bluuebunny/crossref_metadata_2025 huggingface dataset:
Reliability of zygapophysial (Z) joint measurements taken from MRIs of subjects with acute low back pain
Authors: Cramer Gregory D. , Cantu Joe A. , Pocius Judith D. , Balester Frank , Simpson Derek R.J. , Horner Tyra B. , Healy Joshua , McKinnis Ray A.
Published: June 2021

The central anterior to posterior (A‐P) distance of the left and right L4/L5 and L5/S1 zygapophysial (Z) joint space is being measured from MRI scans to assess changes following spinal manipulation. This IRB approved study assesses the reliability of those measurements. An independent radiologist chose 6 subject scans. Three observers measured these scans (24 measurements, rounded to 0.1mm using a backlit digitizer) on two separate occasions separated by a minimum of 4 weeks. Observers were blinded to each other and their previous work. Intra‐ and interobserver reliability was calculated by means of intra‐class correlation coefficients (ICCs), and mean differences. ICCs showed intraobserver reliabilities of 0.95 (95%CI 0.87‐0.98), 0.83 (0.62‐0.92), and 0.92 (0.83‐0.96) for each of the 3 observers; and interobserver reliabilities of 0.90 (0.82‐0.95), 0.79 (0.61‐0.90), and 0.84 (0.75‐0.90) for the first and second measurements and overall reliability, respectively. The mean difference between the first and second measurements was ‐0.04 (+1.96 SD= ‐0.37 ‐ 0.29), 0.23 (‐0.48 ‐ 0.94), 0.25 (‐0.24 ‐ 0.75), and 0.15 (‐0.44 ‐ 0.74) for each of the 3 observers and the overall agreement, respectively. A mean difference of <+0.4 was considered clinically acceptable. Consequently, the measurements were highly reliable. Support: NIH/NCCAM (Grant# 2R01 AT000123).
Anatomy Leading the Competition – Variety is the Spice of Life
Authors: Evans Darrell JR
Published: June 2021

Ever wondered whether your students are getting the most out of their anatomical experience? With extensive changes to medical curricula it has been necessary to reassess the way we teach anatomy so students are well equipped for the challenges beyond university. At one UK medical school we have explored ways to promote anatomical learning. Variety is truly the spice of life and we have designed our program to include both traditional and contemporary teaching methods, all aimed at stimulating deep learning. A varied approach has helped us to effectively include all students in the learning experience and encompass different learning needs of our diverse student population. Whether students spend time in the dissecting room, engaged in interactive tutorial sessions or participating in technology‐enhanced living anatomy classes, a building block approach to learning is used which draws together form, function and clinical relevance. Our approach to anatomy education has had to take on board many conflicting demands, but the learning achievements of our students and their feedback would suggest we are leading the competition in this respect.
Apin participates in establishment of the incompletely‐differentiated junctional epithelium around teeth
Authors: Nishio Clarice , Wazen Rima , Moffat Pierre , Nanci Antonio
Published: June 2021

A novel gene, Apin (Odam), belonging to a gene cluster encoding for proteins involved in the regulation of mineralization, was cloned in our laboratory. Initial studies suggested that Apin might mediate adhesion of the portion of the gingiva that attaches to the tooth surface (junctional epithelium; JE). It is also expressed in many epithelial cancers, including odontogenic tumors.

Purpose

To validate the implication of Apin in JE formation by comparing its distribution in normal and regenerating gingiva. Materials and methods: Gingivectomies were done on the maxillae of adult rats. The contralateral maxilla was left intact as a control. Animals were sacrificed on days 0‐7 after surgery and maxillae were processed for paraffin embedding and immunolabeling for Apin.

Results

In control samples, Apin was localized among cells of the JE and along its interface with the tooth. No labeling was seen in the residual gingiva at day 0. Over time, Apin labeling reappeared in the regenerating JE emerging from the gingiva. By day 7, the labeling pattern was similar to controls.

