Small Substrates Research Articles

Exploring the Proteolysis Mechanism of the Proteasomes.

The proteasome is a key protease in the eukaryotic cells which is responsible for various important cellular processes such as the control of the cell cycle, immune responses, protein homeostasis, inflammation, apoptosis, and the response to proteotoxic stress. Acting as a major molecular machine for protein degradation, proteasome first identifies damaged or obsolete regulatory proteins by attaching ubiquitin chains and subsequently utilizes conserved pore loops of the heterohexameric ring of AAA+ (ATPases associated with diverse cellular activities) to pull and mechanically unfold and translocate the misfolded protein to the active site for proteolysis. A detailed knowledge of the reaction mechanism for this proteasomal proteolysis is of central importance, both for fundamental understanding and for drug discovery. The present study investigates the mechanism of the proteolysis by the proteasome with full consideration of the protein's flexibility and its impact on the reaction free energy. Major attention is paid to the role of the protein electrostatics in determining the activation barriers. The reaction mechanism is studied by considering a small artificial fluorogenic peptide substrate (Suc-LLVY-AMC) and evaluating the activation barriers and reaction free energies for the acylation and deacylation steps, by using the empirical valence bond method. Our results shed light on the proteolysis mechanism and thus should be important for further studies of the proteasome action.

Effects of Cathode Voltage Pulse Width in High Power Impulse Magnetron Sputtering on the Deposited Chromium Thin Films

Environmentally-safe high-power impulse magnetron sputtering (HiPIMS) technology was utilized to deposit chromium films. This research focused on the influences of the HiPIMS pulse widths on the microstructure of films deposited at different deposition pressures and substrate bias voltages. Under the conditions of the same average HiPIMS power and duty cycle, the deposition rate of the Cr thin film at working pressure 0.8 Pa is slightly higher than at 1.2 Pa. Also, the difference between deposition rates under two pressures decreases with the discharge pulse width. The deposition rate of the short pulse width 60 μs is lowest, but those of 200 and 360 μs are approximately the same. With no or small direct current substrate biasing, the microstructure of films coated at short pulse width is similar to the typical magnetron sputtering deposited films. Elongating the pulse width enhances the ion flux toward the substrate and changes the film structure from individual prism-like columns into tangled 3-point/4-point star columns. Substantial synchronized substrate biasing and longer pulse width changes the preferred orientation of Cr films from Cr (110) to Cr (200) and Cr (211). The films deposited at longer pulse width exhibit a higher hardness due to the reducing of intercolumn voids.

Seasonal influence and local factors affecting macroinvertebrate structure in a high-altitude Andean stream

Small water bodies are critical for maintaining freshwater biodiversity, but are among the least investigated aquatic environments. We examined physical and chemical variables at two reaches in Arroyo Tambillos, a small, high-elevation Andean stream, in NW Mendoza province, Argentina, across four seasons to examine how local factors and seasonality affected the structure of the macroinvertebrate community. The Arroyo Tambillos community was numerically dominated by ephemeropterans (mainly Massartellopsis irarrazavali) while the dipteran Chironomidae were the most species rich. Total macroinvertebrate abundance was highest in summer, driven mainly by taxa that were secondary in dominance (Austrelmis sp. and Andesiops peruvianus), while richness did not differ between seasons. However, benthic composition was different in Fall 2013 (March) compared to other seasons, largely because of the increased abundance of Chironomidae. Canonical correspondence analysis discriminated Chironomidae species distributions in Fall 2013 by substrate type (i.e., big and small boulder substrate). By contrast, discharge, velocity, and depth were the variables which most affected the macroinvertebrate abundance and distributions. Contrary to our expectations, most community changes observed occurred in fall instead of summer. Nivo-kryal stream communities like the one described here have become increasingly important for conserving mountain stream biodiversity as anthropogentic impacts and climate change increasingly impact lower stream reaches. Therefore, monitoring high-altitude streams like the Arroyo Tambillos may be critical for preventing the future loss of unique and sensitive stream biota.

