bluuebunny/crossref_metadata_2025 huggingface dataset:
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 Information

This 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 microscope

Figure 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

Introduction

There 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%.

Methods

The 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.

Results

Since 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.

Conclusion

The 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 Information

Children’s Hospital New Orleans

Anterior surgical approach and exposure of L5‐S1 disc for discectomy

Figure 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 Information

NIH grant R 01 GM 054226

E coli topoI‐Crystal Structure

Figure 1

Thermal shift assay

Figure 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 Information

Cancer Prevention and Research Institute of Texas (CPRIT)

Y187C mutant is exonuclease inactive

Figure 1

Affinity for dsDNA binding is lower with Y187C mutation

Figure 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 Information

This 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 abstract

Figure 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 fibrillization

Figure 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 Information

The 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 1

Substrate 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 Methods

Formalin‐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.

Results

Although 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.

Conclusion

Although 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 Information

This 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 1

Bronchial speed‐of sound (mean±SD) among young, middle, and old‐aged groups after actinase digestion.

Figure 2
TMEM49 induced ovarian cancer via apoptosis pathway
Authors: Hong Jinni , Zheng Lili , Lu Yajing , Jia Jin , Chen Jie
Published: April 2020

Ovarian cancer was one of the most lethal types of reproductive system tumors and a leading cause of cancer‐related death in women globally. The mechanism of ovarian cancer is poorly defined, in which apoptosis might play an important role. TMEM49, a 406 amino‐acid multi‐pass membrane protein localized to the endoplasmic reticulum‐Golgi intermediate compartment membrane, was identified to be involved in cancer‐relevant processes, and was demonstrated to inhibit proliferation and pulmonary metastasis of hepato‐cellular carcinoma, but little is known about the expression pattern and biological function of TMEM49 in ovarian cancer. Thus, in this study, we tested whether TMEM49 was associated with ovarian cancer and focused on the underlying mechanism by knocking down TMEM49 expression with siRNA in ovarian cells. In vivo, immuno‐histochemistry staining was performed and results showed TMEM49 was highly up‐regulated ovarian tissue of ovarian cancer patient when compared to patient with benign ovarian tumor. In viro,, with flow cytometry and CCK8 methods, SiRNA TMEM49 was demonstrated to inhibit cell proliferation, arrest G1/S transition, and induce cell apoptosis. Furthermore, western blot showed increased expression of apoptosis‐related protein such as Caspase3, Bad, and Bax, while expression metastasis‐related protein such as MMP2, KLF10, and CXCL12 were down‐regulated by TMEM49 knockdown. All these suggested that TMEM49‐related apoptosis play a role in ovarian cancer, and TMEM49 may be a potential therapeutic target in ovarian cancer.

Over‐expression of TMEM49 in ovarian cancer. a The mRNA level of TMEM49 in ovarian and normal tissues. b TMEM49 expression was significantly increased inovarian tumor tissues when compared with normal tissues of patients from TCGA dataset. c TMEM49 expression was evaluated by immunohistochemistry inovarian and adjacent tissues.

Figure 1
Revealing the function of a linker in the multi‐functional regulatory protein—proline utilization A from Escherichia coli
Authors: Mao Yizi , Arentson Benjamin W. , Becker Donald F.
Published: April 2020

