bluuebunny/crossref_metadata_2025_split huggingface dataset:
β‐Hairpins as stability tags for recombinant expression of recalcitrant small helical proteins (951.1)
Authors: Lokensgard Melissa , Love John 
Published: June 2021

We have empirically established different expression levels in E. coli for two recombinant proteins with high similarity in sequence, size, and native function. Streptococcal Protein G (Gβ1) and many mutants can be easily expressed in high yield in BL21 (DE3) E. coli. Staphylococcal Protein A (SpAB) can only be expressed with amino‐terminal tags or in periplasmic expression vectors. The primary difference between Gβ1 and SpAB is secondary structure composition‐‐Gβ1 contains two short beta‐hairpins and an alpha helix, and SpAB is a three‐helix bundle. Reasoning that the beta‐hairpins may impart unique stability, several chimeras of SpAB and the Gβ1 beta‐hairpins were created and induced expression levels analyzed by SDS‐PAGE. While the addition the very stable C‐terminal hairpin to the N‐terminus of SpAB (CH‐PA) results in higher yield, this same tag at the C‐terminus of SpAB (PA‐CH) does not. However, addition of 12 amino acids of the N‐terminal hairpin of Gβ1 to either terminus of SpAB (NH‐PA and PA‐NH) enhances recombinant expression significantly. Interestingly, both CH‐PA and NH‐PA sequences begin with hydrophobic residues, but do not appear to be significantly degraded despite well‐established correlations between N‐terminal hydrophobics and proteolysis. An expression yield increase relative to the untagged protein by addition of NH to either terminus is also observed for two entirely unrelated proteins of similar size and secondary structure to SpAB. All of the mutants containing the beta‐hairpin sequence are soluble at neutral pH. Our result may be due to decreased degradation of the chimeric host E. coli proteolytic machinery. Current and future work will explore what changes in structure and physical properties account for the observed yield increase.Grant Funding Source: NSF
Vitamin D status and metabolism in pregnant, lactating, and nonpregnant women consuming controlled intakes of vitamin D (959.7)
Authors: Park Hey Jun , Brannon Patsy , West Allyson , Yan Jian , Jiang Xinyin , Perry Cydne , Malysheva Olga , Mehta Saurabh , Caudill Marie 
Published: June 2021

As part of a 10‐wk controlled feeding study, we investigated the impact of reproductive state on circulating levels of vitamin D metabolites among pregnant (n=26, 27wk gestation), lactating (n=28, 5wk postpartum), and nonpregnant (n=21) women consuming a daily intake of 300 IU vitamin D derived from food (90IU/d) and a prenatal supplement (200IU/d). At study‐end, serum 25‐hydroxyvitamin D (25[OH]D, mean±SD nmol/L) was higher in pregnant women (98±32) than lactating (81±19, P=0.044) and nonpregnant (78±25, P=0.032) women. Similar results were achieved after controlling for confounding factors such as season in a multivariate model. Notably, 300 IU (half of the current RDA) achieved 25(OH)D levels above the RDA target value of 50nmol/L in most of study participants (92%). Study‐end plasma 1,25‐dihydroxyvitamin D (mean [95% CI] pmol/L) was ~2 times higher in pregnant women (303 [252‐364]) than lactating (139 [116‐167], P<.0001) and nonpregnant (163 [139‐191], P<.0001) women. Similarly, study‐end vitamin D binding protein levels (mean [95% CI] mcg/mL) were ~1.8 times higher in pregnant women (370 [291‐470]) than lactating (161 [131‐198], P<.0001) and nonpregnant (205[166‐255], P=0.001) women. In sum, pregnancy dramatically influenced circulating vitamin D metabolites under controlled intakes of vitamin D and related nutrients (e.g., calcium and phosphorus).Grant Funding Source: USDA, AEB‐ENC and the Beef Checkoff
Thermodynamics and kinetics of calmodulin/roponic chimeras (969.1)
Authors: Wei Chin‐Chuan , Jensen Drake , Bennett Katherine 
Published: June 2021

