicians have gone to prison for submitting false health care claims.
A few case examples of FCA violations from the U.S. Department of Health & Human Services Office of Inspector General's publication, "Avoiding Medicare and Medicaid Fraud and Abuse" include:
- A psychiatrist was fined $400,000 and permanently excluded from participating in the Federal health care programs for misrepresenting that he provided therapy sessions requiring 30 or 60 minutes of face-to-face time with the patient, when he had provided only medication checks for 15 minutes or less.
- A dermatologist was sentenced to 2 years of probation and 6 months of home confinement and ordered to pay $2.9 million for falsifying lab tests and backdating letters to referring physicians to substantiate false diagnoses to make the documents appear that his patients had Medicare covered conditions when they did not.
- A cardiologist paid the Government $435,000 and entered a 5-year Integrity Agreement with OIG to settle allegations he knowingly submitted claims for consultation services not supported by patient medical records and did not meet the criteria for a consultation.
The AKS [42 U.S.C. § 1320a-7b(b)] is a criminal law that prohibits the knowing and willful payment of compensation to (1) encourage or reward patient referrals or (2) generate business involving any item or service payable by the Federal health care programs including drugs, supplies, or health care services for Medicare or Medicaid patients. Compensation includes anything of value such as cash, free rent, expensive hotel stays and meals, and excessive payment for medical directorships or consultancies. The statute covers the payers of kickbacks; those who offer or pay remuneration and the recipients of kickbacks; those who solicit or receive remuneration. Criminal penalties and administrative sanctions for violating the AKS include fines, jail terms, and exclusion from participation in the Federal health care programs.
Physicians are attractive targets for kickback schemes because they are naturally a great source of referrals for fellow physicians or other health care providers and suppliers. Physicians choose what drugs their patient's use, which specialists they see and what health care services and supplies they receive. Many people and companies want the physician to steer their patients to them and will pay to send that business their way. And remember, it's a two-way street and it is just illegal for the physician to take money from providers and suppliers in return for the referral of Medicare and Medicaid patients as it is for the physician to pay others to refer those patients to them.
A few case examples of kickback violations from the U.S. Department of Health & Human Services Office of Inspector General's publication, "Avoiding Medicare and Medicaid Fraud and Abuse" include:
- Nine cardiologists paid the Government over $3.2 million for engaging in a kickback scheme. The cardiologists received salaries under clinical faculty services agreements with a hospital under which, the Government, they did not provide some or the services. In exchange, the cardiologists referred their patients to the hospital for cardiology services.
- A physician paid the Government $203,000 to settle allegations he violated the physician self-referral prohibition in the Stark law for routinely referring Medicare patients to an oxygen supply company he owned.
- Four orthopedic device manufacturers paid $311 million to settle kickback and false claims allegations that the companies bribed surgeons to recommend their hip and knee surgical implant products. The companies allegedly would award physicians with vacations, gifts, and annual "consulting fees" as high as $200,000 in return for the physicians' endorsements of their implants or use in operations.
The Stark Act prohibits physicians from referring patients to receive designated health services payable by Medicare or Medicaid from entities with which the physician or an immediate family member has a financial relationship, unless an exception applies. Its terms can be a complicated trap for the unwary, particularly in circumstances where physicians are selling their practices, but intend to refer patients or provide services to the purchaser.
As stated: it is easy for physicians to run afoul of numerous federal and state laws when operating or selling their medical practice. An experienced health care lawyer can analyze your issues and provide a legal evaluation and risk analysis of the proposed venture, relationship, or arrangement.

subset: commoncrawl


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News Archive
Offensive Reactions
November 11 2002
By Bryan Hersh of 49ers Paradise
THIS CONTENT IS COPYWRITED, REDISTRIBUTION OF IT (including copy/pasting it to a message board, forum or bbs) IS PROHIBITED AND COULD RESULT IN LEGAL ACTIONS - feel free to quote up to 1 paragraph providing a source link to http://www.49ersparadise.cjb.net is included
Only able to muster 17 points against one of the leagues worst defenses, the 49ers offense was certainly a disappointment. The offense played a good first half of football, but as the blocking began to break down in front of Garcia, the offense began to stifle. The game, also should have given the 49ers a good reason not to shift Tai Streets out of the second receiver position even when Stokes enters the lineup.
Starting on the offensive line, which gave up three sacks on Jeff Garcia. The line did a good job establishing its self in the first half, picking up a fourth and one conversion, but they were unable to give Jeff Garcia the time in the second half. Rookie Eric Heitmann was pounded on all day. This is the second consecutive week of spending lots of time on the field for the entire offense, and it has to be hurting the larger men on this side of the ball.
Quarterback Jeff Garcia did play anywhere near the level he did against the Raiders. While Garcia started the team out pretty well, taking the short completions that the defense was giving him. Garcia threw his fifth interception in scoring position at the end of the second quarter, a pass that should have been thrown away ended up in the hands of a Chief, ending any chance of putting more points on the board before the end of the half. His game, seemed to fall from there. Im a firm believer that Garcias drop in ability this game stemmed from the offensive line and from moving Streets down in the progressions when Stokes was on the field.
The 49ers receivers did nothing special in this game. Terrell Owens added what was his tenth unofficial dropped pass this season, nearly one fifth of all catchable passes seem to fall to the turf. Tai Streets was shifted to the third receiver position when Stokes was on the field, and while in theory this may make sense, given where these two players have spent most of their career practicing, in practice, the theory predictably failed. The 49ers have to bring Stokes in as the third receiver and not drop Streets in the progression just because Stokes is on the field.
