bluuebunny/crossref_metadata_2025_split huggingface dataset:
A new stem-neopterygian fish from the Middle Triassic (Anisian) of Yunnan, China, with a reassessment of the relationships of early neopterygian clades
Authors: Xu Guang-Hui 
Published: April 2020

AbstractNeopterygii is a taxonomically diverse group of ray-finned fishes, including Teleostei, Holostei and their closely related fossil taxa. Here, a new small-sized stem-neopterygian with extraordinarily long teeth, Louwoichthys pusillus gen. et sp. nov., is described based on 14 well-preserved specimens from the Middle Triassic (Anisian) marine deposits of Yunnan, China. Louwoichthys is closely related to Luopingichthys from the same fossil beds and Ctenognathichthys from the Middle Triassic of the Monte San Giorgio area in Europe; the three genera are grouped into the family Louwoichthyidae fam. nov., which is recovered as sister to the ‘perleidiform’ Pseudobeaconiidae. Given that ‘Perleidiformes’ is notoriously paraphyletic, a new order, Louwoichthyiformes ord. nov., is introduced to group both families. The louwoichthyiform monophyly is strongly supported by a series of derived features, such as a relatively short maxilla, an anteriorly inclined preopercle, a subopercle with a prominent anteroventral extension, and two or three pairs of branchiostegal rays. Louwoichthys is one of the smallest louwoichthyiforms, having a largest standard length of 39 mm. It is deduced to be a scavenger that might use its long and sharp teeth to grasp and tear the prey from the substrate or to bite a piece from a larger prey item.
Kindlin‐3 organizes a ring of clustered high affinity β2 integrins during human neutrophil spreading under flow
Authors: Fan Zhichao , Wen Lai , Yeh Yi-Ting , Kiosses William B. , Gutierrez Edgar , Groisman Alex , Francois Joshua , Lasheras Juan C. , del Alamo Juan C. , Chien Shu , Ginsberg Mark H. , Petrich Brian G. , Ley Klaus 
Published: April 2020

Neutrophils are vital for inflammation and immune defense. Dependent on β2 integrins, spherical neutrophils spread on vascular endothelium after arrest, which is critical for their recruitment from circulation to resist high shear flow and to initiate intravascular crawling. Here, we use high‐resolution quantitative dynamic footprinting microscopy to monitor neutrophil spreading on a substrate of recombinant ICAM‐1 and P‐selectin under flow. A homogenous binding assay using the conformation‐reporter antibodies mAb24 (reporting high‐affinity β2, H+) and KIM127 (reporting extended β2, E+) showed three conformations of activated β2 integrins. E−H+ β2 integrins increased before E+H− and E+H+ conformations at the beginning of neutrophil spreading. Integrin extension depended on Syk‐mediated integrin outside‐in signaling. The ring of E−H+ and E+H+, but not E+H− β2 integrins was fully formed during late neutrophil spreading just before migration. Using kindlin‐3‐GFP fusion proteins, a ring of kindlin‐3 was observed before the ring of H+ integrins appeared. These findings show spatially coordinated integrin activation during spreading. The previously unrecognized E−H+ conformation is the pioneer integrin for neutrophil spreading and appears to be organized by kindlin‐3.Support or Funding InformationThis research was supported by funding from the National Institutes of Health, USA (NIH, HL078784 and R01HL145454), WSA postdoctoral fellowships and a Career Development Award from the American Heart Association, USA (AHA, 16POST31160014, 19POST34450228, and 18CDA34110426).Integrin activation on a spread neutrophil footprint. Red is the mAb KIM127, which reports integrin extension; Green is the mAb24, which reports integrin high‐affinity; Blue is conformational non‐specific aLb2 integrins.Figure 1Kindlin‐3‐GFP molecules formed a ring after the spreading of a neutrophil‐like HL60 cell.Figure 2
Inhibitory effect of fructo‐oligosaccharides on inflammation‐associated apoptosis in GLP‐1 secreting L cells via inhibition of iNOS and cleaved caspase‐3 expression
Authors: Wongkrasant Preedajit , Muanprasat Chatchai 
Published: April 2020

