Antibodies against a portion of the rat 5-HT2C receptor third intracellular loop were generated and used to identify receptors solubilized from cell lines and rat brain. Western blots of ...CHAPS-soluble proteins were probed with affinity-purified anti-2C antibodies. The specificity of anti-2C was demonstrated with extracts prepared from NIH/3T3 fibroblasts which stably express functional rat 5-HT2C or 5-HT2A receptors. Extracts from the 5-HT2C cell line, but not the 5-HT2A cell line, contained immunoreactive proteins with masses of 51-52 kDa and 58-68 kDa. In the brain, immunoreactive proteins were identified from choroid plexus extracts with masses of 51 kDa and 58-62 kDa. The major 58-62 kDa and minor 51 kDa proteins were not detected in extracts prepared from the hippocampus, striatum, or frontal cortex using the same amount of CHAPS-soluble protein. These results are consistent with previous studies demonstrating that 5-HT2C receptor binding sites and mRNA are most abundant in choroid plexus. The association of asparagine-linked (N-linked) oligosaccharides with the receptors was examined next. The 5-HT2C receptor cell line (3T3/2C) was grown in the presence of tunicamycin to metabolically inhibit N-linked glycosylation. Proteins from the cell extracts were detected with masses of 40 and 41 kDa. Extracts prepared from 3T3/2C cells (grown in the absence of tunicamycin) and from choroid plexus were incubated with N-glycosidase F to enzymatically remove available N-linked sugars. Immunoreactive proteins were detected with masses of 41 and 42 kDa from 3T3/2C cells and 41 kDa from choroid plexus. Neuraminidase, which cleaves sialic acid (N-acetylneuraminic acid) residues from glycoproteins, reduced the mass of the 51 and 58-62 kDa proteins from the choroid plexus to 50 and 54-58 kDa. In contrast, the 51-52 and 58-68 kDa proteins from 3T3/2C cells were not affected by treatment with neuraminidase. These results demonstrate that 5-HT2C receptors contain N-linked sugars and suggest that sialic acid residues associate with 5-HT2C receptors in the choroid plexus. The oligosaccharide moieties, which contribute up to approximately 30% of the relative mass as judged by SDS-polyacrylamide gel electrophoresis, may impart functional properties to 5-HT2C receptors.
A leucine residue, Leu-98, lines the O(2)-binding pocket in all known hemerythrins. Leu-98 in recombinant Phascolopsis gouldii hemerythrin, was mutated to several other residues of varying sizes ...(Ala, Val), polarities (Thr, Asp, Asn), and aromaticities (Phe, Tyr, Trp). UV-visible and resonance Raman spectra showed that the di-iron sites in these L98X Hrs are very similar to those in the wild type protein, and several of the L98X hemerythrins formed stable oxy adducts. Despite the apparently tight packing in the pocket, all of the L98X Hrs except for L98W, had second order O(2) association rate constants within a factor of 3 of the wild type value. Similarly, the O(2) dissociation rate constant was essentially unaffected by substitutions of larger (Phe) or smaller (Val, Thr) residues for Leu-98. L98Y Hr showed a 170-fold decrease in the O(2) dissociation rate constant and a large D(2)O effect on this rate, which are attributed to a hydrogen-bonding interaction between the Tyr-98 hydroxyl and the bound O(2). Significant increases in autoxidation rates were observed for all of the L98X Hrs other than X = Tyr. These increases in autoxidation rates are attributed to increased solvent access to the binding pocket caused by inefficient packing (Phe), smaller size (Val, Ala), or increased polarity (Thr, Asp, Asn) of the residue 98 side chain. A leucine at position 98 appears to have the optimal size, shape, and hydrophobicity for inhibition of solvent access. Thus, "gating" of small molecule access to the binding pocket of Hr by Leu-98 is not evident for O(2), but is evident for solvent.