The running backs had a decent day. Both Barlow and Hearst got into the endzone and Fred Beasley contributed by adding a few first downs. The group still isnt putting together the rushing performances they were earlier this year, and that could be because the offensive line is tiring after playing so much offense over the past two weeks. Garrison Hearst looked to be much more explosive with his injury healing up.
This was not an impressive offensive performance despite converting 61% of their third down opportunities. The team was playing against a very week defense, and they simply couldnt put the points on the board. It may however be the wakeup call this team needs to get ready for the Chargers.


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Don't get left behind! We have a GREAT train, this month. Here's the easiest way to make sure you grab all the great freebies in this train.
- visit each designer's blog or FB posting for their preview & special link to their MM store
- click on the link provided
- place their gift in your shopping cart
- check out when you have all the gifts collected
My Memories is a great software for your digital scrap booking, craft and printable projects. BRAND NEW Version 8 is awesome!
Here is a link to download the trial version or to purchase if you are ready. LINK
Here is a link to MM Suite software tutorials on You-tube: LINK
Ready to Purchase? Use code: STMMMS13734 – LINK
BE SURE AND USE MY COUPON CODE TO RECEIVE $10 off YOUR PURCHASE PRICE PLUS THESE OTHER SPECIAL OFFERS !!
Buy My Memories Suite now and get: 30% off a photobook or photo gift order! Plus get the 1st Month Free to the Design Club!
This is Memory Mosaic's Contribution this month. Just click here or on the image below.
Here are the rest of the links for this train!!
ADB Designs
Over the Fence Designs
PattyB Scraps
Kathryn Estry Designs
Polka Dot Chicks
Happy Scrap Arts
A Beautiful Mess
Albums to Remember
Louise L
You are here >> Memory Mosaic << You are here
KJD Designs
Carolyn's Scrap Creations
QueenBrat Digital Designs
Ostrea Designs
Lins Creations
Snickerdoodle Designs
Marniejo's House of Scraps
scrap n' designs by Rv MacSouli
Designs by Lisa Minor
Becky's Creations
Sugar Moon Designs
Staffy Scraps Designs by Stina
Marieh H Designs
Triple J Designs
Moore Blessings
Sunshine Inspired Designs
Day Dreams n' Designs
Syrena's Scrap Art
Sweet Pea Designs
Elizabeth's Market Cross
Caroline B


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London Lettings & Property Management
Founded on traditional values, Chambré is a specialist residential London Lettings and Property Management firm for the private landlord and professional adviser.
Focused on properties that rent between £600 and £6,000+ per week, whether you have one property or a portfolio of properties, we take huge pride in providing you with an exceptional level of service.
With over £100 million of property under management, Chambré are the pragmatic alternative to the high street agents, providing bespoke solutions at competitive prices.
Our clients have complete confidence in entrusting their properties with us because of our pro-active and common sense approach, trusted advice and exceptional service levels.
What else do we offer?
We are your people on the ground in London to support you with all property related matters. Other areas we can assist you with include buying and selling property, refurbishment services and property probate management. By becoming a client of Chambré you will gain access, by means of introduction, to our carefully collated address book of UK residential and commercial property experts who share our values.
"I started Chambré in 2005 having listened all too often to the frustrations that landlords and professional advisers were having with the service they were receiving from some high street agents. I spotted a gap in the market for a specialist lettings and property management agency that understood what all property owners and investors require – expert advice, good quality tenants and an exceptional property management service. London property is an incredibly valuable asset and should be handled as such."
Tom Chambré, Managing Director
'Chambré are extremely proud to announce that our clients have given us an overall rating of 93.5% in the category of 2018 ESTAS Best Letting Agent'


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OtherTheia Alison 880613
- Label Size:
- 10
- Street Size:
- 10
OtherTheia Alison 880613
- Label Size:
- 10
- Street Size:
- 10
This listing has expired.
This Theia Alison 880613 Cocktail wedding dress is Brand New with its Hang Tag attached! This Ivory, 100% Silk Petals gown has an A-line silhouette, is Sleeveless with a V-neck neckline. Such a fun and flirty cocktail dress! The silk fabric petals adorned with seed pearls create such a beautiful, delicate look. In addition to being a gorgeous cocktail dress, it's perfect for a destination wedding, rehearsal dinner, reception dress or formal event.
Actual Gown Measurements:
Bust: 38″ (*please note, this is not the same as your bra size)
Waist: 30 1/2″
Hips: 40 1/2″ (If your hip measurement is 42″ or less, this dress will work with your figure)
This is just one of the many designer couture bridal gowns that we sell at Your Dream Dress. This gown has Never been worn to an actual wedding/event. Please visit the 'About Our Dresses' section on our website for more details. As with any dress/gown, alterations are usually needed for the best fit. Please make sure to measure yourself (with undergarments only) and compare to the above measurements. Keep in mind that Evening/Bridal dresses usually run differently (usually smaller) than daywear.
We offer a wonderful NO RISK Return Policy!
Our No Risk, Return Policy:
When you receive the gown or accessory, you will need to contact us by email or telephone within (3) three days of receiving (signing for) to state if you will be keeping the item or returning it. The item would then need to be shipped back by UPS, FedEx or USPS, fully insured (for your protection) and be received by us within (7) seven business days in the same condition as it was sent. Once we receive the item back at our location we will inspect it and, if in the same condition as the item was sent to you, we will refund the price of the item. The shipping to you and from you would not be refunded, only the cost of the item.
If we would not hear from you within (3) three days of receiving (signing for) the item, we would consider the item satisfactory and no returns/refunds would be accepted. Please feel free to call us at 717-506-0343, if you have any further questions, and we will be happy to help! We are in the office Monday to Friday from 10:00 a.m. to 5:00 p.m. EST.