Proline metabolism is critical for several cellular processes such as serving as carbon and nitrogen sources, preventing osmotic stress especially in plants, scavenging reactive oxygen species, and promoting pathogen virulence. In all organisms proline is oxidized into glutamate by the coordinated activities of the flavin‐dependent enzyme L‐proline dehydrogenase (PRODH) and NAD+‐dependent enzyme L‐glutamate‐γ‐semialdehyde (GSAL) dehydrogenase (GSALDH). In gram‐negative bacteria, these two enzymes are encoded on one polypeptide called proline utilization A (PutA). PutA from certain bacteria such as Escherichia coli (EcPutA), also includes a ribbon‐helix‐helix (RHH) DNA binding domain at the N‐terminus that is tethered to the PRODH domain by a 35 amino acid linker corresponding to residues 50–85 of the total 1320 in EcPutA. The RHH domain enables PutA to function as a transcription repressor of the put regulon (putP and putA genes) in addition to its enzymatic roles. This study aims to understand the role of the linker in the redox regulation of PutA and its functions. As conformational changes are required for PutA to switch between transcriptional repressor and enzymatic functions, the linker is proposed to be important for optimal arrangement of the RHH and PRODH domains. Partial and total linker deletion mutants, Δ50–73 EcPutA and Δ50–85 EcPutA, respectively, were characterized. Purified Δ50–85 EcPutA was shown to behave similarly to wild‐type PutA as assessed by enzyme activity and DNA binding assays. The Δ50–85 EcPutA mutant depleted of the full linker region, however, was unable to function as a transcription repressor in cell based assays indicating that the linker is necessary for EcPutA to bind to DNA in vivo. Work is ongoing to determine whether the transcription repressor activity of EcPutA is restored in partial linker mutant Δ50–73 EcPutA.

Support or Funding Information

NIH grants R01GM061068 and P30GM103335. Department of Biochemistry and Redox Biology Center, University of Nebraska‐Lincoln.

Model for regulation of put regulon.s

Figure 1

Domain structure and small angle X‐ray scattering model of EcPutA. Adapted from "Structure, function, and mechanism of proline," by L. Liu, J. J. Tanner, and D. F. Becker. 2017, Archives of Biochemistry and Biophysics, 632, p. 142. Copyright 2019 by 2019 Elsevier B.V.

Figure 2
Exosome‐Encapsulated Persistent Luminescence Nanoparticles Enabled Medicinal Product‐Based Drug Delivery System
Authors: Chan Ming-Hsien , Lee L. James , Hsiao Michael
Published: April 2020

Introduction

Recent studies have found that exosome or extracellular vesicles (EVs) are associated with cancer metastasis, disease progression, diagnosis and treatment, making cell research a hot emerging research area. Instead, cells would send them out via exocytosis. This has also been observed for other rigid nanoparticles such as silica and carbon. Relying on the superior targeting function and biocompatibility of cancer exosomes, they were able to deliver the drug to cancer stem cells deep inside the tumor in mouse models.

Challenge

Despite significant efforts made in this relatively new field of exosome research, progress has been held back by challenges such as inefficient separation methods, difficulties in characterization, and lack of specific biomarkers. Moreover, the applications of exosome still need more evaluation for the true condition.

Action

Persistent luminescence nanoparticles (PLNs) result in their powerful in vivo near‐infrared fluorescence, thus generating remarkable tumors imaging tracking ability in subcutaneous, orthotopic and metastatic tumors. Our study provides an approach for cancer therapy by using exosome‐biomimetic nanoparticles named PLN@tExo exocytosed by the Transwell channel electroporation (TEP) process. The PLN@tExo can be applied for drug carriers to efficiently deliver the anticancer drug into the center of cancer cells.

Resolution

The biocompatible exosome‐sheathed PLNs (Dox‐PLN@tExo) have been synthesized for targeted cancer chemotherapy. Dox‐PLN@tExos are exocytosed from cells after the TEP process. Following intravenous injection, Dox‐PLN@tExos actively accumulate in the tumor site and cross‐reactive cellular uptake by cancer cells, resulting in augmented in vivo DOX enrichment in tumor cells. Our study clearly demonstrates that exosome‐biomimetic nanoparticles have potential as drug carriers to improve anticancer efficacy.

Support or Funding Information

This research was supported by Academia Sinica [AS‐SUMMIT‐108] to MH and Academia Sinica Joint Program Office [AS‐NTU‐108‐01] to MH.

The scheme figure shows the exocytotic pathway can generate therapeutic exosome and encapsulate the nanoparticles as a targeting anticancer nanovehicle.

Figure 1

The confocal image shows the NEP process actively sends the nanoparticles into the cytosol. Moreover, the transport effect is better than incubated‐nanoparticle directly.

