GPCR kinase knockout cells reveal the impact of individual GRKs on arrestin binding and GPCR regulation.

G protein-coupled receptors (GPCRs) activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and the formation of receptor-arrestin complexes. However, the impact of individual GRKs on arrestin binding is not clear. We report the creation of eleven combinatorial HEK293 knockout cell clones lacking GRK2/3/5/6, including single, double, triple and the quadruple GRK knockout. Analysis of ß-arrestin1/2 interactions for twelve GPCRs in our GRK knockout cells enables the differentiation of two main receptor subsets: GRK2/3-regulated and GRK2/3/5/6-regulated receptors. Furthermore, we identify GPCRs that interact with ß-arrestins via the overexpression of specific GRKs even in the absence of agonists. Finally, using GRK knockout cells, PKC inhibitors and ß-arrestin mutants, we present evidence for differential receptor-ß-arrestin1/2 complex configurations mediated by selective engagement of kinases. We anticipate our GRK knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and ß-arrestin complex formation.

Posterolateral plate fixation with Pantalarlock® is more stable than nail fixation in tibiotalocalcaneal arthrodesis in a biomechanical cadaver study.

BACKGROUND: Despite the promising results of ankle joint arthroplasty, the tibiotalocalcaneal (TTC) arthrodesis remains an established procedure in treatment of combined pathology of the ankle and subtalar joint. Despite the promising results in biomechanical investigations, nonunion rates of up to 24% are described in recent studies. The objective of this work was a comparative study of the biomechanical properties of the posterolateral plate fixation with retrograde intramedullary nail fixation. METHODS: Twenty four fresh-frozen human lower leg specimens (12 pairs) were used for the comparative biomechanical testing. Every specimen was preconditioned with 100 N over 200 cycles. After every 250 cycles the force was increased by 50 N from 200 to 600 N. This was followed by cyclic loading in dorsi-/plantiflexion with 800 N for 3000 cycles. All specimens were subjected to bone densitometry (DXA) and computed tomography. RESULTS: Significantly higher number of spacimens with nails (4) failed during the cycling testing in dorsi-/plantarflexion and futher two during the cyclic testing with 800 N. Two specimens with plates failed during the cyclic testing with 800 N. Statistical analysis showed that the specimens with the plate were significantly more stable in each test direction. The Pearson correlation demonstrated for the specimens with plate a linear relationship between the stiffness and the determined bone density. CONCLUSIONS: The results demonstrate a significantly superior stiffness of the Pantalarlock®-plate in all testing directions compared with the HAN nail. Probably the position of the plate on the tension side of the joint and the combination of locking and lag screws provide the higher stiffness of the plate system. The correlation of the stiffness with bone density leads to more predictable results of the plate arthrodesis. We hope for a reduction of the pseudarthrosis rate and shorten the postoperative treatment phase. The authors expect advantages in the treatment of high risk patients with severe deformity of the ankle, bone defects, neuropathic deformity, poor bone quality and osteoporosis.

Seroprevalence of antibodies to Pestivirus infections in South Australian sheep flocks.

OBJECTIVE: Bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) are of the genus Pestivirus. They are known to cause significant reproductive and production losses, with BVDV acknowledged as a major source of economic loss to the Australian cattle industry. Very little is currently known about the prevalence and effect of pestiviruses in the Australian sheep industry. The present study aimed to examine the seroprevalence and effect of both BVDV and BDV in South Australian sheep flocks. METHODS: In total, 875 breeding ewes on 29 properties were serologically tested by ELISA, AGID and VNT assays for the presence of Pestivirus-specific antibodies. RESULTS: Three (0.34%) individual animals returned serological results suggestive of previous BDV infection. All three positive animals were collected from one property, giving a property level seroprevalence of 3.45% and a within-flock seroprevalence of 10%. CONCLUSION: The results suggested that BDV infection is present, albeit at a very low incidence, in the South Australian sheep flock and BVDV infection appears to be absent. Consequently, pestiviruses are unlikely to impair production in South Australian sheep populations.

