bluuebunny/crossref_metadata_2025_split huggingface dataset:
Substrate‐Bound Complex of DesD, a Model Enzyme in the Virulence‐Related NIS Synthetase Family
Authors: Hoffmann Katherine 
Published: April 2020

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
Accurate Annotation of Protein‐coding Small Open Reading Frames in the Human Genome
Authors: Martinez Thomas F. , Chu Qian , Donaldson Cynthia , Tan Dan , Shokhirev Maxim N. , Saghatelian Alan 
Published: April 2020

Functional protein‐coding small open reading frames (smORFs) are emerging as an important class of genes. Several smORF‐encoded microproteins have been characterized and implicated in a variety of critical processes, including regulation of mRNA decay, DNA repair, and muscle formation. Thus, rigorous and comprehensive annotation of protein‐coding smORFs is of fundamental importance. Here, we integrate de novo transcriptome assembly and Ribo‐Seq into an improved workflow that overcomes obstacles with previous methods to more confidently annotate thousands of novel smORFs across multiple human cell lines. Over 1,500 smORFs are found in two or more cell lines, and ~40% lack a canonical AUG start codon. Evolutionary conservation analyses suggest that hundreds of smORF‐encoded microproteins are likely functional. Additionally, monitoring RNA expression and translational efficiency during cellular stress revealed regulated smORFs, demonstrating an approach for identifying biologically relevant smORFs. We also find that smORF‐derived peptides are detectable on human leukocyte antigen complexes, positioning smORFs as a source of novel antigens. The annotation of protein‐coding smORFs radically alters the current view of the human genome’s coding capacity and will provide a rich pool of unexplored, functional human genes.Support or Funding InformationThis research was supported by NIH/NIGMS (R01 GM102491, A.S.), Leona M. and Harry B. Helmsley Charitable Trust grant (A.S.), Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.), NIH/NIGMS postdoctoral fellowship (F32 GM123685, T.F.M.), George E. Hewitt Foundation for medical research (Q.C.), and the Pioneer Fellowship (D.T.). This work was also supported by the Razavi Newman Integrative Genomics and Bioinformatics Core and the Next Generation Sequencing Core Facilities of the Salk Institute with funding from the NIH‐NCICCSG (P30 014195) and the Chapman Foundation.Figure 1
Glycosylation of Ganoderic Acid A via Recombinant Glycosyltransferase of Bacillus subtilis Under Acidic Operating Condition
Authors: Wu Jiumn-Yih , Chang Te-Sheng , Chiang Chien-Min , Wang Tzi-Yuan 
Published: April 2020

Ganoderic acid A (GAA) is a triterpenoid with identified effective activity, isolated from Ganoderma lucidum. Glycosylation of triterpenoid might improve its anti‐toxin bioactivity and also increase both water solubility and physical‐chemical stability. Glycosylation is carried out by glycosyltransferases (GT, EC 2.4.x.y), a type of enzyme that utilize a nucleotide‐activated sugar donor, such as uridine diphosphate (UDP)‐glucose, to transfer the sugar portion to a sugar‐linked molecule. We have utilized Bacillus sp. GA A07, isolated from intestinal bacteria of Zebrafish, to biotransform GAA to GAA‐15‐O‐β‐glucoside
1. Our previous study also showed that the Bacillus subtilis, ATCC 6633 strain, could biotransform GAA to compound, GAA‐15‐O‐b‐glucoside, and one minor compound in Fig.
1. We selected five GT genes‐BsGT110, BsGT292, BsGT296, BsGT398, and BsGT489 from the used strains, and successfully overexpressed and cloned them in Escherichia coli. Two glycosyltransferases (BsGT398 and BsGT489) have shown the capability to catalyze GAA to GAA‐15‐O‐β‐glucoside under some specific conditions. However, the chemical structure of minor compound
2 and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the used B. subtilis strain, for the biotransformation of GAA into this compound under acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a designed operating production, this product
2 was successfully isolated using preparative high‐performance liquid chromatography and was identified to be a new triterpenoid glucoside (GAA‐26‐O‐β‐glucoside) by mass and nuclear magnetic resonance spectroscopy. We identified that BsGT110 is an unique GT that have a specific activity on the glycosylation of triterpenoid at the C‐26 position under acidic conditions (in Fig.
2) but lost most of its activity at alkaline ones, suggesting that acidic solutions may enhance the catalysis activity of this and similar types of GTs toward triterpenoids.Support or Funding InformationThis research was financially supported by the grants of the Ministry of Science and Technology, Taiwan (Project No. MOST 108‐2221‐E‐507 ‐008 ‐ and MOST 108‐2221‐E‐024 ‐008 ‐MY2).UPLC analysis of the biotranformation of GAA by BsGT110Figure 1Glycosylation of GAA by BsGT110 under the acidic conditionFigure 2
Hair fiber morphology and function: at the intersection of anthropology, physiology, and engineering
Authors: Lasisi Tina , Smallcombe James , Shriver Mark D. , Kenney W. Lawrence , Havenith George , Jablonski Nina G. 
Published: April 2020

