Solid organic-coated ammonium sulfate particles at high relative humidity in the summertime Arctic atmosphere.

SignificancePhysical and chemical properties of individual atmospheric particles determine their climate impacts. Hygroscopic inorganic salt particles mixed with trace amounts of organic material are predicted to be liquid under typical tropospheric conditions in the summertime Arctic. Yet, we unexpectedly observed a significant concentration of solid particles composed of ammonium sulfate with an organic coating under conditions of high relative humidity and low temperature. These particle properties are consistent with marine biogenic-derived new particle formation and growth, with particle collision hypothesized to result in the solid phase. This particle source is predicted to have increasing relevance in the context of declining Arctic sea ice and increasing open water, with impacts on clouds, and therefore climate.

Investigating the Allometric Relationship for Total Mercury in Male and Female Signal Crayfish (Pacifastacus leniusculus) from Three Regions in the Columbia River Basin, USA.

The objective of this study was to evaluate the relationship between clawless body mass (CBM) and total mercury concentration (THg) in male and female crayfish captured from three different sites in the Columbia River Basin: the Spokane River, Boise River and Hangman Creek. It was found that the allometric relationships for THg in male and female crayfish did not differ from one another at any of the three locations (Spokane River: ANCOVA, F1,49 = 0.001, p = 0.63; Hangman Creek: ANCOVA, F1,73 = 0.007, p = 0.93; Boise River: ANCOVA, F1,38 = 0.02, p = 0.88. Furthermore, the slopes of the CBM and THg regression lines were not significantly different from one site to the next (ANCOVA, F2,166 = 1.5 p = 0.24), despite considerable differences in mean mass-adjusted THg across locations (Spokane River: 26.8 ± 2.6; Hangman Creek: 75.2 ± 2.1; Boise River: 99.0 ± 2.9). For signal crayfish (Pacifastacus leniusculus) from the Columbia River Basin, size and sex standardization can be accomplished with a single linear relationship.

PROTACs, molecular glues and bifunctionals from bench to bedside: Unlocking the clinical potential of catalytic drugs.

The vast majority of currently marketed drugs rely on small molecules with an 'occupancy-driven' mechanism of action (MOA). Therefore, the efficacy of these therapeutics depends on a high degree of target engagement, which often requires high dosages and enhanced drug exposure at the target site, thus increasing the risk of off-target toxicities (Churcher, 2018 [1]). Although small molecule drugs have been successfully used as treatments for decades, tackling a variety of disease-relevant targets with a defined binding site, many relevant therapeutic targets remain challenging to drug due, for example, to lack of well-defined binding pockets or large protein-protein interaction (PPI) interfaces which resist interference (Dang et al., 2017 [2]). In the quest for alternative therapeutic approaches to address different pathologies and achieve enhanced efficacy with reduced side effects, ligand-induced targeted protein degradation (TPD) has gained the attention of many research groups both in academia and in industry in the last two decades. This therapeutic modality represents a novel paradigm compared to conventional small-molecule inhibitors. To pursue this strategy, heterobifunctional small molecule degraders, termed PROteolysis TArgeting Chimeras (PROTACs) have been devised to artificially redirect a protein of interest (POI) to the cellular protein homeostasis machinery for proteasomal degradation (Chamberlain et al., 2019 [3]). In this chapter, the development of PROTACs will first be discussed providing a historical perspective in parallel to the experimental progress made to understand this novel therapeutic modality. Furthermore, common strategies for PROTAC design, including assays and troubleshooting tips will be provided for the reader, before presenting a compendium of all PROTAC targets reported in the literature to date. Due to the recent advancement of these molecules into clinical trials, consideration of pharmacokinetics and pharmacodynamic properties will be introduced, together with the biotech landscape that has developed from the success of PROTACs. Finally, an overview of subsequent strategies for targeted protein degradation will be presented, concluding with further scientific quests triggered by the invention of PROTACs.

Apportioned primary and secondary organic aerosol during pollution events of DISCOVER-AQ Houston.