Investigation of infectious reproductive pathogens of large ruminants: Are neosporosis, brucellosis, leptospirosis and BVDV of relevance in Lao PDR?

N. caninum, bovine viral diarrhoea virus, Brucella abortus and Leptospira interrogans serovar Hardjo are globally significant reproductive pathogens that cause abortion and reproductive loss in large ruminants. Prevalence information is lacking in Lao People's Democratic Republic (Laos) despite the poor reproductive performance of cattle and buffalo. Serological examination of frozen cattle (n=90) and buffalo (n=61) sera by commercially available enzyme-linked immunosorbent assays provided the first reported screening of some of these pathogens in Laos. Seroprevalence differed amongst these large ruminant species, with N. caninum, BVDV and L. interrogans serovar Hardjo antibodies found in 68.9% (95% CI±11.6), 4.9% (95% CI±5.4) and 3.3% (95% CI±4.5) of buffalo sera, respectively, and in 7.8% (95% CI±5.5), 10.0% (95% CI±6.2) and 22.2% (95% CI±8.6) of cattle sera, respectively. Buffalo sera had a significantly higher seroprevalence of N. caninum compared to cattle (p<0.001) and cattle sera had a significantly higher seroprevalence of L. interrogans serovar Hardjo compared to buffalo (p=0.003). Variability was also observed across provinces for N. caninum in buffalo (p=0.007) and for L. interrogans serovar Hardjo in cattle (p=0.071), suggesting provincial risk factors conducive to pathogen transmission. BVDV and N. caninum seropositivity were negatively associated in buffalo (p=0.018) and cattle (p=0.003). In buffalo, L. interrogans serovar Hardjo and BVDV seropositivity were associated (p=0.035, p=0.039). The identification of antibodies against three major abortifacient pathogens in Laos prompts further research to determine if infection is associated with low reproductive efficiency and the risk factors for infection. This is needed for the development of evidence based prevention strategies for improved large ruminant reproductive management among smallholders in Laos.

EFhd2/Swiprosin-1 is a common genetic determinator for sensation-seeking/low anxiety and alcohol addiction.

In many societies, the majority of adults regularly consume alcohol. However, only a small proportion develops alcohol addiction. Individuals at risk often show a high sensation-seeking/low-anxiety behavioural phenotype. Here we asked which role EF hand domain containing 2 (EFhd2; Swiprosin-1) plays in the control of alcohol addiction-associated behaviours. EFhd2 knockout (KO) mice drink more alcohol than controls and spontaneously escalate their consumption. This coincided with a sensation-seeking and low-anxiety phenotype. A reversal of the behavioural phenotype with ß-carboline, an anxiogenic inverse benzodiazepine receptor agonist, normalized alcohol preference in EFhd2 KO mice, demonstrating an EFhd2-driven relationship between personality traits and alcohol preference. These findings were confirmed in a human sample where we observed a positive association of the EFhd2 single-nucleotide polymorphism rs112146896 with lifetime drinking and a negative association with anxiety in healthy adolescents. The lack of EFhd2 reduced extracellular dopamine levels in the brain, but enhanced responses to alcohol. In confirmation, gene expression analysis revealed reduced tyrosine hydroxylase expression and the regulation of genes involved in cortex development, Eomes and Pax6, in EFhd2 KO cortices. These findings were corroborated in Xenopus tadpoles by EFhd2 knockdown. Magnetic resonance imaging (MRI) in mice showed that a lack of EFhd2 reduces cortical volume in adults. Moreover, human MRI confirmed the negative association between lifetime alcohol drinking and superior frontal gyrus volume. We propose that EFhd2 is a conserved resilience factor against alcohol consumption and its escalation, working through Pax6/Eomes. Reduced EFhd2 function induces high-risk personality traits of sensation-seeking/low anxiety associated with enhanced alcohol consumption, which may be related to cortex function.

