Mass Exchange Research Articles

Identification of an intrinsically disordered region (IDR) in arginyltransferase 1 (ATE1).

Arginyltransferase 1 (ATE1) catalyzes arginylation, an important post-translational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while post-translational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small angle X-ray scattering (SAXS), and hydrogen deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR likely facilitates the formation of the complex between ATE1 and tRNAArg, adding a new complexity to ATE1 structure and providing new insights for future studies of ATE1 functions.

Electronic regulation of hcp-Ru by d-d orbital coupling for robust electrocatalytic hydrogen oxidation in alkaline electrolytes

Bimetallic alloys hold exceptional promise as candidate materials because they offer a diverse parameter space for optimizing electronic structures and catalytic sites. Herein, we fabricate ruthenium-cobalt alloy nanoparticles uniformly dispersed within hollow mesoporous carbon spheres (hcp-RuCo@C) via impregnation and pyrolysis strategies. The intriguing hollow mesopore structure of hcp-RuCo@C facilitates efficient contact between active sites and reactants, thereby accelerating hydrogen oxidation reaction (HOR) kinetics. As anticipated, the hcp-RuCo@C showcases remarkable exchange current density and mass activity of 3.73 mA cm−2 and 2.8 mA μgRu-1, respectively, surpassing those of commercial Pt/C and documented Ru-based electrocatalysts. Notably, hcp-RuCo@C demonstrates robust resistance to 1000 ppm CO, a trait lacking in Pt/C catalysts. Comprehensive experimental results reveal that the alloying-induced d-d electronic interactions between Ru and Co species significantly optimizes hydrogen binding energy (HBE) and hydroxide binding energy (OHBE). This optimization promotes the vital Volmer step, ameliorating the alkaline HOR properties of hcp-RuCo@C.

Understanding and manipulating the recognition of necrosis-inducing secreted protein 1 (NIS1) by BRI1-associated receptor kinase 1 (BAK1)

Necrosis-inducing secreted protein 1 (NIS1) is a core effector of Ascomycota and Basidiomycota fungi. They inhibit the immune responses of host plants mainly through interaction with the multi-functional coreceptor BRI1-associated receptor kinase 1 (BAK1). However, the structural mechanism of the NIS1 family and how they are recognized by BAK1 are unknown. Herein, we report the first crystal structure of the NIS1 family protein, the Magnaporthe oryzae NIS1 (MoNIS1), analyze the recognition mechanism of NIS1s by BAK1, and explore regulation of the NIS1-BAK1 interaction by a chemical compound. MoNIS1 exists as a β barrel formed by eight β strands, a folding mode that has not been reported. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) assay suggested that β4-β5 loop and β5 strand of MoNIS1 participate in OsBAK1 interaction, which was supported by further single-point mutational assays. For OsBAK1, HDX-MS assay suggested four regions involved in MoNIS1 interaction. Additionally, we identified a compound that blocks MoNIS1-OsBAK1 interaction in vitro and inhibits the virulence of M. oryzae on rice. Collectively, we determined the first structure of NIS1 family effectors, presented the recognition mechanism of NIS1 by BAK1, and showed that blocking NIS1-BAK1 interaction could be a new target for fungicide development.

Quantitative Hydrogen-Deuterium Exchange Mass Spectrometry for Simultaneous Structural Characterization and Affinity Indexing of Single Target Drug Candidate Libraries.

Hydrogen-deuterium eXchange mass spectrometry (HDX-MS) is increasingly used in drug development to locate binding sites and to identify allosteric effects in drug/target interactions. However, the potential of this technique to quantitatively analyze drug candidate libraries remains largely unexplored. Here, a collection of 13 WDR5-targeting small molecules with surface plasmon resonance (SPR) dissociation coefficients (KD) ranging from 20 nM to ∼116 μM were characterized using differential HDX-MS (ΔHDX-MS). Conventional qualitative analysis of the ΔHDX-MS data set revealed the binding interfaces for all compounds and allosteric effects where present. We then demonstrated that ΔHDX-MS signal-to-noise (S/N) not only can rank library-relative affinity but also can accurately predict KD from a calibration curve constructed from high-quality SPR data. Three methods for S/N calculation are explored, each suitable for libraries with different characteristics. Our results demonstrate the potential for ΔHDX-MS use in drug candidate library affinity validation and/or determination while simultaneously characterizing structure.

