Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II.

Variable temperature electrospray mass spectrometry is useful for multiplexed measurements of the thermal stabilities of biomolecules, but the ionization process can be disrupted by aggregation-prone proteins/complexes that have irreversible unfolding transitions. Resistively heating solutions containing a mixture of bovine carbonic anhydrase II (BCAII), a CO2 fixing enzyme involved in many biochemical pathways, and cytochrome c leads to complete loss of carbonic anhydrase signal and a significant reduction in cytochrome c signal above ∼72 °C due to aggregation. In contrast, when the tips of borosilicate glass nanoelectrospray emitters are heated with a laser, complete thermal denaturation curves for both proteins are obtained in <1 minute. The simultaneous measurements of the melting temperature of BCAII and BCAII bound to bicarbonate reveal that the bicarbonate stabilizes the folded form of this protein by ∼6.4 °C. Moreover, the temperature dependences of different bicarbonate loss pathways are obtained. Although protein analytes are directly heated by the laser for only 140 ms, heat conduction further up the emitter leads to a total analyte heating time of ∼41 s. Pulsed laser heating experiments could reduce this time to ∼0.5 s for protein aggregation that occurs on a faster time scale. Laser heating provides a powerful method for studying the detailed mechanisms of cofactor/ligand loss with increasing temperature and promises a new tool for studying the effect of ligands, drugs, growth conditions, buffer additives, or other treatments on the stabilities of aggregation-prone biomolecules.

Enhanced weathering in the US Corn Belt delivers carbon removal with agronomic benefits.

Terrestrial enhanced weathering (EW) of silicate rocks, such as crushed basalt, on farmlands is a promising scalable atmospheric carbon dioxide removal (CDR) strategy that urgently requires performance assessment with commercial farming practices. We report findings from a large-scale replicated EW field trial across a typical maize-soybean rotation on an experimental farm in the heart of the United Sates Corn Belt over 4 y (2016 to 2020). We show an average combined loss of major cations (Ca2+ and Mg2+) from crushed basalt applied each fall over 4 y (50 t ha-1 y-1) gave a conservative time-integrated cumulative CDR potential of 10.5 ± 3.8 t CO2 ha-1. Maize and soybean yields increased significantly (P < 0.05) by 12 to 16% with EW following improved soil fertility, decreased soil acidification, and upregulation of root nutrient transport genes. Yield enhancements with EW were achieved with significantly (P < 0.05) increased key micro- and macronutrient concentrations (including potassium, magnesium, manganese, phosphorus, and zinc), thus improving or maintaining crop nutritional status. We observed no significant increase in the content of trace metals in grains of maize or soybean or soil exchangeable pools relative to controls. Our findings suggest that widespread adoption of EW across farming sectors has the potential to contribute significantly to net-zero greenhouse gas emissions goals while simultaneously improving food and soil security.

The Weighted Toxicity Score: Confirmation of a Simple Metric to Communicate Toxicity in Randomized Trials of Systemic Cancer Therapy.

INTRODUCTION: FDA's Project Optimus was developed in part to better identify appropriate dose and schedule of cancer therapeutics. The tabular method to summarize patients' maximum toxicity in a clinical trial does not allow for ready comparison to the treatment's benefit. In this manuscript, we apply a simple tool, the weighted toxicity score (WTS), to trials involving lung cancer immunotherapy and chemotherapy, as well as those cited in a recent publication as examples of trials that represent successful reduction of the appropriate dose of anti-cancer agents. METHODS: PubMed was queried for randomized controlled trials of therapy involving immune checkpoint inhibitors in lung cancer. Trial data from studies highlighting initial success with dose adjustments after FDA approval also were assembled and analyzed according to the WTS procedure described previously, compared to clinical outcomes data. RESULTS: The WTS provided, with the clinical outcome(s), a data pair that leads to easy interpretation of the expected benefit versus relative toxicity of studies involving immunotherapy or chemoimmunotherapy in lung cancers. The WTS was consistent with the conclusions of the primary studies, helping to quantitate the toxicity difference between treatments in a previously unavailable way. CONCLUSION: The WTS provides a tool to show the cost in toxicity of therapy in a randomized clinical trial, with applicability to studies involving chemotherapy, immunotherapy, or kinase-directed therapy. Inclusion of a running tally of WTS during conduct of a trial could serve as one means to adjust dosing or to provide feedback during data safety monitoring of a clinical trial.