Understanding the drivers for high ozone (O3) and atmospheric particulate matter (PM) concentrations is a pressing issue in urban air quality, as this understanding informs decisions for control and mitigation of these key pollutants. The Houston, TX metropolitan area is an ideal location for studying the intersection between O3 and atmospheric secondary organic carbon (SOC) production due to the diversity of source types (urban, industrial, and biogenic) and the on- and off-shore cycling of air masses over Galveston Bay, TX. Detailed characterization of filter-based samples collected during Deriving Information on Surface Conditions from Column and VERtically Resolved Observations Relevant to Air Quality (DISCOVER-AQ) Houston field experiment in September 2013 were used to investigate sources and composition of organic carbon (OC) and potential relationships between daily maximum 8 h average O3 and PM. The current study employed a novel combination of chemical mass balance modeling defining primary (i.e. POC) versus secondary (i.e. SOC) organic carbon and radiocarbon (14C) for apportionment of contemporary and fossil carbon. The apportioned sources include contemporary POC (biomass burning [BB], vegetative detritus), fossil POC (motor vehicle exhaust), biogenic SOC and fossil SOC. The filter-based results were then compared with real-time measurements by aerosol mass spectrometry. With these methods, a consistent urban background of contemporary carbon and motor vehicle exhaust was observed in the Houston metropolitan area. Real-time and filter-based characterization both showed that carbonaceous aerosols in Houston was highly impacted by SOC or oxidized OC, with much higher contributions from biogenic than fossil sources. However, fossil SOC concentration and fractional contribution had a stronger correlation with daily maximum 8 h average O3, peaking during high PM and O3 events. The results indicate that point source emissions processed by on- and off-shore wind cycles likely contribute to peak events for both PM and O3 in the greater Houston metropolitan area.

Urban Trees Are Sinks for Soot: Elemental Carbon Accumulation by Two Widespread Oak Species.

Urban trees could represent important short- and long-term landscape sinks for elemental carbon (EC). Therefore, we quantified foliar EC accumulation by two widespread oak tree species-Quercus stellata (post oak) and Quercus virginiana (live oak)-as well as leaf litterfall EC flux to soil from April 2017 to March 2018 in the City of Denton, Texas, within the Dallas-Fort Worth metropolitan area. Post oak trees accumulated 1.9-fold more EC (299 ± 45 mg EC m-2 canopy yr-1) compared to live oak trees (160 ± 31 mg EC m-2 canopy yr-1). However, in the fall, these oak species converged in their EC accumulation rates, with ∼70% of annual accumulation occurring during fall and on leaf surfaces. The flux of EC to the ground via leaf litterfall mirrored leaf-fall patterns, with post oaks and live oaks delivering ∼60% of annual leaf litterfall EC in fall and early spring, respectively. We estimate that post oak and live oak trees in this urban ecosystem potentially accumulate 3.5 t EC yr-1, equivalent to ∼32% of annual vehicular EC emissions from the city. Thus, city trees are significant sinks for EC and represent potential avenues for climate and air quality mitigation in urban areas.

Cerebrospinal fluid γδ T cell frequency is age-related: a case-control study of 435 children with inflammatory and non-inflammatory neurological disorders.