Chemical Synthesis and Structure-Activity Relationship Studies of the Coagulation Factor Xa Inhibitor Tick Anticoagulant Peptide from the Hematophagous Parasite Ornithodoros moubata.

Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure-activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen-Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles.

Structures of human PTP1B variants reveal allosteric sites to target for weight loss therapy.

Protein Tyrosine Phosphatase 1B (PTP1B) is a negative regulator of leptin signaling whose disruption protects against diet-induced obesity in mice. We investigated whether structural characterization of human PTP1B variant proteins might reveal precise mechanisms to target for weight loss therapy. We selected 12 rare variants for functional characterization from exomes from 997 people with persistent thinness and 200,000 people from UK Biobank. Seven of 12 variants impaired PTP1B function by increasing leptin-stimulated STAT3 phosphorylation in cells. Using room-temperature X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, and computational modeling, we determined that human variants modulate the 3-dimensional structure of PTP1B through distinct allosteric conduits that energetically link distal, highly ligandable structural regions to the active site. These studies inform the design of allosteric PTP1B inhibitors for the treatment of obesity.

Real Time Tracking of Nanoconfined Water-Assisted Ion Transfer in Functionalized Graphene Derivatives Supercapacitor Electrodes.

Water molecules confined in nanoscale spaces of 2D graphene layers have fascinated researchers worldwide for the past several years, especially in the context of energy storage applications. The water molecules exchanged along with ions during the electrochemical process can aid in wetting and stabilizing the layered materials resulting in an anomalous enhancement in the performance of supercapacitor electrodes. Engineering of 2D carbon electrode materials with various functionalities (oxygen (─O), fluorine (─F), nitrile (─C≡N), carboxylic (─COOH), carbonyl (─C═O), nitrogen (─N)) can alter the ion/water organization in graphene derivatives, and eventually their inherent ion storage ability. Thus, in the current study, a comparative set of functionalized graphene derivatives-fluorine-doped cyanographene (G-F-CN), cyanographene (G-CN), graphene acid (G-COOH), oxidized graphene acid (G-COOH (O)) and nitrogen superdoped graphene (G-N) is systematically evaluated toward charge storage in various aqueous-based electrolyte systems. Differences in functionalization on graphene derivatives influence the electrochemical properties, and the real-time mass exchange during the electrochemical process is monitored by electrochemical quartz crystal microbalance (EQCM). Electrogravimetric assessment revealed that oxidized 2D acid derivatives (G-COOH (O)) are shown to exhibit high ion storage performance along with maximum water transfer during the electrochemical process. The complex understanding of the processes gained during supercapacitor electrode charging in aqueous electrolytes paves the way toward the rational utilization of graphene derivatives in forefront energy storage applications.

A high-titer scalable Chinese hamster ovary transient expression platform for production of biotherapeutics.

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells offers a route to accelerate biologics development by delivering material weeks to months earlier than what is possible with conventional cell line development. However, low productivity, inconsistent product quality profiles, and scalability challenges have prevented its broader adoption. In this study, we develop a scalable CHO-based TGE system achieving 1.9 g/L of monoclonal antibody in an unmodified host. We integrated continuous flow-electroporation and alternate tangential flow (ATF) perfusion to enable an end-to-end closed system from N-1 perfusion to fed-batch 50-L bioreactor production. Optimization of both the ATF operation for three-in-one application-cell growth, buffer exchange, and cell mass concentration-and the flow-electroporation process, led to a platform for producing biotherapeutics using transiently transfected cells. We demonstrate scalability up to 50-L bioreactor, maintaining a titer over 1 g/L. We also show comparable quality between both transiently and stably produced material, and consistency across batches. The results confirm that purity, charge variants and N-glycan profiles are similar. Our study demonstrates the potential of CHO-based TGE platforms to accelerate biologics process development timelines and contributes evidence supporting its feasibility for manufacturing early clinical material, aiming to strengthen endorsement for TGE's wider implementation.