Payment Methods: Paypal, Visa, Mastercard, & Discover
We ship Worldwide! (Except a few select countries, please contact the seller.)
Your Dream Dress believes in personalized customer service, so if you have any questions, please do not hesitate to contact us. Also, if you would like additional gown photos, please let us know. We will be happy to help!
Terms Of Sale: Returns Accepted
Location: Enola, PA, United States
Will Ship to: Other
Payment Methods Accepted: Other


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Tests at 41 Weeks
The Non-Stress Test, which takes about 20-30 minutes, assesses your baby's heart rate and how it changes. You will lie back on a recliner and a fetal monitor will be attached to your abdomen. The baby's heart rate and movements will be recorded on a long strip of paper. The heart rates of babies increase as they move, and your doctor will assess the results to make sure your baby's heart rate was high enough and increased with movement.
The Biophysical Profile is an ultrasound that checks how your baby is moving, the amount of amniotic fluid around the baby, the muscle tone, fetal breathing and the number of times the baby moves.


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Ductless Mini-Split A/C Systems
Delivering exceptional ductless HVAC installation, repair & maintenance to families and businesses of Atlanta for over 20 years
In recent years, a growing number of homeowners and businesses are discovering the many benefits of ductless air conditioning.
Often referred to as "mini splits," ductless A/C offers an alternative to traditional residential and commercial cooling technology that provides a more cost-effective and attractive solution for applications where the use of ducted air conditioning machinery would be impractical or unaffordable.
Talk to the experienced residential and commercial ductless HVAC contractors at Gagne to find out how much it will cost to install, schedule a maintenance visit and more.CONTACT US TODAY
Reviews of Our HVAC Contractors with Ductless Repair & Installation Experience
How Does Ductless A/C Work?
Similar to a central air conditioning setup, a ductless mini-split system has an outdoor unit that contains the compressor and condenser. Interior blowers are installed inside to provide cooling and heating (if the system is setup with a heat pump). The blowers are connected to the outside unit through an exterior wall conduit and operate independently of one another.
Most modern ductless A/C units come standard with several energy-saving features, such as:
- Programmable timer and sleeper mode
- Multiple fan speeds for dehumidification
- Reusable filter
- Auto changeover if configured as a heat pump
- Wireless controller
Because of these features and more—not to mention a lack of ducts, which can contribute to 30% of a home's energy consumption due to leaks—ductless A/C is one of the most energy efficient cooling systems in the market today.
Help, My Ductless A/C Unit is Broken!
Is your ductless split system in need of repair? Our emergency A/C repair team is composed of NATE-certified, Georgia-licensed, factory-trained contractors who are ready to respond promptly and professionally to your residential or commercial HVAC needs - including broken or malfunctioning ductless cooling and heating systems.
Though modern ductless A/C technology is highly reliable, every once in awhile a unit or part may malfunction or break down like with any other air conditioning system. Common ductless split system problems include:
- Compressor issues
- Indoor blower problems
- Refrigerant or condensate drain line leaks
- Poor drainage
- Clogged air filter
- Worn out bearings
Whether you need an emergency repair or seasonal maintenance for your ductless A/C system, our Atlanta residential and commercial HVAC contractors are here to help.
When Do You Need a Ductless A/C Split System?
Ductless split systems are highly versatile and can be great solutions for a number of different scenarios. For instance, perhaps you already have a primary whole-home air conditioner, but dust and debris that naturally accumulates in ductwork is aggravating your allergies or asthma. Or maybe you have a basement that gets too cold and are looking for a more comfortable and safer solution than a space heater.
Many businesses and organizations also take advantage of ductless A/C by supplementing their main heating and cooling system. This reduces their energy expenditure overall and increases cost savings in the long run.
Common applications for ductless a/c systems include:
- Room additions. When central A/C is unable to sufficiently cool an add-on room of your house, a mini-split system can be an affordable and effective option.
- Garage, attic, or basement renovation. For basements, attics or garages, sometimes installing A/C ductwork is impractical or unwanted. If you're doing a home renovation and want to cool off or heat up your basement, attic or garage, consider installing a mini-split AC system.
- Supplemental cooling. When your central air conditioner is undersized or the ductwork is defective, a ductless split system may be the answer.
- Whole house. In new construction, split A/C can be combined with the architecture of the building or home to improve indoor comfort levels, eliminate hot/cold spots and maximize energy efficiency.
Regardless of your residential or commercial challenge, the installation of a ductless split system can often be a seamless solution.
What Are the Benefits of Ductless Heating & Cooling?
- Improve efficiency. A/C duct leaks, holes and loose seals are responsible for up to 30% of energy consumption in the average home or office. By getting rid of ducts altogether, homeowners and businesses can eliminate this common energy waster.
- Easy installation. Compared to other HVAC systems, getting a ductless A/C system professionally installed is a simple, straightforward and relatively inexpensive process.
- Reduce your footprint. Eliminating wasted energy and maximizing efficiency is not only good for your pocket book, it's also environmentally friendly!
- Save money. Greater energy efficiency directly translates to financial savings on your monthly energy bill. While paying to have a new ductless split system installed requires upfront expense, this investment will quickly pay itself off through consistently lower heating and cooling bills.
- Greater control. With central heating and cooling system, you typically only have one thermostat that controls the temperature of the entire home or building. Ductless A/C puts more control at your fingertips by having the ability to easily change the temperature in each room or zone and avoid those pesky hot/cold spots.
How is a Ductless Split System Installed?
Installing ductless A/C is a fairly straightforward process. It doesn't involve a major renovation or having to sacrifice a nice view for a bulky window unit. Like central air conditioners, split systems have two primary components: an outdoor compressor and an indoor air-handling unit (blower).
