Allostery and Conformational Dynamics in Protein Kinase ERK2

Extracellular signal-regulated kinase 2 (ERK2) is a mitogen-activated protein kinase (MAP kinase) responsible for regulation of cellular processes such as proliferation, survival, and adhesion. Activation by dual phosphorylation of ERK2 (0P-ERK2&rarr;2P-ERK2), as reported by NMR, causes 2P-ERK2 to access a new structural conformer that is in slow (300 s-1) equilibrium with the dominant conformer of 0P-ERK2. I assigned NMR resonances to activation loop residues, and then used these assignments to show by NMR relaxation-dispersion experiments that the activation loop is conformationally mobile on a ms timescale. The dynamics of the loop match those of previously reported global motions in the kinase core that enable productive nucleotide binding. Mutations in the kinase that altered global motions also changed the dynamics of the activation loop. Conversely, mutations at residues in the loop perturbed global motions in the core domain. These findings provide evidence for coupling between motions in the activation loop and the catalytic site in the active state of the kinase. Thus, the activation loop segment in dual-phosphorylated ERK2 undergoes exchange between conformers separated by a small energetic barrier, serving as part of a dynamic allosteric network. I further explored the allosteric regulation of ERK2, by using NMR to examine complexes that mimic catalytic intermediates formed during enzyme turnover. Measurements of NMR resonance chemical shift perturbations (CSP) revealed that the binary complex of 2P-ERK:ANP (ATP analogue) assumes a unique binding mode that differs from the 2P-ERK:ADP complex. A model peptide substrate, Elk1D, containing a D-recruitment site (DRS) motif, induced only local changes at its DRS binding site in 0P-ERK2 and 2P-ERK2 apoenzymes and in 2P-ERK2:ANP, but induced long-range perturbations when complexed with 2P-ERK2:ADP. This unexpected instance of allostery points toward an unknown regulatory mechanism in ERK2&rsquo;s catalytic cycle. CPMG relaxation dispersion experiments on the 2P-ERK2:ANP and 2P-ERK2:ADP binary complexes revealed that global dynamics were largely unchanged upon addition of nucleotide, except at discrete sidechain clusters which appeared to be uncoupled from global motions. These data reveal differential effects of nucleotides and substrates towards the dynamics of ERK2.</p

Recommendations for Implementing Hepatitis C Virus Care in Homeless Shelters: The Stakeholder Perspective.

Compared with the general population, homeless individuals are at higher risk of hepatitis C infection (HCV) and may face unique barriers in receipt of HCV care. This study sought the perspectives of key stakeholders toward establishing a universal HCV screening, testing, and treatment protocol for individuals accessing homeless shelters. Four focus groups were conducted with homeless shelter staff, practice providers, and social service outreach workers (n&nbsp;=&nbsp;27) in San Francisco, California, and Minneapolis, Minnesota. Focus groups evaluated key societal, system, and individual-level facilitators and barriers to HCV testing and management. Interviews were transcribed and analyzed thematically. The societal-level barriers identified were lack of insurance, high-out-of-pocket expenses, restriction of access to HCV treatment due to active drug and/or alcohol use, and excessive paperwork required for HCV treatment authorization from payers. System-level barriers included workforce constraints and limited health care infrastructure, HCV stigma, low knowledge of HCV treatment, and existing shelter policies. At the individual level, client barriers included competing priorities, behavioral health concerns, and health attitudes. Facilitators at the system level for HCV care service integration in the shelter setting included high acceptability and buy in, and linkage with social service providers. Conclusion: Despite societal, system, and individual-level barriers identified with respect to the scale-up of HCV services in homeless shelters, there was broad support from key stakeholders for increasing capacity for the provision of HCV services in shelter settings. Recommendations for the scale-up of HCV services in homeless shelter settings are discussed

Transit Hunt for Young and Maturing Exoplanets (THYME). VIII. A Pleiades-age Association Harboring Two Transiting Planetary Systems from Kepler