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5' hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

Glyoxyl-Activated Agarose as Support for Covalently Link Novo-Pro D: Biocatalysts Performance in the Hydrolysis of Casein

This study aimed to evaluate the performance of a commercial protease (Novo-Pro D (NPD)), both in soluble and immobilized forms, in the hydrolysis of proteins (using casein as model protein). Immobilization of the protease NPD on 6% agarose activated with glyoxyl groups for 24 h at 20 °C and pH 10.0 allowed preparing immobilized biocatalyst with around 90% immobilization yield, 92% recovered activity versus small substrate, and a thermal stability 5.3-fold higher than the dialyzed soluble enzyme at 50 °C and pH 8.0. Immobilization times longer than 24 h lead to a decrease in the recovered activity and did not improve the biocatalyst stability. At 50 °C and pH 6.5, the immobilized NPD was around 20-fold more stable than the dialyzed soluble protease. Versus casein, the immobilized NDP presented a 10% level of activity, but it allowed hydrolyzing casein (26 g/L) at 50 °C and pH 6.5 up to a 40% degree of hydrolysis (DH) after 2 h reaction, while under the same conditions, only a 34% DH was achieved with soluble NPD. In addition, the immobilized NPD showed good reusability, maintaining the DH of casein for at least ten 2h-reaction batches.

High Throughput Screening for New Fungal Polyester Hydrolyzing Enzymes.

There is a strong need for novel and more efficient polyester hydrolyzing enzymes in order to enable the development of more environmentally friendly plastics recycling processes allowing the closure of the carbon cycle. In this work, a high throughput system on microplate scale was used to screen a high number of fungi for their ability to produce polyester-hydrolyzing enzymes. For induction of responsible enzymes, the fungi were cultivated in presence of aliphatic and aromatic polyesters [poly(1,4-butylene adipate co terephthalate) (PBAT), poly(lactic acid) (PLA) and poly(1,4-butylene succinate) (PBS)], and the esterase activity in the culture supernatants was compared to the culture supernatants of fungi grown without polymers. The results indicate that the esterase activity of the culture supernatants was induced in about 10% of the tested fungi when grown with polyesters in the medium, as indicated by increased activity (to >50 mU/mL) toward the small model substrate para-nitrophenylbutyrate (pNPB). Incubation of these 50 active culture supernatants with different polyesters (PBAT, PLA, PBS) led to hydrolysis of at least one of the polymers according to liquid chromatography-based quantification of the hydrolysis products terephthalic acid, lactic acid and succinic acid, respectively. Interestingly, the specificities for the investigated polyesters varied among the supernatants of the different fungi.

Large and Small Substrate Binding to DesD via ITC Assay Development

Desferrioxamines (“dfo”s) are iron‐chelating compounds which have been used medicinally in cases of iron overload. The synthesis of dfoE involves an enzyme called DesD which is a dimer that has an active site on each chain, but the mechanism of it binding both large or small substrates is unknown. We are studying DesD as a model enzyme for the non‐ribosomal peptide synthetase independent siderophore (NIS) pathway and as a potential new drug target. The purpose of this research is to analyze the co‐factor specificity and large substrate verse small substrate specificity of DesD by comparing substrate dissociation constants to analog dissociation constants (KD, where a smaller number correlates to tighter binding). Future drugs will have to out‐compete the substrates in binding, so we will use these studies to determine which substrate components contribute to maximum binding. Techniques in analytical chemistry will be used to regenerate deferoxamines that were bound to cofactors in previous studies with the DesD enzyme. The binding properties of DesD were explored in a 25% glycerol Isothermal Titration Calorimetry (ITC) buffer. Prior experimentation with this protein was conducted in a buffer that resulted in precipitation of DesD out of solution, thus prompting the need for repeated binding assays in a buffer that maintains synthase solubility. GraphPad Prism 8.0 will be used to analyze the KD and occupancy of DesD. High Performance Liquid Chromatography (HPLC) will be used to separate deferoxamines from substrates they were bound to. An Ocean Optics Spectrometer will be used to quantify the deferoxamines by pairing these substrates to a metal compound that will allow us to identify its absorbance and determine if isolation was effective.We confirmed previous results that the KD of DesD binding to ATP is in the M range, compared to non‐ATP cofactors which result in a KD in the M range. Future studies will compare dfoG (substrate) binding to DesD compared to analog dfoB or product dfoE, and also explore smaller substrates, hydroxysuccinylcadaverine (HSC), vs. analogs. Troubleshooting runs have indicated that isolation of the deferoxamines is feasible. It is expected that substrates like dfoG will be separated from ATP and ADP cofactors. We have confirmed that DesD has a strong binding preference for ATP over non‐ATP cofactors in a buffer that maximizes solubility. This data indicates that the phosphates are essential to tight binding in any future competitive inhibitors. Regeneration of each deferoxamine appeared to be a cost‐effective assay that will allow for these compounds to be used in later experimentation. Future work will expand our understanding of critical regions in the binding site, and aid in developing new inhibitors that could block the NIS pathways and be lead drugs for a new class of antibiotics.