Initial Validation of a Soil-Based Mass-Balance Approach for Empirical Monitoring of Enhanced Rock Weathering Rates.

Enhanced rock weathering (ERW) is a promising scalable and cost-effective carbon dioxide removal (CDR) strategy with significant environmental and agronomic co-benefits. A major barrier to large-scale implementation of ERW is a robust monitoring, reporting, and verification (MRV) framework. To successfully quantify the amount of carbon dioxide removed by ERW, MRV must be accurate, precise, and cost-effective. Here, we outline a mass-balance-based method in which analysis of the chemical composition of soil samples is used to track in situ silicate rock weathering. We show that signal-to-noise issues of in situ soil analysis can be mitigated by using isotope-dilution mass spectrometry to reduce analytical error. We implement a proof-of-concept experiment demonstrating the method in controlled mesocosms. In our experiment, a basalt rock feedstock is added to soil columns containing the cereal crop Sorghum bicolor at a rate equivalent to 50 t ha-1. Using our approach, we calculate rock weathering corresponding to an average initial CDR value of 1.44 ± 0.27 tCO2eq ha-1 from our experiments after 235 days, within error of an independent estimate calculated using conventional elemental budgeting of reaction products. Our method provides a robust time-integrated estimate of initial CDR, to feed into models that track and validate large-scale carbon removal through ERW.

A hybrid method of correcting CBCT for proton range estimation with deep learning and deformable image registration.

Objective. This study aimed to develop a novel method for generating synthetic CT (sCT) from cone-beam CT (CBCT) of the abdomen/pelvis with bowel gas pockets to facilitate estimation of proton ranges.Approach. CBCT, the same-day repeat CT, and the planning CT (pCT) of 81 pediatric patients were used for training (n= 60), validation (n= 6), and testing (n= 15) of the method. The proposed method hybridizes unsupervised deep learning (CycleGAN) and deformable image registration (DIR) of the pCT to CBCT. The CycleGAN and DIR are respectively applied to generate the geometry-weighted (high spatial-frequency) and intensity-weighted (low spatial-frequency) components of the sCT, thereby each process deals with only the component weighted toward its strength. The resultant sCT is further improved in bowel gas regions and other tissues by iteratively feeding back the sCT to adjust incorrect DIR and by increasing the contribution of the deformed pCT in regions of accurate DIR.Main results. The hybrid sCT was more accurate than deformed pCT and CycleGAN-only sCT as indicated by the smaller mean absolute error in CT numbers (28.7 ± 7.1 HU versus 38.8 ± 19.9 HU/53.2 ± 5.5 HU;P≤ 0.012) and higher Dice similarity of the internal gas regions (0.722 ± 0.088 versus 0.180 ± 0.098/0.659 ± 0.129;P≤ 0.002). Accordingly, the hybrid method resulted in more accurate proton range for the beams intersecting gas pockets (11 fields in 6 patients) than the individual methods (the 90th percentile error in 80% distal fall-off, 1.8 ± 0.6 mm versus 6.5 ± 7.8 mm/3.7 ± 1.5 mm;P≤ 0.013). The gamma passing rates also showed a significant dosimetric advantage by the hybrid method (99.7 ± 0.8% versus 98.4 ± 3.1%/98.3 ± 1.8%;P≤ 0.007).Significance. The hybrid method significantly improved the accuracy of sCT and showed promises in CBCT-based proton range verification and adaptive replanning of abdominal/pelvic proton therapy even when gas pockets are present in the beam path.

Lighting Up at High Potential: Effects of Voltage and Emitter Size in Nanoelectrospray Ionization.