Calmodulin (CaM) and Troponic C (TnC) belong to an EF‐hand protein family which contains 4 EF‐hands and share very similar primary sequences and three dimension structures. However, their specificities to target proteins are quite different. For example, enzymes activated or recognized by CaM are generally not activated by TnC. In the past, chimera proteins containing EF‐hands from CaM and TnC have been used to understand the function of individual EF‐hands of CaM, assuming that the sequence difference between CaM and TnC plays the major role in target recognition. However, it is unclear whether such EF‐hand switching affects the structure and function. Here we generated four chimera proteins (1TnC, 2TnC, 3TnC, and 4TnC), in which the particular EF‐hand of CaM is replaced with the corresponding EF‐hand of TnC, and investigated their structural change upon Ca2+ binding using isothermal titration calorimetry, fluorescence, different scanning calorimetry, and circular dichroism. Furthermore, the interaction of these chimera proteins to the peptides corresponding to the CaM binding sequence of CaM dependent kinase II, Nitric Oxide Synthase, Orai, and Fas receptor, was performed in a similar manner and the resulting data were used to compare to structures built from molecular modeling.Grant Funding Source: This work is supported in part by National Science Foundation.
Fluorescent biosensors for probing CDK4/cyclin D activity and developing non‐ATP pocket Inhibitors for melanoma, lung cancer and lymphoma (972.2)
Authors: Morris May , Prevel Camille , Pellerano Morgan , Van Thi Nhu Ngoc 
Published: June 2021

CDK4/cyclin D kinase constitutes an attractive pharmacological target in lung cancer, melanoma and lymphoma, associated with mutation or amplification of CDK4, cyclin D or p16INK4a, but efforts to develop tools for detection of this kinase in its native environment, as well as selective inhibitors for therapeutic purposes have remained limited.To this aim we have engineered a fluorescent polypeptide biosensor that reports on CDK4/cyclin D activity in a sensitive and continuous fashion in vitro, in living cells and in biopsies and which allows to monitor response to therapeutics in animal tumour models. We have further designed two biosensors which have been applied to identify competitors of essential protein/protein interfaces between CDK4 and Cyclin D, and allosteric inhibitors that perturb the conformational dynamics of CDK4, respectively, by high throughput screening. These studies highlight the importance of fluorescent biosensors for fundamental research, biomedical developments and drug discovery programmes, providing novel and sensitive approaches to monitor cancer‐associated alterations in protein kinase activities and develop non‐ATP pocket inhibitors.Grant Funding Source: Grants to MCM “Chercheuse d’Avenir” Région Languedoc‐Roussillon, ARC and INCA
Factors affecting manganese accumulations in gill mitochondria of Crassostrea virginica (996.1)
Authors: Shoneye Temitope , Nuhar Ahmed , Catapane Edward , Carroll Margaret 
Published: June 2021

Manganese (Mn) is a neurotoxin causing Manganism in people exposed to high environmental levels. Oxidative stress is implicated as a factor of Mn toxicity. The mechanisms is attributed to toxic levels of free radicals inducing mitochondrial dysfunction. Reports indicate Mn accumulates in mitochondria, however controversy exists to the extent of Mn accumulation in mitochondria and whether this contributes to the cause of Manganism. We have been using the oyster, Crassostrea virginica, to study Mn neurotoxicty. We found Mn disrupts the animals’ dopaminergic system and mitochondrial respiration. Mn is believed to use the mitochondrial Ca2+ uniporter and H+ or Na+/Ca2+ exchanger for movements in and out of mitochondria. We hypothesize Ca2+ channel blockers will alter mitochondrial Mn accumulations. We prepared mitochondrial fractions from gills. Mitochondria were treated with Ca2+ channel blockers prior to Mn exposure, pelleted and washed, then digested in HNO3 and analyzed in an AA. Mitochondria treated with Mn (1 ‐ 5 mM) exhibited a dose dependent accumulation of up to 1000% compared to controls. The channels blockers, LaCl3, diltiazem, verapamil and ruthenium red (0.25 ‐ 2 mM) reduced accumulations. LaCl3 was the most effective. The study shows mitochondria accumulate Mn and the mechanism of influx and efflux can be affected by drugs that interact with mitochondrial Ca2+ uniporter and H+ or Na+/Ca2+ exchanger.Grant Funding Source: NYSED‐ 0516041171, NSF‐0622197
Human DNA Polymerase β’s endonuclease activity prevents mitotic chromosome instability (LB119)
Authors: Carron Emily , Chen Desheng , Makridakis Nick 
Published: June 2021

