bluuebunny/crossref_metadata_2025_split huggingface dataset:
K‐12 STEM Outreach: Ushering in the Next Generation of Scientists
Authors: Mulligan Kimberly 
Published: June 2021

Students likely to major in and earn college degrees in Science, Technology, Engineering, and Math (STEM) fields are those whose curiosity about STEM careers is piqued at an early age. At the K–12 level, this interest can be enhanced through hands‐on learning, projects with real life context and relevance, and contact with mentors working in these fields.The Interdisciplinary Science and Research (ISR) program at Stratford STEM Magnet High School in Nashville, TN has capitalized on a unique school/university partnership that utilizes a co‐teaching model pairing high school science teachers with Vanderbilt University scientists. The primary teaching strategy is inquiry‐based, incorporating multiple science and math disciplines into the coursework, promoting a facilitated learning model that relies heavily on the scientific method. In a school where the population is 74% minority and 81% of the students are considered economically disadvantaged, ISR provides a rigorous elective to challenge students in STEM disciplines, utilizing project‐based, real world learning.Students in ISR have consistently shown gains when compared to their peers including a 2.7 point increase on the ACT. Additionally, 69% of these students scored Proficient or Advanced on Algebra I and 59% on Biology I end of course exams compared to 33% and 13% of their peers, respectively. When surveyed, students report gains in critical thinking skills, improved public speaking ability, and increased adeptness in working collaboratively. This data emphasizes the need at the K‐12 level for students to be exposed to STEM professionals enhancing STEM content knowledge. While many scientists focus solely on the advancement of discovery and understanding of scientific knowledge, the value of sharing this knowledge with K‐12 students cannot be ignored.
“Protein Un‐Design”: Evolutionary Origins of Native DNA‐Binding Architectures in Unfolded Polypeptides
Authors: Weiss M.A. , Racca J.D. , Chen Y‐S. , Phillips N.B. 
Published: June 2021

We seek to test the hypothesis that the evolutionary origins of globular proteins, however exquisite in their structural organization, lie within the conformational repertoire of unstructured polypeptides. A model is provided by the high‐mobility‐group (HMG) box, an L‐shaped a‐helical motif of sharp DNA bending that represents a primordial innovation at the root of Eukarya. Whereas the modern HMG box exhibits conserved architectural features (such as an “aromatic buttress” underlying its angular protein surface; Racca, J., et al. J. Biol. Chem. E‐pub (2014)), we demonstrate that natively unfolded variants with defective buttresses can, as a seeming paradox, exhibit native DNA binding and DNA bending. Canonical structure is regained on specific DNA binding as inferred from CD and 1H‐15N NMR. Coupled DNA‐dependent protein folding and DNA bending suggest that primordial boxes could readily have evolved from unstructured precursors, driven by selection for architectural gene regulation. Functional “proof of principle” is provided by unfolded variants of human testis‐determining factor SRY. In a rat embryonic gonadal‐ridge cell line such variant HMG boxes are able to regulate the principal target gene in the nascent testis (Sox9), activating a male transcriptional program. We envisage that progressive stabilization of nascent folds represents a fundamental mechanism for the rapid evolution of autonomous protein structures, yielding the modern landscape of DNA‐binding architectures. In this view extant unstructured DNA‐ and RNA‐binding motifs represent relics of the transition between RNA and protein worlds.Acknowledgements. We thank L. Labeots, A. Jansco‐Radek, J. Radek, and G. Chan for their participation in the early stages of this work.
The Reversal of the Anticoagulant and Anti‐hemostatic Effect of LMWH by rLopap
Authors: de Andrade Sonia , Carrijo‐Carvalho Linda , Sato Ana , de Abreu Maffei Francisco , Chudzinski‐Tavassi Ana 
Published: June 2021