Effects of electrospray voltage on cluster size and abundance formed from aqueous CsI were investigated with emitter tip diameters between 260 ± 7 nm and 2.45 ± 0.30 µm. Cluster size increases with increasing voltage, increasing solution concentration and increasing emitter diameter consistent with formation of larger initial droplet sizes. For emitters with tip diameters above ∼1 µm, varying the voltage either up or down leads to reproducible voltage-dependent extents of cluster formation. In contrast, higher voltages with submicron diameter emitters can lead to only Cs+ and Cs(H2O)+ and no clusters. This change in ion formation reproducibly occurs at spray potentials >1.3 kV for 260 nm emitters and appears to be induced by a corona discharge and material build-up at the emitter tip. Under conditions where abundant Cs+ is observed and no clusters are formed, ions such as K+ and Cu1+ are also observed but ions with more negative solvation energies, such as Ba2+, are not. Similarly, ions from bradykinin and ubiquitin are observed predischarge but not post discharge. Ions with more positive solvation energies can desorb directly from the air-water interface that is created at the tip of these emitters, whereas ions with more negative solvation energies as well as peptide and protein ions do not. These results indicate that ion desorption directly from solution can occur, and similar experiments with even smaller emitters may lead to new insights into ion formation in electrospray ionization.

Accurate Sizing of Nanoparticles Using a High-Throughput Charge Detection Mass Spectrometer without Energy Selection.

The sizes and shapes of nanoparticles play a critical role in their chemical and material properties. Common sizing methods based on light scattering or mobility lack individual particle specificity, and microscopy-based methods often require cumbersome sample preparation and image analysis. A promising alternative method for the rapid and accurate characterization of nanoparticle size is charge detection mass spectrometry (CDMS), an emerging technique that measures the masses of individual ions. A recently constructed CDMS instrument designed specifically for high acquisition speed, efficiency, and accuracy is described. This instrument does not rely on an ion energy filter or estimates of ion energy that have been previously required for mass determination, but instead uses direct, in situ measurements. A standardized sample of ∼100 nm diameter polystyrene nanoparticles and ∼50 nm polystyrene nanoparticles with amine-functionalized surfaces are characterized using CDMS and transmission electron microscopy (TEM). Individual nanoparticle masses measured by CDMS are transformed to diameters, and these size distributions are in close agreement with distributions measured by TEM. CDMS analysis also reveals dimerization of ∼100 nm nanoparticles in solution that cannot be determined by TEM due to the tendency of nanoparticles to agglomerate when dried onto a surface. Comparing the acquisition and analysis times of CDMS and TEM shows particle sizing rates up to ∼80× faster are possible using CDMS, even when samples ∼50× more dilute were used. The combination of both high-accuracy individual nanoparticle measurements and fast acquisition rates by CDMS represents an important advance in nanoparticle analysis capabilities.

Laser Heating Nanoelectrospray Emitters for Fast Protein Melting Measurements with Mass Spectrometry.

Temperature-controlled nanoelectrospray ionization has been used to measure heat-induced conformational changes of biomolecules by mass spectrometry, but long thermal equilibration times associated with heating or cooling an entire emitter limit how fast these data can be acquired. Here, the tip of a borosilicate electrospray emitter is heated using 10.6 µm light from an unfocused CO2 laser. At 1.2 W, the solution inside the emitter tip can be heated from room temperature to a steady-state temperature of 78.2 ± 2.5 °C in less than 0.5 s and cools from 82.6 ± 0.6 °C back to room temperature within 4 s. The time required to establish a steady-state temperature is more than 100-fold faster than that required for a resistively heated emitter due to the low thermal mass. Protein unfolding curves measured as a function of laser power can be acquired in ∼40 s compared to a resistively heated apparatus that required ∼21 min to acquire similar data. Laser power is calibrated to temperature by comparisons of the average charge state of the protein cytochrome c measured with laser heating and with resistive heating. This laser heating method is applied to a three-component protein mixture to demonstrate the ability to rapidly acquire melting temperatures of proteins in mixtures. The ability to rapidly assess the thermal stabilities of multiple proteins simultaneously shows significant promise for coupling temperature-controlled electrospray ionization (ESI) to separation techniques, providing a high-throughput method for determining the effects of solution composition, drug binding, or sequence mutations on protein thermal stability.

Tips on Making Tiny Tips: Secrets to Submicron Nanoelectrospray Emitters.

