Outer retinal transduction by AAV2-7m8 following intravitreal injection in a sheep model of CNGA3 achromatopsia.

Sheep carrying a mutated CNGA3 gene exhibit diminished cone function and provide a naturally occurring large animal model of achromatopsia. Subretinal injection of a vector carrying the CNGA3 transgene resulted in long-term recovery of cone function and photopic vision in these sheep. Research is underway to develop efficacious vectors that would enable safer transgene delivery, while avoiding potential drawbacks of subretinal injections. The current study evaluated two modified vectors, adeno-associated virus 2-7m8 (AAV2-7m8) and AAV9-7m8. Intravitreal injection of AAV2-7m8 carrying enhanced green fluorescent protein under a cone-specific promoter resulted in moderate photoreceptor transduction in wild-type sheep, whereas peripheral subretinal delivery of AAV9-7m8 resulted in the radial spread of the vector beyond the point of deposition. Intravitreal injection of AAV2-7m8 carrying human CNGA3 in mutant sheep resulted in mild photoreceptor transduction, but did not lead to the clinical rescue of photopic vision, while day-blind sheep treated with a subretinal injection exhibited functional recovery of photopic vision. Transgene messenger RNA levels in retinas of intravitreally treated eyes amounted to 4-23% of the endogenous CNGA3 levels, indicating that expression levels >23% are needed to achieve clinical rescue. Overall, our results indicate intravitreal injections of AAV2.7m8 transduce ovine photoreceptors, but not with sufficient efficacy to achieve clinical rescue in CNGA3 mutant sheep.

Improvement of dyskinesias with L-dopa infusion in advanced Parkinson's disease.

OBJECTIVES: We wanted to investigate whether continuous intrajejunal levodopa-carbidopa intestinal gel (LCIG) therapy has an antidyskinetic effect in patients with Parkinson's disease (PD) and troublesome dyskinesias. We also sought to examine the effect of LCIG therapy on motor function and health-related quality of life (HRQoL). MATERIALS AND METHODS: This open-label pilot study used a single group pre-post design with follow-up at 6 months. Nine patients with PD who reported to spend at least 3 h per day in on with troublesome dyskinesia were included. The patients were examined at baseline using clinical and self-assessment measures and then switched from peroral/transdermal pharmacotherapy to LCIG therapy. Data collection was repeated 6 months after the pharmaceutical intervention. Nonparametric statistical methods were used for data analyses. RESULTS: The mean time spent in on with troublesome dyskinesia per day after 6 months of LCIG therapy decreased by 47% (P < 0.05). This observation was paralleled by a 112% increase in mean time spent in on without troublesome dyskinesia (P < 0.01). Patient self-assessment of dyskinesia intensity on the visual analog scale displayed a 90% reduction of mean dyskinesia intensity (P < 0.01) and patients also exhibited less dyskinesia during standardized levodopa tests. Furthermore, we noted improvements in motor function and HRQoL. CONCLUSIONS: In this pilot study, we found indications that LCIG therapy has a substantial antidyskinetic effect and could be an alternative also for PD patients with dyskinesias as a major symptom. However, further studies with blinded evaluation and larger numbers of patients are warranted to confirm the findings.

Development of an automatic cow body condition scoring using body shape signature and Fourier descriptors.

Body condition evaluation is a common tool to assess energy reserves of dairy cows and to estimate their fatness or thinness. This study presents a computer-vision tool that automatically estimates cow's body condition score. Top-view images of 151 cows were collected on an Israeli research dairy farm using a digital still camera located at the entrance to the milking parlor. The cow's tailhead area and its contour were segmented and extracted automatically. Two types of features of the tailhead contour were extracted: (1) the angles and distances between 5 anatomical points; and (2) the cow signature, which is a 1-dimensional vector of the Euclidean distances from each point in the normalized tailhead contour to the shape center. Two methods were applied to describe the cow's signature and to reduce its dimension: (1) partial least squares regression, and (2) Fourier descriptors of the cow signature. Three prediction models were compared with manual scores of an expert. Results indicate that (1) it is possible to automatically extract and predict body condition from color images without any manual interference; and (2) Fourier descriptors of the cow's signature result in improved performance (R(2)=0.77).

