KLHL12 promotes non-lysine ubiquitination of the dopamine receptors D-4.2 and D-4.4, but not of the ADHD-associated D-4.7 variant

Dopamine D-4 Receptor Polymorphism : The dopamine D-4 receptor has an important polymorphism in its third intracellular loop that is intensively studied and has been associated with several abnormal conditions, among others, attention deficit hyperactivity disorder. KLHL12 Promotes Ubiquitination of the Dopamine D-4 Receptor on Non-Lysine Residues : In previous studies we have shown that KLHL12, a BTB-Kelch protein, specifically interacts with the polymorphic repeats of the dopamine D-4 receptor and enhances its ubiquitination, which, however, has no influence on receptor degradation. In this study we provide evidence that KLHL12 promotes ubiquitination of the dopamine D-4 receptor on non-lysine residues. By using lysine-deficient receptor mutants and chemical approaches we concluded that ubiquitination on cysteine, serine and/or threonine is possible. Differential Ubiquitination of the Dopamine D-4 Receptor Polymorphic Variants : Additionally, we show that the dopamine D-4.7 receptor variant, which is associated with a predisposition to develop attention deficient hyperactivity disorder, is differentially ubiquitinated compared to the other common receptor variants D-4.2 and D-4.4. Together, our study suggests that GPCR ubiquitination is a complex and variable process

Methods to maximise recovery of environmental DNA from water samples

<div><p>The environmental DNA (eDNA) method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (<i>Misgurnus anguillicaudatus</i>) as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3–5 days). This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.</p></div

The ecology of the two-spined blackfish Gadopsis Bispinosus (Pisces: Gadopsidae)

The Gadopsidae is an endemic mono-generic freshwater fish family of south-eastern Australia. The family was thought to be mono-specific, containing the River Blackfish Gadopsis marmoratus, until 1984 when a second species, the Two-spined Blackfish G. bispinosus was described (Sanger 1984). Due its relatively recent description, little information was available on the ecology or conservation status of G. bispinosus in NSW and the ACT. The distribution and abundance of G. bispinosus in southern NSW and the ACT was investigated with the species recorded at 16 of 119 sites surveyed. At most sites where they were recorded, G. bispinosus was abundant, and was invariably found in association with one of the trout species. G. bispinosus was found to be restricted to cool, clear upland streams with rocky, cobble bottoms and relatively intact forest vegetation. It was hypothesised that the presence of the species in the upper Murrumbidgee drainage was due to stream capture, in which the headwater streams from the Murray drainage have been captured by the Murrumbidgee drainage. Interpretation of historical reports of blackfish distribution in NSW indicated that G. bispinosus had suffered declines in some rivers with the species now apparently absent from the Yarrangobilly River and the Murrumbidgee, Naas, and Paddys rivers in the ACT. The decline is thought to have been due to habitat degradation, particularly sediment addition, which has reduced the cover available by filling the interstitial spaces in the cobble substrate favoured by G. bispinosus. The invariable association of G. bispinosus with introduced trout species and habitats with abundant cover, suggests that trout may have played some part in the current distribution of G bispinosus by excluding them from sub-optimal habitats. The movements of G. bispinosus was found to be very restricted with a home range of approximately 15 metres estimated for adult fish. Recapture rates were high, particularly in adult fish, in comparison with other studies of freshwater fish, indicating that adult G. bispinosus are particularly sedentary. The home ranges of G. bispinosus were found to be stable from year to year with fish able to maintain their position in the stream over the high flow periods of winter and spring. On the basis of aquarium observations of pugnacious and aggressive behaviour between adult G. bispinosus, it was considered that these home ranges may be considered territories under the definition of (Gerking 1953) who defined a territory as "any defended area". The diet of G. bispinosus was investigated with distinct seasonal and ontogenetic differences apparent in the diet. Juvenile fish consumed predominantly smaller items such as early instar ephemeropterans and chironomid larvae with some trichopterans present in the diet. The proportion of trichopterans in the diet increased with increasing fish size, with the importance of ephemeropterans and dipterans inversely related to fish size. terrestrial items were not present at all in the stomachs of juvenile fish, were of minor importance to immature fish, but were a major dietary item of adults. Terrestrial items were most abundant in the diet of both immature and adult fish in summer and autumn. The diet of Rainbow Trout 0. mykiss was also examined, with significant dietary overlap apparent between 0. mykiss and G. bispinosus. As with G. bispinosus, the diet of 0. mykiss was dominated by ephemeropterans, trichopterans and terrestrial items. Dietary overlap was greatest between similar size classes of both species, with some seasonal pattern evident. Significant overlap occurred between the non-adult G. bispinosus and juvenile 0. mykiss all seasons except autumn. Overlap was greatest between mature blackfish and non-juvenile trout with significant dietary overlap recorded in all seasons. It was considered that the consistent significant overlap values indicate the dietary competition is likely between these two fish species. The reproductive ecology of G. bispinosus was found to be similar to that recorded for G. marmoratus by Jackson (1978a). Both blackfish species deposit large, yolky, adhesive, demersal eggs in late spring/early summer when water temperatures exceed 16-17 °C. Fecundity in G. bispinosus is low with less than 300 eggs carried by most females. The natural spawning site was not located but is thought to be in the interstitial spaces between cobble and boulders on the river bed. Artificial P.V.C. spawning tubes proved successful with a total of 15 egg masses deposited in them over the course of the study. The numbers of eggs in each egg mass were within the fecundity estimates of individual fish, and all eggs within a mass were at the same stage of development , indicating that a single fish is probably responsible for each egg mass. A large adult male was present with each egg mass. Eggs hatched after approximately 15-17 days, with the embryo emerging from the chorion but the yolk sac remaining inside, effectively tethering the young to the spawning substrate. Parental care by the male continued for approximately 3-4 weeks after hatching by which time the yolk sac was almost fully utilised and young blackfish could swim well