Determining the Kinetics of Desferroxiamine D from Streptomyces coelicolor using Isothermal Titration Calorimetry

The long‐term objective for this research is to create a new class of antibiotics that targets the non‐ribosomal peptide synthetase independent siderophore (NIS) pathway in bacteria. The focus of my current research is to quantify the first and second order kinetic constants and obtain the kinetics curve of our model system, Desferroxiamine D (DesD), from Streptomyces coelicolor. DesD reacts with ATP as a cofactor, and either the small (hydroxysuccinylcadaverine, HSC), medium or large (desferrioxamine G, or dfoG) substrates in an iterative manner. We have so far collected activity curves with ATP and dfoG limiting, and are pursuing HSC curves. To do this we used Isothermal Titration Calorimetry (ITC) to collect data and GraphPad Prism 8.2 along with Malvern MicroCal Peaq ITC software to fit the kinetics curves and determine the kinetics constants. We successfully gathered and analyzed ITC data for both the catalytic (wild type, Wt) and non‐catalytic (mutant, R306Q) forms of DesD and determined the optimal buffer conditions for solubility. We also gathered data regarding the activity of our Wt DesD enzyme in order to determine the lifespan and the time of optimal catalytic activity. We have found that our enzyme is still active within 96 hours where it was previously thought to have a 36–48 hour catalytic lifespan.In conducting these experiments, we observed that an industry standard amount of magnesium chloride (MgCl2) in our solution caused an oscillating signal during equilibration of the ITC prior to enzyme injection. We hypothesized that the data reflects metal chelation of the Mg by our substrate (dfoG). We plan on conducting a binding experiment using the ITC in order to quantify the substrate (dfoG) and MgCl2 interaction, and ultimately recommend a new standard amount of MgCl2 to the field for future experiments. Other future directions include determining the kinetic constants of Wt DesD interacting with its small substrate HSC (which must be synthesized and purified,) and determining the catalytic role of other residues in the active site by site‐directed mutagenesis and ITC. This experimental assay and data will be the foundation for future drug design targeting DesD.Support or Funding InformationThis research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.), and by the John Stauffer Charitable Trust.

Substrate‐Bound Complex of DesD, a Model Enzyme in the Virulence‐Related NIS Synthetase Family

The understudied NIS Synthetase family have been increasingly associated with virulence in bacterial species, due to their key role in the synthesis of hydroxamate and carboxylate “stealth” siderophores. We have identified a model family member, DesD from Streptomyces coelicolor, to characterize structurally using a combination of wild‐type and Arg306Gln variant in apo, cofactor product (AMP)‐bound with analog cadaverine, and cofactor bound with analog dfoB complexes. Kinetics in the family have been limited by solubility and reporter‐based assays, so we have developed a label‐free kinetics assay utilizing an unusual single‐injection isothermal titration calorimetry based method. We report second order rate constants 50‐fold higher than the previous estimations for DesD in both the cofactor‐limiting and dfoG substrate limiting curves. Our Arg306Gln DesD variant was also tested in identical buffer and substrate conditions, and confirmed undetectable activity. This is the first complete complex structure (cofactor, amine and carboxylate) reported for an NIS synthetase, and the first label‐free assay to unambiguously determine kinetics for the NIS synthetase family. Our future directions aim to quantify binding thermodynamics, characterize the small substrates (two molecules of hydroxysuccinylcadaverine, which must be synthesized) and delineate the active site mechanism with site‐directed mutagenesis.Support or Funding InformationThis research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.), by the John Stauffer Charitable Trust.The DesD dimer has two active sites, only one of which is filled with substrate in the structures (left). The small substrate analog cadaverine (yellow atom coloring) is shown bound to wild‐type DesD at the bottom right, and the large substrate dfoB (magenta atom coloring) is bound to Arg306Gln in the top right. Both structures are shown with cofactor ATP (teal atom coloring) bound as well.Figure 1