Wave-driven current and vortex patterns at an open beach: Insights from phase-resolving numerical computations and Lagrangian measurements

Wave-driven currents have a substantial impact on local circulation patterns in and across the surf zone, and are responsible for cross-shore and longshore exchange of mass and momentum over a broad range of spatial and temporal scales. Nearshore currents may drive sediment transport, lead to beach erosion, and also affect the spread of bacteria and other marine microorganisms, as well as the distribution of pollutants such as chemicals and microplastics. In addition, surf zone currents can cause hazardous conditions for beach-goers in the form of rip currents.It is known from previous work (Chen et al., 2003; Feddersen et al., 2011; Hally-Rosendahl and Feddersen, 2016) that Boussinesq-type models in combination with appropriate boundary conditions and wave breaking capabilities can function as powerful tools for the analysis of circulation patterns in the surf zone. In the present work, data from a recent field campaign reported on in Bjørnestad et al. (2021) are used to further validate the capability of Boussinesq systems to simulate nearshore dynamics.The numerical model is then used to study the influence of tidal elevation, peak direction and directional spread of the incoming wavefield on the quantity, extent, and circulatory magnitude of the nearshore circulation. In addition, fundamental features such as horizontal eddies are investigated, and comparisons are made to solid-body rotation and irrotational vortices.Overall, it is observed that local variations in the bathymetry across the surf zone are the controlling factor regarding the size of these circulations, and an increasing tidal level, which can be seen as a uniform offset to the bathymetry, favors the generation of larger vortex patterns. For a given tidal stage, the directional spread of the incoming wavefield has the most pronounced influence on the size and strength of the nearshore eddies while the peak direction has the strongest effect on the total number of circulations.

Abstract We012: Sheet like extracellular matrix secreting single cell microvesicles for reprogrammed endothelial cell therapy towards vascular regeneration

To enhance cell survival and engraftment for cell therapy, cell encapsulated injectable biomaterials have been widely used. However, the challenges were hypoxia and nutrient shortage in the core, inflammatory cell infiltration and limited distribution of cells from injection sites. Encapsulation of single cells in sub-100-micron microgels with higher surface to volume ratios provides more efficient oxygen and mass exchange. However, typical production of single-cell-laden microgels by microfluidics devices results in cell damage and compromised biological properties. We hypothesized that stimuli responsive amphiphilic copolymer bearing RGD peptides may self-assemble around single cells induced by cell adhesion moieties would result in single cell microvesicles (MV). A stimuli responsive amphiphilic copolymer GPM (Gelatin Poly glycerol sebacate Methacrylate) was synthesized by grafting hydrophobic synthetic polymer PGS into hydrophilic natural polymer gelatin via methacrylation followed by aza-Michael addition reaction. Single cell microvesicles of endothelial cells (EC-MVs) were fabricated by self-assembly of GPM at room temperature. For in vivo studies, sub-50-micron EC-MVs made of reprogrammed endothelial cells (rECs) were clustered into 200 micron spheres (mGPM) using sodium alginate and injected into a hindlimb ischemia model to investigate cell survival and biological function. The mGPM microspheres functioned as a barrier from immune cell infiltration and also enhanced cellular distribution. The encapsulated rECs were released over 2-4 wks in vivo and were shown robust engraftment, migration, and survival in limb tissues, promoting blood flow recovery and sustained vascular regeneration over 6 months. Interestingly, in in vitro culture, rEC-MVs secreted sheet like extra cellular matrix (ECM) which appeared to make an important role for vessel formation (neovascularization) in vivo (Figure 1). The current study clearly demonstrated the favorable biological applicability of GPM single cell MVs toward vascular regeneration by reprogrammed ECs.