Lopap, found in the bristles of the caterpillar<em style=“mso‐bidi‐font‐style: normal;”> Lonomia obliqua, is the first exogenous prothrombin activator that shows serineprotease‐like activity and only lipocalin reported to interfere with hemostasis mechanisms. The aim was to assess the effect of recombinant Lopap, obtained using an E. coli expression system, as an exogenous hemostatic factor in reversing bleeding induced by LMWH .The experimental protocol was approved by the Ethics Committee for the Use of Animals of the Teaching and Research Institute, Hospital Sírio‐Libanês. Briefly, anesthesia was induced with ketamine HCl (40 mg/kg, im) and xylazine (5 mg/kg, im) and was maintained by intermittent injections of the same mixture. Rabbits were allocated to 4 groups and received 1800IU/kg LMWH iv over 2 min, followed by iv administration of saline (SG) or rLopap at a dose of 1 μg/kg (LG1) or 10 μg/kg (LG10), 10 min after the injection of LMWH. The reversal of the anticoagulant and anti‐hemostatic effect of LMWH by rLopap were assessed in terms of bleeding time (BT) and laboratory tests; activated partial thromboplastin time (aPTT), prothrombin time (TP), prothrombin fragment F1+2, Thrombin generation, Thrombin‐Antithrombin complex (TAT) and fibrinogen. LG10 animals showed a decreased BT near normal baseline. The levels of prothrombin fragment F1+2 and measured thrombin were inversely proportional to BT. rLopap can be a possible template for the reversal of bleeding and anticoagulant events.Supported by FAPESP
Improved Accuracy of Proteome Quantification by MS/MS Fragment Intensity
Authors: Wu Qi , Shan Yichu , Zhang Lihua , Zhang Yukui , Fenton Robert 
Published: June 2021

Quantitative proteomics facilitates the discovery of potential biomarkers. Development of novel algorithms to improve the accuracy of proteome quantification is of vital importance. MS/MS fragment intensity has arisen as a novel abundance feature with unique advantages.In isobarically labelled proteome samples, the increase of MS/MS spectra complexity has adverse effect on the identification and coverage of quantification. A paired ion based scoring algorithm (PISA) based on Morpheus, was proposed to address this issue by scoring the matches using the number plus the fraction of intensity of matched paired ions. It was applied to the identification of Lys‐C digest of protein samples isobarically labelled with two different strategies involving both in‐vivo and chemical labeling. Compared to commercially available and open source software Mascot and Morpheus, more peptide spectrum matches, distinct peptides, and proteins can be identified at a 1% false discovery rate with the increase ranging from 22 % to 60 %. Moreover, 100% quantification coverage was achieved.Summed MS/MS Total ion current (SMT) cumulates all fragment intensities of all MS/MS spectra assigned to a protein. Spectral count in Absolute Protein Expression profiling (APEX) algorithm was replaced by SMT to enhance the accuracy of absolute quantification of proteomes in label‐free manner. The combination of APEX and SMT (abbreviated as APEX‐SMT) was capable of improving the accuracy of absolute quantification by reducing the average relative deviation by ~55‐85% compared to that of APEX, through a series of tests on Universal Proteomics Standard sample with a dynamic range of 5 orders of magnitude (UPS2).
DHA up‐regulated expression of lipogenic and endoplasmic reticulum stress related genes in pig adipose tissues
Authors: Huang Chao‐Wei , Chen Yu‐Jen , Mersmann Harry , Ding Shih‐Torng 
Published: June 2021

Endoplasmic reticulum (ER) is the main site for protein and lipid synthesis. In our previous study, DHA promotes triacylglycerol (TAG) accumulation in pig adipose tissues, which may correlate with ER stress. Several studies have shown the link between ER stress and lipogenesis. However, the function of n‐3 or n‐6 PUFA in regulating ER stress in adipose tissues has not been demonstrated.We determined the effect of dietary supplementation of soy bean oil (SB oil, rich in n‐6 PUFA) and docosahexaenoic acid oil (DHA oil, rich in n‐3 PUFA) on ER stress of porcine adipose tissues. 8‐week‐old weaned pigs (18.4 ± 0.3 kg) were fed with diets supplemented with 2% (as‐fed basis) of SB oil or DHA oil for 30 days with feed and water provided ad libitum. After 30 days of feeding, we measured the body compositions by EchoMRI, adipose histological section, and analyzed the expression of lipogenic and ER stress‐related genes in the adipose tissue. Body weight and body compositions were not affected by different sources of PUFA. The average size of adipocytes in DHA‐fed pigs were 1.6‐fold greater than SB‐fed pigs. Furthermore, the lipogenic related genes, CCAAT/enhancer binding protein β, hypoxia‐inducible factors and cyclooxygenase 3 were elevated by DHA treatment. Furthermore, ER‐stress related genes including activating transcription factor 6, glucose regulated protein 78 and x‐box binding protein1 are also significantly higher in DHA‐fed than SB‐fed pigs. These results indicate that DHA supplementation may increase lipogenisis and ER stress in pig adipose tissue. Whether the observed phenomena is a mechanism for animals to cope with dietary challenges awaits further investigation.
Investigating the Binding Affinity of Nod2 and Soluble Bacterial Cell Wall Dimers
Authors: Genova Lauren , Grimes Catherine 
Published: June 2021

