Architecture and membrane interactions of the EGF receptor.

Dimerization-driven activation of the intracellular kinase domains of the epidermal growth factor receptor (EGFR) upon extracellular ligand binding is crucial to cellular pathways regulating proliferation, migration, and differentiation. Inactive EGFR can exist as both monomers and dimers, suggesting that the mechanism regulating EGFR activity may be subtle. The membrane itself may play a role but creates substantial difficulties for structural studies. Our molecular dynamics simulations of membrane-embedded EGFR suggest that, in ligand-bound dimers, the extracellular domains assume conformations favoring dimerization of the transmembrane helices near their N termini, dimerization of the juxtamembrane segments, and formation of asymmetric (active) kinase dimers. In ligand-free dimers, by holding apart the N termini of the transmembrane helices, the extracellular domains instead favor C-terminal dimerization of the transmembrane helices, juxtamembrane segment dissociation and membrane burial, and formation of symmetric (inactive) kinase dimers. Electrostatic interactions of EGFR's intracellular module with the membrane are critical in maintaining this coupling.

Oncogenic mutations counteract intrinsic disorder in the EGFR kinase and promote receptor dimerization.

The mutation and overexpression of the epidermal growth factor receptor (EGFR) are associated with the development of a variety of cancers, making this prototypical dimerization-activated receptor tyrosine kinase a prominent target of cancer drugs. Using long-timescale molecular dynamics simulations, we find that the N lobe dimerization interface of the wild-type EGFR kinase domain is intrinsically disordered and that it becomes ordered only upon dimerization. Our simulations suggest, moreover, that some cancer-linked mutations distal to the dimerization interface, particularly the widespread L834R mutation (also referred to as L858R), facilitate EGFR dimerization by suppressing this local disorder. Corroborating these findings, our biophysical experiments and kinase enzymatic assays indicate that the L834R mutation causes abnormally high activity primarily by promoting EGFR dimerization rather than by allowing activation without dimerization. We also find that phosphorylation of EGFR kinase domain at Tyr845 may suppress the intrinsic disorder, suggesting a molecular mechanism for autonomous EGFR signaling.

California's methane super-emitters.

Methane is a powerful greenhouse gas and is targeted for emissions mitigation by the US state of California and other jurisdictions worldwide1,2. Unique opportunities for mitigation are presented by point-source emitters-surface features or infrastructure components that are typically less than 10 metres in diameter and emit plumes of highly concentrated methane3. However, data on point-source emissions are sparse and typically lack sufficient spatial and temporal resolution to guide their mitigation and to accurately assess their magnitude4. Here we survey more than 272,000 infrastructure elements in California using an airborne imaging spectrometer that can rapidly map methane plumes5-7. We conduct five campaigns over several months from 2016 to 2018, spanning the oil and gas, manure-management and waste-management sectors, resulting in the detection, geolocation and quantification of emissions from 564 strong methane point sources. Our remote sensing approach enables the rapid and repeated assessment of large areas at high spatial resolution for a poorly characterized population of methane emitters that often appear intermittently and stochastically. We estimate net methane point-source emissions in California to be 0.618 teragrams per year (95 per cent confidence interval 0.523-0.725), equivalent to 34-46 per cent of the state's methane inventory8 for 2016. Methane 'super-emitter' activity occurs in every sector surveyed, with 10 per cent of point sources contributing roughly 60 per cent of point-source emissions-consistent with a study of the US Four Corners region that had a different sectoral mix9. The largest methane emitters in California are a subset of landfills, which exhibit persistent anomalous activity. Methane point-source emissions in California are dominated by landfills (41 per cent), followed by dairies (26 per cent) and the oil and gas sector (26 per cent). Our data have enabled the identification of the 0.2 per cent of California's infrastructure that is responsible for these emissions. Sharing these data with collaborating infrastructure operators has led to the mitigation of anomalous methane-emission activity10.

Strong methane point sources contribute a disproportionate fraction of total emissions across multiple basins in the United States.