Nanoelectrospray ionization emitters with submicron tip diameters have significant advantages for use in native mass spectrometry, including the ability to produce resolved charge-state distributions for proteins and macromolecular complexes from standard biochemical buffers that contain high concentrations of nonvolatile salts and to prevent nonspecific aggregation that can occur during droplet evaporation. We report on various factors affecting the tip morphology and provide suggestions for producing and using emitters with submicron tips. Effects of pulling parameters for a Sutter Instrument P-87 tip puller on the resulting tip diameter and morphology are shown. The "Pull" parameter has the largest effect on tip diameter, followed by "Velocity", "Pressure", and "Heat", whereas the "Time" parameter has minimal effect beyond a lower threshold. High "Pull" values generate emitters with multiple tapers, whereas high "Velocity" values generate a tip with only a single tapered region. A protocol for producing reproducible emitters in the submicron size range, as well as guidelines and tips for using these emitters with standard biochemical buffers that contain high concentrations of nonvolatile salts, is presented with the aim of expanding their use within the native mass spectrometry community.

Patient Satisfaction with Pharmacist-Provided Health-Related Services in a Primary Care Clinic.

(1) Background: Patient satisfaction plays an important role in the perceived value, sustained utilization, and coverage of healthcare services by payers and clinics. (2) Methods: A 33-question survey was designed to assess patient satisfaction and perceived value for healthcare services provided by a clinical pharmacist in a single primary care facility. It included general items from validated patient satisfaction surveys (i.e., PROMIS®, CAHPS) and pharmacist-specific items identified in selected literature. It was offered to all patients who were presenting for a new, unique visit with the clinical pharmacist at the medical clinic between May 2019 and April 2020. (3) Results: A total of 66 patients agreed to take the survey (RR = 100%), and the responses were overwhelmingly positive. However, men were more likely than women to report higher satisfaction (X2(1, n = 920) = 0.67, p = 0.027), and new patients reported higher satisfaction than existing patients (X2(1, n = 1211) = 1.698, p = 0.037). (4) Conclusions: The findings of this study indicate a high degree of patient satisfaction with pharmacist-provided healthcare services in the primary care setting.

Homochiral preference of serine octamer in solution and formed by dissociation of large gaseous clusters.

The ability of electrospray emitters with submicron tip diameters to significantly reduce and even eliminate aggregation of analyte molecules that can occur inside evaporating droplets was recently demonstrated to show that serine octamer exists in bulk solution, albeit in low abundance. Results using 222 nm emitter tips for D-serine and deuterium labeled L-serine show that the serine octamer that exists in 100 µM solution has a strong homochiral preference. Dissociation of large multiply protonated clusters results in formation of protonated octamer through a doubly protonated decamer intermediate. Remarkably, dissociation of the doubly protonated decamer from solution, which has a heterochiral preference, results in protonated octamer with strong homochiral preference. This homochiral preference is higher when protonated octamer is formed from larger clusters and approaches the chiral preference of the octamer in solution. These results show that the doubly protonated decamer has a different structure when formed from solution than when formed by dissociation of larger clusters. These results indicate that the unusually high abundance of protonated homochiral octamer formed by spray ionization methods that has been reported previously can be largely attributed to aggregation of serine that occurs in rapidly evaporating droplets and from dissociation of large clusters that form abundant protonated octamer at an optimized effective temperature.

A comprehensive dataset for the thermal conductivity of ice Ih for application to planetary ice shells.

Contemporary models representing the thermal conductivity of ice Ih as a function of temperature are based on data from published experiments that span over a century. Each model is derived using specific datasets with distinct experimental setups, temperature ranges, and uncertainties. Model errors introduced by inaccurate digitization and biased datapoints are challenging to trace due to a lack of transparency of the primary data. This dataset is a collection of published thermal conductivity data for ice Ih, including both tabulated and digitized data, presented in the original units. Specific samples or pressure conditions are noted where applicable. The dataset also includes a survey of published thermal conductivity models, providing the valid temperature range, accuracy and uncertainty (where noted in the original publication), and the primary data sources. Importantly, the dataset includes notes that were contained in the original publication or subsequent publications that provide additional context for the data. This dataset is used to derive a new thermal conductivity model which best represents the thermal conductivity of ice Ih for temperatures greater than 30 K. Statistics are provided to evaluate the fit of each thermal conductivity model in the survey of published models to the comprehensive dataset presented here. This dataset is constructed in support of the work "New insights into temperature-dependent ice properties and their effect on ice shell convection for icy ocean worlds" [1].

Dissociation of large gaseous serine clusters produces abundant protonated serine octamer.