Triple P is an integrated, multi-level system of evidence-based parenting support designed to promote the well-being of children and families to reduce prevalence rates of social, emotional, and ...behavioral problems in children and adolescents and to prevent child maltreatment. The system developed gradually over four decades to address the complex needs of parents and children from diverse family, socioeconomic and cultural backgrounds. It blends universal and targeted programs, a focus on developing parental self-regulation capabilities, and adopts a life span perspective with a population health framework. The Triple P system is used as a case example to discuss the past, present and future challenges, and opportunities involved in developing, evaluating, adapting, scaling and maintaining a sustainable system of evidence-based parenting intervention. Seven stages of program development are outlined from initial theory building and development of the core parenting program through to the sustained deployment of the intervention system delivered at scale. The importance of ongoing research and evaluation is highlighted so that different programs within the system evolve and adapt to address the contemporary concerns and priorities of families in diverse cultural contexts. A well-trained workforce is essential to deliver evidence-based programs, in a need-responsive manner that blends both fidelity of delivery and flexibility and is tailored to respond to the needs of individual families and local context. Programs need to be gender-sensitive, culturally informed, and attuned to the local context including relevant policies, resources, cultural factors, funding, workforce availability and their capacity to implement programs.
•E-scooters used more for transport than recreation, potentially filling a niche.•E-scooters viewed as convenient, faster, and better in hot weather than walking.•Non-white non-riders significantly ...more likely to intend to try e-scooters.•E-scooters disproportionately replace walking and bicycling for all trip types.•Women significantly more likely to cite safety-related barriers to e-scooter use.
E-scooters are rapidly changing transportation in US cities and university campuses. Hailed as a convenient, inexpensive solution for “last mile” and other short trips, e-scooters are available in over 100 US cities and were used for nearly forty million trips in 2018. Yet relatively little is known about e-scooter use, including who uses them, for which types of trips, and the perceived benefits and barriers related to e-scooters. This information is particularly important in light of concerns about safety and the loss of physical activity (PA) due to replacing walking and biking with e-scooting.
In this paper, we aim to characterize trends in the barriers and benefits related to e-scooter use within a professional population. We surveyed 1,256 university staff in Tempe, AZ, finding that 36% of respondents had ridden e-scooters and 40% indicated that they would do so outside of campus in the next year. Overwhelmingly, e-scooters are seen as a convenient way to travel, particularly in the heat and compared to walking. However, demographic differences were notable, particularly regarding barriers. African American and non-white Hispanic respondents were significantly more likely than non-Hispanic white respondents to intend to try e-scooters and to be unhappy with current transportation options. E-scooters are also associated with concerns about traffic safety – particularly for women – and barriers related to being able to find working equipment when needed. These findings suggest that e-scooters fill an important transportation niche and may contribute to transportation equity, and that efforts to address barriers could further enhance that contribution.
Biological oceanic processes, principally the surface production, sinking and interior remineralization of organic particles, keep atmospheric CO
lower than if the ocean was abiotic. The ...remineralization length scale (RLS, the vertical distance over which organic particle flux declines by 63%, affected by particle respiration, fragmentation and sinking rates) controls the size of this effect and is anomalously high in oxygen minimum zones (OMZ). Here we show in the Eastern Tropical North Pacific OMZ 70% of POC remineralization is due to microbial respiration, indicating that the high RLS is the result of lower particle fragmentation by zooplankton, likely due to the almost complete absence of zooplankton particle interactions in OMZ waters. Hence, the sensitivity of zooplankton to ocean oxygen concentrations can have direct implications for atmospheric carbon sequestration. Future expansion of OMZs is likely to increase biological ocean carbon storage and act as a negative feedback on climate change.
The spin of Cygnus X-1 is measured by fitting reflection models to Suzaku data covering the energy band 0.9-400 keV. The inner radius of the accretion disc is found to lie within 2 gravitational ...radii (r
g=GM/c
2), and a value of
is obtained for the dimensionless black hole spin. This agrees with recent measurements using the continuum fitting method by Gou et al. and of the broad iron line by Duro et al. The disc inclination is measured at
, which is consistent with the recent optical measurement of the binary system inclination by Orosz et al. of 27°± 0°.8. We pay special attention to the emissivity profile caused by irradiation of the inner disc by the hard power-law source. The X-ray observations and simulations show that the index q of that profile deviates from the commonly used, Newtonian, value of 3 within 3r
g, steepening considerably within 2r
g, as expected in the strong gravity regime.
AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is ...stimulated more than 100-fold by phosphorylation of threonine 172 (Thr
). Binding of AMP to the γ subunit allosterically activates the kinase. Additionally, many small molecules, e.g. 991, have been identified that bind between the kinase domain and the carbohydrate-binding module of the β subunit, stabilising their interaction and leading to activation. It was reported recently that non-phosphorylated Thr
AMPK is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated Thr
AMPK in complex with AMP and 991. This structure reveals that the activation loop, as well as the complex overall, is similar to the Thr
phosphorylated complex. We find that in the presence of AMP and 991 non-phosphorylated Thr
, AMPK is much less active than the Thr
phosphorylated enzyme. In human cells, the basal level of Thr
phosphorylation is very low (∼1%), but is increased 10-fold by treatment with 2-deoxyglucose. In cells lacking the major Thr
kinases, LKB1 and CaMKKβ, Thr
phosphorylation is almost completely abolished, and AMPK activity is virtually undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr
AMPK can induce an ordered, active-like, conformation of the activation loop explaining how AMPK activity can be measured
without Thr
phosphorylation. However, in a cellular context, phosphorylation of Thr
is critical for significant activation of AMPK.
Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and ...coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.
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•Stress granule formation requires RNA-binding nodes with high network connectivity•Capping of nodes by ligands lacking connectivity prevents condensation•Protein disorder and RNA-binding specificity play non-essential, modulatory roles•Competition of RNP networks for connecting nodes controls multiphase organization
With sufficient RNA-binding interfaces, diverse protein complexes can trigger stress-dependent multiphase condensates, whose composition and spatial organization is determined by overlapping interaction networks.
A gluten-free diet is the only treatment option of coeliac disease, but recently an increasing number of trials have begun to explore alternative treatment strategies. We aimed to review the ...literature on coeliac disease therapeutic trials and issue recommendations for outcome measures.
Based on a literature review of 10 062 references, we (17 researchers and 2 patient representatives from 10 countries) reviewed the use and suitability of both clinical and non-clinical outcome measures. We then made expert-based recommendations for use of these outcomes in coeliac disease trials and identified areas where research is needed.
We comment on the use of histology, serology, clinical outcome assessment (including patient-reported outcomes), quality of life and immunological tools including gluten immunogenic peptides for trials in coeliac disease.
Careful evaluation and reporting of outcome measures will increase transparency and comparability of coeliac disease therapeutic trials, and will benefit patients, healthcare and the pharmaceutical industry.
Abstract
Background
Assessing the duration of immunity following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a first priority to gauge the degree of protection ...following infection. Such knowledge is lacking, especially in the general population. Here, we studied changes in immunoglobulin isotype seropositivity and immunoglobulin G (IgG) binding strength of SARS-CoV-2–specific serum antibodies up to 7 months following onset of symptoms in a nationwide sample.
Methods
Participants from a prospective representative serological study in the Netherlands were included based on IgG seroconversion to the spike S1 protein of SARS-CoV-2 (N = 353), with up to 3 consecutive serum samples per seroconverted participant (N = 738). Immunoglobulin M (IgM), immunoglobulin A (IgA), and IgG antibody concentrations to S1, and increase in IgG avidity in relation to time since onset of disease symptoms, were determined.
Results
While SARS-CoV-2–specific IgM and IgA antibodies declined rapidly after the first month after disease onset, specific IgG was still present in 92% (95% confidence interval CI, 89%–95%) of the participants after 7 months. The estimated 2-fold decrease of IgG antibodies was 158 days (95% CI, 136–189 days). Concentrations were sustained better in persons reporting significant symptoms compared to asymptomatic persons or those with mild upper respiratory complaints only. Similarly, avidity of IgG antibodies for symptomatic persons showed a steeper increase over time compared with persons with mild or no symptoms (P = .022).
Conclusions
SARS-CoV-2–specific IgG antibodies persist and show increasing avidity over time, indicative of underlying immune maturation. These data support development of immune memory against SARS-CoV-2, providing insight into protection of the general unvaccinated part of the population.
Clinical Trials Registration
NL8473 (the Dutch trial registry).
In this nationwide sample, immunoglobulin (Ig) G antibodies were sustained in 92% of the participants after 7 months after onset of symptoms of SARS-CoV-2 infection, whereas IgM and IgA antibodies wane. Concentrations are higher in symptomatic persons and avidity increases with time.
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