Ca2+/calmodulin-dependent protein kinase IIα (αCaMKII) controls the activity of the dopamine transporter: Implications for angelman syndrome

The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/ threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP+) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII T305D), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP+ efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome

Mechanism and control of the eye formation in cheese

The production of Swiss-type cheeses with a typical number, size, and distribution of eyes is a difficult task, especially when bactofuged or microfiltrated milk is utilised. In this study, the potential of microparticles (plant origin) to influence eye formation in cheese, was assessed. Eight experimental Emmental cheeses were produced with one replicate from microfiltrated milk with addition of 0.0625–4.000 mg of powdered hay to the milk (90 L) and ripened for 130 days. Eye formation was quantified by means of X-ray computed tomography (between 30 and 130 days). The contents of fat, water, citric acid, lactic acid, and volatile carboxylic acids were determined at 130 days. The results demonstrate that microparticles of plant origin act as eye nuclei that control the number (P < 0.001) and size of the eyes in cheese in a dose-dependent manner. The findings also provide new insights into the formation of eye defects

Membrane permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release.

The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate the role of the DAT C terminus in AMPH-evoked DA efflux using cell-permeant dominant-negative peptides. A peptide, which corresponded to the last 24 C-terminal residues of DAT (TAT-C24 DAT) and thereby contained the Ca(2+)-calmodulin-dependent protein kinase IIα (CaMKIIα) binding domain and the PSD-95/Discs-large/ZO-1 (PDZ)-binding sequence of DAT, was made membrane-permeable by fusing it to the cell membrane transduction domain of the HIV-1 Tat protein (TAT-C24WT). The ability of TAT-C24WT but not a scrambled peptide (TAT-C24Scr) to block the CaMKIIα-DAT interaction was supported by co-immunoprecipitation experiments in heterologous cells. In heterologous cells, we also found that TAT-C24WT, but not TAT-C24Scr, decreased AMPH-evoked 1-methyl-4-phenylpyridinium efflux. Moreover, chronoamperometric recordings in striatum revealed diminished AMPH-evoked DA efflux in mice preinjected with TAT-C24WT. Both in heterologous cells and in striatum, the peptide did not further inhibit efflux upon KN-93-mediated inhibition of CaMKIIα activity, consistent with a dominant-negative action preventing binding of CaMKIIα to the DAT C terminus. This was further supported by the ability of a peptide with perturbed PDZ-binding sequence, but preserved CaMKIIα binding (TAT-C24AAA), to diminish AMPH-evoked DA efflux in vivo to the same extent as TAT-C24WT. Finally, AMPH-induced locomotor hyperactivity was attenuated following systemic administration of TAT-C24WT but not TAT-C24Scr. Summarized, our findings substantiate that DAT C-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects

Status quo in requirements engineering: a theory and a global family of surveys

This paper reports on the second run of the Naming the Pain in Requirements Engineering (NaPiRE) initiative that has the goal to characterise requirements engineering practice and problems and was published in the ACM Transactions on Software Engineering and Methodology in 2019. Requirements Engineering (RE) has established itself as a software engineering discipline over the past decades. While researchers have been investigating the RE discipline with a plethora of empirical studies, attempts to systematically derive an empirical theory in context of the RE discipline have just recently been started. However, such a theory is needed if we are to define and motivate guidance in performing high quality RE research and practice. We aim at providing an empirical and externally valid foundation for a theory of RE practice, which helps software engineers establish effective and efficient RE processes in a problem-driven manner. We designed a survey instrument and an engineer-focused theory that was first piloted in Germany and, after making substantial modifications, has now been replicated in 10 countries world-wide. We have a theory in the form of a set of propositions inferred from our experiences and available studies, as well as the results from our pilot study in Germany. We evaluate the propositions with bootstrapped confidence intervals and derive potential explanations for the propositions. In this article, we report on the design of the family of surveys, its underlying theory, and the full results obtained from the replication studies conducted in 10 countries with participants from 228 organisations. Our results represent a substantial step forward towards developing an empirical theory of RE practice. The results reveal, for example, that there are no strong differences between organisations in different countries and regions, that interviews, facilitated meetings and prototyping are the most used elicitation techniques, that requirements are often documented textually, that traces between requirements and code or design documents are common, that requirements specifications themselves are rarely changed and that requirements engineering (process) improvement endeavours are mostly internally driven. Our study establishes a theory that can be used as starting point for many further studies for more detailed investigations. Practitioners can use the results as theory-supported guidance on selecting suitable RE methods and techniques.<br/

Status Quo in Requirements Engineering: A Theory and a Global Family of Surveys

While researchers have been investigating the Requirements Engineering (RE) discipline with a plethora of empirical studies, attempts to systematically derive an empirical theory in context of the RE discipline have just recently been started. We aim at providing an empirical and externally valid foundation for a theory of RE practice, which helps software engineers establish effective and efficient RE processes in a problem-driven manner. We designed a survey instrument and an engineer-focused theory that has been conducted in 10 countries. We have a theory in the form of a set of propositions inferred from our experiences and available studies, as well as the results from our pilot study in Germany. We evaluate the propositions with bootstrapped confidence intervals and derive potential explanations for the propositions

Ruxolitinib for Glucocorticoid-Refractory Chronic Graft-versus-Host Disease

Background: Chronic graft-versus-host disease (GVHD), a major complication of allogeneic stem-cell transplantation, becomes glucocorticoid-refractory or glucocorticoid-dependent in approximately 50% of patients. Robust data from phase 3 randomized studies evaluating second-line therapy for chronic GVHD are lacking. In retrospective surveys, ruxolitinib, a Janus kinase (JAK1-JAK2) inhibitor, showed potential efficacy in patients with glucocorticoid-refractory or -dependent chronic GVHD. Methods: This phase 3 open-label, randomized trial evaluated the efficacy and safety of ruxolitinib at a dose of 10 mg twice daily, as compared with the investigator's choice of therapy from a list of 10 commonly used options considered best available care (control), in patients 12 years of age or older with moderate or severe glucocorticoid-refractory or -dependent chronic GVHD. The primary end point was overall response (complete or partial response) at week 24; key secondary end points were failure-free survival and improved score on the modified Lee Symptom Scale at week 24. Results: A total of 329 patients underwent randomization; 165 patients were assigned to receive ruxolitinib and 164 patients to receive control therapy. Overall response at week 24 was greater in the ruxolitinib group than in the control group (49.7% vs. 25.6%; odds ratio, 2.99; P18.6 months vs. 5.7 months; hazard ratio, 0.37; P<0.001) and higher symptom response (24.2% vs. 11.0%; odds ratio, 2.62; P = 0.001). The most common (occurring in ≥10% patients) adverse events of grade 3 or higher up to week 24 were thrombocytopenia (15.2% in the ruxolitinib group and 10.1% in the control group) and anemia (12.7% and 7.6%, respectively). The incidence of cytomegalovirus infections and reactivations was similar in the two groups. Conclusions: Among patients with glucocorticoid-refractory or -dependent chronic GVHD, ruxolitinib led to significantly greater overall response, failure-free survival, and symptom response. The incidence of thrombocytopenia and anemia was greater with ruxolitinib. (Funded by Novartis and Incyte; REACH3 ClinicalTrials.gov number, NCT03112603.)