Copepod community in the coastal waters off Kochi (India) signify the enhanced estuarine flushing during the Southwest Monsoon

Even though many studies refer to copepods as potential indicators of hydrographical settings and/or water masses, focused studies on this aspect are very scarce in Indian waters. Hence, this study assesses the hydrography and the copepod composition in a cross-shore transect (7–30 m depth zone) off Kochi along the southwest coast of India during different seasons to understand how seasonal hydrography alters the composition of copepods. The copepod community in the study area was found to consist of 36 species (26 dominant) during the Southwest Monsoon [(SWM) June to September], 31 species (16 dominant) during the Northeast Monsoon [(NEM) November to February], and 39 species (26 dominant) during the Pre-Monsoon [(PRM) March to May]. The salient observations in this study are (a) a higher contribution of cyclopoids (45–50%) during the oligotrophic PRM when the microbial food web was dominant; (b) the dominance of several truly estuarine copepods (Acartia plumosa, Acartiella keralensis, Pseudodiaptomous annandalei, and P. serricaudatus) during the SWM when the flushing of the adjacent estuarine system (Kochi backwaters) was the seasonal highest; and (c) the dominant occurrence of Temora turbinata, commonly referred to as an indicator of coastal upwelling, during both the SWM and the NEM indicates their conducive conditions in the study area during both seasons and (d) the occurrence of copepods Acartia danae, A. erythraea, Acrocalanus gibber, A. gracilis, Centropages orsinii, C. tenuiremis, Corycaeus catus, C. speciosus, Oithona similis, O. spinirostris, Oncaea venusta, Paracalanus parvus, and Undinula vulgaris in the study area regardless of seasons. The study concludes that, while the presence of truly estuarine copepods in the study area can be attributed to estuarine watermass indicators, the presence of several other dominant species found regardless of the season cannot, as their occurrence is widespread in the eastern Arabian Sea and the western Bay of Bengal, owing to seasonal water mass exchange between the two regions.

Reactivity of N terminal histidine of peptides towards excipients/impurity of excipients: A case study of liraglutide excipient compatibility study

The selection of quality excipients is a crucial step in peptide formulation development. Apart from excipient incompatibility, process-related impurities or degradants of an excipient can interact with peptide-active pharmaceutical ingredients, forming the interaction products. The formaldehyde has been reported as an impurity of excipient in polyethylene glycol, glycerol, magnesium stearate, microcrystalline cellulose, mannitol, etc. The peptide contains various amino acids such as histidine, lysine, and arginine having free amine groups. These amine groups act as strong nucleophile and can increase the reactivity of peptides. PLGA is the most widely used biodegradable polymer in sustained-release formulations. The hydrolysis of PLGA generates glycolic acid and lactic acid impurities, which can form the interaction product with the amines of peptides. During the formulation development of Liraglutide, we have found few interaction products. The systematic characterization and mechanistic understanding of these interaction products lead us to imidazopyrimidine, glycolyl, and lactolyl moieties. These interaction products have been characterized thoroughly with the use of LC-HRMS, MS/MS, and hydrogen-deuterium exchange mass studies. The study revealed that the reactivity of N-terminal histidine must be considered for formulation development. Moreover, the quality of excipients with respect to presence of impurities must be considered as critical material attributes.

Structure of calcineurin bound to PI4KA reveals dual interface in both PI4KA and FAM126A.