Studies of cerebrospinal fluid (CSF) γδ T cells in children are limited, due especially to the lack of control data. In adults, gamma/delta T cells (TCR-γδ) residing in the intrathecal space are sometimes involved in neuroinflammation. To evaluate the possible role of γδ T cells in paediatric neuroinflammation, we immunophenotyped cerebrospinal fluid (CSF) and blood lymphocytes using flow cytometry in a case-control study of 100 children with non-inflammatory neurological disorders (NIND), 312 with opsoclonus-myoclonus (OMS) and 23 with other inflammatory neurological disorders (OIND). In NIND, the negative correlation between CSF γδ T cell frequency and patient age was striking: median frequency of 27% in infants and 3·3% in teens. Interindividual variations were largest in the youngest. There was no gender effect. In all OMS, after correcting for age, only a small effect of OMS severity remained. Measurement of markers for γδ T cell activation [human leucocyte antigen D-related (HLA-DR)], maturation (CD45RA, CD45RO) or intracellular cytokine staining [interleukin (IL)-4, interferon (IFN)-γ] failed to discriminate OMS and NIND groups. Of seven OMS immunotherapies/combinations, none altered the frequency of total CSF γδ T cells or subsets significantly. In OIND, the CSF γδ T cell frequency was < 10% for single samples of other paraneoplastic disorders [anti-neuronal nuclear antibody (ANNA)-1, PCA-1, teratoma-associated syndrome], cerebellar ataxia (post-infectious, ataxia-telangiectasia), acute disseminated encephalomyelitis, neuroborreliosis and encephalitis. This study provides new insights into CSF γδ T cells in the paediatric population. Although their role in CSF remains elusive, the negative age correlation, resistance to immunotherapy and our age cut-off references for NIND are important findings for the design of future paediatric studies.

6-Mercaptopurine modifies cerebrospinal fluid T cell abnormalities in paediatric opsoclonus-myoclonus as steroid sparer.

The purpose of this study was to evaluate the capacity of 6-mercaptopurine (6-MP), a known immunosuppressant, to normalize cerebrospinal fluid (CSF) lymphocyte frequencies in opsoclonus-myoclonus syndrome (OMS) and function as a steroid sparer. CSF and blood lymphocytes were immunophenotyped in 11 children with OMS (without CSF B cell expansion) using a comprehensive panel of cell surface adhesion, activation and maturation markers by flow cytometry, and referenced to 18 paediatric controls. Drug metabolites, lymphocyte counts and liver function tests were used clinically to monitoring therapeutic range and toxicity. In CSF, adjunctive oral 6-MP was associated with a 21% increase in the low percentage of CD4+ T cells in OMS, restoring the CD4/CD8 ratio. The percentage of CD4+ T cells that were interferon (IFN)-γ+ was reduced by 66%, shifting the cytokine balance away from T helper type 1 (Th1) (proinflammatory) predominance. The percentage of natural killer (NK) cells decreased significantly in CSF (-32%) and blood (-67 to -82%). Low blood absolute lymphocyte count was more predictive of improvement in CSF lymphocyte proportions (correlated with % CD4+ T cells) than the 6-thioguanine level (no correlation). 6-MP was difficult to titrate: 50% achieved the target absolute lymphocyte count (< 1·5 K/mm); 20%, the 'therapeutic' 6-thioguanine level; and 40% the non-toxic 6-methylmercaptopurine level. Side effects and transaminase elevation were mild and reversible. Clinical steroid-sparing properties and lowered relapse frequency were demonstrated. 6-MP displayed unique pharmacodynamic properties that may be useful in OMS and other autoimmune disorders. Its steroid sparer capacity is limited to children in whom the therapeutic window can be reached without limiting pharmacokinetic factors or side effects.

Urban impacts on regional carbonaceous aerosols: case study in central Texas.