Humans are unique among mammals in having a nearly naked body but a fur‐covered head. Furthermore, scalp hair is one of the most variable traits among human populations. However, the evolutionary emergence of scalp hair, the factors contributing to its evolution, and its subsequent diversification remain largely unknown. Among the few discussions of human scalp hair evolution is the hypothesis that hairy scalps may have evolved as protective barriers against solar radiation in bipedal savannah dwellers. Managing thermal load would be particularly important in the context of a large thermogenic and thermosensitive brain. And as the presence and morphology of fur, or hair, is generally of great importance to mammalian thermoregulation, we set out to test the thermal effect of scalp hair (morphology). To do this, we developed methods for the quantification of hair fiber morphology and experimentally tested the effect of scalp hair (and variation in its morphology) on heat gain from solar radiation. Thermal manikins are human models designed for scientific testing of thermal environments and to simulate heat transfer between the skin and the environment in a range of temperatures. Using one such thermal manikin, we collected data on heat flux when the manikin was equipped with no wig, and wigs of various hair textures. Our preliminary results show that tightly curled scalp hair, in particular, minimizes the heat gained from solar radiation (Figure
1). The study is being expanded and experiments repeated to enable statistical testing. We plan to use these data in mathematical models of human physiological response to estimate the impact scalp hair might have had in early human evolution. In this research we combined anthropological theory, physiological models and engineering methods to answer a longstanding question of human evolution ‐ why did humans evolve scalp hair and why does it vary? The innovative work stemming from this collaboration provided the opportunity to tackle old questions in new ways and pave the way for more potential interdisciplinary work.Support or Funding InformationThis work is supported by the National Science Foundation (award number 1847845); the Wenner‐Gren Foundation (grant number 9911); and the Africana Research Center at The Pennsylvania State University.Solar heat gain in different hair textures at three windspeeds.Figure 1
PKA RIα phase separation drives cAMP compartmentation and suppresses tumorigenic signaling
Authors: Zhang Jason Z. , Lu Tsan-Wen , Tenner Brian , Yang Jessica , Zhang Jin-Fan , Taylor Susan , Mehta Sohum , Zhang Jin 
Published: April 2020

The fidelity of intracellular signal transduction hinges on the organization of signaling molecule activities into a dynamic architecture. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G‐protein‐coupled receptors can regulate specific cellular processes despite converging on the ubiquitous second messenger 3’,5’‐cyclic adenosine monophosphate (cAMP). Recent work has challenged the textbook model that cAMP compartmentation is achieved by its local degradation, yet the specific mechanisms responsible for spatially constraining this diffusible messenger remain elusive. We address this longstanding question by identifying the formation of biomolecular condensates of the type I regulatory subunit of cAMP‐dependent kinase (PKA), RIα, as a key driver of cAMP compartmentation. RIα undergoes liquid‐liquid phase‐separation at endogenous levels as a function of dynamic cAMP signaling to form RIα bodies harboring high levels of cAMP and PKA activity. Importantly, we show that this active cAMP sequestration is critical for effective cAMP compartmentation in cells. The pathophysiological relevance of this compartmentation system is illustrated by its key role in the etiology of the atypical liver cancer fibrolamellar carcinoma (FLC). We show that an FLC‐linked oncoprotein fusion between DnaJB1 and the PKA catalytic subunit (PKAcat) potently blocks RIα phase separation and induces aberrant cAMP signaling. Furthermore, loss of RIα phase separation independent of the fusion oncoprotein in normal hepatocytes increased cell proliferation and resulted in cell transformation. Our work reveals liquid‐liquid phase separation as a principle organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.Support or Funding InformationThis work is supported by NIH R35 CA197622, R01 DK073368 (to J.Z). J.Z.Z. is supported by a predoctoral fellowship from the National Science Foundation (DGE‐1650112).In healthy cells, endogenous RIα acts as a dynamic cAMP buffer via phase separation, allowing for PDE‐mediated cAMP compartmentation. For fibrolamellar carcinoma (FLC) patients, DnaJB1‐PKAcat fusion oncogene disrupts RIα phase separation, thus disrupting cAMP compartmentation and leading to tumorigenesis.Figure 1
Development of “Sharkavir”: A Hypothetical Inhibitor for HIV‐1 Protease
Authors: Hunt Matthew , Abbas Feza , Abbas Lyla , Ghali Mina , Ghali Helana , Chabbra Alesa , Schmitt Lavin Emily F. , Sikora Arthur 
Published: April 2020