Phosphatidylinositol 4-kinase alpha (PI4KA) maintains the phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine pools of the plasma membrane. A key regulator of PI4KA is its association into a complex with TTC7 and FAM126 proteins. This complex can be regulated by the CNAβ1 isoform of the phosphatase calcineurin. We previously identified that CNAβ1 directly binds to FAM126A. Here, we report a cryoelectron microscopic (cryo-EM) structure of a truncated PI4KA complex bound to calcineurin, revealing a unique direct interaction between PI4KA and calcineurin. Hydrogen deuterium exchange mass spectrometry (HDX-MS) and computational analysis show that calcineurin forms a complex with an evolutionarily conserved IKISVT sequence in PI4KA's horn domain. We also characterized conserved LTLT and PSISIT calcineurin binding sequences in the C terminus of FAM126A. These dual sites in PI4KA and FAM126A are both in close proximity to phosphorylation sites in the PI4KA complex, suggesting key roles of calcineurin-regulated phosphosites in PI4KA regulation. This work reveals novel insight into how calcineurin can regulate PI4KA activity.

Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience.

TLRs initiate innate immune signaling pathways via Toll/IL-1R (TIR) domains on their cytoplasmic tails. Various bacterial species also express TIR domain-containing proteins that contribute to bacterial evasion of the innate immune system. Bacterial TIR domains, along with the mammalian sterile α and TIR motif-containing protein 1 and TIRs from plants, also have been found to exhibit NADase activity. Initial X-ray crystallographic studies of the bacterial TIR from Acinetobacter baumannii provided insight into bacterial TIR structure but were unsuccessful in cocrystallization with the NAD+ ligand, leading to further questions about the TIR NAD binding site. In this study, we designed a Course-Based Undergraduate Research Experience (CURE) involving 16-20 students per year to identify amino acids crucial for NADase activity of A. baumannii TIR domain protein and the TIR from Escherichia coli (TIR domain-containing protein C). Students used structural data to identify amino acids that they hypothesized would play a role in TIR NADase activity, and created plasmids to express mutated TIRs through site-directed mutagenesis. Mutant TIRs were expressed, purified, and tested for NADase activity. The results from these studies provide evidence for a conformational change upon NAD binding, as was predicted by recent cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry studies. Along with corroborating recent characterization of TIR NADases that could contribute to drug development for diseases associated with dysregulated TIR activity, this work also highlights the value of CURE-based projects for inclusion of a diverse group of students in authentic research experiences.

Experimental and numerical investigations of the mixing processes of pollutants at notched weirs in rivers

This study experimentally and numerically investigated the mixing processes at a notched weir to examine the relationship between the mass exchange coefficient and geo-hydraulic factors of notched weirs. Laboratory experiments on idealized notched weirs with variable notch spaces, notch heights, and hydraulic factors were performed to measure the flow field dynamics and depth-averaged concentration fields of the solute tracer. The experiment results demonstrated that flow separations occurred upstream of the notched weir. These flow structures controlled the horizontal storage zone and tracer mixing between the main flow and storage zone. The tracer cloud exhibited a longer retention time as the storage zone area increased. The normalized value of measured mass exchange coefficients by mean flow velocity, ke /Umean ranged from of 0.11 to 0.25, approximately one order larger than the values obtained in previous studies on lateral transient storage zones, such as lateral groin fields, and cavities, which implied that the notched weir in this study generated a storage zone with a fast exchange process and relatively weak storage effect. Numerical simulations with expanded geometric and hydraulic conditions revealed that the mass exchange coefficient was strongly correlated with the geometric factors, such as the notch space and height, and hydraulic factors, such as the Froude and Reynolds numbers. An empirical relationship applicable to rivers with notched weir structures under low-flow conditions (Fr < 0.1) was proposed based on the experiment and numerical results. The performance of the proposed empirical relationship showed a satisfactory level of R2 of 0.87. This relationship can be applied as a tool for estimating the mass exchange coefficient of notched weirs using easily measurable field data in rivers.