Rural and background sites provide valuable information on the concentration and optical properties of organic, elemental, and water-soluble organic carbon (OC, EC, and WSOC), which are relevant for understanding the climate forcing potential of regional atmospheric aerosols. To quantify climate- and air quality-relevant characteristics of carbonaceous aerosol in the central United States, a regional background site in central Texas was chosen for long-term measurement. Back trajectory (BT) analysis, ambient OC, EC, and WSOC concentrations and absorption parameters are reported for the first 15 months of a long-term campaign (May 2011-August 2012). BT analysis indicates consistent north-south airflow connecting central Texas to the Central Plains. Central Texas aerosols exhibited seasonal trends with increased fine particulate matter (< 2.5 microm aerodynamic diameter, PM2.5) and OC during the summer (PM2.5 = 10.9 microg m(-3) and OC = 3.0 microg m(-3)) and elevated EC during the winter (0.22 microg m(-3)). When compared to measurements in Dallas and Houston, TX, central Texas OC appears to have mixed urban and rural sources. However central Texas EC appears to be dominated by transport of urban emissions. WSOC averaged 63% of the annual OC, with little seasonal variability in this ratio. To monitor brown carbon (BrC), absorption was measured for the aqueous WSOC extracts. Light absorption coefficients for EC and BrC were highest during summer (EC MAC = 11 m2 g(-1) and BRC MAE365 = 0.15 m2 g(-1)). Results from optical analysis indicate that regional aerosol absorption is mostly due to EC with summertime peaks in BrC attenuation. This study represents the first reported values of WSOC absorption, MAE365, for the central United States. Implications: Background concentration and absorption measurements are essential in determining regional potential radiative forcing due to atmospheric aerosols. Back trajectory, chemical, and optical analysis of PM2.5 was used to determine climatic and air quality implications of urban outflow to a regional receptor site, representative of the central United States. Results indicate that central Texas organic carbon has mixed urban and rural sources, while elemental carbon is controlled by the transport of urban emissions. Analysis of aerosol absorption showed black carbon as the dominant absorber, with less brown carbon absorption than regional studies in California and the southeastern United States.

Advanced gastric cancer with peritoneal oligometastases treated with adaptive radiotherapy and concurrent chemotherapy.

BACKGROUND: There are no reports of concurrent chemoradiotherapy for gastric cancer with peritoneal oligometastases. CASE DESCRIPTION: A 70-year-old man with gastric cancer and peritoneal oligometastases received concurrent adaptive radiotherapy and oral S-1. After radiotherapy, S-1 was discontinued, and 2 years later the tumor had completely regressed, with no recurrence or metastasis 6 years after radiotherapy. CONCLUSION: Peritoneal oligometastatic gastric cancer may be a candidate for curative treatment with concurrent adaptive radiotherapy and oral S-1.

An Assessment of Clinical Accuracy of Vital Sign-based Triage Tools Among U.S. and Coalition Forces.

INTRODUCTION: Early appropriate allocation of resources for critically injured combat casualties is essential. This is especially important when inundated with an overwhelming number of casualties where limited resources must be efficiently allocated, such as during mass casualty events. There are multiple scoring systems utilized in the prehospital combat setting, including the shock index (SI), modified shock index (MSI), simple triage and rapid treatment (START), revised trauma score (RTS), new trauma score (NTS), Glasgow Coma Scale + age + pressure (GAP), and the mechanism + GAP (MGAP) score. The optimal score for application to the combat trauma population remains unclear. MATERIALS AND METHODS: This is a secondary analysis of a previously described dataset from the Department of Defense Trauma Registry from January 1, 2007 through March 17, 2020. We constructed univariable analyses to determine the area under the receiving operator characteristic (AUROC) for the scoring systems of interest. Our primary outcomes were early death (within 24 hours) or early massive transfusion, as defined by ≥3 units. RESULTS: There were 12,268 casualties that met inclusion criteria. There were 168 (1%) who died within the first 24 hours and 2082 (17%) that underwent significant transfusion within the first 24 hours. When assessing the predictive capabilities for death within 24 hours, the AUROCs were 0.72 (SI), 0.69 (MSI), 0.89 (START), 0.90 (RTS), 0.83 (NTS), 0.90 (GAP), and 0.91 (MGAP). The AUROCs for massive transfusion were 0.89 (SI), 0.89 (MSI), 0.82 (START), 0.81 (RTS), 0.83 (NTS), 0.85 (MGAP), and 0.86 (GAP). CONCLUSIONS: This study retrospectively applied seven triage tools to a database of 12,268 cases from the Department of Defense Trauma Registry to evaluate their performance in predicting early death or massive transfusion in combat. All scoring systems performed well with an AUROC >0.8 for both outcomes. Although the SI and MSI performed best for predicting massive transfusion (both had an AUROC of 0.89), they ranked last for assessment of mortality within 24 hours, with the other tools performing well. START, RTS, NTS, MGAP and GAP reliably identified early death and need for massive transfusion, with MGAP and GAP performing the best overall. These findings highlight the importance of assessing triage tools to best manage resources and ultimately preserve lives of traumatically wounded warfighters. Further studies are needed to explain the surprising performance discrepancy of the SI and MSI in predicting early death and massive transfusion.