Metabolomics Profiling in Response to a Cold Pressor Test

The cold pressor test (CPT) has been widely used to assess both cardiovascular and hemodynamic responses to stress. Despite the broad utilization of the CPT, there have been few if any studies in which metabolomic profiling was used to study the metabolic response to such a stimulus. Metabolomics allows insight into varying chemical processes, displaying detailed profiles of metabolites, small molecule substrates, intermediates and products of metabolism. The purpose of this study was to investigate the influence of sympathetic activation induced by a 1‐minute cold‐water immersion CPT, upon muscle metabolic responses, through metabolomics profiling of muscle interstitial fluid in adults. Five healthy adults (2 males, 3 females; 29.4 ± 9.4 yrs.; 77.6 ± 19.3 kg) were recruited for this pilot study. Three microdialysis probes (CMA 20, CMA/Microdialysis, Stockholm, Sweden) were percutaneously inserted into the vastus lateralis muscle of the dominant leg using sterile technique. Probes were then perfused with sterile saline containing 5 mM ethanol through the inlet tubing at a rate of 2.0 μL/min, and dialysate from the outlet tubing was collected in 150 μL polyethylene collection vials. One hour (3 × 20‐minute collections) of basal dialysates were collected and pooled. Participants then completed, in a seated position, a CPT in which the right hand was immersed up to the wrist in ice water for 1 minute. A 20‐min dialysate sample was collected during and immediately following the CPT. Microdialysis samples were sent to the West Coast Metabolomics Center (UC Davis Genome Center, Davis, CA, USA) for metabolomic profiling. Enzymatic‐fluorometric ethanol assay was also used to measure ethanol outflow: inflow (O:I) ratio to determine microvascular blood flow. The percent change from pre‐CPT to the CPT sample was calculated for every metabolite found. Data were then sorted on a magnitude basis, from smallest to largest change from basal levels. From the 413 metabolites detected, 207 were found to increase and 206 were found to decrease from baseline to post‐CPT. Nine metabolites were found within the 95th to 100th percentile of positive change from baseline to CPT, including: 1‐monostearin (median: 50.4%; range: 10.0–183.1), 3‐aminoisobutyric acid (median: 52.5%; range: 39.2–95.4), palmitoleic acid (median: 92.2%; range: 35.4–204.5); and 6 “unknown metabolites.” Furthermore, 5 “unknown metabolites” were also detected within the 95th to 100th percentile of negative change (decrease from baseline). Ethanol O:I ratio increased baseline to CPT, indicating decreased blood flow (0.483 ± 0.088 to 0.547 ± 0.027, mean ± SEM). These results indicate that the CPT can elicit both vasoconstriction and a notable metabolic response in muscle tissue, identifiable by the use of ethanol analysis and metabolomics of dialysate from microdialysis of skeletal muscle. Of specific interest, 1‐monostearin and palmitoleic acid were found to be in the group of molecules that increased the most in response to CPT. Although speculative, increased adrenergic stimulation resulting from CPT‐induced sympathetic activation may have resulted in liberation of 1‐monostearin and palmitoleic acid from membranes or other locations, or from increases in desaturase activities.

Analisis Parameter Fisika-Kimia untuk Kepentingan Rehabilitasi Ekosistem Mangrove di Pesisir Pulau Bungkutoko Kota Kendari