Human immunodeficiency virus (HIV) affects approximately 37 million people worldwide and results in over 1 million deaths annually. A class of drugs first developed in 1995 inhibits the enzyme HIV‐1 protease, thus preventing the maturation of an HIV precursor protein. There are now over 10 protease inhibitors available to treat HIV. Multiple mutations in the protein have made this treatment less effective. The CREST (Connecting Researchers, Educators, and STudents) team at Nova Southeastern University modeled the interaction between HIV‐1 protease and its competitive inhibitors. Darunavir is currently one of the strongest competitive inhibitors, as it binds effectively to the substrate envelope and has yielded a lower resistance for patients. The most effective treatment is a combination of two of these inhibitors: Darunavir and Ritonavir. In order to explain how these drugs work, the active site of the non‐mutated wild‐type HIV‐1 protease was depicted as a binding box model. The protease inhibitors Darunavir, Ritonavir, and our hypothetical drug, “Sharkavir” were 3D printed to show how they fit into the protease active site. Details of the wild‐type HIV‐1 protease, as well as the drugs Darunavir, and Ritonavir, were found in the Protein Data Bank files, 1T3R, 4DQF, and 1N49, respectively. Both structures were imported into Jmol: a protein visualization software. The new protease inhibitor molecule, “Sharkavir”, was designed as a combination of Darunavir and Ritonavir using Marvin Sketch: a software used to manipulate molecular structures. The molecular structure of the hypothetical drug “Sharkavir” is shown below.Support or Funding InformationThis project is funded in part by NSF‐IUSE #1725940 for the CREST Project.Molecular Structure of "Sharkavir”Figure 1
Investigating the Kinetics of β1 and β2 Adrenoceptor Effector Recruitment using Luciferase Complementation Assays
Authors: Dijon Nicola , Holliday Nicholas , Charlton Steven 
Published: April 2020

Nanoluc‐based luciferase complementation assays (NanoBiT;
Dixon et al, 2016) provide new approaches to measure G protein‐coupled receptor (GPCR) signalling, by assessing the kinetics of receptor subtype recruitment of effector proteins in real‐time. Here, we use NanoBiT systems to compare β adrenoceptor subtype (β1AR/β2AR), recruitment of β‐arrestin2 and an engineered Gαs GTPase domain sensor (mini Gαs; mGαs)(
Wan et al, 2018).HEK293T cells stably expressed SNAP‐tagged βAR (β1AR or β2AR) fused to C‐terminal 18 kDa LgBiT luciferase fragment, and the 11 amino acid SmBiT fused to either β‐arrestin2 or mGαs. NanoBiT assays were conducted in cells in 96 well plates in HBSS/0.1% BSA at 37°C. Cells were pre‐incubated with furimazine (5min; 37°C), before the addition of agonist. Luminescence was monitored for 31 min (BMG Pherastar Platereader, 37°C). Concentration response curves were fitted in GraphPad Prism v7, with pharmacology quoted as mean±s.e.m (n≥3)Both βAR subtypes stimulated transient β‐arrestin2 recruitment upon 10μM isoprenaline stimulation (Figure
1a). At 31 min, both receptor subtypes reported partial agonism of salbutamol and salmeterol, with increased potency and efficacy (relative to isoprenaline) at β2AR compared to β1AR (Figure
1c–d). 10μM Isoprenaline also stimulated recruitment of mGas at each subtype, increasing rapidly between 0 and 5 min post agonist addition. Association of β2AR‐mGαs complexes was maintained stably throughout incubation, whilst at β1AR, mGαs recruitment continued to increase over incubation window, reaching 322.0±39.1% by 31 min (%10μM isoprenaline, at 3 min) (Figure
1b).NanoBiT complementation assays report real‐time recruitment of β‐arrestin2 and mGαs protein, at both β1AR and β2AR. Transient β‐arrestin2 recruitment was reported at both receptor subtypes responses distinguished full and partial agonists. Further studies of the continued βAR‐mGαs association will probe whether mGαs sensors remain stably associated with membrane or endosomal pools of adrenoceptors.Support or Funding InformationPhD funded by Biotechnology and Biological Sciences Research Council (BBSRC).NanoBiT luciferase complementation monitors βAR recruitment of signalling effector proteins. Signalling timecourse of (a.) β‐arrestin2 and (b.) mGαs recruitment, at β1AR and β2AR. Concentration response curves of β‐arrestin2 recruitment, at (c.) β1AR and (d.) β2AR. Data points represent mean±s.e.m.; n≥3.Figure 1
Estimation of Cerebral Vasomotor Reactivity with Near Infrared Spectroscopy in Young Adults
Authors: Shang Jing , Tomoto Tsubasa , Cardim Danilo , Zhang Rong 
Published: April 2020