Most tumors have numerical and structural chromosome aberrations that can provide selective growth advantages. It has been reported that overexpression of DNA Polymerase B (Pol β) contributes to chromosome instability by inducing aneuplody, telomere fusions, and a deficient mitotic checkpoint. However, the mechanism is currently unknown. Here, we report that Pol β’s endonuclease activity plays an important role in maintaining chromosome stability by collaborating with Topoisomerase IIα. Our results indicate that Pol β has endonuclease activity, and that this activity is required to prevent chromosomal instability during mitosis as deletion of Pol β induces aneuploidy, the formation of anaphase bridges and micronuclei. Moreover, cells expressing the endonuclease deficient E216K mutant accumulate more anaphase bridges and micronuclei than those expressing wild‐type Pol β. Consistent with these observations, Pol β localizes near the centromere in metaphase. This data suggests that Pol β’s endonuclease activity plays a role in mitotic chromosome segregation.Grant Funding Source: DOD grant PC094628, and NIH grant 8 P20 GM103518 (to NM
Comparative metacarpal volume and relative thumb muscle mass in humans and non‐human primates (LB35)
Authors: Perez Adela , Patel Biren 
Published: June 2021

Fine precision (pad‐to‐pad) grasping is facilitated by having a relative long thumb and short fingers. Power grasping is aided by having large thumb muscle forces generated by large thumb muscles (e.g., adductor pollicis). Reconstructing precision grasping in fossils is easily done by examining bone lengths. Reconstructing muscle force is more difficult because soft tissue seldom preserves in fossils. The goal of this study was to find a metric that can estimate thumb muscle mass from skeletal elements. Accordingly, we examined the relationship between thumb muscle mass and metacarpal 1 (Mc1) volume in a sample (>220) of human and non‐human primates (>20 species). Relative thumb muscle masses (all thumb muscles, thenar muscles only, adductor pollicis only) were calculated as a percentage of total hand muscles mass. Relative Mc1 volume was calculated as a percentage of Mc2‐5 volume. Metacarpal volumes were obtain from 3D surfaces models derived from CT scanning. Regression analyses demonstrated a significant relationship (p<0.01) between relative Mc1 volume and each measure of relative thumb muscle mass. Relative muscle volumes calculated for the fossil hominin Ardipithecus ramidus are lower than modern humans, further supporting the claims that its hands were more ape‐like in function.Grant Funding Source: Supported by the National Science Foundation (BCS 1317047) and USC URAP
Therapeutic efficacy of cerium oxide nanoparticles in treatment of sepsis induced renal failure (LB608)
Authors: Manne Nandini , Arvapalli Ravikumar , Nepal Niraj , Nandyala Geeta , Rice Kevin , Shinichi Asano , Blough Eric 
Published: June 2021

Severe sepsis is a complex disease of multifactorial etiology that is characterized by systemic inflammation which can rapidly progress to multi‐organ dysfunction and death. Although most of the deaths due to sepsis are related to the release of inflammatory mediators, treatments to date have largely centered on the administration of antibiotics, i.v. fluids, NSAIDS, and vasopressors. Studies have shown that the release of inflammatory mediators is mediated, at least in part, through the generation of reactive oxygen species (ROS). Other work has shown that cerium oxide (CeO2) nanoparticles can function to scavenge ROS and that these particles exhibit anti‐bacterial activity. On the basis of these data, we hypothesized a CeO2 nanoparticle based treatment strategy would diminish sepsis‐induced mortality, sepsis‐associated inflammation and that these improvements would be associated with improved renal function. Male Sprague Dawley rats were subjected to polymicrobial sepsis and treated with 0.5mg/kg of CeO2 nanoparticles intravenously. Our preliminary data suggests that the CeO2 nanoparticle intervention improved animal survivability and promoted the restoration of core body temperature. Changes in survivability and thermoregulation were associated with an attenuation in the sepsis‐induced release of β2 microglobulin, cystatin, gluthatione‐s transferase alpha, gluthatione‐s transferase mu, kidney injury molecule‐1, osteopontin and a reduction in serum total ROS. Histological analysis demonstrated that CeO2 nanoparticles decreased sepsis‐induced proximal tubular dilatation and loss of brush border. Taken together, these data suggest that CeO2 nanoparticle based interventions may exhibit potential for the treatment of sepsis‐induced renal failure and death.Grant Funding Source: This work was supported in part from DOE grant (DE‐PS02‐09ER‐01 to E.R.B)
Characterization of YML020W in Saccharomyces cerevisiae
Authors: Shoemaker Katherine , Keeney Jill 
Published: June 2021