Antibodies to bovine viral diarrhoea virus (BVDV) in water buffalo (Bubalus bubalis) and cattle from the Northern Territory of Australia.

BACKGROUND: Farmed and feral water buffaloes (Bubalus bubalis) populations often coexist with cattle in the Northern Territory of Australia, but their level of exposure to bovine viral diarrhoea virus (BVDV) is unknown. METHODS: Water buffalo (n = 245) and cattle (n = 184) serum samples were collected by the NT Government as part of an ongoing disease surveillance scheme at varying intervals between 1993 and 2001. All samples were frozen and stored at -80°C until testing. Water buffalo samples from farming properties were identified as 'farmed' animals and the remaining samples as 'feral' populations. Serum samples were analysed using commercially available ELISAs to test for the presence of BVDV antibodies. RESULTS: Testing of historical water buffalo sera for BVDV antibodies revealed a low level of exposure, with 4.5% (95% CI ± 2.6%) being sero-positive; cattle from the same geographical area and time period had higher levels of exposure at 74.5% (95% CI ± 6.3%). DISCUSSION: This survey showed that water buffalo are susceptible to infection with BVDV. No persistently infected water buffalo were identified in this study.

Cross-sectional observational survey of serum biochemistry values in a population of 69 adult female alpacas (Vicugna pacos) in South Australia.

Blood samples were collected from 69 'healthy' female alpacas aged ≥12 months from 11 properties in South Australia. The 10-90 percentile ranges of the 16/19 analytes measured in this sample population were within the published ranges of four healthy alpaca populations from other geographic locations. Marginal exceptions were glutamate dehydrogenase and bicarbonate. Potassium was notably elevated, probably because of haemolysis of some samples. The sample size was insufficient to provide the appropriate statistical power to define diagnostic references ranges according to international standards. The health status of the sample population of alpacas was presumptive based on a physical examination.

Serological survey for antibodies against bovine viral diarrhoea virus and Neospora caninum in a population of South Australian alpacas (Vicugna pacos).

OBJECTIVE: Bovine viral diarrhoea virus (BVDV) and Neospora caninum may cause clinical disease in alpacas. Both diseases are present in the Australian cattle population. The objective of this study was to perform a serological prevalence survey for BVDV and N. caninum exposure in a regional alpaca population of South Australia. METHODS: Serum samples were taken from 182 alpacas on 10 farms, which had a combined population of 1308 alpacas. Serological analysis for BVD antibodies was performed using a competitive BVDV antibody ELISA kit. Serological analysis for N. caninum was performed using an anti-Neospora ELISA with a protein G conjugate. RESULTS: Of the 182 alpacas sampled, 5 animals located on three properties were positive for BVDV antibodies, constituting a prevalence of 2.7% (95% confidence interval 1-6%). All samples tested negative for N. caninum antibodies. CONCLUSION: There is a low BVDV seroprevalence and N. caninum is currently either absent or present at a very low prevalence in this population of alpacas in South Australia. There is serological evidence for the presence of both organisms in South Australian beef and dairy cattle herds. Appropriate biosecurity protocols to minimise the risk of introduction and exposure should be a high priority to maintain this favourable status.

Survey of farmer knowledge and attitudes to endemic disease management in South Australia, with a focus on bovine viral diarrhoea (bovine pestivirus).

OBJECTIVE: We aimed to establish the attitudes of South Australian cattle farmers towards endemic animal disease prevention and control, with a particular focus on the awareness of and attitudes towards bovine viral diarrhoea (BVD). METHODS: This cross-sectional postal survey involved mailing a questionnaire to all South Australian cattle owners with 35 or more head of cattle. RESULTS: Worms and lice were the most common animal disease concerns. Less than half of responding farmers were adequately vaccinating their herds against clostridial diseases, but 53.0% stated that they utilised quarantine procedures. Less than 20% of respondents had actively taken part in BVD educational opportunities, or had vaccinated or tested their herd for BVD; less than 20% of respondents were actively involved in any systematic control of Johne's disease. Overall, farmers' actual knowledge of BVD was lower than their perceived understanding, although their interest in BVD and its control was high. CONCLUSIONS: Disease prevention measures such as vaccination, quarantine and participation in systematic control schemes were used by a minority of respondents. The results suggest that respondents acknowledge BVD as an important and relevant disease, despite many believing it was not a problem in their herd. Interest in BVD appears to be high and it is likely that an education program would be well received.