Protonated serine octamer is especially abundant in spray ionization mass spectra of serine solutions under a wide range of conditions. Although serine octamer exists in low abundance in solution, abundant clusters, including octamer, can be formed by aggregation inside evaporating electrospray droplets. A minimum cluster size of 8 and 21 serine molecules was observed for doubly protonated and triply protonated clusters, respectively, formed by electrospray ionization of a 10 mM serine solution. Dissociation of these clusters results in charge separation to produce predominantly protonated serine dimer and some trimer and the complimentary charged ion. Dissociation of clusters significantly larger than the minimum cluster size occurs by sequential loss of serine molecules. Dissociation of all large clusters investigated leads to protonated octamer as the second most abundant cluster (protonated dimer is most abundant) at optimized collision energies. All larger clusters dissociate through a combination of charge separation and neutral serine loss to form small doubly protonated clusters, and the vast majority of protonated octamer is produced by dissociation of the doubly protonated decamer by charge separation. Protonated octamer abundance is optimized at a uniform energy per degrees of freedom for all clusters indicating that simultaneous dissociation of all large clusters will lead to abundant protonated octamer at an optimum ion temperature. These results provide evidence for another route to formation of abundant protonated octamer in spray ionization or other methods that promote formation and subsequent dissociation of large clusters.

Effects of Electrospray Droplet Size on Analyte Aggregation: Evidence for Serine Octamer in Solution.

Spraying solutions of serine under a wide variety of conditions results in unusually abundant gaseous octamer clusters that exhibit significant homochiral specificity, but the extent to which these clusters exist in solution or are formed by clustering during droplet evaporation has been debated. Electrospray ionization emitters with tip sizes between 210 nm and 9.2 µm were used to constrain the number of serine molecules that droplets initially contain. Protonated octamer was observed for all tip sizes with 10 mM serine solution, but the abundance decreases from 10% of the serine population at the largest tip size to ∼5.6% for the two smallest tip sizes. At 100 µM, the population abundance of the protonated serine octamer decreases from 1% to 0.6% from the largest to the smallest tip size, respectively. At 100 µM, fewer than 10% of the initial droplets should contain even a single analyte molecule with 210 nm emitter tips. These results indicate that the majority of protonated octamer observed in mass spectra under previous conditions is formed by clustering inside the electrospray droplet, but ≤5.6% and ∼0.6% of serine exists as an octamer complex in 10 mM and 100 µM solutions, respectively. These results show that aggregation occurs in large droplets, but this aggregation can be eliminated using emitters with sufficiently small tips. Use of these emitters with small tips is advantageous for clearly distinguishing between species that exist in solution and species formed by clustering inside droplets as solvent evaporation occurs.

The biosecurity benefits of genetic engineering attribution.

Biology can be misused, and the risk of this causing widespread harm increases in step with the rapid march of technological progress. A key security challenge involves attribution: determining, in the wake of a human-caused biological event, who was responsible. Recent scientific developments have demonstrated a capability for detecting whether an organism involved in such an event has been genetically modified and, if modified, to infer from its genetic sequence its likely lab of origin. We believe this technique could be developed into powerful forensic tools to aid the attribution of outbreaks caused by genetically engineered pathogens, and thus protect against the potential misuse of synthetic biology.

Root morphology and exudate availability are shaped by particle size and chemistry in Brachypodium distachyon.

Root morphology and exudation define a plants' sphere of influence in soils. In turn, soil characteristics influence plant growth, morphology, root microbiome, and rhizosphere chemistry. Collectively, all these parameters have significant implications on the major biogeochemical cycles, crop yield, and ecosystem health. However, how plants are shaped by the physiochemistry of soil particles is still not well understood. We explored how particle size and chemistry of growth substrates affect root morphology and exudation of a model grass. We grew Brachypodium distachyon in glass beads with various sizes (0.5, 1, 2, 3 mm), as well as in sand (0.005, 0.25, 4 mm) and in clay (4 mm) particles and in particle-free hydroponic medium. Plant morphology, root weight, and shoot weight were measured. We found that particle size significantly influenced root fresh weight and root length, whereas root number and shoot weight remained constant. Next, plant exudation profiles were analyzed with mass spectrometry imaging and liquid chromatography-mass spectrometry. Mass spectrometry imaging suggested that both, root length and number shape root exudation. Exudate profiles were comparable for plants growing in glass beads or sand with various particles sizes, but distinct for plants growing in clay for in situ exudate collection. Clay particles were found to sorb 20% of compounds exuded by clay-grown plants, and 70% of compounds from a defined exudate medium. The sorbed compounds belonged to a range of chemical classes, among them nucleosides, organic acids, sugars, and amino acids. Some of the sorbed compounds could be desorbed by a rhizobacterium (Pseudomonas fluorescens WCS415), supporting its growth. This study demonstrates the effect of different characteristics of particles on root morphology, plant exudation and availability of nutrients to microorganisms. These findings further support the critical importance of the physiochemical properties of soils when investigating plant morphology, plant chemistry, and plant-microbe interactions.