Understanding, prioritizing, and mitigating methane (CH4) emissions requires quantifying CH4 budgets from facility scales to regional scales with the ability to differentiate between source sectors. We deployed a tiered observing system for multiple basins in the United States (San Joaquin Valley, Uinta, Denver-Julesburg, Permian, Marcellus). We quantify strong point source emissions (>10 kg CH4 h-1) using airborne imaging spectrometers, attribute them to sectors, and assess their intermittency with multiple revisits. We compare these point source emissions to total basin CH4 fluxes derived from inversion of Sentinel-5p satellite CH4 observations. Across basins, point sources make up on average 40% of the regional flux. We sampled some basins several times across multiple months and years and find a distinct bimodal structure to emission timescales: the total point source budget is split nearly in half by short-lasting and long-lasting emission events. With the increasing airborne and satellite observing capabilities planned for the near future, tiered observing systems will more fully quantify and attribute CH4 emissions from facility to regional scales, which is needed to effectively and efficiently reduce methane emissions.

Students' Experiences of Peer Observed Teaching: A Qualitative Interview Study.

PHENOMENON: Development of teaching skills is an important aspect of medical student training. One method of developing teaching skills is participation in peer teaching with observation and feedback from peers. This study aims to explore student teachers' experiences of peer observation of teaching and how they intend to utilize this feedback. APPROACH: We conducted individual semi-structured interviews with peer tutors who had experienced peer observation of their small group teaching and subsequent feedback. The interviews were conducted by a medical student peer not involved in the peer observation of teaching scheme. They were audio recorded and transcribed. The pseudonymised transcripts were coded independently by two researchers using thematic analysis. FINDINGS: Nine students participated in interviews lasting a mean of 42 minutes. We identified three main themes: motivations for observation, experiences of observation, and responses to feedback. Students were motivated to have their teaching observed by both intrinsic and extrinsic factors: to develop their skills and competence as a teacher, in recognition of the important role this plays in their career, to provide reassurance that they are providing good quality teaching, to ensure the content of their teaching is appropriate and accurate, and to provide evidence of engagement in, and development of, teaching. Students described feeling nervous before the observations and preparing more for their teaching than they might normally, however, during the observations they felt more comfortable which they attributed to the peer-peer relationship. Students described finding the narrative feedback more useful than the quantitative elements as it provided more detail as to how they might improve. Several students described how they have used the feedback they have received on their teaching to improve subsequent sessions. INSIGHTS: Peer observation of teaching is a useful and acceptable method of providing feedback on student teaching and recipients intend to use this feedback to improve their teaching.

Binucleate germ cells in Caenorhabditis elegans are removed by physiological apoptosis.

Cell death plays a major role during C. elegans oogenesis, where over half of the oogenic germ cells die in a process termed physiological apoptosis. How germ cells are selected for physiological apoptosis, or instead become oocytes, is not understood. Most oocytes produce viable embryos when apoptosis is blocked, suggesting that physiological apoptosis does not function to cull defective germ cells. Instead, cells targeted for apoptosis may function as nurse cells; the germline is syncytial, and all germ cells appear to contribute cytoplasm to developing oocytes. C. elegans has been a leading model for the genetics and molecular biology of apoptosis and phagocytosis, but comparatively few studies have examined the cell biology of apoptotic cells. We used live imaging to identify and examine pre-apoptotic germ cells in the adult gonad. After initiating apoptosis, germ cells selectively export their mitochondria into the shared pool of syncytial cytoplasm; this transport appears to use the microtubule motor kinesin. The apoptotic cells then shrink as they expel most of their remaining cytoplasm, and close off from the syncytium. Shortly thereafter the apoptotic cells restructure their microtubule and actin cytoskeletons, possibly to maintain cell integrity; the microtubules form a novel, cortical array of stabilized microtubules, and actin and cofilin organize into giant cofilin-actin rods. We discovered that some apoptotic germ cells are binucleate; the binucleate germ cells can develop into binucleate oocytes in apoptosis-defective strains, and appear capable of producing triploid offspring. Our results suggest that the nuclear layer of the germline syncytium becomes folded during mitosis and growth, and that binucleate cells arise as the layer unfolds or everts; all of the binucleate cells are subsequently removed by apoptosis. These results show that physiological apoptosis targets at least two distinct populations of germ cells, and that the apoptosis machinery efficiently recognizes cells with two nuclei.

