A Network of Sites and Upskilled Therapists to Deliver Best-Practice Stroke Rehabilitation of the Arm: Protocol for a Knowledge Translation Study.

Implementation of evidence-informed rehabilitation of the upper limb is variable, and outcomes for stroke survivors are often suboptimal. We established a national partnership of clinicians, survivors of stroke, researchers, healthcare organizations, and policy makers to facilitate change. The objectives of this study are to increase access to best-evidence rehabilitation of the upper limb and improve outcomes for stroke survivors. This prospective pragmatic, knowledge translation study involves four new specialist therapy centers to deliver best-evidence upper-limb sensory rehabilitation (known as SENSe therapy) for survivors of stroke in the community. A knowledge-transfer intervention will be used to upskill therapists and guide implementation. Specialist centers will deliver SENSe therapy, an effective and recommended therapy, to stroke survivors in the community. Outcomes include number of successful deliveries of SENSe therapy by credentialled therapists; improved somatosensory function for stroke survivors; improved performance in self-selected activities, arm use, and quality of life; treatment fidelity and confidence to deliver therapy; and for future implementation, expert therapist effect and cost-effectiveness. In summary, we will determine the effect of a national partnership to increase access to evidence-based upper-limb sensory rehabilitation following stroke. If effective, this knowledge-transfer intervention could be used to optimize the delivery of other complex, evidence-based rehabilitation interventions.

A serial analysis of gene expression profile of the Alzheimer's disease Tg2576 mouse model.

Serial analysis of gene expression (SAGE), a technique that allows for the simultaneous detection of expression levels of the entire genome without a priori knowledge of gene sequences, was used to examine the transcriptional expression pattern of the Tg2576 mouse model of Alzheimer's disease (AD). Pairwise comparison between the Tg2576 and nontransgenic SAGE libraries identified a number of differentially expressed genes in the Tg2576 SAGE library, some of which were not previously revealed by the microarray studies. Real-time PCR was used to validate a panel of genes selected from the SAGE analysis in the Tg2576 mouse brain, as well as the hippocampus and temporal cortex of sporadic AD and normal age-matched controls. NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 5 (NDUFA5) and FXYD domain-containing ion transport regulator 6 (FXYD6) were found to be significantly decreased in the Tg2576 mouse brain and AD hippocampus. PTEN-induced putative kinase 1 (PINK1), phosphatidylethanolamine binding protein (PEBP), crystalline mu (CRYM), and neurogranin (NRGN) were significantly decreased in AD tissues. The gene ontologies represented in the Tg2576 data were statistically analyzed and demonstrated a significant under-representation of genes involved with G-protein-coupled receptor signaling and odorant binding, while genes significantly over-represented were focused on cellular communication and cellular physiological processes. The novel approach of profiling the Tg2576 mouse brain using SAGE has identified different genes that could subsequently be examined for their potential as peripheral diagnostic and prognostic markers for Alzheimer's disease.

Developmental impact of a familial GABAA receptor epilepsy mutation.

OBJECTIVE: A major goal of epilepsy research is to understand the molecular and functional basis of seizure genesis. A human GABA(A) gamma2 gene mutation (R43Q) is associated with generalized epilepsy. Introduction of this mutation into a mouse by gene targeting recapitulates the human phenotype demonstrating a strong genotype to phenotype link. GABA(A) receptors play a role in the moment-to-moment control of brain function and also on the long-term wiring of the brain by directing neuronal development. Our objective was to determine whether developmental expression of the mutation alters seizure susceptibility later in life. METHODS: A tetracycline-based conditional model for activation of a hypomorphic Q43 disease allele was created and validated. Seizure susceptibility was assessed using the subcutaneous pentylenetetrazole model. RESULTS: Seizure susceptibility was significantly reduced in mice where the Q43 allele was suppressed during development. INTERPRETATION: These results demonstrate that a human epilepsy-causing mutation impacts network stability during a critical developmental period. These data suggest that identification of presymptomatic children may provide a window for therapeutic intervention before overt symptoms are observed, potentially altering the course of epileptogenesis.