The innate immune system is the body's first line of defense against pathogens. The innate immune system is triggered by pathogen associated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs) such as Toll‐like receptors (TLRs) and Nod‐like receptors (NLRs). This research project focuses on providing a better understanding of how the innate immune system senses and responds to the presence of bacteria. Specifically, our group is interested in the relationship between the nucleotide‐binding oligomerization domain‐containing protein 2 (Nod2), an NLR protein found in the cytosol of mammalian host cells, and muramyl dipeptide (MDP), the smallest bacterial cell wall fragment known to elicit an immunological response. When Nod2 is mutated, the signaling pathway becomes disrupted and uncontrollable inflammation arises, leading to chronic inflammatory bowel disorders such as Crohn's disease. To discover how to better treat these diseases, it is imperative to learn more about how Nod2 and MDP interact, a mechanism which is currently unknown. The Grimes Lab has previously shown that Nod2 binds to MDP in vitro; however, research suggests that a heightened immunological response may be elicited in a host if molecules containing multiple MDP's are used, suggesting multivalency is at play. To test this hypothesis, a variety of novel MDP dimers were synthesized and assessed via in vitro SPR binding assays and cell‐based assays.This research has been funded by the Howard Hughes Medical Institute.
Proinflammatory Environmental Exposures in Asthma
Authors: Cheah Cheryl , Daines Michael , Morgan Wayne 
Published: June 2021

Asthma is a chronic inflammatory disease that affects over 35 million people in the United States, and is a leading cause of hospitalization in children. While asthma‐causing allergens like fungi, mites, and insects represent biologically diverse organisms, they have some biological similarities that may underlie their potential to cause asthma. One of these shared biological features is the production of chitin. Studies have shown that chitin impacts lung function through the innate immune system. There are no available chitin assays for assessing environmental samples. Creation of a quantitative assay for chitin is complicated because it is a modified polysaccharide and not a protein. This makes it difficult to make antibodies that will specifically recognize it. Chitin is also poorly soluble in water and many standard solvents, which makes developing adequate standards challengingAfter successfully creating a chitin quantification assay with a detection limit of about 20ng/mg dust and validating commercially available assays for endotoxins and lipopolysaccharides, we acquired 387 household dust samples from the Inner‐City Anti‐IgE Therapy for Asthma (ICATA) clinical trial and quantified MAMPS like chitin, endotoxins and lipopolysaccharides in those samples. Quantifying chitin, endotoxins and glucans in household dust samples gave us a better understanding of the microbiome profile in those samples. Overall, this study contributes to our understanding of the mechanisms in human asthma and allergy. It also has the potential to improve existing techniques in the treatment and prevention of a disease that affects millions of people worldwide.
ADP‐ribosylargininyl Activity of Cholix Toxin Is Mediated Through Diffusible Intermediates
Authors: Sung Vicky , Tsai Chia‐Lun 
Published: June 2021

OBJECTIVEWe discovered and characterized the ADP‐ribosylargininyl activity of the cholix toxin catalytic fragment, which was first identified as a diphthamide‐dependent ADP‐ribosyltransferase.METHODSWe utilized genetic approach to construct direct and reverse mutant enzymes and substrates in conjunction with biochemical assays including fluorescence/non‐fluorescence based enzymatic assays, protein/peptide‐2D‐electrophoresis analysis.RESULTSOur studies show that the transfer of ADP‐ribose to toxin itself takes place by a predominantly intramolecular mechanism. Multiple arginine residues, located in the proximity of the catalytic site but also at distal sites, can be the ADP‐ribose acceptor in the auto‐reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially modifies arginine residues in proximity to the NAD+ binding pocket. ADP‐ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates, such as oligo arginine peptides and eEF2(H715R) mutant. Auto‐ADP‐ribosylation of cholix toxin appears to have negative regulatory effect on its enzymatic activities. However, at the presence of both endogenous and exogenous substrates, the ADP‐ribosylation of exogenous substrate occurred more efficiently than endogenous substrate.CONCLUSIONSThe lifetime of the hypothetical intermediate exceeds recorded and predicted lifetime for the cognate oxocarbenium ion. We therefore hypothesize a strained form of NAD+ intermediate generated through an error‐prone mechanism of the enzyme to react with arginine residues in a proximity dependent manner.
An Old Cofactor in a New Light: Adenosylcobalamin in Light‐Dependent Gene Regulation
Authors: Jost Marco , Padmanabhan S. , Elíaz‐Arnanz Montserrat , Drennan Catherine 
Published: June 2021