A Framework for (Tele-) Monitoring of the Rehabilitation Progress in Stroke Patients: eHealth 2015 Special Issue.

BACKGROUND: Preservation of mobility in conjunction with an independent life style is one of the major goals of rehabilitation after stroke. OBJECTIVES: The Rehab@Home framework shall support the continuation of rehabilitation at home. METHODS: The framework consists of instrumented insoles, connected wirelessly to a 3G ready tablet PC, a server, and a web-interface for medical experts. The rehabilitation progress is estimated via automated analysis of movement data from standardized assessment tests which are designed according to the needs of stroke patients and executed via the tablet PC application. RESULTS: The Rehab@Home framework's implementation is finished and ready for the field trial (at five patients' homes). Initial testing of the automated evaluation of the standardized mobility tests shows reproducible results. CONCLUSIONS: Therefore it is assumed that the Rehab@Home framework is applicable as monitoring tool for the gait rehabilitation progress in stroke patients.

Milk as a diagnostic sample for a commercially available ELISA to identify bovine viral diarrhoea (BVD) antibodies in dairy herds.

OBJECTIVE: The aims of this study were to evaluate a commercially available ELISA for the detection of bovine viral diarrhoea virus (BVDV)-specific antibodies in individual milk compared with individual serum samples, and in bulk milk samples compared with within-herd antibody prevalence and bulk milk quantitative reverse transcription polymerase chain reaction (qRT-PCR) results. METHODS: Paired individual serum and individual milk samples were collected from 125 lactating cows and tested by ELISA; 96 bulk milk samples were also tested. Within-herd antibody prevalence was calculated based on milk ELISA results for 25 individual cows in each herd. Additionally, 167 bulk milk samples were tested for BVDV-specific antibodies by ELISA and for the presence of BVDV by qRT-PCR to establish the correlation between antibody result and virus presence. RESULTS: Good agreement was observed between individual milk and serum results (Kappa = 0.865). The ELISA was observed to detect BVDV-specific antibodies in individual milk samples with a relative sensitivity of 96.6% and specificity of 89.2%. The bulk milk samples revealed a strong (r(2) = 0.95) relationship between the ELISA result and the within-herd antibody prevalence. The proportion of herds that tested positive by bulk milk qRT-PCR increased as the bulk milk antibody S/P ratio increased. CONCLUSION: Commercially available ELISA testing of individual and bulk milk samples is an appropriate alternative to serum testing with good test performance in these samples. Determining a threshold for the detection of herds containing active BVD infection by testing bulk milk is a novel use for an antibody ELISA kit and provides more practical, relevant test results.

Bovine viral diarrhoea virus ('pestivirus') in Australia: to control or not to control?

BACKGROUND: Acute infection with bovine viral diarrhoea virus (BVDV) usually causes only mild clinical disease in cattle, but infection of animals of breeding age can result in immune suppression (resulting in an increased incidence and severity of secondary disease) and decreased reproductive performance. If infection occurs during pregnancy, the virus may cross the placenta and either cause abortion, establish immunotolerance and persistent infection (PI) in the fetus or cause congenital deformities. These outcomes depend on the stage of pregnancy at the time of infection. INTERNATIONAL PERSPECTIVE: BVDV is recognised as a disease of significant financial impact in a number of countries. As a result, national and regional BVDV control programs are now in place in several regions around the world. In Europe, these programs largely rely on the identification and removal of the PI animals, whereas vaccination has tended to be the chosen method of control in the United States. BVD IN AUSTRALIA: BVDV is endemic in Australian cattle populations, with more than 80% of herds surveyed showing some level of exposure to the pathogen. The cost to the national industry is estimated to be AUD57.9 million annually. This review identifies and discusses the challenges to BVDV control in Australia, including farmer attitudes, herd size, sheep as a potential reservoir host and diagnostic capabilities. We conclude that systematic BVDV control in Australia is, or soon will be, an option; however, detailed cost-benefit analyses will need to be undertaken.