Figure 2
Cause and Consequence of MAGEB2 expression in cancer
Authors: Ramanathan Saumya , Colemon Ashley , Jones Angelle , Jackson Richala
Published: April 2020

Melanoma Antigen Genes (MAGEs) are a family of tumor‐associated genes. MAGE‐A, ‐B and ‐C genes belong to the Type I MAGE subfamily. They are typically expressed in the male germline and then aberrantly expressed in many cancers and therefore referred to as cancer‐testis antigens. Their expression in tumors is often associated with poor patient prognosis. There is a significant gap in understanding the mechanisms that regulate the expression of Type I MAGE genes. While cancers often express more than one MAGE gene, this study focusses on MAGEB2, an exemplary member of the Type I gene family and a bona fide cancer‐testis antigen. We hypothesized that epigenetic mechanisms such as DNA methylation, which regulates the expression of many germline genes also regulates MAGEB2 gene expression. Using bioinformatics and ChIP assays, we have determined the transcription factor networks that regulate MAGEB2 expression. In addition, we show that expressing MAGEB2 gene provides non‐transformed cells with a proliferative advantage by dampening TGFb signaling. Taken together our data indicate that cells use epigenetic memory to express MAGEB2 resulting in shift of the cells’ gene signature to a pro‐proliferative, anti‐apoptotic state that firmly places cells in the path to transformation.

Support or Funding Information

NSF Research Initiation Award HRD1764201 to S.R.

Graphical Abstract

Figure 1
Intracranial Pressure After Soccer Heading
Authors: Lee Justin Hyunwoo , Grace Evan James , Sieker Jeremy , Hargens Alan , Petersen Johan C. G. , Petersen Lonnie G.
Published: April 2020

OBJECTIVE

To evaluate the effect of soccer heading on intracranial pressure (ICP) in trained athletes.

METHODS

Eight subjects (all male, 21.7±1.7 years) were recruited from the UC San Diego soccer team. ICP and cardiovascular variables were recorded before and after 6 repeats of heading a soccer ball kicked from 35 yards away, into a goal. ICP was estimated non‐invasively from acoustical evoked tympanic membrane displacement (Vm), where a more negative volume displacement is correlated with higher ICP. Mean arterial pressure (MAP), heart rate (HR; Nexfin, Netherlands), and ICP were recorded in three different postures, 15° head up tilt (HUT), 0° supine, −15° head down tilt (HDT), in a randomized order.

RESULTS

Mean Vm at 15° HUT was 37.81±133.21 nL at baseline and −20.82±158.27 nL immediately post soccer heading (P=0.0482, paired t test), analogous to an increased ICP. At 0° supine, mean Vm was −46.19±178.50 nL at baseline and −84.27±183.55 nL after impact (P=0.0321, paired t test). At −15° HDT, mean Vm was −148.48±196.07 nL at baseline and −187.02±186.19 nL immediately post soccer heading (P=0.1176, paired t test). MAP and HR did not significantly change between the baseline and post soccer heading measurements.

DISCUSSION

After repeated soccer ball heading, ICP significantly increased at 15° HUT and 0° supine, while ICP at −15° HDT, MAP and HR did not significantly change. The relative increase in ICP associated with HDT is comparable to previously described trends. Although the measurement technique currently employed does not provide an absolute ICP baseline, the technique has been established as useful in evaluating relative changes. Under that interpretation, these results reflect what may be an elevated ICP related to soccer heading impacts. Accordingly, the results of this study suggest a potential health risk of participation in head contact sports.

Support or Funding Information

NASA (80NSSC19K0020) and Novo Nordic Foundation (NNF16OC0019196)

Soccer Heading Increases Intracranial Pressure at Head Up Tilt and Supine Positions

Volume displacement of tympanic membrane (Vm), measured non‐invasively from acoustical evoked tympanic membrane displacement, indicated an increase in intracranial pressure (ICP) after soccer heading at 15° head up tilt (HUT) and 0° supine (n = 8, paired t test, *P < 0.05). No significant increase in ICP at −15° head down tilt (HDT) was observed after soccer heading.