The indoor unit is strategically mounted high on the wall of each target room or zone where it is out of the way and blends neatly into your space. Once placed, it quietly and efficiently pumps cool, treated air from the outside compressor into every corner of the room. In commercial spaces, there are options for ceiling-mounted or in-ceiling systems as well.
While you can certainly attempt to install a ductless split system on your own using one of the many DIY installation guides found online, we recommend leaving it to a professional HVAC installer. Trying to install a complex indoor air comfort equipment without proper experience or knowledge can result in poor efficiency levels (at best) or a broken system (at worst).
What's Involved in DIY Mini-Split AC Maintenance for Homeowners?
With regular maintenance and care, your ductless mini split HVAC system should last anywhere from 12 to 15 years, at least. Homes in areas with higher humidity or salt levels may have to replace their ductless system sooner. Mini split AC systems work best in temperatures from 14°F to 109°F. The best way to prolong the lifespan of your mini split AC equipment is to change and replace your air filters regularly. Doing so will ensure that your system runs efficiently, prevent sudden breakdowns, and help keep the air in your home clean during all seasons.
Other DIY maintenance and cleaning tips for your ductless mini-split air conditioner include:
- Trimming up vegetation and picking up debris around your outside unit (compressor)
- Ensuring there is a sufficient place for channelling excess condensation
- Wiping down your compressor with a cloth (they can overheat when excessively dirty)
- Pressing the reset button (often at the bottom of the control) if your remote stops working
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Today, companies must also find ways to define and deliver unique experiences, together with users, in order to survive. However, this requires a paradigm shift – both a change in mindset and adjustments to current working practices.In this report, the process of defining unique experiences together with users is referred to as user-driven innovation. User-driven innovation encompasses both an understanding of true user needs and a systematic involvement of users in the innovation process.The report explains a number of market forces and academic underpinnings, and presents an overview of the context regarding user-driven innovation in each of the five Nordic countries. The report also presents concrete examples of how companies employ user-driven innovation processes. Finally, the report suggests a number of policy-level activities which could be undertaken to strengthen both the understanding and practical competencies related to user-driven innovation.Project duration: September 2007 - June 2008
Nordic focus on user-driven innovationIn November 2006, the Nordic Innovation Centre (NICe) launched a call for proposals under the theme of 'user-driven innovation' (UDI). This is one of six projects that were selected and approved for financing. These projects were kicked-off in June 2007. For more information on the other five projects, see links below.
Consultant and Research Fellow, Research Policy Institute, Sweden
Copyright © 2010 Nordic Innovation All rights reserved. webmaster @ nordicinnovation.orgNordic Innovation | Stensberggt. 25 | NO-0170 Oslo | Telefon +47 47 61 44 00 | info @ nordicinnovation.org


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- Updated model 2017: LG SJ950V
The LG UH950V is the best TV LED LG for 2016-2017. It is a high-end TV so the specs are excellent, including a 10bit panel compatible with HDR. The frequency is 100Hz panel and PMI 2700. The panel is LED Display Quantum SuperUHD 4K IPS and although it behaves quite well, but is far contrast models with VA panel. The UH950V also supports 3D with passive glasses. The Smart Platform is the new webOS 3.0 that leads this aspect today. Except for the 10-bit panel we do not belive that the UH950V is better than the UH850V.
Technical / Specifications:Screen Size: 55 and 65 inches.
Technology: IPS Edge LED TrueBlack, Local Dimming, Ultra Luminance, ...
Resolution: 4K UHD 3840x2160
HDR: 10bit
3D: yes, passive
PMI: 2700Hz
Smart TV: webOS 3.0
- 3x HDMI
- 3x USB
- Magic Remote Control
- Audio 40W
Design LG UH950V:
Other TV like LG UH950V?
Do not forget to check out out "TV Buying Guide 2017"
Our opinion LG UH950V (veredict):Our opinion on the LG UH950V is quite good, which is not enough for a TV of this price. The Smart is the best, working fluid and set design is spectacular: the slimmest. The image quality should be the priority, at least for most buyers and this TV though 4K and a panel 10-bit IPS, so the contrast is somewhat lower than other models in the same price range. The IPS panel offers the possibility of watching TV from the sides without losing image quality, but we lose contrast, if we watch TV in the dark. Our advice is to buy the LG UH950V if you want an excellent TV (very expensive) even see with the whole family, enjoy a good Smart and rarely watch movies with the lights off.
Model Name: What's the meaning of the name of LG 2016 TV?
You can check our List of all models 2016 LG TV


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Twentieth Sunday After Pentecost/Lectionary 26C, October 2nd, 2016
Lamentations 1:1-6, Lamentations 3:19-26, 2 Timothy 1:1-14, Luke 17:5-10
Preached by Pastor Anna C. Haugen, Augustana and Birka Lutheran Churches, Underwood, ND
May the words of my mouth, and the meditations of my heart, be acceptable in your sight, my rock and my redeemer.
Grace and peace to you from God our Father, and the Lord Jesus Christ. Amen.
Today's first reading and psalm come from the book of Lamentations. A lament is a passionate expression of grief and sorrow. A lament is when mere tears are not enough. A lament is when every inch of your body and soul cry out within you. When no consolation is possible. There are times for songs of joy and hope, but there are also times for songs of sadness and despair. There is a time for grief. The book of Lamentations is a whole book filled with lament.