Adenosylcobalamin (AdoCbl, coenzyme B12) is one of Nature's most complex enzyme cofactors and enables difficult radical chemistry via cleavage of its covalent cobalt‐carbon bond. In a recently discovered bacterial pathway, however, AdoCbl is used in a completely new function: it is used as a light sensor in light‐dependent gene regulation. Here, AdoCbl binds to the transcription factor CarH to regulate expression of the carotenoid biosynthetic genes. Gene expression is only activated upon light exposure, leading to carotenoid production and protection of the cell from light‐induced damage. This elegant response mechanism to sunlight is found in hundreds of bacterial species.Here, we present the full structural characterization of the light‐sensing transcription factor CarH. In the dark, AdoCbl mediates formation of a CarH tetramer that positions the DNA‐binding domains on the DNA operator, blocking transcription. Exposure to light cleaves AdoCbl's Co‐C bond, triggering a conformational change that causes tetramer disassembly, dissociation from DNA, and transcription activation. Thus, CarH harnesses the light sensitivity of AdoCbl to drive a light‐dependent gene expression switch. These studies provide fundamental insight into a new mode of light‐dependent gene regulation and provide a new biological role for AdoCbl.This work is supported by the NIH (CLD), the Ministerio de Ciencia e Innovación–Spain (SP and ME‐A), and a MIT Poitras fellowship (MJ). CLD is a HHMI investigator.
Investigation of the evolutionary relationship between Rio2 kinases and the canonical eukaryotic protein kinases using x‐ray crystallography
Authors: Bahmanjah Soheila , LaRonde Nicole 
Published: June 2021

Rio proteins are serine kinases that contain few of the conserved residues of typical protein kinases and are thus categorized as atypical protein kinases. Rio proteins are required for the cleavage of 20S rRNA to 18S rRNA. Canonical protein kinases are very similar to Rio2 proteins but are more active in ATPase and atuophosphorylation activity and they are onlyobserved in archaea. Computational studies have revealed that the arginine residue at position 228 highly affects the activity of canonical protein kinases. This residue in Rio family is replaced by either alanine or glycine. We were interested to study mutation at 228 of Rio2, and have found out how this mutation would affect the structure and activity of the Rio2 family. We have investigated the evolutionary relationship between Rio kinases and the canonical eukaryotic protein kinases. Our observation is that canonical eukaryotic protein kinases have evolved from Rio kinases. We have made several mutations in a key differentiating position of the kinase domain sequence of Ct‐ Rio2, G228, and have collected biochemical data to test the ability of the mutated Rio kinase to autophosphorylate and carry out ATP hydrolysis. We have also co‐crystallized Ct‐Rio2 G228 point mutants with ATP in order to determine the structural differences between the mutant and wild‐type Hence, wild type Ct‐Rio2 and five mutants of Ct‐Rio2 were purified and co‐crystalized with ATP/Mg2+. These mutants include G228A, G228D, G228N, G228R, and G228V and x‐ray diffraction data sets were collected.
Radical Chemistry in the Human Gut: Discovery of Choline Trimethylamine‐Lyase
Authors: Bodea Smaranda , Balskus Emily 
Published: June 2021

