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.     

Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs

Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA-protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells.     

Three research priorities for just and sustainable urban systems: Now is the time to refocus

Now is the time to refocus efforts in urban research and design. A changing climate and extreme weather events are presenting unique challenges to urban systems around the world. These challenges illuminate the social barriers that accompany disruptive events such as resource inequities and injustices. In this perspective, we provide three research priorities for just and sustainable urban systems that help to address these matters. The three research priorities are: (1) social equity and justice, (2) circularity, and (3) digital twins. Conceptual context and future research directions are provided for each. For social equity and justice, the future directions are mandatory equity analysis and inclusionary practices, understanding and reconciling historical injustices, and intentional integration with diverse community stakeholders. For circularity applications, they are better metrics for integration, more robust evaluation frameworks, and dynamic modeling at multiple spatial and temporal scales. Future directions for digital twins include developing principles to reduce complexity, integrating model and system components, and reducing barriers to data access. These research priorities are core to meeting several of the United Nations Sustainable Development Goals (i.e., 1—No Poverty, 8—Decent Work and Economic Growth, 10—Reduced Inequalities, and 11—Sustainable Cities and Communities). Useful social and technical matters are discussed throughout, where we highlight the importance of prioritizing localized research efforts, provide guidance for community-engaged research and co-development practices, and explain how these priorities interact to align with the evolving field of industrial ecology.     

Haemagglutinin substitutions N125D,D127E,D222G and R223Q improve replicative fitness and vaccine effectiveness of an A/H1N1pdm09 live attenuated influenza vaccine virus by enhancing α-2,6 receptor binding

During 2013–14 and 2015–16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.     

A phenomenological computational model of the evoked action potential fitted to human cochlear implant responses

There is a growing interest in biomedical engineering in developing procedures that provide accurate simulations of the neural response to electrical stimulus produced by implants. Moreover, recent research focuses on models that take into account individual patient characteristics.We present a phenomenological computational model that is customized with the patient’s data provided by the electrically evoked compound action potential (ECAP) for simulating the neural response to electrical stimulus produced by the electrodes of cochlear implants (CIs). The model links the input currents of the electrodes to the simulated ECAP.Potentials and currents are calculated by solving the quasi-static approximation of the Maxwell equations with the finite element method (FEM). In ECAPs recording, an active electrode generates a current that elicits action potentials in the surrounding auditory nerve fibers (ANFs). The sum of these action potentials is registered by other nearby electrode. Our computational model emulates this phenomenon introducing a set of line current sources replacing the ANFs by a set of virtual neurons (VNs). To fit the ECAP amplitudes we assign a suitable weight to each VN related with the probability of an ANF to be excited. This probability is expressed by a cumulative beta distribution parameterized by two shape parameters that are calculated by means of a differential evolution algorithm (DE). Being the weights function of the current density, any change in the design of the CI affecting the current density produces changes in the weights and, therefore, in the simulated ECAP, which confers to our model a predictive capacity.The results of the validation with ECAP data from two patients are presented, achieving a satisfactory fit of the experimental data with those provided by the proposed computational model.     

Effects of grass species and grass growth on atmospheric nitrogen deposition to a bog ecosystem surrounded by intensive agricultural land use

We applied a ¹⁵N dilution technique called “Integrated Total Nitrogen Input” (ITNI) to quantify annual atmospheric N input into a peatland surrounded by intensive agricultural practices over a 2-year period. Grass species and grass growth effects on atmospheric N deposition were investigated using Lolium multiflorum and Eriophorum vaginatum and different levels of added N resulting in increased biomass production. Plant biomass production was positively correlated with atmospheric N uptake (up to 102.7 mg N pot⁻¹) when using Lolium multiflorum. In contrast, atmospheric N deposition to Eriophorum vaginatum did not show a clear dependency to produced biomass and ranged from 81.9 to 138.2 mg N pot⁻¹. Both species revealed a relationship between atmospheric N input and total biomass N contents. Airborne N deposition varied from about 24 to 55 kg N ha⁻¹ yr⁻¹. Partitioning of airborne N within the monitor system differed such that most of the deposited N was found in roots of Eriophorum vaginatum while the highest share was allocated in aboveground biomass of Lolium multiflorum. Compared to other approaches determining atmospheric N deposition, ITNI showed highest airborne N input and an up to fivefold exceedance of the ecosystem-specific critical load of 5–10 kg N ha⁻¹ yr⁻¹.     

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.     

Lithium Effects on Inositol Phospholipids and Inositol Phosphates: Evaluation of an In Vivo Model for Assessing Polyphosphoinositide Turnover in Brain

Administration of lithium chloride to rats injected intracerebrally with [3H]inositol led to time- and dose-dependent increases in levels of labeled inositol monophosphates in brain. Quantitative analysis of the inositol phosphates by ion chromatography revealed 37- and 20-fold increases in the mass of myo-inositol 1-phosphate and 4-phosphate, respectively, at 4 h intraperitoneal after injections of 6 mEq/kg of lithium chloride. Albeit to a much lesser extent, lithium administration also resulted in an increase in the level of myo-inositol, 1,4-bisphosphate in brain. The lithium-induced increase in content of labeled inositol monophosphates was marked by a concomitant decrease in content of labeled inositol, and after injections of high doses of lithium, e.g., 10 mEq/kg, this was followed by a general decrease in labeling of the inositol phospholipids. In general, animals injected with [3H]inositol but not lithium did not reveal obvious differences in labeling of inositol monophosphates on stimulation by mecamylamine or pilocarpine. However, when animals were injected with [3H]inositol and then lithium, there were large increases in the levels of labeled inositol monophosphates on administration of these compounds. Administration of atropine to the lithium-treated mice led to a partial reduction in the amount of labeled inositol monophosphates accumulated due to the administration of lithium alone. Furthermore, atropine was able to block the pilocarpine-induced increase in level of labeled inositol monophosphates. These results demonstrate the suitable use of the radiotracer technique together with lithium administration for assessing the effects of drugs and receptor agonists on the signaling system involving polyphosphoinositide turnover in brain.