Modulation of T and B cell function in Granulomatosis with polyangiitis:Targeting Kv1.3 potassium channels

Granulomatosis with polyangiitis (GPA) is a chronic systemic autoimmune disease characterized by inflammation of small blood vessels predominantly in the upper and lower respiratory tract and/or kidney. Current treatment with immunosuppressants succeeds in establishing disease remission in most patients. However, 50% of patients experience disease relapses during their disease course. Each relapse is accompanied by increased organ damage and loss of quality of life. In particular, generalized immunosuppressive therapies used induce adverse events and cause increased risk for opportunistic infections. This emphasizes the need for novel immunomodulators with more specific and less toxic treatment regimens for GPA-patients. Considerable evidence supports a key role for B- and T-cells in the pathogenesis of GPA. In this thesis, we have demonstrated that the distribution of effector memory T- and B-cells in the circulation of GPA-patients is altered. Differences in effector memory T-cell phenotypes were associated with various clinical presentations of the disease which were influenced by CMV infection. Interestingly, it has been shown that effector T- and B-cells express high levels of a specific Kv1.3 potassium channel that may serve as a selective therapeutic target. Indeed, in vitro blockade of these channels with the highly potent Kv1.3 channel inhibitor ShK-186 modulated T-cell function in GPA-patients resulting in normalization of pro-inflammatory cytokine production. For B-cells, antibody dependent and independent effector functions were suppressed by ShK-186, skewing B-cell responses towards a more prominent regulatory response. In conclusion, selective Kv1.3 channel blockade using ShK-186 may be an attractive therapeutic option for T and B effector subset-selective immunomodulation in GPA

Distinguished Brief

The Government of the Republic of Bretoria and the Kingdom of Pagonia have agreed to submit by Special Agreement the present controversy for final solution to the International Court of Justice pursuant to Article 36, paragraph 1 of the Statute of the International Court of Justice, in relation to Article 40, paragraph 1, of the Statute of the Court