Neospora caninum serostatus is affected by age and species variables in cohabiting water buffaloes and beef cattle.

The aim of this study was to investigate how Neospora caninum serostatus may be affected by variables such as host species (water buffaloes or cattle) and age in animals cohabiting in the same ranch. A convenience cross-sectional study was performed on four ranches in the Northeast of Argentina, where water buffalo are cohabitating with beef cattle. Blood samples were collected from 1350 female water buffaloes (Bubalus bubalis) and 880 female beef cattle (Bos taurus and Bos indicus crossbreeds) from four ranches. Calving and weaning percentages at herd level for each ranch were also recorded. N. caninum antibody levels were measured by an indirect fluorescent antibody test (IFAT) (reciprocal antibody titers ≥ 100). Serological results were classified into 2 categories (0: negative; 1: positive). A logistic regression model was used to describe the relationship between N. caninum serostatus and specie (water buffalo or cattle), age or ranch and their interactions. Likelihood ratio tests were used to assess the significance of the model and their terms. Odds ratios were estimated and 95% profile likelihood (LR) and Wald confidence intervals (CI) obtained. Overall, specific antibody titers were found in 43.3% (584/1350) of water buffaloes and 28.6% (252/880) of cattle. Seropositive water buffaloes and cattle were observed on all ranches. Age was statistically significant (p=0.01) with an overall estimate of logit (log odds) of age of 0.03 for both species. This indicates that for every one year increase in age, the expected change in log odds of being seropositive increased by 0.03. On three of four ranches a water buffalo was 4.48, 1.54 and 2.25 times more likely to be seropositive than cattle for animals of the same age. The N. caninum serostatus was affected by age in the first place, but also by species on at least three of the four ranches. Calving and weaning percentages were higher in water buffaloes than in beef cattle (p<0.05). Even though the low pathogenicity that N. caninum seems to have in water buffaloes, this study reinforces the importance of this specie as maintenance of the disease.

Sphingolipids in psychiatric disorders and pain syndromes.

Despite the high prevalence and devastating impact of psychiatric disorders, little is known about their etiopathology. In this review, we provide an overview on the participation of sphingolipids and enzymes responsible for their metabolism in mechanisms underlying psychiatric disorders. We focus on the pathway from sphingomyelin to proapoptotic ceramide and the subsequent metabolism of ceramide to sphingosine, which is in turn phosphorylated to yield anti-apoptotic sphingosine-1-phosphate (S1P).The sphingomyelinase/ceramide system has been linked to effects of reactive oxygen species and proinflammatory cytokines in the central nervous system as well as to synaptic transmission. Compared to ubiquitously expressed acid sphingomyelinase, acid and neutral ceramidase and neutral sphingomyelinase are highly active in brain regions. Depressed patients show elevated plasma ceramide levels and increased activities of acid sphingomyelinase which is functionally inhibited by many anti-depressive drugs. Exposure to alcohol is associated with an activation of acid and neutral sphingomyelinase observed in cell culture, mouse models and in alcohol-dependent patients and with increased concentrations of ceramide in various organs.Levels of sphingomyelin and ceramide are altered in erythrocytes and post-mortem brain tissues of schizophrenic patients in addition to changes in expression patterns for serine palmitoyltransferase and acid ceramidase leading to impaired myelination. After induction of anxiety-like behavior in animal models, higher serum levels of S1P were reported to lead to neurodegeneration. Correspondingly, S1P infusion appeared to increase anxiety-like behavior. Significantly upregulated levels of the endogenous ceramide catabolite N,N-dimethylsphingosine were observed in rat models of allodynia. Conversely, rats injected intrathecally with N,N-dimethylsphingosine developed mechanical allodynia. Moreover, S1P has been implicated in spinal nociceptive processing.The increasing interest in lipidomics and improved analytical methods led to growing insight into the connection between psychiatric and neurological disorders and sphingolipid metabolism and may once provide new targets and strategies for therapeutic intervention.