In Southeast Sulawesi rehabilitation of mangrove areas that have been damaged but in reality not all mangrove rehabilitation activities were successful, this was allegedly caused by a mismatch in the type of mangrove and incompatibility of rehabilitation techniques used with environmental conditions or parameters of the local coastal environment. This study is aimed to analyze the condition of coastal environmental parameters in Bungkutoko island, district of Abeli in rehabilitation proposed of mangrove ecosystem and to find a proper rehabilitation strategy for it can be applied in that area. This study was carried on June to July 2009 in the coastal of Bungkutoko island, Abeli district, Kendari Town. Data in this study is analyzed as descriptively for giving common view of that area. The measurement results of several physical-chemical parameters on the coast of Bungkutoko island at stations I, II and III are suitable for mangrove ecosystem rehabilitation activities, which have a slope of the base: flat and sloping, particle size: small substrate, binding capacity of substrate particles: moderate to loose, confinement coastline: protected and semi protected and open, wave: relatively small, sea level deviation: moderate, tidal type: mixture tends to double daily, current speed: weak, sediment suspension: normal and salinity: 25-35 ppt. Also pay attention to the right planting time on the condition of mangrove tree is in having fruits and calm water condition of sea. And for planting technic is propaguls directly planted to the ground and using seeds on the polybags.

77 GHz Metamaterial Absorbers Composed of Crossed Dipoles on Grounded Dielectric Substrate for Automotive Application

In addressing the needs of automotive radar designers, this study investigates the 77 GHz radar absorbing materials (RAMs) suitable for the automotive environment. A metamaterial structure is composed of metallic conductors of cross pattern on a grounded dielectric substrate (FR4). Computational tools (ANSYS HFSS 13.0) were used to model the interaction between electromagnetic waves and the metamaterial structures. The simulation output included the reflection coefficient. Another parameter of interest was the surface currents and electric fields, which were used to depict the resonating behavior of the metallic portions of the metamaterials. Magnetic resonance resulting from antiparallel currents between the strip conductors on front surface and ground bottom plane was observed at the frequency of minimum reflection loss (-23 dB at 77 GHz) with a small substrate thickness as low as 0.2 mm. The simulated resonance frequency and reflection loss can be explained well on the basis of the circuit theory of an LC resonator.

Identification and characterization of the gene encoding an extracellular protease from haloarchaeon Halococcus salifodinae

Extracellular proteases from haloarchaea (halolysins) can resist high salt conditions. In this study, the gene encoding a halolysin from Halococcus salifodinae was identified. The hlyA gene encoded an active halolysin with the classical Asp-His-Ser catalytic triad of serine proteases. Site-directed mutagenesis showed that the three cysteine residues in the catalytic domain were important for the extracellular proteolytic activity and displayed an additive effect on the activity. Truncation mutants of the C-terminal extension (CTE) domain displayed very low or almost no extracellular protease activity towards milk and small peptide substrates, indicating its importance for the function of HlyA. CTE can be functionally interchangeable among halolysins. Additionally, the HlyA expressing strain as a starter culture for fish sauce fermentation significantly increased the peptide release and total free amino acid content in fish sauce. This study enriches our knowledge of the key amino acid residues and domains of halolysins, and provides an opportunity for applications of halolysins in fish sauce fermentation.

The Central Role of Small Vertical Substrates for the Origin of Grasping in Early Primates.

The manual and pedal grasping abilities of primates, characterized by an opposable hallux, flat nails, and elongated digits, constitute a unique combination of features that likely promoted their characteristic use of arboreal habitats. These hand and foot specificities are central for understanding the origins and early evolution of primates and have long been associated with foraging in a fine-branch milieu. However, other arboreal mammals occupy similar niches, and it remains unclear how substrate type may have exerted a selective pressure on the acquisition of nails and a divergent pollex/hallux in primates or in what sequential order these traits evolved. Here, we video-recorded 14,564 grasps during arboreal locomotion in 11 primate species (6 strepsirrhines and 5 platyrrhines) and 11 non-primate arboreal species (1 scandentian, 3 rodents, 3 carnivorans, and 4 marsupials). We quantified our observations with 19 variables to analyze the effect of substrate orientation and diameter on hand and foot postural repertoire. We found that hand and foot postures correlate with phylogeny. Also, primates exhibited high repertoire diversity, with a strong capability for postural adjustment compared to the other studied groups. Surprisingly, nails do not confer an advantage in negotiating small substrates unless the animal is large, but the possession of a grasping pollex and hallux is crucial for climbing small vertical substrates. We propose that the divergent hallux and pollex may have resulted from a frequent use of vertical plants in early primate ecological scenarios, although nails may not have resulted from a fundamental adaptation to arboreal locomotion.