A large airborne survey of Earth's visible-infrared spectral dimensionality.

The intrinsic spectral dimensionality indicates the observable degrees of freedom in Earth's solar-reflected light field, quantifying the diversity of spectral content accessible by visible and infrared remote sensing. The solar-reflected regime spans the 0.38 - 2.5 µm interval, and is captured by a wide range of current and planned instruments on both airborne and orbital platforms. To date there has been no systematic study of its spectral dimensionality as a function of space, time, and land cover. Here we report a multi-site, multi-year statistical survey by NASA's "Classic" Airborne Visible Near InfraRed Spectrometer (AVIRIS-C). AVIRIS-C measured large regions of California, USA, spanning wide latitudinal and elevation gradients containing all canonical MODIS land cover types. The spectral uniformity of the AVIRIS-C design enabled consistent in-scene assessment of measurement noise across acquisitions. The estimated dimensionality as a function of cover type ranged from the low 20s to the high 40s, and was approximately 50 for the combined dataset. This result indicates the high diversity of physical processes distinguishable by imaging spectrometers like AVIRIS-C for one region of the Earth.

Developmental Coordination of Gamete Differentiation with Programmed Cell Death in Sporulating Yeast.

The gametogenesis program of the budding yeast Saccharomyces cerevisiae, also known as sporulation, employs unusual internal meiotic divisions, after which all four meiotic products differentiate within the parental cell. We showed previously that sporulation is typically accompanied by the destruction of discarded immature meiotic products through their exposure to proteases released from the mother cell vacuole, which undergoes an apparent programmed rupture. Here we demonstrate that vacuolar rupture contributes to de facto programmed cell death (PCD) of the meiotic mother cell itself. Meiotic mother cell PCD is accompanied by an accumulation of depolarized mitochondria, organelle swelling, altered plasma membrane characteristics, and cytoplasmic clearance. To ensure that the gametes survive the destructive consequences of developing within a cell that is executing PCD, we hypothesized that PCD is restrained from occurring until spores have attained a threshold degree of differentiation. Consistent with this hypothesis, gene deletions that perturb all but the most terminal postmeiotic spore developmental stages are associated with altered PCD. In these mutants, meiotic mother cells exhibit a delay in vacuolar rupture and then appear to undergo an alternative form of PCD associated with catastrophic consequences for the underdeveloped spores. Our findings reveal yeast sporulation as a context of bona fide PCD that is developmentally coordinated with gamete differentiation.

Portable Remote Imaging Spectrometer coastal ocean sensor: design, characteristics, and first flight results.

The design, characteristics, and first test flight results are described of the Portable Remote Imaging Spectrometer, an airborne sensor specifically designed to address the challenges of coastal ocean remote sensing. The sensor incorporates several technologies that are demonstrated for the first time, to the best of our knowledge, in a working system in order to achieve a high performance level in terms of uniformity, signal-to-noise ratio, low polarization sensitivity, low stray light, and high spatial resolution. The instrument covers the 350-1050 nm spectral range with a 2.83 nm sampling per pixel, and a 0.88 mrad instantaneous field of view, with 608 cross-track pixels in a pushbroom configuration. Two additional infrared channels (1240 and 1610 nm) are measured by a spot radiometer housed in the same head. The spectrometer design is based on an optically fast (F/1.8) Dyson design form coupled to a wide angle two-mirror telescope in a configuration that minimizes polarization sensitivity without the use of a depolarizer. A grating with minimum polarization sensitivity and broadband efficiency was fabricated as well as a slit assembly with black (etched) silicon surface to minimize backscatter. First flight results over calibration sites as well as Monterey Bay in California have demonstrated good agreement between in situ and remotely sensed data, confirming the potential value of the sensor to the coastal ocean science community.

Quantifying methane emissions from United States landfills.