Glucagon‐like peptide 1 (GLP‐1) released from enteroendocrine L‐cells regulate insulin secretion. Intestinal inflammation and GLP‐1 secretion impairment are the dominant phenotype in type 2 diabetes mellitus (T2DM) patients. Fructo‐oligosaccharides (FOS) is a prebiotic known to be associated with GLP‐1 release and glucose homeostasis. This study aimed to investigate effect of FOS on inflammation‐associated GLP‐1 unresponsiveness. Herein, using cell death assays, immunofluorescence staining, real time PCR and western blot analyses, we demonstrated that FOS suppressed the effect of tumor necrosis factor‐α (TNF‐α), a key pathogenic cytokine in T2DM, on L‐cells apoptosis. Interestingly, FOS did not suppress TNF‐α‐induced NF‐κB nuclear translocation, but inhibited expression of IL‐6, IL‐1β and TNF‐α mRNA levels and iNOS and cleaved caspase‐3. Of particular importance, GLP‐1 secretion assay revealed that FOS rescued TNF‐α‐mediated GLP‐1 unresponsiveness in L cells. In conclusion, our study demonstrated the beneficial effects of FOS in maintaining GLP‐1 level and its mechanistic insights. Therefore, FOS exerts a novel class of drug candidate for the treatment of T2DM, at least in part, by targeting GLP‐1 signaling.Support or Funding InformationThis work is supported by the Thailand Research Fund (DBG6180029), Faculty of Science Mahidol University and Faculty of Medicine Ramathibodi Hospital Mahidol University. Financial support from the Thailand Research Fund and Mahidol University through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0045/2558) to P.W. and C.M.Figure 1
3D Imaging of Cleared Tissues
Authors: Zhao Hu 
Published: April 2020

Tissue clearing technique enables visualization of opaque organ and tissue in 3‐dimension by turning tissue transparent. Current tissue clearing methods are restricted by limited types of tissue that can be cleared with each individual protocol, which inevitably led to the presence of blind‐spots within whole body or body parts imaging. Hard tissues including bones and teeth are still the most difficult organs to be cleared. In addition, endogenous fluorescence loss remains to be a major concern for solvent based clearing methods. Here, we developed a polyethylene glycol (PEG) Associated Solvent System (PEGASOS), which rendered nearly all types of tissue transparent and preserved endogenous fluorescence. Bones and teeth could be turned to nearly invisible after clearing. The PEGASOS method turned the whole adult mouse body transparent and we were able to image an adult mouse head composed of bones, teeth, brain, muscles and other tissues with no blind areas. Hard tissue transparency enabled us to reconstruct intact mandible, teeth, femur or knee joint in 3‐D. We were able to image intact mouse brain at sub‐cellular resolution and to trace individual neurons and axons over a long distance. We were able to visualize dorsal root ganglions directly through vertebrae. Finally, we revealed the neural network distribution pattern in 3‐dimension within the long bone marrow space. These results suggest that the PEGASOS method is a useful tool for general biomedical research.Support or Funding InformationThis work was supported by startup funding from Texas A&M University, NIH/NIDCR grant K08DE025090, NIH/NIDCR grant R21 DE027928 and NIH/NIDCR grant R01 DE 028291 to Hu Zhao.Thy1‐EGFP spinal cord imaged with a tiling light sheet microscopeFigure 1
The Evaluation of a Combined Surgical Approach Without Decompression for the Management of Grade 3 and 4 Spondylolisthesis in Adolescents
Authors: Wheeler Austin Wayne , Brown Jarrod 
Published: April 2020

IntroductionThere are multiple surgical approaches for correcting high grade spondylolisthesis (slip percentage greater than or equal to 50%) in adolescents, however there is no consensus on treatment. We hypothesize that a combined, two step approach, consisting of an anterior lumbar interbody fusion (ALIF) at L5‐S1 followed by a posterior spinal fusion at the pedicles of L5 and S1 will improve patient outcomes. The purpose of this study is to determine if this surgical approach can effectively decrease the patient’s slip angle and reduce the slip percentage below 50%.MethodsThe initial approach to surgery is performing an ALIF at the level of L5‐S1. A pfannenstiel incision is followed by transperitoneal exposure of the L5‐S1 disc between the common iliac vessels. The surgeon completes a discectomy at L5 and inserts a lordotic cage. The lordotic cage is filled with recombinant human bone morphogenetic protein 2 (RHBMP2). The second approach involves posterior exposure of L5‐S1 through a midline incision. There is no removal of the posterior elements of L5, or nerve root decompression. Pedicle screws with any necessary instrumentation are inserted at L5 and S1 only. The segment is mildly compressed to prevent column elongation. The L5‐S1 vertebrae and posterior instrumentation are fused with donor bone graft.ResultsSince 2003, this unique surgical approach has been performed successfully on fourteen patients (mean age 15.5 +/− 3.5 years). All had preoperative MRI scans that showed normal signal in the L4/L5 disc. Follow up confirmed a slip angle reduction in all fourteen patients. The slip percentage was successfully reduced from high grade to a grade of I (0–25%) or II (25–50%). There was no loss of reduction or cage displacement seen post‐operatively. At follow up appointments, patients did not report experiencing any pseudoarthrosis, symptoms of nerve root pain or lesion, cauda equina lesions, sexual dysfunction, retrograde ejaculation, nor the need for a re‐operation. All patients had a solid anterior fusion visualized on x‐ray during follow up. There were two surgical wound infections reported, which required incision and drainage or wound dehiscence. No other surgical complications were reported.ConclusionThe results of this study demonstrate that a combined, anterior‐posterior surgical approach, with anterior insertion of lordotic cage, posterior pedicle screw fixation, and bone graft fusion, effectively reduces slip percentage (to <50%) and restores a normal slip angle. Decompression and fusion of the healthy L4‐L5 segment can be avoided.Support or Funding InformationChildren’s Hospital New OrleansAnterior surgical approach and exposure of L5‐S1 disc for discectomyFigure 1
Inter‐domain Protein‐Protein Interaction through a Unique α‐Helix in the Hinge Region of E coli Topoisomerase I Leads to the Conformational Change of the Enzyme
Authors: Dasgupta Tumpa , Tan Kemin , Tse-Dinh Yuk-Ching 
Published: April 2020