We don't know how to lament, these days. We are uncomfortable with grief and sorrow. When someone suffers a loss, we don't often cry with them. How often have I seen this: someone is overcome with grief, and we pat them on the shoulder, tell them we're praying for them, and then explain why they shouldn't cry. God wanted another angel in heaven. She's in a better place. You'll feel better soon. God's got a plan—and don't you trust God? Shouldn't you be over it by now? We tell ourselves that these platitudes are to comfort the one who grieves; yet all too often what they really do is just shut them up. In big ways and small ways, our culture tells us that we can't grieve too much. We can't be too extravagant in our tears, and we can't take too long. It makes people uncomfortable. As Christians, especially, there is a pressure to hide our grief and recover quickly, to put a good face on our sorrows. After all, don't we have God? Isn't God supposed to take care of us? Isn't God supposed to supply us with all good things all the time? If our suffering is too great, if our sorrow is too deep, well. Maybe we're not being faithful enough. Maybe we just don't have the right attitude. And yet, here in the Bible is an entire book filled with grief and pain and anger and fear and sorrow and all the emotions that rage through us in the darkest times.
The book of Lamentations was written after the Babylonians destroyed the country of Judah, and its capital the city of Jerusalem, in 587BC. And by destroyed I don't just mean they conquered it. They tore down the Temple to its very foundations. They took a large portion of the population away in chains to live as hostages to the good behavior of those left behind, and to be forced to serve the very empire that had destroyed their home. A large portion of Judah's population, including the prophet Jeremiah, fled to Egypt, where they lived as refugees watching from afar as their enemy destroyed their homes. To add insult to injury, the Babylonians resettled people from other parts of their empire in Judah, to make doubly sure that even Judah's culture would be destroyed.
Imagine that. Put yourself in their shoes. How would you feel? Imagine that America was conquered by a foreign power. Imagine that an occupying army patrolled the streets of Bismarck every day, and swept through Underwood regularly. Imagine that they destroyed the church, the city hall, the pharmacy, the grocery store. Imagine that they took your friends and family away at gunpoint, and took them somewhere else—you didn't know where. Imagine that they were coming for you, and so you gathered your family and what you could carry on your back and slipped out of town at night, heading for Mexico, in the hopes that you would be safe there. Imagine arriving with nothing, terrified and alone, in a place you didn't speak the language, a place where no one liked you and no one wanted you. Imagine waiting every day for news from home, hoping that the invaders would be destroyed and you could go back, but only hearing more stories of pain and suffering. How would you feel?
That's what the book of Lamentations is all about. That despair. That pain. That sorrow. "How lonely sits the city that once was full of people! … she weeps bitterly in the night with tears on her cheeks … all her friends have dealt treacherously with her. Judah has gone into exile with suffering and hard servitude." They sang these songs, Jeremiah and the rest of the refugees in Egypt, and they cried, and they wept. There is no platitude that will fix this, no consolation that will make it all worth it, no sweet, pious words that will make things better. And you know what? It was okay to be honest about that. It was okay to be honest about the depth of their pain and their grief. It was okay to scream and yell and rage at God. God knew what was in their hearts. Putting a brave face on it and pretending to be okay would not fool God; all it would do is bottle all that emotion up where it could do nothing but fester. God is big enough to take all of us, even the ugly parts, even the grief and the pain and the anger and the fear and the sorrow.
And yes, the captives and the exiles and the refugees were partly to blame for their own misfortunes. As a nation, they had turned away from God, taking his love and protection for granted, seeking after other gods and allowing injustice free reign in their communities. If they hadn't done that, if they had remained as faithful to God as he was to them, even all the might of Babylon would not have prevailed against them. By turning away from God, they had removed his protecting hand from them, and so the Babylonians had come. I imagine that must have made things ten times worse, to look back and wonder what they might have done differently, what might have been possible if they had been more faithful.
But even in the midst of that grief, God was with them. As they grieved the destruction of their homes, as they took responsibility for the things they had done leading up to the fall of their country, God was there. He wasn't there with a magic bullet to take away their pain and make things better. He wasn't there with greater rewards to make the destruction of their homeland and the deaths and kidnappings of so many of their loved ones unimportant. He wasn't there to tell them to get over it. He was there in the midst of their pain to hold them as they cried. He was there in a million small ways, giving them strength to get through each day and courage to start building new lives. "The thought of my affliction and my homelessness is wormwood and gall! My soul thinks continually of it and is bowed down within me. But this I call to mind, and therefore I have hope: the steadfast love of the Lord never ceases, his mercies never come to an end."
I hope and pray that we never suffer what they suffered, but there are people today who suffer that and worse. Between imperialistic nations, terrorists, gang violence, and environmental disasters, there are more refugees in the world today than there have been since the end of World War II. But there is no Olympics of grief: no scale to weigh things out and go, well, this grief is worse than that one, so you can't be too upset about that one. There is death in this community. There are broken relationships and broken homes in this community. There is abuse and rape and homelessness and suicide in this community. There is loss and grief and pain. And you know what? It's okay to lament. It's okay to not be okay. If grief overwhelms you and fear and pain and doubt and anger and sorrow drag at your footsteps and threaten to drown you, that's okay. It doesn't mean you're a bad Christian, and it doesn't mean your faith isn't strong enough, and it doesn't mean that God isn't there with you, helping you along and giving you strength.
Things may never be the same. There may be no happy shining thing that makes what you have suffered all worth it in the end. Sometimes things get better; sometimes, there is a dramatic recovery and change of fortune and everything becomes almost perfect. And we rejoice when that happens and celebrate it. But that doesn't mean the pain wasn't real, and it doesn't mean that there's something wrong with you or your faith if that never happens.
Because God is with us. You, me, every person who suffers loss, every person who celebrates a joy. God is here. With us. God is always faithful; his steadfast love never ceases; his mercies never come to an end. Even in the darkest parts of our lives, when we can do nothing but lament and wail at our suffering, God is with us, and God will never let us fall. You are not alone. We are not alone, not any of us, for God is with us. Thanks be to God.