Quantifiler observations of relevance to forensic casework.

The Quantifiler (QF) kit is regularly used by forensic scientists for DNA quantitation. We performed in-house validation studies which revealed some interesting observations. The QF standard displayed a two-fold difference between two different lot numbers which suggests that every standard should be tested prior to use. The Promega K562 DNA standard works well with the QF kit. c. 41% of samples that inhibited the internal PCR control (IPC) system within the QF kit still produced good Profiler Plus reactions. QIAquick was effective at removing inhibitors. The presence of dyes within casework samples were observed not to inhibit QF amplifications. Template DNA greater than 100 ng/muL appeared to inhibit the IPC. Close to identical concentration results were obtained when alternative analysis settings were used. These validation findings will assist DNA processes involved in forensic casework.

The kidney is resistant to chronic hypoglycaemia in late-gestation fetal sheep.

We imposed a sustained reduction in glucose supply to late-gestation fetal sheep to see whether the reduction in glucose and insulin levels affected renal growth, renin expression and synthesis, and renal function. Maternal glucose concentrations were lowered to 1.7-1.9 mmol/L for 12-13 days by i.v. insulin infusion (n = 9, 121 days gestation, term = 150 days). Control ewes (n = 7) received vehicle. Maternal and fetal glucose concentrations were 40% and 31% lower than in controls (p < 0.001), respectively. Fetal plasma insulin levels fell 36% +/- 7% by day 7 (p < 0.05); IGF-I levels were unchanged. Arterial PO2 and pH increased and PCO2 fell (p < 0.05). Renal function was largely unaffected. Longitudinal growth was 28% slower and spleen weights were 36% smaller (p < 0.05); body and kidney weights were not affected. Renal renin levels and renin, angiotensinogen, and angiotensin receptor mRNA levels were similar to those of controls. Plasma renin levels increased from 2.1 +/- 0.6 to 7.6 +/- 2.8 ng angiotensin I.mL-1.h-1 (p = 0.01). Thus reductions in fetal glucose and insulin levels in late gestation that were sufficient to retard skeletal growth had no effect on kidney growth or function or the renal renin-angiotensin system, possibly because IGF-I levels were not reduced. There was, however, increased activity of the circulating renin-angiotensin system similar to that seen during insulin-induced hypoglycaemia.

Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping.

Multiple displacement amplification (MDA) can generate large quantities of genomic DNA product from small amounts of template. We investigated the ability of MDA to amplify samples containing very small amounts of target DNA (5 pg to 1 ng) in the presence of a second, larger DNA sample for downstream short tandem repeat (STR) multiplex genotyping. We observed that STR amplification success of the minor fraction was increased in these mixed samples when compared with standard PCR only or MDA containing only the single trace DNA sample. Increased numbers of alleles were detected, with less amplification bias between loci than in single source samples undergoing the same protocol. To improve the STR genotyping accuracy, animal DNA was substituted for the additional human DNA, maintaining the increase in the number and quality of human-specific STR loci amplified. Polyethylene glycol 400, a commonly used crowding agent, was used as a replacement for the added genomic DNA in the MDA reaction and produced very similar results. Therefore, we suggest that additional DNA is acting as a molecular crowding agent during MDA. Performing MDA on trace amounts of DNA under crowded conditions results in greater numbers of alleles being amplified and more balanced amplification occurring between alleles.

Effects of 7-day amino acid infusion on renal growth, function, and renin-angiotensin system in fetal sheep.