Design of Nonlinear Autoregressive Neuro-Computing Structure for Bioconvective Micropolar Nanofluidic Model

In the present arena, the tools of artificial intelligence (AI) play a significant role in research across various fields by enabling advanced data analysis, pattern recognition and decision-making. This research work presents the numerical investigation of bioconvective micropolar nanofluidic model (BCMNFM) by employing the knacks of AI-based nonlinear autoregressive (NAR) approach with a combination of backpropagated Levenberg–Marquardt neural networks (BLMNNs) represented as NAR-BLMNNs. This research work investigates the flow design to highlight the attributes of mass and heat exchange. A dataset for BCMNFM is created by applying the Adam numerical procedure by variation of unsteadiness parameter ([Formula: see text], magnetic field parameter ([Formula: see text] thermophoresis parameter (Nt), Brownian motion parameter (Nb), bioconvection Peclet number (Pe) and spin gradient viscosity parameter ([Formula: see text] The skills of AI-based NAR-BLMNNs technique is then utilized on the dataset created for BCMNFM to investigate the approximate solutions. The achieved and impactful values of performance consistently range between [Formula: see text] and [Formula: see text] across all scenarios of BCMNFM. The precision and the validation of predicted approach NAR-BLMNNs is exceptionally established by the graphical demonstration for all scenarios of MSE, regression metrics, error histograms and time series graphs. The numerical calculations attained through AI-based NAR-BLMNNs technique further rationalize the precision of the proposed methodology for solving the BCMNFM effectively and efficiently.

HDXBoxeR: an R package for statistical analysis and visualization of multiple Hydrogen-Deuterium Exchange Mass-Spectrometry datasets of different protein states.

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a powerful protein characterization technique that provides insights into protein dynamics and flexibility at the peptide level. However, analyzing HDX-MS data presents a significant challenge due to the wealth of information it generates. Each experiment produces data for hundreds of peptides, often measured in triplicate across multiple time points. Comparisons between different protein states create distinct datasets containing thousands of peptides that require matching, rigorous statistical evaluation, and visualization. Our open-source R package, HDXBoxeR, is a comprehensive tool designed to facilitate statistical analysis and comparison of multiple sets among samples and time points for different protein states, along with data visualization. HDXBoxeR is accessible as the R package (https://cran.r-project.org/web//packages/HDXBoxeR) and GitHub: mkajano/HDXBoxeR.

Small-scale vortical motions in cool stellar atmospheres

Small-scale vortices in the solar atmosphere have received considerable attention in recent years. These events are considered potential conduits for the exchange of energy and mass between the solar atmospheric layers from the convective surface to the corona. Similar events may occur in the atmospheres of other stars and play a role in energy transfer within their atmospheres. Our aim is to study the presence and properties of small-scale swirls in numerical simulations of the atmospheres of cool main-sequence stars. Our particular focus is on understanding the variations in these properties for different stellar types and their sensitivity to the surface magnetic field. Furthermore, we aim to investigate the role of these events in the energy transport within the simulated atmospheres. We analyzed three-dimensional, radiative-magnetohydrodynamic, box-in-a-star, numerical simulations of four main-sequence stars of spectral types K8V, K2V, G2V, and F5V. These simulations include a surface small-scale dynamo responsible for amplifying an initially weak magnetic field. Thus, we can study models characterized by very weak, or, in near equipartition magnetic fields. To identify small-scale vortices in horizontal layers of the simulations, we employed the automated algorithm SWIRL. Small-scale swirls are abundant in the simulated atmospheres of all the investigated cool stars. The characteristics of these events appear to be influenced by the main properties of the stellar models and by the strength of the surface magnetic field. In addition, we identify signatures of torsional Alfv\'enic pulses associated with these swirls, which are responsible for a significant vertical Poynting flux in the middle photospheres of the simulated stellar models. Notably, this flux is particularly significant in the K8V model, suggesting that if sim 70&lt;!PCT!&gt; of it is dissipated in the low chromosphere, small-scale vortical motions may play a role in the enhanced basal Ca ii H and K fluxes observed in the range of $B-V$ color index $1.1 B - V 1.4$. Finally, we present a simple analytical model, along with an accompanying scaling relation, to explain a peculiar result of the statistical analysis that the rotational period of surface vortices increases with the effective temperature of the stellar model. Our study shows that small-scale vortical motions are not unique to the solar atmosphere and that their interplay with the stellar surface magnetic field may effect the observable chromospheric activity of main-sequence cool dwarf stars.