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hey those last four photos of the harbour bridge are heaps cool . They are some of the best photos of the Habour Bridge I have ever seen. Good Work!!!
hey those last four photos of the harbour bridge are heaps cool . They are some of the best photos of the Habour Bridge I have ever seen. Good Work!!!
I love photography and film, but it's only when I've got a bit of time and inspiration that I'll actually get any good shots. I'm hoping that 4U English will give me a little freedom with my video camera.
I'm not good at photography, no matter how hard I try.
God, what can I do?
Ba. Art Education, COFA
man, I hate art
...if I had my very own camera.
Hopefully I will be getting one soon.
Your pics are awesome!! I like photography, im no pro at it and i don't have a fancy camera but its a hobby i enjoy ^^
visit my website for my works if you like::
I am into Photography, Do it just about every day, I mostly do Landscapes and the occasional Youth event etc.
I love taking photo's of Landscapes and Macro shooting, I recenty went down to a lake and took some of a Sunset, Absolutely beautiful the photo's turned out.
Full 8MP, Highest Quality, Sad thing is I didn't have time to adjust the settings myself so I just used Auto, but even that turned out great!!!
I'll post some up when I Watermark them and fiddle with them through the week.
I always forget about this forum, so you must help me remember! Email me, send private messages, whatever!!
Help me to never forget about it
Your picture are beautifully taken. I like photography, but I am not an expert however take good photo are my hobbies and I love it as I can use my creativity to take and than edit the picture. The outcome is fulfilling and enjoyable...
Travel Advises: Budget Airlines
that is good
the knights have won
rd 1 25-6
rd 2 70-32
rd 3 46-22
hay those photos you took are amazing! i'm really passionate about photography also and am looking at universities for next year for photography does anyone have any recommendations about any good uni's, ?
thanx xoxo amelia
My first camera was given to me when i was 6.
Today I am using an analog but want to purchase digital due to the fact that I am doing alot of practical work and i have to document it. On an analog I cant really tell if it came out.
So sometime next week with my buddy ( who i took photos for last years 150th anniversary of the railways ) going to buy a camera. My photos are a bit random. I love the influence of light and natural Items in photos.
let me play the fiddle for ye amongst the pining trees and moonlike swoon
I used to love photography when I was a kid. Then I forgot about it a bit and lately I've started it again.
You can check my stuff out here:
But right now deviantart is down. So I was just looking around BOS for photography and came across this.
El Monster
Photography is great. Mainly playing around with my Canon S3-IS at the moment.. very interested in the EOS 400D.
I've also always wanted a good pocket camera.. but the entire range of the IXUS series seems to have full manual mode missing. That is, shutter-speed priority seems to be hidden somewhere in the functions menu and no idea about aperture. The G7 is funky too.
I just bought myself a Polaroid Sun LMS 600 camera a few weeks ago. Trying out different styles. I'd really like to try out the Holga. It sounds interesting.
Used to when I did art in high school, now just don't have the time for it.
I had Canon s45, great camera. Now I've got Panasonic FX7, great image stabilization without flash (which I hate in compact cameras)
and of cos, sexy leica lens
Wanna get a cheap film SLR for b/w photography
i <3333333333333333 developing my own film!
so... anyone? =D
400d is a very sexy entry level slr digital camera, though it may sound vain avoid the silver finish as it scratches and the black looks far more pro (I have the lowly 300d, which i've had for nearly 2 years now). Invest in a couple of 1gb cards if you can too, look around in the internet for good deals, I have a super fast one I got for like $30. Man I spend way too much money on photography related products (processing, accessories etc).Originally Posted by Collin
The S3-13 is also a very commendable digital point and shoot camera and i'd probably get one of those if I could afford it as a party/everyday camera.
Canono's Ixus range is merely for sleek looks and families/people just wanting snapshots I personally believe, you're far better off with the smaller sized Powershot series (excl. S3-13). The Powershot series has the manual function which is quite interesting though nothing compares to a dSLR!! That or the G7 (hot, but like i'm an SLR/dSLR girl at heart :mad1. I've used the IXUS a few times and I agree about the complicated "manual" settings.
If I could get any of the cameras, i'd get
dSLR - EOS 1Ds Mark II (Aim to get this before i'm 21)
Compact - Powershot G7
Super Compact - Top IXUS [there's not alot of variation between them is there?]
B. Teaching/B. Fine Arts (2010)
Originally Posted by (^o^)
Mmm I love wet photography too... just wish i had greater access to a darkroom!
I also love underwater photography (i used it in my major HSC artwork)
B. Teaching/B. Fine Arts (2010)
I just got the 400D the other day! Personal preference I guess, but I thought the silver finish looked more sexay.. so I got that one. The black finish would probably look more 'professional'.. but then you've gotta consider that alot of Canon's EF telephoto zoom lens are stark white.. and I think in terms of aesthetics a big fat white lens with a black body just looks a bit odd.Originally Posted by luscious-llama
Oh and for humour's sake, go check out the EF 1200mm prime:
I got a decently fast 1GB CF the other day, but I'm gonna look into 4GB soon. I'm gonna get a battery grip for the camera, and with two standard NB-2LHs, some people talk about getting 700-800 shots. Now the standard 10.1MP JPEG on the 400D takes up about 3.8MB and the equivalent RAW around 10MB; I'm gonna need atleast 4GB for each dual-battery discharge cycle.