Periparturient dairy cows do not exhibit hepatic insulin resistance, yet adipose-specific insulin resistance occurs in cows prone to high weight loss.

The periparturient period in dairy cows is associated with alterations in insulin action in peripheral tissues; however, the molecular mechanism underlying this process is not completely understood. The objective was to examine the response to a glucose tolerance test (GTT) and to analyze insulin signaling in liver and adipose tissues in pre- and postpartum dairy cows. Liver and adipose tissue biopsies were taken before and after GTT, at 17d prepartum and again at 3 to 5d postpartum from 8 high-yielding Israeli Holstein dairy cows. Glucose clearance rate after GTT was similar pre- and postpartum. Basal insulin concentrations and the insulin response to GTT were approximately 4-fold higher prepartum than postpartum. In accordance, phosphorylation of the hepatic insulin receptor after GTT was higher prepartum than postpartum. Across periods, a positive correlation was observed between the basal and peak plasma insulin and phosphorylated insulin receptor after GTT in the liver. Hepatic phosphorylation of protein kinase B after GTT was elevated pre- and postpartum. Conversely, in adipose tissue, phosphorylation of protein kinase B after GTT pre- and postpartum was increased only in 4 out of 8 cows that lost less body weight postpartum. Our results demonstrate that hepatic insulin signaling is regulated by plasma insulin concentrations as part of the homeorhetic adjustments toward calving, and do not support a model of hepatic insulin resistance in periparturient cows. Nevertheless, we suggest that specific insulin resistance in adipose tissue occurs pre- and postpartum only in cows prone to high weight loss. The different responses among these cows imply that genetic background may affect insulin responsiveness in adipose tissue pre- and postpartum.

Performance and welfare of high-yielding dairy cows subjected to 5 or 8 cooling sessions daily under hot and humid climate.

The objectives were to determine the effects of cooling of high-yielding dairy cows under a hot and humid climate on intake, milk yield, rumination time, and welfare parameters. Forty-two multiparous Israeli Holstein dairy cows were divided into 2 treatment groups and were housed in an open barn divided into 2 pens. The groups were subjected to different cooling schedules, in a crossover design as follows: cows were exposed to 5 or 8 cooling sessions per day (designated 5CS and 8CS, respectively) in the holding area of the milking parlor. Each period lasted 4 wk, and then treatments were switched for another 4-wk period. Each cooling session lasted 45 min, comprising cycles of 30s of showering and 4.5 min of ventilation without showering. Respiration rate and rectal temperature were recorded twice per week (Monday and Thursday) at 0630 and 1600 h. Rumination and lying times were recorded automatically. Rectal temperatures were 0.16 and 1.08°C lower in 8CS than in 5CS cows in the morning and afternoon, respectively. Respiration rate was lower in 8CS than in 5CS cows in the morning (49.1 and 54.6 breaths/min, respectively), and more so in the afternoon (50.0 and 83.0 breaths/min, respectively). Dry matter intake and milk yields were 9.3 and 9.6% higher in the 8CS than in the 5CS cows (27.0 vs. 24.7 and 40.1 vs. 36.6 kg/d, respectively), with no differences in milk fat and protein contents. Daily rumination time was 7.4% longer in the 8CS than in the 5CS (440.1 and 409.6 min/d, respectively); however, rumination time per unit of dry matter or neutral detergent fiber consumed was higher in the 5CS than in the 8CS cows. Although the 8CS cows moved 3 times more to the milking area for extra cooling sessions than the 5CS ones, they spent 9.9 min/d more than the 5CS ones in lying down (484.4 and 474.5 min/d, respectively), and used more of their free time (excluding milking and feeding time) in resting than the 5CS cows: 52.0 and 43.9%, respectively. It appears that increasing the cooling frequency from 5 to 8 times per day improved their feeling of welfare, so they could spend more time lying and ruminating. In conclusion, increasing the cooling frequency of high-yielding dairy cows under hot and humid conditions from 5 to 8 times a day increased their intake and milk yield, and lowered their respiration rate and rectal temperature. Moreover, the 8CS cows spent more time resting than 5CS cows, an indication that increasing cooling frequency improved animal welfare.