E coli topoisomerase I (EcTopI) is a type IA topoisomerase that relaxes negatively supercoiled DNA to prevent the inhibition of vital cellular processes such as transcription. Type IA topoisomerase polypeptide folds into a torus structure to catalyze the breaking and rejoining of a DNA single‐strand coupled with DNA strand passage, thus maintain the DNA topology. Previously elucidated full‐length crystal structure of EcTopI consisting nine domains (D1–D9), bound with an ssDNA to the C‐terminal domain revealed that D6 has an unusual 21‐amino acids insert in the middle of the first β‐strand. A part of the insert (12 amino acids) forms a unique alpha helix (α1), which is present in the vicinity of the N‐terminal domain at the hinge region of D2 and D4. We hypothesized that the C‐terminal element D6 domain interacts with the N‐terminal elements D2 and D4 domains hinge region through this α1‐helix and aids in the separation of D3 from D4 and D1, thus creating an opening to the toroid hole for DNA strand passage in the catalytic cycle. Site‐directed mutagenesis has been employed to create two mutant EcTopI proteins with deletion of D6‐ α1 helix (residues 647–658, Mut‐1) and substitutions of A651G and A655G to perturb α1‐helix structure (Mut‐2). Biochemical analysis of these mutant proteins showed significantly reduced relaxation activity with negatively supercoiled DNA as substrate, and the kinetics of DNA negative supercoil removal by mutants EcTopI showed clear decrease in the rate of relaxation at different time points versus wild‐type EcTopI. To test the effect of these mutations on protein’s thermal stability, thermal shift assay has been carried out and the result suggested that the mutations affected the inter‐domain interactions without significant destabilization of the polypeptide structure as only the first melting peak is shifted to lower temperature but no shift in second peak has been observed compared to wild‐type. Our findings suggest that D6 α1‐helix supports a functional role in the hinge regions between D2 and D4 where its movement (e.g. a push or a pull) as an arm against the hinge will cause the rotation of D2 in respect to D4 leading to the enzyme undergoing a large conformational change required for catalysis. The results of this study have provided a more in‐depth mechanistic insight into the mechanism of type IA topoisomerases that is required for maintaining DNA topology.Support or Funding InformationNIH grant R 01 GM 054226E coli topoI‐Crystal StructureFigure 1Thermal shift assayFigure 2
Studies of an Mre11 separation of function mutation give insight into the mechanism of double stranded DNA damage repair
Authors: RAHMAN SAMIUR , Beikzadeh Mahtab , Canny Marella , Latham Michael 
Published: April 2020