Methane emissions from solid waste may represent a substantial fraction of the global anthropogenic budget, but few comprehensive studies exist to assess inventory assumptions. We quantified emissions at hundreds of large landfills across 18 states in the United States between 2016 and 2022 using airborne imaging spectrometers. Spanning 20% of open United States landfills, this represents the most systematic measurement-based study of methane point sources of the waste sector. We detected significant point source emissions at a majority (52%) of these sites, many with emissions persisting over multiple revisits (weeks to years). We compared these against independent contemporaneous in situ airborne observations at 15 landfills and established good agreement. Our findings indicate a need for long-term, synoptic-scale monitoring of landfill emissions in the context of climate change mitigation policy.

Ultra-high optical absorption efficiency from the ultraviolet to the infrared using multi-walled carbon nanotube ensembles.

The optical absorption efficiencies of vertically aligned multi-walled (MW)-carbon nanotube (CNT) ensembles are characterized in the 350-7000 nm wavelength range where CNT site densities > 1 × 10(11) /cm(2) are achieved directly on metallic substrates. The site density directly impacts the optical absorption characteristics, and while high-density arrays of CNTs on electrically insulating and non-metallic substrates have been commonly reported, achieving high site-densities on metals has been challenging and remains an area of active research. These absorber ensembles are ultra-thin (<10 µm) and yet they still exhibit a reflectance as low as ∼0.02%, which is 100 times lower than the reference; these characteristics make them potentially attractive for high-sensitivity and high-speed thermal detectors. In addition, the use of a plasma-enhanced chemical vapor deposition process for the synthesis of the absorbers increases the portfolio of materials that can be integrated with such absorbers due to the potential for reduced synthesis temperatures. The remarkable ruggedness of the absorbers is also demonstrated as they are exposed to high temperatures in an oxidizing ambient environment, making them well-suited for extreme thermal environments encountered in the field, potentially for solar cell applications. Finally, a phenomenological model enables the determinatiom of the extinction coefficients in these nanostructures and the results compare well with experiment.

Accurate and efficient integration for molecular dynamics simulations at constant temperature and pressure.

In molecular dynamics simulations, control over temperature and pressure is typically achieved by augmenting the original system with additional dynamical variables to create a thermostat and a barostat, respectively. These variables generally evolve on timescales much longer than those of particle motion, but typical integrator implementations update the additional variables along with the particle positions and momenta at each time step. We present a framework that replaces the traditional integration procedure with separate barostat, thermostat, and Newtonian particle motion updates, allowing thermostat and barostat updates to be applied infrequently. Such infrequent updates provide a particularly substantial performance advantage for simulations parallelized across many computer processors, because thermostat and barostat updates typically require communication among all processors. Infrequent updates can also improve accuracy by alleviating certain sources of error associated with limited-precision arithmetic. In addition, separating the barostat, thermostat, and particle motion update steps reduces certain truncation errors, bringing the time-average pressure closer to its target value. Finally, this framework, which we have implemented on both general-purpose and special-purpose hardware, reduces software complexity and improves software modularity.

Principles of conduction and hydrophobic gating in K+ channels.

We present the first atomic-resolution observations of permeation and gating in a K(+) channel, based on molecular dynamics simulations of the Kv1.2 pore domain. Analysis of hundreds of simulated permeation events revealed a detailed conduction mechanism, resembling the Hodgkin-Keynes "knock-on" model, in which translocation of two selectivity filter-bound ions is driven by a third ion; formation of this knock-on intermediate is rate determining. In addition, at reverse or zero voltages, we observed pore closure by a novel "hydrophobic gating" mechanism: A dewetting transition of the hydrophobic pore cavity-fastest when K(+) was not bound in selectivity filter sites nearest the cavity-caused the open, conducting pore to collapse into a closed, nonconducting conformation. Such pore closure corroborates the idea that voltage sensors can act to prevent pore collapse into the intrinsically more stable, closed conformation, and it further suggests that molecular-scale dewetting facilitates a specific biological function: K(+) channel gating. Existing experimental data support our hypothesis that hydrophobic gating may be a fundamental principle underlying the gating of voltage-sensitive K(+) channels. We suggest that hydrophobic gating explains, in part, why diverse ion channels conserve hydrophobic pore cavities, and we speculate that modulation of cavity hydration could enable structural determination of both open and closed channels.