Figure 1
Human‐derived Bifidobacterium dentium Metabolites Modulate the Mammalian Serotonergic System
Authors: Engevik Melinda A. , Luk Berkley , Ihekweazu Faith D. , Danhof Heather A. , Chang-Graham Alexandra A. , Haag Anthony A. , Britton Robert A. , Hyser Joseph M. , Versalovic James A.
Published: April 2020

Background

Recent work has identified an essential role for the gut microbiota in regulating host‐derived serotonin (5‐hydroxytryptamine; 5‐HT) from enterochromaffin cells. Studies using germ‐free mice have revealed impaired 5‐HT levels and signaling in the absence of microbes. While it is known that addition of microbial communities to germ‐free mice restores 5‐HT levels, information on which microbes directly influence 5‐HT remains unknown. Commensal Bifidobacterium species produce acetate, a short chain fatty acid known to stimulate 5‐HT release in vitro. We hypothesize that B. dentium, a model Bifidobacterium species, secretes acetate and modulates the serotonergic system.

Methods & Results

Adult germ‐free mice were mono‐associated with acetate producing B. dentium, propionate producing Bacteroides ovatus, or heat killed B. dentium. Mass spectrometry of cecal and fecal samples revealed elevated levels of acetate in B. dentium mono‐associated mice and increased ileal, colonic, fecal and serum 5‐HT compared to all other groups. No increases in acetate or 5‐HT were observed in heat‐killed B. dentium or B. ovatus treated mice compared to germ‐free controls, as assessed by mass spec, immunostaining, and qPCR. Using mouse and human enteroids, we observed that addition of B. dentium metabolites and acetate stimulated release of 5‐HT in a dose‐dependent manner. Depletion of acetate in B. dentium secreted products reduced the amount of 5‐HT released from enteroids. We also confirmed our findings using a novel NGN3 over‐expression system in human enteroids, which increased the number of enterochromaffin cells. In addition to 5‐HT alterations, we found increased expression of 5‐HT receptors 2a and 4 in B. dentium mono‐associated mouse ileum and colon. RNA in situ hybridization revealed a similar increase in 5‐HT receptor 2a in the hippocampus of B. dentium treated mice compared to germ‐free controls. Functionally, live B. dentium modulated species typical behavior previously shown to be linked to 5‐HT receptor 2a.

Conclusion

Collectively, these data indicate that B. dentium, and the bacterial metabolite acetate, regulate key components of the serotonergic system.

B. dentium colonizes the intestinal mucus layer (left) and it’s metabolites stimulate serotonin release in mouse organoids (right).

Figure 1
Microbiome Signatures Associated with Rodent Model of Traumatic Brain Injury vs. Psychological Stress
Authors: Chakraborty Nabarun , Gautam Aarti , Demar James , Hammamieh Rasha , Long Joseph , Jett Marti
Published: April 2020

Traumatic brain injury (TBI) and post‐traumatic stress disorder (PTSD) are two signature illnesses of modern warfare. Discrimination between psychological stress and TBI using a knowledge‐driven unbiased panel of biomarker signatures will be essential for designing precise care management. Recent data suggest a role of alterations of the gut micobiome architecture susceptible to both brain injury and psychological disorders. The present study was conducted to identify factors discriminating between psychological stress and TBI in the fecal microbiome of rats and further to study the effect of diets enriched with varied polyunsaturated fat compositions.

A closed‐head TBI model consisting of blast overpressure (BOP) wave exposure coupled with a weight drop concussion (Marmarou method) was used on a group of adult male rats (N=12 each exposed and sham). In parallel, an independent group of rats (N=12 each exposed and sham) was subjected to an underwater trauma (UWT) stressor model that consisted of 30s of swimming and habituation, followed by 30s of forced whole body immersion. Prior to the stress types (BOP or UWT), animals were maintained for six weeks and continued thereafter on two different diets (N=4 per diet group): Standard house chow and custom chow. Unlike the house chow, the custom diets contained no long chain ω‐3 polyunsaturated fatty acids to offset their high Linoleic acid content. The fecal bacterial populations were characterized by identification of 16S ribosomal RNA.