Young planets provide a window into the early stages and evolution of planetary systems. Ideal planets for such research are in coeval associations, where the parent population can precisely determine their ages. We describe a young association (MELANGE-3) in the Kepler field, which harbors two transiting planetary systems (KOI-3876 and Kepler-970). We identify MELANGE-3 by searching for kinematic and spatial overdensities around Kepler planet hosts with high levels of lithium. To determine the age and membership of MELANGE-3, we combine new high-resolution spectra with archival light curves, velocities, and astrometry of stars near KOI-3876 spatially and kinematically. We use the resulting rotation sequence, lithium levels, and color-magnitude diagram of candidate members to confirm the presence of a coeval 105 ± 10 Myr population. MELANGE-3 may be part of the recently identified Theia 316 stream. For the two exoplanet systems, we revise the stellar and planetary parameters, taking into account the newly determined age. Fitting the 4.5 yr Kepler light curves, we find that KOI-3876b is a 2.0 ± 0.1 R⊕ planet on a 19.58 day orbit, while Kepler-970 b is a 2.8 ± 0.2 R⊕ planet on a 16.73 day orbit. KOI-3876 was previously flagged as an eclipsing binary, which we rule out using radial velocities from APOGEE and statistically validate the signal as planetary in origin. Given its overlap with the Kepler field, MELANGE-3 is valuable for studies of spot evolution on year timescales, and both planets contribute to the growing work on transiting planets in young stellar associations

Initial Steps Towards a Clinical FLASH Radiotherapy System: Pediatric Whole Brain Irradiation with 40 MeV Electrons at FLASH Dose Rates

In this work, we investigated the delivery of a clinically acceptable pediatric whole brain radiotherapy plan at FLASH dose rates using two lateral opposing 40-MeV electron beams produced by a practically realizable linear accelerator system. The EGSnrc Monte Carlo software modules, BEAMnrc and DOSXYZnrc, were used to generate whole brain radiotherapy plans for a pediatric patient using two lateral opposing 40-MeV electron beams. Electron beam phase space files were simulated using a model of a diverging beam with a diameter of 10 cm at 50 cm SAD (defined at brain midline). The electron beams were collimated using a 10-cm-thick block composed of 5 cm of aluminum oxide and 5 cm of tungsten. For comparison, a 6-MV photon plan was calculated with the Varian AAA algorithm. Electron beam parameters were based on a novel linear accelerator designed for the PHASER system and powered by a commercial 6-MW klystron. Calculations of the linear accelerator's performance indicated an average beam current of at least 6.25 µA, providing a dose rate of 115 Gy/s at isocenter, high enough for cognition-sparing FLASH effects. The electron plan was less homogenous with a homogeneity index of 0.133 compared to the photon plan's index of 0.087. Overall, the dosimetric characteristics of the 40-MeV electron plan were suitable for treatment. In conclusion, Monte Carlo simulations performed in this work indicate that two lateral opposing 40-MeV electron beams can be used for pediatric whole brain irradiation at FLASH dose rates of >115 Gy/s and serve as motivation for a practical clinical FLASH radiotherapy system, which can be implemented in the near future

FLASH Irradiation Results in Reduced Severe Skin Toxicity Compared to Conventional-Dose-Rate Irradiation

Radiation therapy, along with surgery and chemotherapy, is one of the main treatments for cancer. While radiotherapy is highly effective in the treatment of localized tumors, its main limitation is its toxicity to normal tissue. Previous preclinical studies have reported that ultra-high dose-rate (FLASH) irradiation results in reduced toxicity to normal tissues while controlling tumor growth to a similar extent relative to conventional-dose-rate (CONV) irradiation. To our knowledge this is the first report of a dose-response study in mice comparing the effect of FLASH irradiation vs. CONV irradiation on skin toxicity. We found that FLASH irradiation results in both a lower incidence and lower severity of skin ulceration than CONV irradiation 8 weeks after single-fraction hemithoracic irradiation at high doses (30 and 40 Gy). Survival was also higher after FLASH hemithoracic irradiation (median survival >180 days at doses of 30 and 40 Gy) compared to CONV irradiation (median survival 100 and 52 days at 30 and 40 Gy, respectively). No ulceration was observed at doses 20 Gy or below in either FLASH or CONV. These results suggest a shifting of the dose-response curve for radiation-induced skin ulceration to the right for FLASH, compared to CONV irradiation, suggesting the potential for an enhanced therapeutic index for radiation therapy of cancer