The collection of microorganisms that inhabit the human gastrointestinal tract, our gut microbiota, has an extensive set of metabolic capabilities that directly impact human health. Our research seeks to discover and understand the molecular basis of disease‐associated metabolic pathways from the human microbiome. One example under investigation is the catabolism of the essential nutrient choline by anaerobic microbes. Gut bacteria break down choline in an entirely different manner than human cells, cleaving its C–N bond to produce trimethylamine (TMA), which is subsequently oxidized to trimethylamine N‐oxide by liver enzymes. This microbial‐human co‐metabolic pathway has been linked to several diseases, including non‐alcoholic fatty liver disease and atherosclerosis.Using a genome mining strategy, we recently identified the first gene cluster responsible for anaerobic choline utilization (the cut gene cluster). We verified the functional role of this cluster using both genetic and heterologous expression approaches. These studies revealed that the key C–N bond cleavage step involved in choline breakdown is catalyzed by choline trimethylamine‐lyase (CutC), a new type of glycyl radical enzyme. This reaction is unprecedented chemistry for this enzyme family. We confirmed the activity of the purified choline TMA‐lyase and its activating protein in vitro, observing that CutC is exquisitely specific for choline cleavage. Currently, we are investigating the mechanism of the CutC‐catalyzed reaction, with the ultimate goal of designing small molecule inhibitors for this enzymatic transformation.
Neonatal Antibiotic Exposure Alters Compositional Gut Microbiota Development During the First 6 Months of Life
Authors: Kumar Himanshu , Rautava Samuli , Collado Maria , Borzykh Nikita , Löyttyniemi Eliisa , Isolauri Erika , Salminen Seppo 
Published: June 2021

ObjectiveTo investigate the impact of neonatal antibiotic exposure on gut microbiota development in infants born after 36 weeks of gestationMethodsA case‐control study of 6 neonates exposed to brief (<5 days) empirical antibiotics (penicillin G and gentamicin) during the first 72 hours of life, 6 neonates receiving a 7‐day course of the same antibiotics for documented infection and 24 matched healthy non‐exposed neonates. Gut microbiota composition was assessed at 1 and 6 months of age by qPCR, DGGE and 16S rRNA pyrosequencingResultsNeonatal antibiotic exposure resulted in a significant decrease in fecal bifidobacteria at 1 month of age (p=0.015), particularly in B. bifidum (p=0.0061). The bacterial richness of infants exposed to antibiotics was significantly lower compared to non‐exposed infants. The principal coordinates analysis (PCoA) formed separate clusters when compared between antibiotic‐exposed and non‐exposed infants at 6 months of age. In particular, an increased frequency of Firmicutes and a decrease in Bacteroidetes was observed in antibiotic‐exposed infantsConclusionsAberrant gut microbiota colonization is attributable to antibiotic exposure since it was observed also in antibiotic‐exposed neonates in whom infection was later ruled out. Decreased levels of both bifidobacteria and Bacteroidetes in early infancy have previously been linked to increased risk for non‐communicable disease in later life. Means of accurately detecting bacterial infections in neonates are needed to avoid unnecessary exposure to antibiotics during this vulnerable periodResearch supportFunctional Foods Forum, University of Turku
Cell‐fie of a Cancer Cell: Imaging Metastatic Potential of Single Living Tumor Cells with Fluorescent Biosensors
Authors: Midde Krishna , Sun Nina , Mehlawat Priyanka , Husain Hatim , Ghosh Pradipta 
Published: June 2021

Recognizing the metastatic potential of solid tumors is of utmost importance to personalize treatment plans for cancer patients. Conventional approaches utilize ensemble studies in which read‐outs from a heterogeneous population of cells are averaged to assess tumor aggressiveness. Such averaging obscures precious information on those individual tumor cells that are a minority within any given tumor, but possess the most ominous characteristics that contribute to their intrinsic metastatic potential, e.g., stem‐ness, EMT/invasiveness, drug resistance). Thus, the quest for the Holy Grail in measuring such ‘potential’ continues. Here we developed FRET‐based biosensors that measure the metastatic potential of single living cancer cells by confocal imaging.By design, these biosensors measure functional phosphorylation of Gα‐Interacting and Vesicle Associated Protein (GIV) a multimodular signal transducer and a bona‐fide metastasis‐related protein across a variety of solid tumors. These biosensors allow the generation of spatial and temporal signaling profiles of single cells, downstream of multiple receptors, even at a steady‐state. Such single‐cell signaling profiles are reflective of tumor heterogeneity, are unbiased to a given receptor/pathway, and eliminate the need to know which receptors/pathways drive a given tumor. Using these probes we detected those few cells within the primary lung and breast tumors with the highest metastatic potential, which are subsequently selected during metastasis.These results provide proof‐of‐concept that GIV‐biosensors are effective in monitoring the metastatic potential of single‐cells, in a sensitive and unbiased way, across different tumors.