These experiments examined whether renal growth and the fetal renin-angiotensin system could be stimulated by infusion of amino acids and whether chronic amino acid infusions restored glomerulotubular balance, which had been disrupted during 4-h infusions. Five fetal sheep aged 122 +/- 1 days gestation received an infusion of alanine, glycine, proline and serine in 0.15 M saline at 0.22 mmol/min for 7 days. Six control fetuses were given saline at the same rate (5 ml/h). Kidney wet weights after amino acid infusion were 28% larger than control fetuses (P < 0.05), and renal angiotensinogen mRNA levels were approximately 2.6-fold higher (P < 0.005). Circulating renin levels and renal renin mRNA levels were suppressed (P < 0.05), and renal renin protein levels tended to be lower. Arterial pressure was increased, and there was a marked, sustained natriuresis and diuresis. Glomerular filtration rate and filtered sodium were approximately two-fold higher throughout infusion (P < 0.05). Fractional proximal sodium reabsorption, suppressed at 4 h (from 73.4 +/- 6.5 to 53.7 +/- 10.2%), did not return to control levels (36.1 +/- 3.4% on day 7, P < 0.05). Distal sodium reabsorption was markedly increased (from 79 +/- 25 to 261 +/- 75 mumol/min by day 7, P < 0.005), but this was not sufficient to restore glomerulotubular balance. The resultant high rates of sodium excretion led to hyponatremia and polyhydramnios. In conclusion, long-term amino acid infusions increased renal angiotensinogen gene expression, kidney weight, and distal nephron sodium reabsorptive capacity but failed to restore proximal and total glomerulotubular balance.

Genomic organisation and nervous system expression of radial spoke protein 3.

Following their generation in the germinal zones, young neurons of the neocortex, hippocampus and cerebellum undergo long-distance migration to reach their final destinations. This locomotive activity depends on active deployment of cytoskeletal elements including the microtubule apparatus. In this study, we report the identification and expression of radial spoke protein 3 (RSP3), a member of a protein cluster responsible for anchoring and modifying dynein motor activity known to be crucial to microtubule sliding. The mouse RSP3 gene consists of eight exons and seven introns and spans over 230 kb. The genomic organisations of the human and rat RSP3 genes are similar although they span approximately 23 and 53 kb, respectively. This is in contrast to the Chlamydomonas RSP3 gene, where RSP3 was first isolated, which consists of four exons and three introns and spans approximately 2.7 kb. Despite these differences, the nucleotide and amino acid sequences upstream of, and throughout the RPII-binding domain of RSP3 are highly conserved between all the above-mentioned species. Mouse RSP3 mRNA was restricted to the developing neocortex, hippocampus and cerebellum during the stages when these structures are known to contain large numbers of migratory neurons. Gene expression studies suggest that RSP3 function is consistent with a locomotory role for this protein in migrating young neurons. In addition, expression of RSP3 mRNA in adult neurons point to additional, though still unknown functions. Our data provides the first evidence for the expression of radial spoke proteins in higher eukaryotes, and provides a biological framework for how these proteins may participate in microtubule sliding and neuronal migration in the embryonic brain.

Vasopressin receptor expression in the placenta.

The arginine vasopressin (AVP) type 1a receptor (V1a) is well known to mediate vasoconstriction. In pregnancy, blood flow in the placenta is crucial for sustaining normal growth and development of the fetus. This is the first AVP receptor study in the placenta and fetal membranes. The aim was to compare, quantitatively, the level of V1a gene expression with that of a known marker for vascularization, aquaporin 1 (AQP1). V1a and AQP1 gene expression did not correlate; placental V1a mRNA levels were significantly upregulated at 45 and 66+/-1 compared with 27, 100+/-4, and 140 days (term approximately 150 days). V1a mRNA levels were much lower in fetal membranes in which no significant difference across gestation was observed. In situ hybridization histochemistry localized V1a gene expression in the maternal component of the placenta similar to the receptor-binding studies using 125I-labeled [d(CH2)5, sarcosine7] vasopressin. No AVP gene expression was observed in the placenta and fetal membranes, which eliminates local AVP production. This increase in V1a expression at 45 and 66+/-1 days of gestation correlates with the period of maximal placental growth in the sheep and suggests that AVP and V1a receptors may play a hitherto unrecognized role in placental growth, differentiation, and/or function, particularly in the deleterious effects of heat stress, early in pregnancy, on fetal growth.