Early Cretaceous vegetation and climate change at high latitude: Palynological evidence from Isachsen Formation, Arctic Canada

Quantitative palynology of the marginal marine and deltaic-fluvial Isachsen Formation of the Sverdrup Basin, Canadian Arctic, provides insight into high latitude climate during much of the Early Cretaceous (Valanginian to early Aptian). Detrended Correspondence Analysis of main pollen and spore taxa is used to derive three ecological groupings influenced by moisture and disturbance based on the botanical affinities of palynomorphs: 1) a mixed coniferous assemblage containing both lowland and upland components; 2) a conifer-filicopsid community that likely grew in dynamic lowland habitats; and, 3) a mature dry lowland community composed of Cheirolepidiaceans. Stratigraphic changes in the relative abundance of pollen and spore taxa reflect climate variability in this polar region during the ~20 Mya history of the Isachsen Formation. The late Valanginian was relatively cool and moist and promoted lowland conifer-filicopsid communities. Warming in the Hauterivian resulted in the expansion coniferous communities in well-drained or arid hinterlands. A return to relatively cool and moist conditions in the Barremian resulted in the expansion of mixed lowland communities. This work demonstrates the utility of a multivariate statistical approach to palynology to provide insight into the composition and dynamics of ecosystems and climate of high latitude regions during the Early Cretaceous

In vitro models for the study of osteoarthritis

AbstractOsteoarthritis (OA) is a prevalent disease of most mammalian species and is a significant cause of welfare and economic morbidity in affected individuals and populations. In vitro models of osteoarthritis are vital to advance research into the causes of the disease, and the subsequent design and testing of potential therapeutics. However, a plethora of in vitro models have been used by researchers but with no consensus on the most appropriate model. Models attempt to mimic factors and conditions which initiate OA, or dissect the pathways active in the disease. Underlying uncertainty as to the cause of OA and the different attributes of isolated cells and tissues used mean that similar models may produce differing results and can differ from the naturally occurring disease.This review article assesses a selection of the in vitro models currently used in OA research, and considers the merits of each. Particular focus is placed on the more prevalent cytokine stimulation and load-based models. A brief review of the mechanism of these models is given, with their relevance to the naturally occurring disease. Most in vitro models have used supraphysiological loads or cytokine concentrations (compared with the natural disease) in order to impart a timely response from the cells or tissue assessed. Whilst models inducing OA-like pathology with a single stimulus can answer important biological questions about the behaviour of cells and tissues, the development of combinatorial models encompassing different physiological and molecular aspects of the disease should more accurately reflect the pathogenesis of the naturally occurring disease

An economic and environmental assessment of farmers' practices to propose strategies that alleviate a pertinent agricultural problem in Cuenca, Ecuador

Working with faculty and Master’s students from the Agronomy Department at the University of Cuenca, our project aimed to understand farmers’ lifestyles in the region of San Antonio de Gapal to identify a pertinent problem, and to propose strategies to resolve that problem. To do this, we interviewed 12 farmers in the region, observed their properties, and analyzed collected data to create strategies to mitigate these issues. Our key finding was that water collection and retention during droughts and excessive rain was a critical issue for farm productivity. Lastly, we conducted a participatory SWOT analysis to share our recommendations, which were to increase the number of barrels, investigate the use of fog nets for water collection, and reduce erosion with terraces among others

Solomon Islands Oncology Unit: Sustainability in Terms of Outcomes

Bush et al emphasize that the key to establishing enduring and efficient global health systems lies in prioritizing local stakeholders and, above all, the welfare of patients

*WINNER* Caribbean Delphinidae Click Classifications

This research focuses on identifying animals of the Delphinidae family found in the Caribbean by analyzing acoustic signals. Delphinids produce large numbers of acoustic signals identified as echolocation clicks which provides a useful classification system. Recent studies have established that the fundamental frequency of the majority of whistles used for social communications covers the frequency range of 2–35 kHz and up to 100 kHz for the harmonic frequencies. The challenge in click classification results from recognizing patterns among many sources of variability. Our findings will be presented by figures of network relationships between clicks and a detailed exploratory analysis to highlight clusters and subsets of training and test data along with clustering algorithms