Conclusion

The interfacial localization of Apin suggests that it plays a role in adhesion. Its presence among cells of the JE, an incompletely differentiated epithelium, and its expression in epithelial tumors indicates that Apin may also be implicated in other cellular activities such as differentiation. Support by CIHR and Department of Foreign Affairs.
Analysis of immune cells and proinflammatory substances as biocompatibility markers of dental sealers
Authors: NAKAGOSHI‐CEPEDA AKEMI argelia , SOLIS‐SOTO JUAN MANUEL
Published: June 2021

A variety of studies have demonstrated that root canal sealers induce mild to severe inflammatory reactions. Several factors participate in this immune response. Analysis of these factors can be indicative of biocompatibility.

Objective

In this study immune cells and proinflammatory chemokines and cytokines are analyzed in response to dental sealers of different composition.

Methods

After Apexit (calcium hydroxide based), Portland or MTA were implanted in the dorsal dermis of BALB/c mice, histopathological and immunocytochemical studies were performed to analyze the presence of a) Macrophages and CD8 lymphocytes, b) Cells producing proinflammatory cytokines (IL‐1beta, IL‐6, and TNF‐alpha), and c) Cells producing proinflammatory chemokines (CCL5 and CCL20).

Results

All treated materials produced a mild inflammatory reaction. Apexit was the most biocompatible, with results similar to controls, except for 12 hours post‐implantation, when significative more IL‐6 and CCL20 immunoreactive cells were observed (p<0.05). Macrophages, CCL20 and IL‐6 were in higher amounts in mice implanted with MTA in comparison than Portland (p<0.05).

Conclusions

The calcium hydroxide based dental sealer showed better in situ biocompatibility than calcium silicate oxide sealers.
Functional characterization of Dps homologs from D. radiodurans suggests distinct in vivo roles
Authors: Reon Brian , Bhattacharyya Gargi , Grove Anne
Published: June 2021

Deinococcus radiodurans, a Gram‐positive eubacterium, is widely recognized for its extreme resistance to various DNA‐damaging conditions, such as desiccation and ionizing and ultraviolet radiation. D. radiodurans is one of only a few bacteria which encode two different DNA protection during starvation (Dps) proteins. We report here a comparison of both Dps homologs from D. radiodurans. Dps‐1 was found to bind to DNA at lower concentrations than Dps‐2 and both have the ability to oxidize Fe2+. Dps‐2 protects DNA from degradation by reactive oxygen species (ROS), but is unable to protect DNA from DNase I, while the converse is true for Dps‐1. Dps‐1 was shown to condense DNA in vivo while Dps‐2 is incapable of nucleoid condensation. Notably, in vivo localization assays showed that unlike most previously characterized Dps proteins, Dps‐2 is non‐cytoplasmically localized. While Dps‐1 may serve a function in iron homeostasis or nucleoid organization, the function of Dps‐2 may be to protect against ROS‐mediated hydroxyl radical degradation. Together these proteins may contribute to D. radiodurans' ability to withstand such extreme environments.
Human notch and mastermind proteins activate target genes differently depending on combination used
Authors: Burke Tommy M , White J Brandon
Published: June 2021

Notch signaling plays a crucial role in cell development and proliferation. The Notch receptor is an inducible transcription factor found on the cell surface which is cleaved resulting in the release of the intracellular domain (NICD). The NICD enters the nucleus and complexes with CBF1 and Mastermind (MAM) to activate target genes. In humans, there are four Notch receptors and three Mastermind proteins. We wish to understand if all NICD and MAM proteins contribute equally to transcriptional activation of target genes and if all MAM proteins are capable of degrading the different NICD's. Various combinations of NICD and MAM constructs result in different activation of the HES1 and HES5 promoters depending on the combination used. HES5 was much stronger in activation than HES1. NICD3 and NICD4 paired with any MAM protein weakly activated both HES1 and HES5 promoters. The strongest activation of promoters occurred in the presence of MAM2 with NICD1 and 2. NICD degradation occurred by over‐expression of all three MAM's with the exception of NICD4 which was weakly degraded by the different MAM's. Taken together these results suggest that promoter activation depends strongly on the context of the combination of proteins used and degradation may not necessarily be the deciding factor in strength of promoter activation. Support of research was through NSF‐REU and HHMI‐SCRIBE Programs at San Jose State University to TMB.
Antagonistic gene control through histone H2A and H2B ubiquitylation
Authors: Verrijzer Peter
Published: June 2021