As for the camera.. everything seems good at the moment. The only thing which has been of an issue to me is that I miss the higher depth of field results I get with smaller compact point & shoots with a smaller matrix.. such as with my S3. That's so far only been an issue for my night/dim-lighted indoor shoots, since a smaller aperture compensation forces a slower shutter.. and I don't bring tripods to those events. Another thing is the absence of spot metering.. but that isn't a biggie at all really for me.
Anyway I was thinking of getting the EF 28-105mm IS USM lens.. reviews are generally good but I heard quality can vary quite a bit if you get a 'bad copy'.
The G7 is quite hot indeed.. I LOVE that separate ISO dial. Retro looks funky too. But besides that, alot of people are finding it hard to justify the relatively large price difference over say, the PS A710IS/A640. My friend got an 710IS the other day, they are freaking nice too. The only real disadvantage I felt the G7 had over it was the MP count and the XGA movie mode.. but atm I won't bother with another point and shoot until they release XGA movie mode with 30fps. I took so many VGA 15fps movies on my older A510 whilst overseas a little while ago.. and man, the 30fps VGA + stereo on my S3 really kick butt over it. And I LOVE the dedicated movie recording button on the S3.
As for variation over the IXUS's.. not really. The new 900 Ti (and I think one other IXUS model.. maybe the 850IS) sports the new DIGIC III with face-tracking and other discreet improvements.. but apart from that, the MP count and the XGA recording mode, they seem pretty similar. Sensor quality seems similar too between all the IXUSes in the last 12-18 months. In my opinion though, even though IXUSes are SUCH sexy little cameras with sexy big screens and that little light body - until they get Tv and Av modes.. I wouldn't personally buy them. If they released an IXUS with those modes + full manual along with XGA @ 30fps stereo sound, IS, 4-6x optical zoom and a 9-point AF AND manual 1-point focus with a 3" screen (only one with 3" LCD right now is the IXUS 65 I believe).. I would be pretty eager to buy it.
Last edited by Collin; 8 Oct 2006 at 10:51 PM.
Any of you photography "gurus" have any advice for a not so artsy person trying to get into the hobby? I have a Nikon D50 which I really like, always wanted an SLR camera just for the extra control.
Now I've heard it said that the difference between visual artsy people and sciencey/mathsy people are the former can see a scene as a bunch of shapes, colours, etc, whereas the latter see just actual objects. I can take good photos of huge scenes: cityscapes, landscapes, wide angle shots of uni, but when it comes to individual objects or more asbtract kind of weird closer, smaller scenes my photographs simply suck.
This photo is deadset AMAZING but if I was there I'd be walking along thinking "oh, this is some random bridge, not worth taking a photo of..." what is it that you guys are seeing that I'm not? Is there anything that can be done or am I a lost cause?
Simon. 22/M
The University of New South Wales: I'm there. Are you?
~ 5th year Advanced Mathematics (Pure) / Arts (Philosophy + Linguistics) @ UNSW ~
~ Diploma in Innovation Management @ UNSW ~
"In order not to believe in evolution you must either be ignorant, stupid or insane"
Professor Richard Dawkins
I find that taking photos with someone else helps majorly. Even if you just watch what they're doing/taking photos of, it gives you that bit of a nudge in a direction. Then it's just a matter of taking it one step further and doing your own thing.Originally Posted by Dumsum
Anyways, my photos can be found at http://jhakka.deviantart.com
Some of my personal favourites are:
Road to somewhere:
Bright ideas (3rd in Photosoc [USyd] Abstract [Macro Close-Up] competition 2006):
When, I mean IF i get a SLR it WILL be in black, cos it just looks so sexy silver finish just reminds me of normal compact cameras.
However i'm too poor to afford SLR and all the other stuff like external flash, better tripod, lens cleaning kit etc
Dumsum: You mean you can't "capture the true emotion of people" and all those arty crap? :P
I agree with jhakka, grab a buddy and learn from him/her.
Sometimes, taking photos are also an appreciation of something as well. Try start from taking photos with those golden ratio (squares!?!); look at photography books, walk around and observe things around you, or jsut simply shot things that interest you.
You mean like schoolgirls?
ah, to you maybe...
not my cup of tea tho
Yep absolutely!!
i love photography(and Photoshop!!XD)
i did photography in year 11 and dropped it in year 12 coz it didnt count for uai but i still did art so i submitted my photos in for my major work!!
i have a Sony f828 Cybershot, and iv taken hundreds of shots in the city, the opera house, harbour bridge, and around the Rocks area, with an infra-red lense with a neutral density filter
here are jus 2 out of the hundreds that iv taken
Some photos are really pretty. They just make you think from a different perspective...
B Construction Management & Property (Hons) @ UNSW (2014)
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Aiming,though often failing 'to become all things to all men so that by all possible means I might save some'. Join with me in these reflections,discussions, videos and even humour about how we might become truly authentic in mission:Contextual yet Biblical:Passionate, but also Compassionate:In Word, as well as in Deed.The Spirit of Jesus within is calling each of his followers to reach out and fulfil the Missio Dei in a world of pain and need.
Thursday, 6 October 2011
Sowing to the flesh
stroke it, instead of crucifying it. The seeds we sow are
largely thoughts and deeds. Every time we allow our mind to
harbour a grudge, nurse a grievance, entertain an impure
fantasy, or wallow in self-pity, we are sowing to the flesh.
Every time we linger in bad company whose insidious influence we
know we cannot resist, every time we lie in bed when we ought to
be up and praying, every time we read pornographic literature,
every time we take a risk which strains our self-control, we are
sowing, sowing, sowing to the flesh. Some Christians sow to the
flesh every day and wonder why they do not reap holiness.John Stott
2 comments:
- Ρωμανός ~ Romanós said...