Expression of CXCR3 and its ligands CXCL9, -10 and -11 in paediatric opsoclonus-myoclonus syndrome.

Opsoclonus-myoclonus syndrome (OMS) is a neuroinflammatory disorder associated with remote cancer. To understand more clearly the role of inflammatory mediators, the concentration of CXCR3 ligands CXCL10, CXCL9 and CXCL11 was measured in 245 children with OMS and 81 paediatric controls using enzyme-linked immunosorbent assay (ELISA), and CXCR3 expression on CD4(+) T cells was measured by flow cytometry. Mean cerebrospinal fluid (CSF) CXCL10 was 2·7-fold higher in untreated OMS than controls. Intrathecal production was demonstrated by significantly different CXCL10 CSF : serum ratios. The dichotomized 'high' CSF CXCL10 group had higher CSF leucocyte count (P = 0·0007) and B cell activating factor (BAFF) and CXCL13 concentrations (P < 0·0001). CSF CXCL10 did not correlate with clinical severity or relapse using grouped data, although it did in some patients. Among seven types of immunotherapy, including rituximab or chemotherapy, only adrenocorticotrophic hormone (ACTH) monotherapy showed reduced CSF CXCL10, but prospective longitudinal studies of ACTH combination therapies indicated no reduction in CXCL10 despite clinical improvement (P < 0·0001). CXCL10 concentrations were 11-fold higher in CSF and twofold higher in serum by multiplexed fluorescent bead-based immunoassay than enzyme-linked immunosorbent assay, but the two correlated (r = 0·7 and 0·83). In serum, no group differences for CXCL9 or CXCL11 were found. CXCR3 expression on CD4(+) T cells was fivefold higher in those from CSF than blood, but was not increased in OMS or altered by conventional immunotherapy. These data suggest alternative roles for CXCL10 in OMS. Over-expression of CXCL10 was not reduced by clinical immunotherapies as a whole, indicating the need for better therapeutic approaches.

Benign lymphoid hyperplasia mimicking oligometastasis from non-small cell lung cancer after stereotactic ablative radiotherapy.

BACKGROUND: Benign lymphoid hyperplasia (BLH) is a rare lymphoproliferative disorder of normal polyclonal B lymphocytes, but is sometimes difficult to distinguish from malignancy. CASE: An 87-year-old man with a history of localized non-small cell lung cancer (NSCLC) was referred for evaluation and treatment of an elastic hard tumor in the left supraclavicular fossa one year after stereotactic ablative radiotherapy (SABR). Whole-body PET scan showed high 18F-fluorodeoxyglucose uptake in the left supraclavicular fossa, and a dia-gnosis of oligometastasis was made. The tumor was homogeneously high signal on T2-weighted image with homogeneous enhancement after contrast administration. Since the palpation and MRI findings were inconsistent with those of metastatic NSCLC, a bio-psy was performed. Pathological and immunohistochemical investigation revealed the lesion to be BLH. CONCLUSION: In a patient with suspected oligometastasis after SABR for NSCLC, caution should be exercised before undergoing SABR for oligometastasis because BLH may be present.

Contemporary sources dominate carbonaceous aerosol on the North Slope of Alaska.