Outcomes of Cochlear implantation in early-deafened patients with Waardenburg syndrome: A systematic review and narrative synthesis.

Objective: This systematic review aims to establish the expected hearing and speech outcomes following cochlear implantation (CI) in patients with profound congenital deafness secondary to Waardenburg syndrome (WS). Methods: A systematic review of the literature and narrative synthesis was performed in accordance with the PRISMA statement. Databases searched: Medline, Pubmed, Embase, Web of Science, Cochrane Collection, and ClinicalTrials.gov. No limits were placed on language or year of publication. Results: Searches identified 186 abstracts and full texts. Of these, 16 studies met inclusion criteria reporting outcomes in 179 patients and at least 194 implants. Hearing outcomes of those receiving cochlear implantation were generally good. Five studies included genetic analysis of one or more of the participants. A total of 11 peri/post-operative complications were reported. The methodological quality of included studies was modest, mainly comprising noncontrolled case series with small cohort size. All studies were OCEBM grade III-IV. Conclusion: Cochlear implantation in congenitally deafened children with Waardenburg Syndrome is a well-established intervention as a method of auditory rehabilitation. Due to the uncommon nature of the condition, there is a lack of large-scale high-quality studies examining the use of cochlear implantation in this patient group. However, overall outcomes following implantation are positive with the majority of patients demonstrating improved audiometry, speech perception and speech intelligibility supporting its use in appropriately selected cases.

Attribution of individual methane and carbon dioxide emission sources using EMIT observations from space.

Carbon dioxide and methane emissions are the two primary anthropogenic climate-forcing agents and an important source of uncertainty in the global carbon budget. Uncertainties are further magnified when emissions occur at fine spatial scales (<1 km), making attribution challenging. We present the first observations from NASA's Earth Surface Mineral Dust Source Investigation (EMIT) imaging spectrometer showing quantification and attribution of fine-scale methane (0.3 to 73 tonnes CH4 hour-1) and carbon dioxide sources (1571 to 3511 tonnes CO2 hour-1) spanning the oil and gas, waste, and energy sectors. For selected countries observed during the first 30 days of EMIT operations, methane emissions varied at a regional scale, with the largest total emissions observed for Turkmenistan (731 ± 148 tonnes CH4 hour-1). These results highlight the contributions of current and planned point source imagers in closing global carbon budgets.

Refinement of protein structure homology models via long, all-atom molecular dynamics simulations.

Accurate computational prediction of protein structure represents a longstanding challenge in molecular biology and structure-based drug design. Although homology modeling techniques are widely used to produce low-resolution models, refining these models to high resolution has proven difficult. With long enough simulations and sufficiently accurate force fields, molecular dynamics (MD) simulations should in principle allow such refinement, but efforts to refine homology models using MD have for the most part yielded disappointing results. It has thus far been unclear whether MD-based refinement is limited primarily by accessible simulation timescales, force field accuracy, or both. Here, we examine MD as a technique for homology model refinement using all-atom simulations, each at least 100 µs long-more than 100 times longer than previous refinement simulations-and a physics-based force field that was recently shown to successfully fold a structurally diverse set of fast-folding proteins. In MD simulations of 24 proteins chosen from the refinement category of recent Critical Assessment of Structure Prediction (CASP) experiments, we find that in most cases, simulations initiated from homology models drift away from the native structure. Comparison with simulations initiated from the native structure suggests that force field accuracy is the primary factor limiting MD-based refinement. This problem can be mitigated to some extent by restricting sampling to the neighborhood of the initial model, leading to structural improvement that, while limited, is roughly comparable to the leading alternative methods.

A conserved protonation-dependent switch controls drug binding in the Abl kinase.