Maintenance of gene transcription status plays a critical role in development and disease. Here, we will discuss the antagonistic functions of histone H2A and H2B ubiquitylation. Whereas histone H2A ubiquitylation is associated with gene silencing, histone H2B ubiquitylation correlates with transcription activation. We recently identified dRAF, a novel Polycomb group silencing complex that harbors dKDM2 as a key dRAF subunit that plays a pivotal role in a trans‐histone pathway involving the removal of an active histone H3 methyl mark and formation of the repressive H2Aub mark during PcG silencing. New results on the functioning of dRAF and related complexes will be presented. In addition, we will discuss developmental gene regulation by the GMP synthetase (GMPS) and Ubiquitin Specific Protease 7 (USP7), which directs histone H2Bub de‐ubiquitylation. Both the metabolic enzyme GMPS and the DUB USP7 play remarkably specific roles in the transcriptional regulation of neural development of the optical system. We will discuss the impact of histone ubiquitylation on gene‐specific transcription control.
Repression of Proliferative Transcription Factors by Guanylyl Cyclase‐A/ Natriuretic Peptide Receptor‐A in Mouse Mesangial Cells
Authors: Tripathi Satyabha , Kumar Prerna , Pandey Kailash N.
Published: June 2021

Atrial natriuretic peptide (ANP) is a member of the natriuretic peptide hormone family that maintains cardiovascular homeostasis and exerts antiproliferative effects on various cell types. ANP mediates its effects by binding guanylyl cyclase‐A/natriuretic peptide receptor‐A (GC‐A/NPRA) and activating cGMP‐dependent protein kinase (PKG). The objective of this study was to determine the effects of ANP‐NPRA system on the proliferative transcription factors activating protein‐1 (AP‐1) and cAMP‐response element binding protein (CREB) in mouse mesangial cells (MMCs). Treatment of MMCs with vascular endothelial growth factor (VEGF) stimulated phosphorylation of c‐jun, c‐fos, and CREB by 3‐ to 4‐ fold compared to control cells. ANP treatment significantly reduced VEGF‐stimulated AP‐1 and CREB transcriptional activity in a PKG‐dependent manner. Moreover, treatment of VEGF‐stimulated cells with ANP inhibited in vitro and in vivo binding of AP‐1 (c‐fos and c‐jun) and CREB to DNA, as measured by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The inhibitory effects of ANP were augmented by 60‐70% in NPRA‐transfected relative to vector‐transfected MMCs. The present study delineates a mechanism through which ANP‐NPRA signaling exerts an inhibitory effect on cell proliferation and plays a critical role in responses to cardiovascular disease states.
Yin Yang 1 Associated Factor 2 Interacts with the Yin Yang 1 REPO Domain
Authors: Pratt Heather , Wilkinson Frank
Published: June 2021