As the Arctic continues to change and warm rapidly, it is increasingly important to understand the organic carbon (OC) contribution to Arctic aerosol. Biogenic sources of primary and secondary OC in the Arctic will be impacted by climate change, including warming temperatures and earlier snow and ice melt. This study focuses on identifying potential sources and regional influences on the seasonal concentration of organic aerosol through analysis of chemical and isotopic composition. Aerosol samples were collected at two sites on the North Slope of Alaska (Utqiagvik, UQK, and Oliktok Point, OLK, which is in an Arctic oilfield) over three summers from 2015 to 2017. The elemental carbon (EC) trends at each site were used to understand local combustion influences. Local sources drove EC concentrations at Oliktok Point, where high EC was attributed to oil and gas extraction activity, including diesel combustion emissions. Utqiagvik had very low EC in the summer. OC was more similar in concentration and well correlated between the two sites with high contributions of contemporary carbon by radiocarbon apportionment (UQK = 74%, OLK = 63%), which could include both marine and terrestrial sources of contemporary carbon (e.g. primary and secondary biogenic, biomass burning and/or associated SOA, and bioaerosols). OC concentrations are strongly correlated to maximum ambient temperatures on the NSA during the summer, which may have implications for predicting future OC aerosol concentrations in a warming Arctic. Biomass burning was determined to be an episodic influence at both sites, based on interpretation of combined aerosol composition, air mass trajectories, and remote sensing of smoke plumes. The results from this study overall strongly suggests contribution from regional sources of contemporary organic aerosol on the NSA, but additional analysis is needed to better constrain contributions from both biogenic sources (terrestrial and/or marine) and bioaerosol to better understand temperature-related aerosol processes in the Arctic.

CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery.

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Urban edge trees: Urban form and meteorology drive elemental carbon deposition to canopies and soils.

Urban tree canopies are a significant sink for atmospheric elemental carbon (EC)--an air pollutant that is a powerful climate-forcing agent and threat to human health. Understanding what controls EC deposition to urban trees is therefore important for evaluating the potential role of vegetation in air pollution mitigation strategies. We estimated wet, dry, and throughfall EC deposition for oak trees at 53 sites in Denton, TX. Spatial data and airborne discrete-return LiDAR were used to compute predictors of EC deposition, including urban form characteristics, and meteorologic and topographic factors. Dry and throughfall EC deposition varied 14-fold across this urban ecosystem and exhibited significant variability from spring to fall. Generalized additive modeling and multiple linear regression analyses showed that urban form strongly influenced tree-scale variability in dry EC deposition: traffic count as well as road length and building height within 100-150 m of trees were positively related to leaf-scale dry deposition. Rainfall amount and extreme wind-driven rain from the direction of major pollution sources were significant drivers of throughfall EC. Our findings indicate that complex configurations of roads, buildings, and vegetation produce "urban edge trees" that contribute to heterogeneous EC deposition patterns across urban systems, with implications for greenspace planning.

Protein multi-scale organization through graph partitioning and robustness analysis: application to the myosin-myosin light chain interaction.

Despite the recognized importance of the multi-scale spatio-temporal organization of proteins, most computational tools can only access a limited spectrum of time and spatial scales, thereby ignoring the effects on protein behavior of the intricate coupling between the different scales. Starting from a physico-chemical atomistic network of interactions that encodes the structure of the protein, we introduce a methodology based on multi-scale graph partitioning that can uncover partitions and levels of organization of proteins that span the whole range of scales, revealing biological features occurring at different levels of organization and tracking their effect across scales. Additionally, we introduce a measure of robustness to quantify the relevance of the partitions through the generation of biochemically-motivated surrogate random graph models. We apply the method to four distinct conformations of myosin tail interacting protein, a protein from the molecular motor of the malaria parasite, and study properties that have been experimentally addressed such as the closing mechanism, the presence of conserved clusters, and the identification through computational mutational analysis of key residues for binding.

Metal artifact-free MRI-guided re-irradiation for recurrent spinal metastases from thyroid cancer.

BACKGROUND: Soft tissue tumors near metal implants are sometimes difficult to treat with real-time image-guided radiation therapy. Low-field MRI was utilized to clearly delineate the tumor and spinal cord while avoiding metal artifacts, and re-irradiation was performed for recurrent spinal metastases. CASE DESCRIPTION: A 57-year-old woman with a history of thyroid cancer was referred for re-irradiation for recurrent painful bone metastases in the thoracolumbar spine. She had already undergone conventional radiation therapy followed by stereotactic ablative radiotherapy and multiple fusion surgeries. Since the radiation dose to the spinal cord should have been limited, metal artifact-free low-field MRI-guided re-irradiation was performed with no significant adverse events. CONCLUSION: Low-field MRI-guided re-irradiation may be feasible for recurrent spinal metastases, even after metal implants have been placed.