Validation and evaluation of a commercially available ELISA for the detection of antibodies specific to bovine viral diarrhoea virus (bovine pestivirus).

OBJECTIVE: To compare the performance of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies specific to bovine viral diarrhoea virus (BVDV) with a virus neutralisation test (VNT) and agarose gel immunodiffusion (AGID) test. DESIGN: A total of 125 cattle serum samples were tested by a commercially available ELISA for antibodies specific to BVDV and by a VNT as the reference standard. A comparison between AGID and ELISA for detection of BVDV antibodies was also carried out, using 1182 serum samples from unvaccinated South Australian cattle. METHODS: Two-graph receiver operating characteristics (TG-ROC) analysis was used to confirm that the manufacturer's recommended cut-off value for the ELISA was appropriate. Two-by-two tables were constructed to analyse the concordance of serological results among the three assays. McNemar tests were used to assess the agreement among serological tests. RESULTS AND CONCLUSIONS: Using the manufacturer's cut-off threshold, supported by TG-ROC analysis, the ELISA's sensitivity and specificity were calculated to be 96.7% and 97.1%, respectively, compared with the VNT. Compared with AGID, ELISA with specific BVDV antibodies may be more sensitive and detect 5.8% more samples than AGID. McNemar test also showed a significant difference (P < 0.001) between AGID and ELISA.

Treating Cryptosporidium parvum infection in calves.

The present study evaluated the therapeutic efficacy of azithromycin, co-trimoxazole and kalvangi (Nigella sativa, also known as Black Cumin) against Cryptosporidium parvum infection in calves under field conditions. The experimental calves were treated with azithromycin (group A) at 1500 mg/calf/day, co-trimoxazole (group B) at 30 mg Kg-1 and kalvangi seeds powder (group C) at 750 mg Kg-1 BW orally for 7 days. Calves in the group D were naturally infected with C. parvum , untreated animals (positive control) while the calves in the group E were uninfected negative control animals. A significant decrease (p < 0.05) in oocyst counts for calves in groups A, B and C was observed compared to group D. When the oocyst counts amongst the treatment groups A, B and C were compared, a significant decrease (p < 0.05) was observed in group A. On day 21 post-treatment, the efficacy of azithromycin, co-trimoxazole and kalvangi in calves was 88.2% (95% C.I. ± 15.4), 45% (95% C.I. ± 21.8) and 27.8% (95% C.I. ± 20.7), respectively. This study confirmed previous reports of azithromycin efficacy against C. parvum infection, but found co-trimoxazole and kalvangi to be ineffective for this infection under these treatment regimens.

Prevalence of Neospora caninum antibodies in sheep and goats in Pakistan.

The purpose of the present study was to obtain seroepidemiological information on the Neospora caninum infection status of sheep and goats in different areas of Punjab Province and Azad Kashmir (Pakistan). A cross-sectional study, with the use of a competitive ELISA, showed an overall 27.7% (35 of 128) (95% confidence interval [CI] ± 7.7%) and 8.6% (13 of 142) (95% CI ± 4.6%) seroprevalence of N. caninum antibodies in sheep and goats, respectively. The difference in seroprevalence between sheep and goat populations was statistically significant (P < 0.05). The highest prevalence (37.4% ± 13.2%) was recorded in the tailless breed of sheep.