The MRE11, RAD50, and NBS1 genes encode the proteins of the Mre11–Rad50–Nbs1 (MRN) complex, which is critical for proper maintenance of genomic integrity. The MRN complex acts in DNA double‐strand break repair (DSBR) detection, repair, and signaling. Exo‐ and endonuclease activities of Mre11 may regulate the choice between non‐homologous end‐joining (NHEJ) versus homologous recombination (HR) repair pathways but the exact mechanism is still nebulous. Two Mre11 mutations have been previously described as separation of function mutants: P. furiosus (Pf) H85S is both exo‐ and endonuclease inactive while Pf Mre11 H52S is exonuclease inactive but endonuclease active. Here we describe a new separation of function mutant: Pf Mre11 Y187C, which is also exonuclease inactive and endonuclease active. We have examined the possible causes for the difference in activities in Pf Mre11 Y187C and for the separation of function phenotype in general. Nuclear Magnetic Resonance (NMR) spectroscopy shows that both Y187C and H52S mutations alter the dynamic landscape of Mre11 affecting the DNA binding properties of the protein. We found that there is no change in binding of AMP (a nucleolytic product) by Y187C compared to WT. For DNA binding, Mre11ND constructs showed that Y187C binds both single and double stranded DNA with lower affinity than WT, but once Rad50 is added to the complex, there was no significant difference in affinity. Single site mutations in S. cerevisiae (yeast) established that the analogous Mre11 mutations are capable of DNA damage repair (i.e., survivability on plates with DNA‐damaging drugs), NHEJ repair, and DNA end resection, which is expected based on the previously described in vivo activity studies of MRN/X. Together, our data reveal that an aromatic group at amino acid position 187 in Pf Mre11 is important for nuclease activity, and our yeast data suggest that the presence of Mre11, but not it’s nuclease activity, is required for DSB repair in both repair pathways.Support or Funding InformationCancer Prevention and Research Institute of Texas (CPRIT)Y187C mutant is exonuclease inactiveFigure 1Affinity for dsDNA binding is lower with Y187C mutationFigure 2
Calcitonin Receptor N‐Glycosylation Enhances Peptide Hormone Affinity by Controlling Receptor Dynamics
Authors: Lee Sangmin , Jeong Yejin , Simms John , Warner Margaret L. , Poyner David R. , Chung Ka Young , Pioszak Augen A. 
Published: April 2020

The class B G protein‐coupled receptor (GPCR) calcitonin receptor (CTR) is a drug target for osteoporosis and diabetes. N‐glycosylation of asparagine 130 in its extracellular domain (ECD) enhances calcitonin hormone affinity with the proximal GlcNAc residue mediating this effect through an unknown mechanism. Here, we present two crystal structures of salmon calcitonin‐bound, GlcNAc‐bearing CTR ECD and analyze the mechanism of the glycan effect. The GlcNAc does not contact the hormone. Surprisingly, the structures are nearly identical to a structure of hormone‐bound, N‐glycan‐free ECD. N‐glycosylation of CTR increased calcitonin ligand association rate and decreased its dissociation rate. Hydrogen‐deuterium exchange mass spectrometry and molecular dynamics simulations revealed that the GlcNAc stabilized an adjacent β‐sheet while increasing flexibility of the peptide‐binding site turret loop. The glycan effect extended to RAMP‐CTR amylin receptor complexes and was also conserved in the related CGRP receptor. These results reveal that N‐glycosylation can modulate GPCR function by altering receptor dynamics.Support or Funding InformationThis work was supported by NIH grant R01GM104251 to AAP, the National Research Foundation of Korea grant NRF‐2018R1A2B6001554 to KYC, and use of the crystal imaging microscope was supported by NIH COBRE award P20GM103640. DRP and JS were supported by the BBSRC grant BB/M007529/1.Graphical abstractFigure 1
Interaction of sTREM2 with Amyloid Beta: Implication on the Protective Role of sTREM2 in Alzheimer’s Disease
Authors: Belsare Ketaki , Wu Haifan , DeGrado William 
Published: April 2020

Alzheimer’s disease (AD) is a leading cause of dementia and have two distinct histopathological makers‐amyloid plaques composed of Amyloid‐β (Aβ) peptides and neurofibrillary tangles of phosphorylated tau. AD is presented as dementia and loss of cognitive functions and usually seen in people over the age of 65. Currently there is no treatment available to treat the cause and progression of AD and hence there has been considerable efforts and interest in the study of AD, along with its associated proteins and histopathological markers for the development of diagnostics and therapeutics. Triggering receptor expressed on myeloid cells 2 (TREM2) is a receptor of the innate immune system expressed predominantly in the microglia along with dendritic cells and osteoclasts. Neurobiological functions and the pathological ligands of TREM2 are still not clearly known, but when triggered, it signals through the transmembrane adapter protein called DAP12 to activate phagocytosis of cellular debris (including ApoE, Aβ, and lipids). A rare missense mutation (R47H) in the gene encoding TREM2 confers a significant risk of AD (2–3 folds increase). Soluble TREM2 (sTREM2) which is supposed to be involved in the phagocytosis of Aβ plaques. Mutations in the TREM2 impair this phagocytic activity and therefore contribute to pathogenesis of AD. In this study, we are using various biophysical approaches to study the interaction of sTREM2 with Aβ. Kinetic measurements based on fluorescent ThT measurements showed a profound inhibition in the fibrillization/aggregation of Aβ (1–40) and Aβ (1–42) in presence of sTREM2 WT whereas the AD associated mutant (R47H) causes partial loss of inhibition function. Our experimental findings demonstrate that the sTREM2 affects the secondary pathway in the fibrillization of Aβ implying sTREM2 might affect Aβ seeding and spreading leading to lower Aβ associated neuronal toxicity. Additionally, we have interesting insights from cell infectivity and neuronal toxicity assay regarding the protective role of sTREM2 in AD.sTREM2 affects the secondary pathway of Aβ fibrillizationFigure 1
Characterization of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough
Authors: Bachochin Maxwell James , Barber Rob 
Published: April 2020