Effect of substrate stiffness on human intestinal enteroids' infectivity by enteroaggregative Escherichia coli.

Human intestinal enteroids (HIE) models have contributed significantly to our understanding of diarrheal diseases and other intestinal infections, but their routine culture conditions fail to mimic the mechanical environment of the native intestinal wall. Because the mechanical characteristics of the intestine significantly alter how pathogens interact with the intestinal epithelium, we used different concentrations of polyethylene glycol (PEG) to generate soft (~2 kPa), medium (~10 kPa), and stiff (~100 kPa) hydrogel biomaterial scaffolds. The height of HIEs cultured in monolayers atop these hydrogels was 18 µm whereas HIEs grown on rigid tissue culture surfaces (with stiffness in the GPa range) were 10 µm. Substrate stiffness also influenced the amount of enteroaggregative E. coli (EAEC strain 042) adhered to the HIEs. We quantified a striking difference in adherence pattern; on the medium and soft gels, the bacteria formed clusters of > 100 and even > 1000 on both duodenal and jejunal HIEs (such as would be found in biofilms), but did not on glass slides and stiff hydrogels. All hydrogel cultured HIEs showed significant enrichment for gene and signaling pathways related to epithelial differentiation, cell junctions and adhesions, extracellular matrix, mucins, and cell signaling compared to the HIEs cultured on rigid tissue culture surfaces. Collectively, these results indicate that the HIE monolayers cultured on the hydrogels are primed for a robust engagement with their mechanical environment, and that the soft hydrogels promote the formation of larger EAEC aggregates, likely through an indirect differential effect on mucus. STATEMENT OF SIGNIFICANCE: Enteroids are a form of in vitro experimental mini-guts created from intestinal stem cells. Enteroids are usually cultured in 3D within Matrigel atop rigid glass or plastic substrates, which fail to mimic the native intestinal mechanical environment. Because intestinal mechanics significantly alter how pathogens interact with the intestinal epithelium, we grew human intestinal enteroids in 2D atop polyethylene glycol (PEG) hydrogel scaffolds that were soft, medium, or stiff. Compared with enteroids grown in 2D atop glass or plastic, the enteroids grown on hydrogels were taller and more enriched in mechanobiology-related gene signaling pathways. Additionally, enteroids on the softest hydrogels supported adhesion of large aggregates of enteroaggregative E. coli. Thus, this platform offers a more biomimetic model for studying enteric diseases.

Improved green fluorescent protein by molecular evolution using DNA shuffling.

Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.

In vitro selection of peptides acting at a new site of NMDA glutamate receptors.

Oligomeric N-methyl D-aspartate receptor (NMDAR) in brain is a ligand-gated ion channel that becomes selectively permeable to ions upon binding to ligands. For NMDAR channel, the binding of glutamate and glycine results in opening of the calcium permeable channel. Because the calcium influx mediated by NMDAR is important for synaptic plasticity and excitotoxicity, the function of NMDA receptors has been implicated in both health and disease. Native NMDA receptors are thought to be heteromeric pentamers with a central ion conduction pathway. There are five genes (NR1, 2A, 2B, 2C, and 2D) encoding various subunits that have been cloned, and NR1 is thought to be the essential subunit since it forms a functional channel by itself. To study NMDAR structure and function, we have searched for peptide modulators of NR1 using random peptide bacteriophage libraries. The peptides were identified based on their specific association with a purified receptor fusion protein that contains the putative ligand binding domain. We report the identification of one group of cyclic peptides (Mag-1) with a consensus sequence of CDGLRHMWFC. Using biochemical binding analysis and patch clamp electrophysiological recording, we show that the synthetic Mag-1 peptides cause noncompetitive inhibition of the receptor channel activity.