Structural Comparison of Various Silkworm Silks: An Insight into the Structure-Property Relationship.

Silkworm silk has attracted considerable attention in recent years due to its excellent mechanical properties, biocompatibility, and promising applications in biomedical sector. However, a clear understanding of the molecular structure and the relationship between the excellent mechanical properties and the silk protein sequences are still lacking. This study carries out a thorough comparative structural analysis of silk fibers of four silkworm species ( Bombyx mori, Antheraea pernyi, Samia cynthia ricini, and Antheraea assamensis). A combination of characterization techniques including scanning electron microscopy, mechanical test, synchrotron X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), and NMR spectroscopy was applied to investigate the morphologies, mechanical properties, amino acid compositions, nanoscale organizations, and molecular structures of various silkworm silks. Furthermore, the structure-property relationship is discussed by correlating the molecular structural features of silks with their mechanical properties. The results show that a high content of ß-sheet structures and a high crystallinity would result in a high Young's modulus for silkworm silk fibers. Additionally, a low content of ß-sheet structures would result in a high extensibility.

Informatics Approaches to Address New Challenges in the Classification of Lymphoid Malignancies.

Purpose: Lymphoid malignancies are remarkably heterogeneous, with variations in outcomes and clinical, biologic, and histologic presentation complicating classification according to the World Health Organization guidelines. Incorrect classification of lymphoid neoplasms can result in suboptimal therapeutic strategies for individual patients and confound the interpretation of clinical trials involving personalized, class-based treatments. This review discusses the potential role of pathology informatics in improving the classification accuracy and objectivity for lymphoid malignancies. Design: We identified peer-reviewed publications examining pathology informatics approaches for the classification of lymphoid malignancies, reviewed developments in the lymphoma classification systems, and summarized computational methods for pathologic assessment that can impact practice. Results: Computer-assisted pathology image analysis algorithms in lymphoma most commonly have been applied to follicular lymphoma to address biologic heterogeneity and subjectivity in the process of classification. Conclusion: Objective methods are available to assist pathologists in lymphoma classification and grading, and have been demonstrated to provide measurable benefits in specific contexts. Future validation and extension of these approaches will require datasets that link high resolution pathology images available for image analysis algorithms with clinical variables and follow up outcomes.

Highly Efficient Fumed Silica Nanoparticles for Peptide Bond Formation: Converting Alanine to Alanine Anhydride.

In this work, thermal condensation of alanine adsorbed on fumed silica nanoparticles is investigated using thermal analysis and multiple spectroscopic techniques, including infrared (IR), Raman, and nuclear magnetic resonance (NMR) spectroscopies. Thermal analysis shows that adsorbed alanine can undergo thermal condensation, forming peptide bonds within a short time period and at a lower temperature (∼170 °C) on fumed silica nanoparticle surfaces than that in bulk (∼210 °C). Spectroscopic results further show that alanine is converted to alanine anhydride with a yield of 98.8% during thermal condensation. After comparing peptide formation on solution-derived colloidal silica nanoparticles, it is found that fumed silica nanoparticles show much better efficiency and selectivity than solution-derived colloidal silica nanoparticles for synthesizing alanine anhydride. Furthermore, Raman spectroscopy provides evidence that the high efficiency for fumed silica nanoparticles is likely related to their unique surface features: the intrinsic high population of strained ring structures present at the surface. This work indicates the great potential of fumed silica nanoparticles in synthesizing peptides with high efficiency and selectivity.