Cerebral vasomotor reactivity (CVMR) reflects the ability of cerebral blood vessels to regulate cerebral blood flow (CBF) in response to changes in carbon dioxide (CO2). Transcranial Doppler (TCD) measurement of CBF velocity (CBFV) in the large cerebral arteries has been widely used to evaluate CVMR. However, assessment of CVMR in the cerebral microcirculation using near infrared spectroscopy (NIRS) has not been fully explored. The aims of this study are to characterize CVMR during hypo‐ and hypercapnia using NIRS, and to investigate the relationship between CVMRs assessed by NIRS and TCD. Thirty‐six healthy adults aged 21 to 45 (women: n=23) underwent CBFV and brain tissue oxygenation measurements using both NIRS and TCD during hyperventilation (hypocapnia) and modified CO2 rebreathing (hypercapnia). NIRS was placed on the forehead to measure brain tissue oxygenation index (TOI), TCD was used to measure CBFV in the middle cerebral artery, and capnography was used to measure end‐tidal CO2 (EtCO2). The percentage changes in TOI and CBFV relative to the baseline level in response to changes in EtCO2 were calculated to assess CVMR. Both TOI (−6±2 %) and CBFV (−41±10 %) were reduced during hypocapnia, and elevated during hypercapnia (TOI: 6±2 %, CBFV: 62±20 %) (Fig.
1). CVMR assessed by NIRS was significantly lower than that measured with TCD in both hypocapnia (0.33±0.16 vs. 2.21±0.43 %/mmHg, p< 0.001) and hypercapnia (0.52±0.19 vs. 3.88±1.22 %/mmHg, p< 0.001, Fig.
2). CVMRs assessed by NIRS and TCD were correlated in hypocapnia (r=0.40, p=0.02), but not in hypercapnia (r=0.10, p=0.55). These findings suggest that NIRS measurement of TOI in responses to changes in CO2 likely reflects CVMR of the cerebral microcirculation, which is in difference from that measured in the large cerebral arteries using TCDSupport or Funding InformationThe study was supported by National Institutes of Health (R01AG033106 and R01HL102457). JingShang was supported by the China Scholarship Council.The typical recordings of hyperventilation (hypocapnia) and the modified CO2 rebreathing (hypercapnia).Figure 1Average values of the relative change in CBFV and TOI in 4‐mmHg bins of ΔEtCO2 during hypercapnia induced by CO2 rebreathing. Error bars indicate standard deviation. ΔEtCO2 refers to change in EtCO2 from baseline.Figure 2
Mapping the relationship between proliferation and morphology in the developing mouse face
Authors: Green Rebecca M. , Lo Vercio Lucas , Guo Sihan , Dauter Andreas , Marchini Marta , Xhao Xiang , Marcucio Ralph S. , Hallgrimsson Benedikt 
Published: April 2020

There is a long‐standing prediction that small changes in proliferation and apoptosis during the time frame of facial morphogenesis act to shape the face. Further, many studies show genetic alterations that cause structural birth defects affect local proliferation or apoptosis. Yet, it is unclear how much of local change in regional proliferation would be necessary to cause a defect. Here, we set out to understand the relationship between growth, morphology and proliferation and test that prediction that targeted proliferation shapes the developing face by quantifying proliferation and apoptosis in 3D and relating it to the growth of the face. We use whole mount staining for proliferation and apoptosis markers, whole tissue clearing methods, lightsheet microscopy and atlas and machine learning based quantification methods to identify individual proliferating or apoptotic nuclei within a 3D tissue structure at a set time point. We also employee geometric morphometric analysis of the same tissue structure to quantify overall morphology. By collecting data at various time points across facial development (E9.5–E11.5) and quantifying the age of each embryo, we are able to relate cell biological level growth to tissue level growth and morphological change and relate these two parameters in a way not performed previously.Support or Funding InformationNIH NIDCR R01‐DE019638 to RM and BH, NSERC Discovery to BH, and CIHR Foundation grant to BH and RM, CIHR postdoctoral fellowship to RMG.Atlas based quantification of proliferation: A) Maximum Projection of the embryo highlighting the external morphology ‐ lateral view. B) Proliferation staining (phospho‐Histone H3) ‐ lateral view. C–D) Surface morphology of the atlas (n=5) C ‐ anterior view, D ‐ Lateral view. E–F) Heat map of proliferating cells (no density correction) E ‐ anterior view, F ‐ Lateral view.Figure 1