In many protein kinases, a characteristic conformational change (the "DFG flip") connects catalytically active and inactive conformations. Many kinase inhibitors--including the cancer drug imatinib--selectively target a specific DFG conformation, but the function and mechanism of the flip remain unclear. Using long molecular dynamics simulations of the Abl kinase, we visualized the DFG flip in atomic-level detail and formulated an energetic model predicting that protonation of the DFG aspartate controls the flip. Consistent with our model's predictions, we demonstrated experimentally that the kinetics of imatinib binding to Abl kinase have a pH dependence that disappears when the DFG aspartate is mutated. Our model suggests a possible explanation for the high degree of conservation of the DFG motif: that the flip, modulated by electrostatic changes inherent to the catalytic cycle, allows the kinase to access flexible conformations facilitating nucleotide binding and release.

A Systematic Review of the Effectiveness of Bisphosphonates for Otosclerosis.

OBJECTIVE: To systematically review the evidence for the use of bisphosphonate therapy in otosclerosis through clinically relevant outcomes. DATABASES REVIEWED: MEDLINE, EMBASE, PubMed, and CINAHL databases were searched up to July 12, 2021. METHODS: RCTs and cohort studies investigating the effect of bisphosphate therapy on adults or children diagnosed with otosclerosis were included. The risk of bias within trials was examined using the ROB2 tool for RCTs, and the ROBINS-I for non-RCTs. RESULTS: Three studies reported over five publications were included in the systematic review. Data from one RCT at 6 months did not demonstrate any improvement nor deterioration in audiological outcomes in participants treated with Sodium Alendronate. Data from MRI in this group demonstrated improvements in the SI of the otosclerotic foci at the RAOW compared to participants taking placebo. In another RCT, improvements in audiological outcomes were seen at 12 and 24 months in individuals treated with Etidronate Sodium. Long-term data from a retrospective cohort study demonstrated stabilisation of hearing in individuals with otosclerosis and progressive SNHL. CONCLUSION: There is insufficient evidence to recommend the routine use of bisphosphonates in otosclerosis patients at present. Long-term retrospective data has suggested a role for bisphosphonates in the subset of patients with deteriorating sensorineural hearing loss with the aim of hearing stabilisation. Adequately powered RCTs with long term follow up will be required to evaluate this further.

Atmospheric Lengthscales for Global VSWIR Imaging Spectroscopy.

Future global Visible Shortwave Infrared Imaging Spectrometers, such as the Surface Biology and Geology (SBG) mission, will regularly cover the Earth's entire terrestrial land area. These missions need high fidelity atmospheric correction to produce consistent maps of terrestrial and aquatic ecosystem traits. However, estimation of surface reflectance and atmospheric state is computationally challenging, and the terabyte data volumes of global missions will exceed available processing capacity. This article describes how missions can overcome this bottleneck using the spatial continuity of atmospheric fields. Contemporary imaging spectrometers oversample atmospheric spatial variability, so it is not necessary to invert every pixel. Spatially sparse solutions can train local linear emulators that provide fast, exact inversions in their vicinity. We find that estimating the atmosphere at 200 m scales can outperform traditional atmospheric correction, improving speed by one to two orders of magnitude with no measurable penalty to accuracy. We validate performance with an airborne field campaign, showing reflectance accuracies with RMSE of 1.1% or better compared to ground measurements of diverse targets. These errors are statistically consistent with retrieval uncertainty budgets. Local emulators can close the efficiency gap and make rigorous model inversion algorithms feasible for global missions such as SBG.

How does a drug molecule find its target binding site?

Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, detailed experimental characterization of the process by which such binding occurs has proven challenging. We conducted relatively long, unguided molecular dynamics simulations in which a ligand (the cancer drug dasatinib or the kinase inhibitor PP1) was initially placed at a random location within a box that also contained a protein (Src kinase) to which that ligand was known to bind. In several of these simulations, the ligand correctly identified its target binding site, forming a complex virtually identical to the crystallographically determined bound structure. The simulated trajectories provide a continuous, atomic-level view of the entire binding process, revealing persistent and noteworthy intermediate conformations and shedding light on the role of water molecules. The technique we employed, which does not assume any prior knowledge of the binding site's location, may prove particularly useful in the development of allosteric inhibitors that target previously undiscovered binding sites.