The Yin Yang 1 (YY1) REPO domain was found to be necessary and sufficient to recruit Polycomb proteins to DNA and silence a transgeneic reporter gene (PNAS 2006 103:19296‐19301). The objective in this study was to characterize the molecular mechanisms responsible for Polycomb recruitment by REPO. Yin Yang 1 Associated Factor 2 (Yaf2) was identified as a putative ligand for the REPO domain in a yeast two‐hybrid screen of a mouse 17 day embryonic cDNA library. Full length YY1 (1‐414) and the YY1 REPO domain (YY1 201‐226), but not the YY1 REPO deletion (YY1 1‐414del201‐226), were capable of interacting with Yaf2 in the two hybrid assay. These observations were validated in vivo using a chromatin immunoprecipitation assay. A transgenic Drosophila strain was prepared expressing flag‐tagged Yaf2 under control of the heat shock 70 promoter. This strain was crossed to a strain containing the transgenic reporter element (BGUZ) and either heat shock 70 driven‐Gal4 DNA binding domain (GALDBD) or the GALDBD fused to YY1 201‐226 (GALREPO). FlagYaf2 was immunoprecipitated at the BGUZ locus in the presence of GALREPO but not in the presence of GALDBD. Together, these results indicate that the YY1 REPO recruits Yaf2 to DNA and likely contributes to Polycomb‐mediated silencing. This work was funded by a faculty development grant awarded to F.W from Philadelphia University.
Developmentally‐regulated expression of caspases during cyclic blastogenesis in a colonial urochordate
Authors: Kearney Matthew David , Lauzon Robert J.
Published: June 2021

Botryllus schlosseri is a clonally modular ascidian that reproduces asexually by blastogenesis, and utilizes a unique lifecycle whereby every new asexual generation in a colony resorbs the old adult generation on a weekly basis. During this process, cells from adult tissues and organs synchronously undergo programmed cell death by apoptosis, and cell corpses are engulfed by circulating phagocytes. Resorption of the entire adult generation is completed within 24‐36 hours and occurs simultaneously within the functional emergence of a new asexual generation of individuals. We have previously shown that developing buds and phagocytic cells are critical effectors in this process and as such engineer the reconstitution of a new asexual generation. Based on it unique lifecycle, Botryllus provides an excellent model system to investigate the developmental regulation of organismal apoptosis. Caspases are cysteinyl aspartases that are functionally involved in both the initiation and execution phase of apoptosis in multicellular animals. An expressed sequence tag (EST) sequencing project is currently underway in Botryllus, and has yielded several candidate caspases orthologs. In this study we have investigated their expression patterns during cyclic blastogenesis using semi‐quantitative PCR. Work is currently underway to determine whether caspase expression is up regulated during organismal apoptosis.
Regulation of Germ Cell Promoter Activity in Xenopus Oocytes
Authors: Li Dan , Raza Abbas , DeJong Jeff
Published: June 2021

Here we evaluate the feasibility of using Xenopus laevis oocytes as a model system for assaying the activity and architecture of a germ cell promoter. To test this approach the promoter from the X. laevis ALF transcription factor gene was cloned and evaluated for transcription start site usage and DNA methylation status. Comparison with human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis revealed low homology and demonstrated the difficulty in the computer prediction of regulatory elements. However, the activity of ALF promoter‐luciferase reporter constructs assayed after microinjection into oocytes was very high and could be localized to a 63 base pair segment. Interestingly, promoter activity in oocytes and somatic Xenopus XR epithelial cells were very different, indicating that these cell types are not interchangeable as assay systems. The use of site‐specific promoter mutants and deletion constructs identified two distinct functional elements (A and B) in the promoter, both of which were required for activity and recognized by proteins present in oocyte extracts. Rearrangement of the two elements displayed effects on activity ranging from minimal to severe. Overall, the results show that microinjection of a germ cell‐specific promoter into Xenopus oocytes provides a viable model system for dissecting the biochemistry of germ cell gene regulation.
Functional studies of the Med25 Mediator subunit in Arabidopsis thaliana
Authors: Björklund Stefan , Elfving Nils , Davoine Celine
Published: June 2021