Christ gets only the fleeting moments, the chronic leftovers, of our lives. We stay with Him and in stillness hear His voice as little as we can. Even when we follow Him intentionally, our following is at least half in good intentions not carried out. We excuse in ourselves every little fault and then act surprised when caught in the act. 'I don't know what came over me! I never do that, as a rule!'
Thanks, brother, for posting these wonderful reminders, never to accuse, but to awaken, us to a greater day that is always filled with the grace of Jesus. The Church is what salvation looks like, as Fr Stephen recently put it, and though the story will probably have a happy ending, getting there is anything but smooth sailing.
God grant to us a worthy repentance, and to meet Him with joy and not shame at His coming.
Hope you and your beloved family and friends are well.
- 10 October 2011 at 19:27
- Andrew Kenny said...
Thanks Romanos.I appreciate the comment.
- 11 October 2011 at 12:06


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Increase your happiness and well being with these top de-stressing tips.
Circuit breaker
Take a Break . Try to get your mind off the problem, it's not going anywhere, but you can. Take the dog for a mini walk or walk alone in the park. Revisit your funniest emails/watch comedy shows. Listen to your favourite song. Make your self a cup of herbal tea.
Avoid caffeine
Caffeine is a stimulant that increases adrenaline in the body, the very hormone you are looking to reduce. Avoid coffee (decaf is OK), colas, guarana, chocolate, and more than 2 cups of tea daily.
Eat small meals often
Maintaining steady blood sugar levels is key to stabilizing mood. Ensure you have a small meal every 2-3 hours that contains protein. For example, a hard-boiled egg and lettuce, 10-12 almonds, a small can of tuna and brown rice.
Herbs for stress
Herbs that calm include passionflower, St. John's wort, kava, oats, damiana, chamomile. Those that reduce the effects of stress on the body include Licorice, Siberian ginseng, rhodiola and withania.
The more anxious you become, breathing becomes shallower and higher in the chest. Consciously lengthen your breath, breathing deeper into the belly. Count to three on the in-breath, and four on the out-breath.
Talk to yourself
Affirmations are positive, self-affirming messages to self. In the case of anxiety, calming and positive is required. Try "All will be well", or "I always cope, just breathe and relax".
Tell someone who cares
Phone your mum or a friend. Acknowledging you are finding a situation stressful, and being heard and understood by someone who cares for you, even if they can't physically help, will reduce stress levels.
The sense of smell bypasses the cognitive brain (the bit that does the worrying). Light some incense e.g. nag champa or sandalwood. Burn some aromatherapy oils such as lavender or rose. Or just wear your favourite perfume or cologne.
In the moment you feel totally overwhelmed, gently place both palms side by side on your face, fingers gently resting on your closed eyelids. This cocoons and quietens the mind, until you feel more centred and ready to face the world again.
Seek help
If anxiety and stress has become a significant part your life, seek help from a psychologist or counselor, stress is their specialty. You don't have to do this on your own. It is not a sign of weakness to seek help.


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Les Rêves d'Anaïs - Playpen Mat Cui Cui
Regular price €78,95
Les Rêves d'Anaïs captures the innocence and beauty of a newborn with a refined range of bedding and nursery products. The brand breaths an enchanting elegance that can be found in every single detail. Les Rêves d'anais is characterised by its unique offer of collections. The beautiful series of classic uni collections are enriched with collections that have a subtle, delicate print. Together they provide a refreshing touch to the classical segment.
This playpen mat transfroms your child's playpen into a cozy and soft environment for playing and sleeping.
Playpen Mat
size: 75 x 95 cm
More from: Les rêves d'Anaïs
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Timothy R. Duncan is a partner and business attorney with over twenty-five years of legal experience. Tim's practice focuses upon advising clients on a wide range of business legal needs, from day-to-day general business matters to complex commercial transactions. Tim's practice areas include commercial transactions, merger and acquisitions, business and employment law, construction law, and general commercial litigation. Representative clients range from sole proprietorships, closely held corporations or limited liability companies to publicly traded corporations. He has significant experience in representing small businesses and closely-held corporations in all aspects of their legal and business needs, including corporate formation, buy-sell agreements, shareholder disputes, mergers and acquisitions, contractual drafting (license, employment, non-compete, distributorship/sales, and independent contract agreements), employment issues, collection, and property acquisition.
Client Services:
Business Transactions
Corporate Formation
Employment Law
Mergers and Acquisitions
Contract Review and Drafting
Shareholder Disputes
Admitted to Practice:
United States Court of Appeals for Eighth Circuit
United States District Court for District of Minnesota
Professional Activities:
Member, Minnesota State Bar Association
Member, Hennepin County Bar Association
Member, Minnesota Consulting Engineers Council
Judicial Clerk, Chief Judge D.D. Wozniak, Minnesota Court of Appeals, 1987
Publications and Presentations:
Several publications in the Hamline University Law Review, Minnesota Bench & Bar, Minnesota Continuing Legal Education publications, and local business magazines
Community Activities:
Ski Instructor, Buck Hill, Minnesota
Awards and Recognition:
- Repeatedly recognized since 1999 by the Minnesota Law & Politics magazine in its annual survey as one of top 4.5% of Minnesota attorneys
- Consistently achieved the highest rankings (AV rating) by the Martindale-Hubbell Law Directory
B.B.A., University of Minnesota, 1982
Law School:
Juris Doctorate (High Honors), Hamline University School of Law, 1986
Primary Editor, Hamline University Law Review, 1986
Member, Hamline University National Moot Court Team, 1986
Timothy R. Duncan
866.841.0080 toll free
952.841.0202 direct dial
952.841.0041 facsimile
Jamie Baumgart
952.841.0208 direct dial