Plasma concentrations of key metabolites and insulin in late-pregnant ewes carrying 1 to 5 fetuses.

Ewes bearing more than 1 fetus are more susceptible to pregnancy toxemia than those with a single fetus. Crossbreeding programs in Israel increased the occurrences of ewes bearing more than 2 fetuses; therefore, the aim was to assess the exacerbation in the metabolic status of ewes pregnant with several fetuses. Fifty ewes, genetically developed to achieve multiple-fetus pregnancies, were monitored, on average, from d 115 of pregnancy until lambing for plasma concentrations of several key metabolites and insulin. The numbers of fetuses were examined by ultrasonography at 35 d of pregnancy. Blood samples were collected weekly, and concentrations of glucose, ß-hydroxybutyrate (BHBA), NEFA, triglycerides, cholesterol, total calcium, and insulin were determined. The average litter size was 2.75 (±1.1), and 1 (1F), 2 (2F), 3 (3F), and 4 or more (4F) fetuses were conceived, respectively, by 6 (12%), 17 (34%), 14 (28%), and 13 (26%) ewes. Total birth weights of lambs were 6.1, 9.5, 12.7, and 15.0 kg for 1F, 2F, 3F, and 4F, respectively (P < 0.001). Plasma glucose concentrations in 1F were greater than those in 3F and 4F (P < 0.05) and were similar among 2F, 3F, and 4F. Trends toward increasing plasma concentrations of BHBA and NEFA were observed as the number of fetuses increased and also as lambing approached. Plasma concentrations of BHBA and NEFA were, respectively, 3.7 (P < 0.002) and 2.1 (P < 0.001) times as great in 4F ewes as in 1F ewes. Trends toward decreased concentrations of triglycerides and cholesterol were observed as litter size increased. Insulin concentrations in blood decreased considerably as the numbers of fetuses increased and, on average, they were less by a factor of 5 in the 4F ewes than in the 1F ewes (P < 0.001). Moreover, insulin concentrations during the week before lambing were extremely low (e.g., 0.54 µIU/mL in the 4F ewes). Insulin concentrations were reduced in ewes bearing >3 fetuses, even 5 wk before lambing; this decline apparently began earlier than the last month of gestation. Therefore, it seems that insulin has a pivotal role in the etiology of pregnancy ketonemia in ewes carrying multiple fetuses. The present findings may suggest that the decline in insulin concentrations that apparently occurs in the earlier stages of pregnancy represents a homeorhetic control to spare glucose for the brains and fetoplacental units of the dams. The results clearly demonstrate the increased susceptibility to pregnancy toxemia of ewes carrying multiple fetuses. Appropriate nutritional strategies should be developed for ewes that conceive >3 fetuses, to meet the increased nutritional requirements of the fetoplacental unit.

[Intermittent apomorphine injections as rescue therapy for advanced Parkinson's disease. Consensus statement]. / Intermittierende Apomorphin-Injektionen als Rescue-Therapie beim fortgeschrittenen M. Parkinson. Konsensusstatement.

Intermittent subcutaneous apomorphine therapy should be considered in patients with advanced Parkinson's disease who experience recurrent off periods despite optimised oral treatment (according to guidelines), for reliable and quick reversal of these otherwise refractory periods. Such treatment is also called rescue therapy. At present, apomorphine injections with the apomorphine pen are underutilised, considering its current indications and contraindications. In the present consensus statement, concepts for the use of apomorphine are presented and discussed based on existing study results, indications, and contraindications. Recommendations for a practical approach are also provided.