Formulation Development of a COVID-19 Recombinant Spike Protein-Based Vaccine.

The purpose of this study was to develop a formulation for a recombinant prefusion spike protein vaccine against SARS-CoV-2. It was found that the spike protein was susceptible to aggregation due to mechanical stress. Therefore, formulation studies were initiated focused on screening pharmaceutical excipients capable of preventing this. The screening of a panel of potential stabilizing conditions found that Tween 20 could inhibit mechanically induced aggregation. A concentration-dependent study indicated that a higher concentration of Tween 20 (0.2% v/v) was required to prevent conformational changes in the trimer. The conformational changes induced by mechanical stress were characterized by size exclusion chromatography (SEC) and hydrogen-deuterium exchange mass spectrometry (HDX-MS), indicating the formation of an extended trimeric conformation that was also unable to bind to antibodies directed to the S2 domain. Long-term stability modeling, using advanced kinetic analysis, indicated that the formulation containing 0.2% (v/v) Tween 20 at a neutral pH was predicted to be stable for at least two years at 2 °C to 8 °C. Additional stabilizer screening conducted by thermal shift assay indicated that sucrose and glycerol were able to significantly increase the spike protein melting temperature (Tm) and improve the overall thermostability of the spike protein in a short-term stability study. Thus, while 0.2% (v/v) Tween 20 was sufficient to prevent aggregation and to maintain spike protein stability under refrigeration, the addition of sucrose further improved vaccine thermostability. Altogether, our study provides a systematic approach to the formulation of protein-based COVID-19 vaccine and highlights the impact of mechanical stress on the conformation of the spike protein and the significance of surfactants and stabilizers in maintaining the structural and functional integrity of the spike protein.

Freshwater runoff on the east Greenland shelf

The Greenland Ice Sheet releases fresh water from ice melt, tundra snow melt, and solid ice at an increasing rate during recent decades resulting in an increasing amount of freshwater runoff into the ocean. As a result, freshwater runoff is changing the continental shelf circulation by increasing the amount of fresher water on the shelf that may then enter the deep ocean. Observational studies show that dense water does traverse the east Greenland shelf near the ocean bottom but it is unclear to what extent the transport of near surface fresh water, as a result of runoff, reaches deeper water and enters the Irminger Basin. Using 4 km resolution nested numerical model simulations with and without freshwater runoff, we show freshwater runoff increases salinity variability with increased baroclinicity. While higher salinity variability and baroclinicity suggest a greater potential for water mass exchange across the East Greenland Current, most freshwater runoff along east Greenland remains on the shelf. From freshwater runoff alone, salinity and salt mass decreases by 0.22% on the continental shelf compared to a 0.01% in the rest of the Nordic Seas. There is a 0.05% reduction in salt mass on the Greenland shelf region that makes up 8% of the simulation domain, suggesting little water exits the shelf along the east coast of Greenland. The largest reduction in salt mass occurs around Iceland, where substantial freshwater runoff exists. A calculation of baroclinic conversion rate suggests likely pathways for runoff to exit the continental shelf and enter deep water in the Denmark Strait and over the Greenland/Scottland ridge east of Iceland. Most of the fresh water, however, released along the east coast of Greenland continues towards Cape Farewell, outside our modeling domain, and into the Labrador Sea.