Principal coordinate analysis showed clear separation between taxonomic phylogenetic profiles linked to the stress types from their corresponding shams. Within each stress type, the rodents fed on house chow showed different fecal microbial population from the customized diet. Firmicutes, Proteobacteria, and Cyanobacteria were those phyla of interest that showed significant population variance caused by the stress and diet variants. We integrated this microbial profile with rodent blood and brain genomic profile assayed in parallel. The result suggested a significant contribution of gut‐microbiome in energy networks related to the stress response, and diet emerged as a strong candidate in regulating resilience. This outcome could lead in identifying novel customized treatment of TBI and psychological stress.

Support or Funding Information

The project was funded by DoD‐MOMRP

The differential impact of two neurological stress models on different component of gut‐brain axis

Figure 1
An allosteric hot spot in the tandem SH2 domain of ZAP‐70 regulates T‐cell signaling
Authors: Gangopadhyay Kaustav , Roy Swarnendu , Debnath Olivia , Kumari Sunitha , Das Rahul
Published: April 2020

The T‐Cell signaling commences with the binding of Antigen Presenting Cell with T‐cell Receptor (TCR) Complex leading to the recruitment of a non‐receptor tyrosine kinase, Zeta Associated Protein‐70 (ZAP‐70), to the membrane. ZAP‐70 is comprised of the tandem SH2 domain (tSH2), which is made up of N‐terminal SH2 (N‐SH2) and C‐terminal SH2 (C‐SH2) domains, and a carboxy‐terminal kinase domain. The two SH2 domains cooperatively bind to the doubly‐phosphorylated ITAM (ITAM‐Y2P) motif resulting in a conformational rearrangement in the whole of the regulatory module, thereby releasing the autoinhibitory conformation of the kinase domain. However, the structural mechanism of how the two SH2 domain cross‐talk during ITAM‐Y2P binding and dissociation from the membrane are poorly understood. We observed that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly‐phosphorylated ITAM‐ ζ1 (ITAM‐Y2P‐ ζ1) peptide. The C‐SH2 binds first to one of the phosphotyrosine in ITAM‐Y2P motifs, leading to the formation of an encounter complex which further leads to the binding by N‐SH2 domain. NMR titration and Network analysis studies show that an intra‐residue network, which allosterically interconnects the N‐SH2 and C‐SH2 binding sites, plays a central role in ITAM‐Y2P binding. Further, a mutation in the allosteric network (F117A, W165C) uncouples the two binding sites, causing autoimmune arthritis in SKG mice. The allosteric coupling in ZAP‐70 is found to be unique and thus plays a significant role in T‐cell regulation.

Support or Funding Information

Authors are thankful to Prof. John Kuriyan and Prof. David E. Wemmer for access to the 900 MHz NMR spectrometer at the University of California, Berkeley. Dr. Jeffrey G. Pelton and Dr. Patrick R. Visperas at the University of California, Berkeley for NMR data collection and sample preparation. Authors thank Prof. Gautam Basu and Mr. Barun Majumder at Bose Institute, India for access to 700 MHz NMR spectrometer. Authors are thankful to Dr. Ashima Bhattacharjee, Dr. Pradip K. Tarafdar, and Prof. Pradipta Purkayastha for access to ITC and fluorimeter. Authors thank Prof. Giuseppe Melacini and Prof. Maitrayee DasGupta for helpful discussion. The authors thanks research funding from IISER Kolkata, infrastructural facilities supported by IISER Kolkata and DST‐FIST (SR/FST/LS‐II/2017/93(c)). This work is supported by grant from SERB (ECR/2015/000142) and DBT Ramalingaswami Fellowship (BT/RFF/Re‐entry/14/2014) to Rahul Das.

Figure 1
Effects of an Adenosine Receptor Bitopic Ligand on The Cardiovascular System
Authors: Cooper Samantha Louise , Wragg Edward , March Julie , Hill Stephen , Woolard Jeanette
Published: April 2020

Adenosine regulates physiological and pathophysiological processes via the activation of four G‐protein coupled receptors identified as the A1, A2A, A2B and A3 receptors. All of these receptors are known to be involved in regulation of the cardiovascular system
[1]. Recently, we described the effects of A1‐receptor selective agonists and the novel A1‐receptor bitopic ligand VCP746 on the rat cardiovascular system
[2]. Here, we assess the haemodynamic responses of VCP746 in the presence and absence of selective A2A and A2B‐receptor antagonists (SCH58261 and PSB1115, respectively).