Significance of a family-6 carbohydrate-binding module in a modular feruloyl esterase for removing ferulic acid from insoluble wheat arabinoxylan

Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 23-α-l-arabinofuranosyl-xylotriose (A2XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A2XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6’s preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation.

Fibrinogen-clotting enzyme, pictobin, from Bothrops pictus snake venom. Structural and functional characterization

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friable/porous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor α2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondrial network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold β/β hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease.

Targeting the Regulatory Site of ER Aminopeptidase 1 Leads to the Discovery of a Natural Product Modulator of Antigen Presentation.

ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa that constitutes a submicromolar, highly selective, and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development.

Rainbow Darter (Etheostoma caeruleum, Storer, 1845) predation on early ontogenetic stages of Lake Sturgeon (Acipenser fulvescens, Rafinesque, 1817)

Previous molecular diet analysis identified lake sturgeon (Acipenser fulvescens, Rafinesque, 1817) DNA in the gastrointestinal tracts of stream-resident rainbow darters (Etheostoma caeruleum, Storer, 1845) during the egg incubation, free embryo, and larval drift stages. The objectives of this experimental study were to: (a) quantify levels of predation by rainbow darters on lake sturgeon at the egg and free-embryo stages; and (b) evaluate whether predation varied as a function of substrate size and rainbow darter body size. We conducted experimental trials in 23-L polycarbonate tanks 0.41 m (L) × 0.33 m (W) × 0.30 m (D) with a standardized benthic area of 0.14 m2. The tanks were randomly assigned one of two different substrate size classes: large rock (51.35 mm ± 0.91 mm) or small rock (27.68 mm ± 0.57 mm). We stocked individual rainbow darter, which were deprived of feed for 48 hr, with lake sturgeon (133 individuals/m2) in each of 12 replicates per ontogenetic stage and substrate type. The number of surviving lake sturgeon was quantified following a 24-hr predation exposure period. We used a generalized linear model with a binomial distribution to assess the influence of ontogenetic stage, substrate size, and rainbow darter body size on proportional lake sturgeon survival. Predation on lake sturgeon occurred at both egg (6.25 ± 1.16 individuals, mean ± 2SE) and free embryo (3.08 ± 1.08 individuals, mean ± 2SE) stages. Egg proportional survival was generally lower than at the free embryo stage in both substrate sizes; however, free embryo proportional survival was greater in small substrate trials. Rainbow darter total length did not affect the probability of lake sturgeon survival at either developmental stage. Results demonstrate that rainbow darters prey on early ontogenetic stages of lake sturgeon, corroborating previous results based on genetic diet analysis. Results fill a major knowledge gap concerning the vulnerability of pre-drift sturgeon to predation by an abundant river resident species that was previously discounted as a predator for early ontogenetic stages of lake sturgeon due to its small body size.

Design and Fabrication of Metamaterial Absorbers Used for RF-ID

Radio frequency identification (RF-ID) uses electromagnetic fields to automatically identify and track tags attached to objects. In response to the need to suppress the electromagnetic interference between adjacent RF-ID equipment or systems, this study aims to achieve a thin and multi-resonance absorber using metamaterials composed of patterned grids on a grounded dielectric substrate in the frequency region of UHF and microwaves. A computational tool (ANSYS HFSS) was used to model and estimate the reflection coefficient and surface current distribution. The samples used to measure reflection loss were fabricated by the printed circuit board (PCB) method in which a copper film was deposited on both sides of a photosensitized board. The reflection loss was measured using a free space measurement system, composed of a pair of horn antennas and network analyzer. The grid-patterned metamaterial absorbers exhibited dual-band absorption peaks at 0.88 GHz and 2.45 GHz with a small substrate thickness (about 3.7 mm) that can be usefully applied to electromagnetic compatibility in RF-ID system. Magnetic coupling is achieved via antiparallel currents in the grid conductor on the top layer and the ground conductor on the bottom layer. Since the magnetic response is influenced by the dimension of the resonators, it is possible to achieve two resonances by scaling the resonating structures. The metamaterial absorbers also exhibited good oblique incidence performance. A high level of absorption (above 10 dB) was maintained at up to 30 degrees of incidence angle for both TE and TM polarization.