Saccharomyces cerevisiae is a single‐celled, eukaryotic yeast commonly known as baker's or brewer's yeast. As a model organism, genes can be easily inserted and removed allowing researchers to utilize deletion genetics to better understand the function of open reading frames (ORFs) within the S. cerevisiae genome. ORFs are characterized using gene ontology, which includes three categories: biological processes, cellular components, and molecular functions. The characterization of genes within the S. cerevisiae genome is of importance in order to understand the functionality of these uncharacterized genes. Through a better understanding of how genes and their products interact with one another, we can gain insight into the function of S. cerevisiae as a model organism. The purpose of this study is to characterize the S. cerevisiae ORF YML020W, which was first identified in a screen for resistance to the apoptosis‐inducing drug, KP1019. In order to further the understanding of the structure and function of this ORF, a deletion strain of YML020W was constructed. Genetic interactions with DUN1 and YML020W were characterized through the construction of a double deletion mutant. To determine the location of the gene within the cell, the protein product associated with YML020W was fluorescently labeled at the C‐terminus and observed using fluorescence microscopy where clusters of YML020Wp were visible. These strategies will add details to gene ontology and provide information concerning of the importance of YML020W in S. cerevisiae.Support or Funding InformationThis work was supported by Juniata College, Howard Hughes Medical Institute, and the National Science Foundation
A new approach to characterize the molecular interactions between TiO2 nanoparticles and dietary flavonoids using surface‐enhanced Raman spectroscopy
Authors: Cao Xiaoqiong , Gao Zili , Zheng Jinkai , He Lili , Xiao Hang 
Published: June 2021

TiO2 nanoparticles are commonly presented in food additives (E171), and have been associated with potential adverse effects on health. TiO2 nanoparticles has a specific surface reactivity and bind bidentate ligands from the environment. Flavonoids are widely found in fruits and vegetables, and can act as electro‐donating agents for TiO2 nanoparticles. Knowledge on the molecular interactions between TiO2 nanoparticles and food components such as flavonoid compounds will facilitate the understanding of the fate of TiO2 nanoparticles in food and in the gastrointestinal tract. In this study, we utilized surface‐enhanced Raman spectroscopy (SERS) to develop a rapid and accurate characterization method for the interactions between TiO2 nanoparticles and flavonoids. TiO2 anatase nanoparticles were mixed with seven different flavonoids (i.e. apigenin, luteolin, fisetin, kaempferol, quercetin, myricetin and baicalin) to form a complex that was subjected to SERS analysis. Our results demonstrated that the complex produced distinctive SERS ‘fingerprints’, which allowed further analysis of the molecular interaction. Phenolic groups in the flavonoids played pivotal roles in the interaction between flavonoids and TiO2 nanoparticles. Difference in the numbers and positions of the phenolic groups contributed to the distinctive SERS spectra of different complex. There is no report has been found on the application of SERS in studying the interactions between TiO2 nanoparticles and dietary flavonoids. We successfully developed a simple and accurate SERS‐based method to characterize the molecular interaction between TiO2 nanoparticles and various dietary flavonoid compounds.Support or Funding InformationThis study was partially support by fund from USDA.
Using Mutagenesis and Synthetic Substrates to Explore Regiochemical Control by Soybean‐Lipoxygenase‐1
Authors: Clapp Charles H. , Kahler Kirsten M. , Plumeri Patricia , Karaisz Fred , Dodson Cassidy R. 
Published: June 2021