As described previously
[3], under anaesthetic, rats were instrumented with miniature pulsed Doppler flow probes for the measurement of vascular conductance at the renal, mesenteric and hindquarters vascular beds. Intra‐arterial and intra‐venous catheters were implanted for measurement of heart rate (HR) and mean arterial pressure (MAP), and drug administration, respectively. All procedures were carried out with approval of the University of Nottingham Animal Welfare Ethical Review Board, under Home Office Project and Personal License Authority.

Experimental studies were conducted in conscious, freely‐moving rats. All compounds were prepared in a propylene glycol (PEG) buffer (5% PEG, 2% Tween, 0.9% saline). A 0.1ml bolus of SCH58261 (1.0 mg/kg), PSB1115 (10mg/kg) or vehicle was administered 10 minutes prior to increasing doses of VCP746 (6, 20, 60 μg/kg/min). Doses of VCP746 were administered as 3 minute infusions over a total of 9 minutes at an infusion rate of 0.1 ml/min.

VCP746 produced tachycardic effects, which were associated with renal and mesenteric vasodilatations (Figure). There was little effect in the hindquarter vascular bed and MAP was not significantly affected (Figure). The haemodynamic effects of VCP746 were partially antagonised by SCH58261 and PSB1115 fully attenuated haemodynamic responses to VCP746.

This study suggests that VCP746, the A1‐receptor bitopic ligand, mediates haemodynamic responses through the activation of the A2‐receptors, particularly the A2B‐receptor, rather than the A1‐receptor. This has implications for the design of bitopic ligands in the future.

Support or Funding Information

We would like to thank the BHF and COMPARE for funding.

Cardiovascular responses to VCP746. Rats were dosed with either PSB1115 (0.1 ml bolus dose of 10 mg/kg i.v., n=8) or vehicle (0.1 ml bolus PEG buffer, n=8). Approximately 10 min later, all animals received an infusion of VCP746 (6, 20, and 60 μg/kg/min; each dose infused over 3 minutes). Data presented as mean ± SEM. *P < 0.05 versus baseline (Friedman test). A Wilcoxon test for integrated area under or above curve analysis was used for comparisons between groups (♯ P<0.05).

Figure 1
SPARC: SODA, an interactive software for curating SPARC datasets
Authors: Patel Bhavesh , Srivastava Harshit , Aghasafari Parya , Helmer Karl
Published: April 2020

In this era of data‐intensive research, data sharing and reuse are becoming increasingly important for rapid discovery and innovation. While sharing can be as easy as a few clicks thanks to numerous data sharing platforms, data reuse is still an issue as standards for data curation and annotation are not rigorously or consistently followed by all researchers. As a result, it is often difficult, if not impossible, for researchers to understand and work with data they were not involved with during collection or analysis. To address this concern, the NIH SPARC program has imposed strict guidelines for curating and sharing data resulting from SPARC‐funded research. Complying with the guidelines requires additional time investment from the researchers (as with any data curation and submission standards), and the data curation process could thus become overwhelming as more and more data is generated. The objective of this project was to develop computer software to improve the curation workflow for SPARC researchers.

To achieve that, we are developing a software named SODA (Software for Organizing Data Automatically) to assist SPARC investigators in curating and annotating their datasets. Distributed as an open‐source desktop application (Windows, MAC, Linux), the goal of SODA is to bridge a long‐standing and overlooked gap between comprehensive data standards and their convenient application by researchers. SODA provides an interactive interface that, without requiring any coding knowledge, walks SPARC investigators step‐by‐step through the data organization and sharing requirements, while automating repetitive, complex and/or time‐consuming tasks (Fig
1). An introduction to the software and the algorithms implemented will be given. Quantitative results from ongoing performance evaluation (time to curate a SPARC dataset with and without SODA) by beta testers, all of whom receive funding from the SPARC program, will be presented as well.