Programmed hypertension: kidney, brain or both?

The results from numerous epidemiological studies suggested that there was a link between low birth weight (low for gestational age) and development of high blood pressure in adulthood. More recently, it has been shown that one important determinant is the early exposure of the developing fetus to excess glucocorticoid (GC). Hypertension develops in adult sheep and rats that are exposed to excess GC at a stage in gestation when both kidney and brain are still extremely primitive organs. Here, we propose that permanent changes in gene expression and function of these two organs could be crucial in the development of adult-onset hypertension as a result of prenatal GC exposure.

Angiotensin II receptor (type 1 and 2) expression peaks when placental growth is maximal in sheep.

In sheep, placental size is maximal by midgestation, but blood flow continues to increase until term. No nerves are present and ANG II is thought to be a major regulator of vascular tone. We hypothesized that angiotensin type 2 receptors (AT(2)) would predominate over type 1 (AT(1)) until late in gestation and be primarily expressed in the vasculature. Real-time PCR, hybridization histochemistry, and ligand-binding studies were performed on placentae and fetal membranes at 27, 45, 66 +/- 1, 100 +/- 4, 130, and 140 days of gestation (term approximately 150 days) to determine quantitative changes and localization. The maximum level of AT(1) expression occurred in the 45-day placenta and was located predominantly in the maternal stromal cells. AT(1) receptors were expressed in the endothelial cells of the chorion in the first half of pregnancy, where later in gestation, both AT(1) and AT(2) receptors were predominant in blood vessels. These results suggest that ANG II, via the AT(1) receptor, may have hitherto unsuspected important roles in the growth/function on the ovine placenta during the maximal growth phase.

Programming effects of short prenatal exposure to cortisol.

Recent studies have linked fetal exposure to a suboptimal intrauterine environment with adult hypertension. The aims of this study were twofold: 1) to see whether cortisol treatment administered to the ewe for 2 days at 27 days of gestation (term approximately 150 days) resulted in high blood pressure in offspring; 2) to study the effect of the same treatment on gene expression in the brain at 130 days of gestation and in lambs at 2 months of age. Mean arterial pressure was significantly higher in the adult female and male offspring of sheep treated with cortisol than in the control group (females: 89+/-2 mmHg vs. 81+/-2; P<0.05 and males: 102+/-4 mmHg vs. 91+/-3; P<0.05). Prenatal cortisol treatment led to up-regulation of angiotensinogen, AT1, MR, and GR mRNA in the hippocampus in fetuses at 130 days of gestation but not in the animals at 2 months of age. This is the first evidence that short prenatal exposure to cortisol programmed high blood pressure in the adult female and male offspring of sheep. Altered gene expression in the hippocampus could have a significant effect on the development of the hippocampus, and on postnatal behavior.

Quantitation of the mRNA levels of Epo and EpoR in various tissues in the ovine fetus.