Sulfate‐reducing bacteria play a key role in the global carbon and sulfur cycles; a role realized through complex competitive or syntrophic metabolic relationships. These relationships are established in response to sulfate levels as well as concentrations of other nutrients such as short chain fatty acids (SCFAs). For some prokaryotes, SCFA production or consumption requires an ATP‐dependent reaction catalyzed by a SCFA kinase. In this study, we have determined the biochemical properties of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough heterologously expressed in E. coli. Our experiments characterize optimal conditions for activity as well as kinetic parameters for six SCFA substrates and the nucleotide cofactor ATP. These findings have implications on structure and function relationships as well as the potential physiological role of this enzyme.Support or Funding InformationThe authors would like to thank the Scott and Alice Thomson Fellowship Program for financial support of MB. Further, the authors thank the UW‐Parkside Committee on Research and Creative Activity and the College of Natural and Health Sciences for financial support of this project.Enzymatic Characterization of DvBuk.(A) DvBuk purity assessment was visualized by resolving 10 μg of purified DvBuk on 10% (w/v) polyacrylamide gel and stained with Coomassie Blue. Lane 1: Protein molecular weight markers, Lane 2: recombinant His‐tagged DvBuk. Enzyme activity (%) at various (B) pH and (C) temperature values. (D) Saturation kinetics of DvBuk with butyrate as substrate. (E) Relative activities of DvBuk in the presence of various SCFA substrates.Figure 1Substrate Preference of DvBuk.(A) Relative activities of DvBuk in the presence of various SCFA substrates (relative activities normalized to observed specific activity in the presence of 200 mM valerate = 9.7 U mg−1). (B) Kinetic constants for DvBuk using various SCFA substrates.Figure 2
Alteration of Pulmonary Elasticity and Vulnerability to Protease Digestion with Aging
Authors: Miura Katsutoshi 
Published: April 2020

An aging lung shows decreased elasticity with structural changes. Light microscopy reveals structural changes and speculates functional deterioration. Scanning acoustic microscopy (SAM) can present not only histology but also visualize elasticity of the tissue because of speed‐of‐sound (SOS) through tissues correlated with their stiffness.This study aims to detect pulmonary aging by pulmonary elasticity and vulnerability to protease digestion.Materials and MethodsFormalin‐fixed paraffin‐embedded sections of young (teens), middle (the forties), and old (eighties) were measured SOS through bronchioles, peribronchial vessels, and alveolar walls. An actinase E, which has non‐specific protease activity, was applied to the section and the functional and structural changes were followed by SOS and light microscopy.ResultsAlthough blood vessels of the old‐aged group showed higher SOS values compared to the young and middle‐aged groups, no significant difference in mean SOS values was present in the bronchiolar and alveolar walls among three groups. After actinase digestion, SOS of both bronchial and alveolar walls of young and middle aged‐groups showed a significant decrease while the old aged‐group appeared no considerable decline (Fig.
1. Fig.
2). Collagen fibers constituting alveolar and bronchiolar walls kept original structures in the old‐aged group compared to the young and middle‐aged groups. Blood vessels exhibited a significant reduction according to incubation time in three groups.ConclusionAlthough stiffness of blood vessels tends to increase with aging, pulmonary components of bronchiolar and alveolar walls showed no significant change of stiffness with aging. Bronchiolar and alveolar walls of young and middle‐aged tissues were vulnerable to protease digestion while old‐aged ones were resistant. Modification of collagen fibers may extend in the old‐aged lung. Following SOS images after protease digestion can help evaluate not only the morphological alterations but also the mechanical properties of pulmonary tissues.Support or Funding InformationThis work was supported by a grant from the Japan Society for the Promotion of Science (KAKENHI), Scientific Research Grant Number (c) 15K08375 and (c) 17K1088901.Speed‐of‐sound (SOS) image after actinase E digestion The lower row of the young lung shows gradual decrease in SOS values while the upper low of the old lung exhibits no remarkable changes.Figure 1Bronchial speed‐of sound (mean±SD) among young, middle, and old‐aged groups after actinase digestion.Figure 2