Efficient algorithms to enumerate isomers and diamutamers with more than one type of substituent

In this paper we describe numeric as well as symbolic algorithms for the enumeration of substitutional isomers with an unlimited number of different achiral substituents. We consider three different scenarios: first, the enumeration of diamutamers with a given set of ligand types and ligand multiplicity, second, the enumeration of diamutamer libraries with a given ligand assortment pattern, and, third, the enumerations of libraries with diamutamers exhibiting a limited number of ligands.

N-[trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl]-L-alanyl-D-glutamic acid: a novel immunologically active carbocyclic muramyl dipeptide analogue.

A novel non-pyrogenic carbocyclic muramyl dipeptide (MDP) analogue, N-¿trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl¿-L-alanyl-D-glutamic acid, was obtained by replacement of the N-acetylmuramic acid part and the D-isoglutamine residue of the MDP molecule by a trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl moiety and D-glutamic acid, respectively. The title compound was selected as a promising candidate for further evaluation among several related analogues on the basis of an immunorestoration test in mice. This novel nor-MDP analogue protects mice against the immunosuppressive effect of cyclophosphamide and increases the nonspecific resistance of mice against fungal infection. It is an immunomodulator which enhances the maturation of lymphocytes B to plasma cells and increases the activity of lymphocytes B and lymphocytes T as well as that of macrophages but does not alter the number of these cells.

Syntheses of polymeric vesicles containing partial structures of N-acetyl-muramyl-dipeptide (MDP).

The syntheses of 11-methacroylaminoundecanoyl-L-alanyl-D-isoglutamine (3) and 2-[2-(11-methacroylaminoundecanoyl)aminoethoxy]propanoyl-L- alanyl-D-isoglutamine (13) are described. Each of these monomers has been copolymerized with 11-methacroyloxyundecyltrimethylammonium bromide (16), yielding polymeric vesicles that contain partial structures of N-acetyl muramyl dipeptide (MDP). All products have been characterized by 1H- and 13C-NMR spectroscopy and the polymeric vesicles have been studied by electron microscopy. The potential use of these structures as immunostimulating products is discussed.

[Glucose determination by means of NMR spectrometry]. / Glukosebestimmung mittels NMR-Spektrometrie.

NMR spectroscopy could be a method for non-invasive determination of glucose concentration in the future. In this connection, 13C-NMR spectroscopy is of special interest. In vitro investigation of blood serum samples in 13C-NMR revealed significant changes in signal intensities reflecting the different glucose concentrations in these samples. The further development of noninvasive in vivo glucose determination by means of NMR spectroscopy is closely associated with technological progress.

Severe aortic regurgitation secondary to idiopathic aortitis.

Clinical and morphologic features are described in two relatively young adults with aortic regurgitation secondary to chronic aortitis. The regurgitation in each was severe enough to require aortic valve replacement. Both patients had normochromic, normocytic anemia, considerable weight loss despite congestive cardiac failure, and negative serologic tests for syphilis. These systemic manifestations in association with the aortitis suggest that both had Takayasu's arteritis. In addition, one patient had total occlusion at the origin of one subclavian artery (classic pulseless disease). Takayasu's arteritis must be added to the list of causes of severe aortic regurgitation.

Treatment of digitalis intoxication by charcoal hemoperfusion (CHP).

In the present communication, we demonstrate the removal of digoxin both in vitro and in vivo from digoxin intoxicated dogs by use of hemoperfusion over activated charcoal. In vitro drug removal was 33 ml/min at mean blood flow rates of 160 ml/min. By 30 mins of in vitro perfusion 51% of drug was removed from the reservoir and by 2 1/2 hrs 83%. In vivo clearance in dogs was 55 ml/min at blood flow rates of 150 ml/min. In view of the poor success that has been encountered in the treatment of digoxin intoxication with other modalities, charcoal hemoperfusion may represent an effective method of rapid digoxin removal from blood.