A partial cDNA of the sheep erythropoietin receptor (EpoR) was obtained and used in real-time PCR to quantitate mRNA levels in placenta, liver and kidney throughout development (term=150 days). This was compared with Epo mRNA levels in the same tissues. Both Epo and EpoR mRNA were present in the placenta throughout gestation at low levels from 66 days onwards and these did not vary throughout gestation. Compared with the expression levels in the placenta, the levels of EpoR gene expression in the liver at 66, 99 and 140 days were, median (range)-288 (120-343), 278 (63-541) and 7 (3-15), respectively, reflecting the disappearance of erythropoiesis after 130 days. Low levels of EpoR gene expression were seen in the kidney at 3 (2-5), 5 (2-7), and 7 (2-10) times that in the placenta at 66, 99, and 140 days, respectively. By hybridization histochemistry the EpoR mRNA was located in the proximal tubular cells of the mesonephros and metanephros at 42 days. Epo mRNA levels in the kidney were 215 (116-867), 528 (113-765) and 46 (15-204) times those in the placenta at 69, 99, and 140 days, respectively. In the liver at the same ages the concentrations of mRNA were lower than in the kidney, the liver/placenta ratios being 50 (11-90), 17 (3-39), 9 (5-14). At 130 days Epo/EpoR levels in the hippocampus were 6+/-3 and 8+/-3 times that in the term placenta, respectively. These studies demonstrate that the ovine placenta expresses the Epo gene from at least 66 days of gestation. However, gene expression levels are very low compared with those in the liver and kidney, and even the hippocampus.

Effect of early glucocorticoid treatment on MR and GR in late gestation ovine kidney.

BACKGROUND: The ontogeny of the renal mineralocorticoid (MR) and glucocorticoid (GR) receptors in the ovine fetus, and the effects of early exposure to synthetic or natural glucocorticoids on the expression of these genes in late gestation were examined. METHODS: A partial cDNA sequence for the ovine MR was cloned and used to generate primers and probes to measure MR mRNA expression by real-time polymerase chain reaction (PCR). GR mRNA was also measured. Kidneys were collected from ovine fetuses at various stages of gestation (days 60 to 140), twin ovine fetuses at 130 days, from ewes treated at days 26 to 28 with either saline, dexamethasone or cortisol, and adult sheep. Ligand binding was used to determine both GR and MR protein levels in all 130-day-old fetuses and adults. RESULTS: No significant changes in the expression of either renal MR or GR were detected throughout gestation. Cytosolic protein levels were higher in the fetal kidneys than in the adult. There was a significant increase in both fetal MR and GR mRNA expression, but not protein levels in kidneys from ewes pretreated with dexamethasone. CONCLUSIONS: MR and GR mRNA are expressed throughout development in ovine fetal kidneys. Dexamethasone treatment resulted in increased expression of MR and GR mRNA but not protein levels. The dissociation between fetal mRNA and protein levels, relative to adult kidneys, suggests that it may be confounding to draw conclusions based on mRNA levels alone.

Reduced glycogen availability is associated with an elevation in HSP72 in contracting human skeletal muscle.

To test the hypothesis that a decrease in intramuscular glycogen availability may stimulate heat shock protein expression, seven men depleted one leg of muscle glycogen the day before performing 4-5 h of exhaustive, two-legged knee extensor exercise at 40 % of leg peak power output. Subjects then rested for a further 3 h. Muscle biopsies were obtained from the depleted and control leg before, immediately after and 3 h into recovery from exercise. These samples were analysed for muscle glycogen, and HSP72 gene and protein expression. In addition, catheters were placed in one femoral artery and both femoral veins and blood was sampled from these catheters prior to exercise and at 1 h intervals during exercise and into recovery for the measurement of arterial-venous differences in serum HSP72. Plasma creatine kinase (CK) was also measured from arterial blood samples. Pre-exercise muscle glycogen content was 40 % lower in the depleted compared with the control leg and this difference was maintained throughout the experiment (P < 0.05; main treatment effect). Neither HSP72 gene nor protein expression was different pre-exercise. However, both HSP72 gene and protein increased (P < 0.05) post-exercise in the depleted leg, but not in the control leg. Exercise did not increase plasma CK concentrations and we were unable to detect HSP72 in the serum of any samples. These results demonstrate that while acute, concentric exercise is capable of increasing HSP72 in human skeletal muscle, it does so only when glycogen is reduced to relatively low levels. Hence, our data suggest that HSP72 protein expression is related to glycogen availability. In addition, because CK did not increase and we found no evidence of HSP72 in the venous effluent, our data suggest that skeletal muscle is impermeable to HSP72.