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Plant lipoxygenases oxygenate linoleic acid to produce either 13‐hydroperoxy‐9,11‐octadecadienoic acid (13‐HPOD), 9‐hydroperoxy‐10,12‐octadecadienoic acid (9‐HPOD) or a mixture of both. The factors determining the position of oxygen incorporation are not well understood. Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197) identified one critical residue, corresponding to phenylalanine‐557 in soybean lipoxygenase‐1 (SBLO‐1). These workers observed that lipoxygenases such as SBLO‐1 with phenylalanine or histidine in this position generally produce primarily 13‐HPOD, whereas those with valine or threonine produce primarily 9‐HPOD. In the present work, the F557V mutant of SBLO‐1 has been prepared. Consistent with the trend noted by Hornung et al., this mutant produces 30–40% 9‐HPOD from linoleic acid at pH 7.5, compared with less than 10% for the wild‐type enzyme. Hornung et al. suggested that having a smaller residue in position 557 might enable linoleic acid to bind in a head‐first manner with the carboxylate group of the substrate interacting with arginine‐707. Consistent with head‐first binding, the 9‐HPOD formed by the F557V mutant has predominantly (86%) the S‐configuration at carbon‐9. To further test the model, we have investigated the action of SBLO‐1 on linoleoyldiethylamine (LDMA), which has been previously shown to be a good substrate for wild‐type SBLO‐1 at pH 7.0, where the dimethylamino group of LDMA is expected to be protonated and positively charged, which would disfavor interaction with arginine‐707. With this substrate, the amount of oxygenation at carbon‐9 is greater than observed with linoleic acid. This result argues against the hypothesis that oxygenation at carbon‐9 arises from binding of the polar end of the substrate near arginine‐707. Surprisingly, the F557A mutant produces only 9% 9‐HPOD from linoleic acid. This result indicates that the amount of 9‐oxygenation does not always correlate with the size of the residue at position‐557. In sum, the results confirm the importance of residue 557 in determining the regiochemistry of the products, but the structural basis for these effects remains to be elucidated.Support or Funding InformationNSF CHE‐1213262
The Influence of Conserved Residues and Structural Elements on the Stability and Activity of a Thermophilic Intein
Authors: Minteer Christopher J. , Colelli Katie M. , Reitter Julie N. , Mills Kenneth V. 
Published: June 2021

Protein splicing is a self‐catalyzed reaction where an intein is excised from precursor flanking polypeptides (exteins) and the exteins are ligated together to produce a mature protein. We are interested in how specific conserved residues catalyze this process and how structural domains affect intein structure. The structure of an intein from Pyrococcus abyssi (the Pab PolII intein) has a Hyperthermophilic/Thermophilic Hairpin (HTH), only found in the structures of thermophilic inteins. It may add thermal stability by extending the central beta sheet of the typical intein fold. Mutations to destabilize inter‐and intra‐strand salt bridges in the hairpin region may result in global destabilization, and were studied through assays to assess activity and susceptibility to proteolysis. We also are interested in the influence of conserved intein residues on activity. Residues in intein block B may serve to destabilize the scissile peptide bond at the N‐terminal splice junction. We have observed cleavage of that bond at low pH, independent of splicing steps, and are exploring whether block B residues are responsible.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).
Characterizing Conformational Ensemble of H‐, K‐, and N‐Ras Using Accelerated Molecular Dynamics and Wide‐Angle X‐ray Solution Scattering (WAXS)
Authors: Guterres Hugo , Zhou Hao , Makowski Lee , Mattos Carla 
Published: June 2021

Ras GTPase is involved in cell‐signaling pathways that control cell proliferation, differentiation, and apoptosis. There are three main isoforms of Ras, H‐, K‐, and N‐Ras, which share about 95% sequence identity in their soluble domain. They have very similar three‐dimensional folds, however they have been suggested to display functional specificity. For example, in pancreatic cancer K‐Ras mutations occur at much higher frequency than either H‐ or N‐Ras, whereas in leukemia N‐Ras mutations play the dominant role. These isoforms have also been shown to follow distinct cellular processing pathways and localize to different subcellular compartments. In this study, we combine accelerated Molecular Dynamics (aMD) and wide‐angle X‐ray scattering (WAXS) methods to characterize the structural ensemble of Ras isoforms and measure their dynamic properties. Using aMD, we reduce the energetic barriers to enhance conformational sampling of these proteins. A representative subset of structures from aMD is then experimentally tested using WAXS patterns to quantify their relative population. Each isoform shows at least two major conformations, where one is characterized as a relaxed state and another one as a reactive state, based on their relative potential energy calculated from aMD. These conformations are unique for each of the isoform, where they differ primarily around the active site and regions away from the active site, including helix 3/loop 7. This study will provide significant insights into the conformational features of these small proteins that give rise to their functional specificity.Support or Funding InformationNSF MCB‐1244203