Overall, the beta testers for early versions have acknowledged that SODA has the potential to improve their curation workflow. Data curation is becoming a critical issue, even beyond the SPARC consortium, and SODA could impact the broader research community by providing an exemplar and foundational tool for convenient and time‐efficient curation of data, which could be adopted by other projects. Potential applications, beyond the SPARC program, will be thus discussed as well.

Support or Funding Information

SPARC OT2OD025308‐01S2

Overview of the major features included in SODA during the on‐going development phase

Figure 1
Molecular Basis of 14‐3‐3 Protein Dependent Regulation of Caspase‐2
Authors: Obsilova Veronika , Kalabova Dana , Filandr Frantisek , Alblova Miroslava , Petrvalska Olivia , Horvath Matej , Man Petr , Obsil Tomas
Published: April 2020

Among all species, caspase‐2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164, within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14‐3‐3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14‐3‐3‐dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14‐3‐3 by hydrogen/deuterium mass spectrometry (HDX‐MS) and protein crystallography to determine the molecular basis for 14‐3‐3‐mediated inhibition of C2 activation. Our data reveal that the 14‐3‐3 dimer interacts with proC2 not only through ligand‐binding grooves but also through other regions outside the central channel, thus explaining the isoform‐dependent specificity of 14‐3‐3 protein binding to proC2 and the substantially higher binding affinity of 14‐3‐3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14‐3‐3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14‐3‐3 protein interacts with and masks both the nuclear localization sequence (NLS) and the C‐terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14‐3‐3. This masked region of p12 domain is involved in caspase‐2 dimerization. Therefore, 14‐3‐3 protein likely inhibits proC2 activation by blocking its dimerization surface.

Support or Funding Information

Funding

This study was funded by the Czech Science Foundation (V.O., grant number 19‐00121S), the Czech Academy of Sciences (RVO:67985823 of the Institute of Physiology), project BIOCEV (CZ.1.05/1.1.00.02.0109 and LQ1604) and MEYS CR (LM2015043 CIISB, Biocev, Biophysical methods, Structural Mass Spectrometry).

Crystal structure of the caspase‐2 peptide pS139+pS164 bound to the 14‐3‐3 dimer.

Figure 1
Association between single nucleotide polymorphism rs1044925 in the acyl‐CoA:cholesterol acyltransferase‐1 gene and plasma lipid parameters in ischemic stroke subjects
Authors: Oshiobugie Momoh Johnson
Published: April 2020

Acyl‐coenzyme A: cholesterol acyltransferase‐1 (ACAT‐1) is an intracellular enzyme that catalyzes the synthesis of cholesteryl esters from long‐chain fatty acyl‐CoA and cholesterol in various tissues and plays a major role in cellular cholesterol homeostasis and in atherosclerosis. The present study was carried out to clarify the association of ACAT‐1 rs1044925 single nucleotide polymorphism (SNP) and plasma lipid profiles and lipoproteins levels in patients with ischemic stroke. A total of 100 ischemic stroke patients and 100 controls matched for sex and aged 46–87 were selected. Plasma lipid profiles were measured and lipoprotein ratios were calculated using Excel software. Genotyping of the of the ACAT‐1 rs1044925 SNP was performed by polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) combined with 2% gel electrophoresis and then confirmed by direct sequencing. There were significant difference (P<0.05) in the genotypic and allelic frequencies of ACAT‐1 rs1044925 SNP between the stroke and normolipidemic subjects. The frequencies of AA, AC and CC genotypes of the ACAT‐1 gene were 64%, 33% and 3% for the control and 38%, 57% and 5% for the stroke subjects respectively. The frequencies of A and C alleles were 80.50% and 19.50% for the control and 66.50% and 33.50% for the stroke subjects (P < 0.0001) respectively. The association of genotypes and plasma lipid profiles and lipoprotein ratios were found for both subjects. The A allele carriers had lower plasma TC, TG, VLDL‐C, ApoB levels and other lipid parameters as compared to the C allele carriers for both subjects. The C allele carriers of ACAT‐1 rs1044925 SNP were responsible for increased dyslipidemia and lipid parameters for both subjects. The results of this study suggest that the polymorphism of rs1044925 in the ACAT‐1 gene is associated with plasma lipid profiles and lipoprotein ratios in patients with ischemic stroke.