We recently purified the Mediator complex from Arabidospsis thaliana (At) (B ?ckstr ?m et al., 2007). This is so far the only example of Mediator identified in plants. We could show that the AtMed25 subunit is identical to PFT1 (PHYTOCHROME AND FLOWERING TIME 1). PFT1 was originally identified as a nuclear protein that acts in a phyB pathway and induces flowering in response to suboptimal light conditions (Cerdán and Chory, 2003). In mammalian cells, Med25 is identified as the Mediator subunit that functions as target for the VP16 transcriptional activation domain ([Mittler et al., 2003] and [Yang et al., 2004]). The VP16 was mapped to interact with a region between amino acids 402‐543 of the mammalian Med25 and this domain is conserved in AtMed25. We have used the corresponding region of AtMed25 in yeast 2‐hybrid screening assays to identify potential plant‐specific transcriptional activators involved induction of flowering at suboptimal light conditions by interaction with AtMed25. We have so far identified three transcription factors that operate through AtMed25 and the results indicate that Med25 might function in integration of different signaling pathways that control flowering.

This work was supported by grants from the Swedish Research Councils Formas and Natural and Engineering Sciences.
Nuclear Bmp2 (nBmp2) Alters the Expression of Several Genes
Authors: Davis Evan Drew , Fonhoue Borel S. D. , Felin Jenny E. , McCune Broc T. , Bridgewater Laura C.
Published: June 2021

Bone morphogenetic protein 2 (Bmp2) is a secreted growth factor that modulates many essential physiological and developmental processes through extracellular signaling. Bmp2 signaling is essential in neuronal differentiation, embryonic vasculogenesis, and cardiac development. Recent identification of a novel nuclear variant of Bmp2 challenged its sole classification as a secreted growth factor. Nuclear Bmp2 (nBmp2) is translated from an alternative start codon downstream of the signal peptide sequence, which allows a bipartite nuclear localization signal to direct translocation to the nucleus. In order to elucidate the function of nBmp2, microarray analyses were performed on cells transfected with nBmp2. These analyses indicated that over‐expression of nBmp2 alters the expression levels of hundreds of different genes. From this list, genes were selected for further analysis by quantitative RT‐PCR. This work confirmed that nBmp2 significantly alters the expression levels of several genes that are expressed in neuronal and cardiac tissue. These results suggest a functional role for nBmp2 in development or maintenance of neurological and circulatory systems. This work was supported by the NIH grant AR4883 and by an award from the Fulton Family Foundation to support undergraduate research.
Potential role of distal regulatory elements in ubiquitous induction of the calcitonin/calcitonin gene‐related peptide (CALCA) gene in sepsis
Authors: Park Ki Youb , Russo Andrew
Published: June 2021

The CALCA gene is expressed only in neuroendocrine tissues in a normal condition but it is ubiquitously induced in a septic condition. So far, the molecular mechanism underlying the tissue‐wide induction of the CALCA gene is unknown. Here, I identified DNA regulatory elements that might mediate the abnormal induction of the CALCA gene using adipocytes as a model system. Using a web‐based "evolutionary conserved region (ECR)" browser, I found two ECRs (ECR2 and ECR3) at the 3' end of the CALCA gene. Both ECRs were conserved among mammals, marsupial and bird. ECR2 showed a moderate enhancer activity at a basal condition in CALCA expressing cell line, CA77. ECRs were then, delivered to adipocytes through virus. Treatment of pro‐inflammatory agents increased activity of these virally delivered ECRs in adipocytes, but not in CA77 and Rat2 cells. This supports that ECRs might be involved in the inflammation‐caused CALCA gene induction. Furthermore, I found that ECR2 and ECR3 were bound by acetylated histone H3 independently of pro‐inflammatory stimuli. In silico analysis of the ECR2 sequence provided a putative binding site of hepatocyte nuclear factor (HNF)‐1. This study is the first attempt to identify the regulatory DNA element involved in the abnormal CALCA gene induction during sepsis. This study has been supported from NIH Grant DE016511.
Characterization of ZBP1‐βactin mRNA complex
Authors: Chao Jeffrey Alan , Singer Robert
Published: June 2021

Zipcode binding protein 1 (ZBP1) belongs to a family of highly conserved RNA‐binding proteins that have been linked to several steps in the post‐transcriptional regulation of gene expression including mRNA stability, localization and translation. This family of proteins functions during embryogenesis and is often found over‐expressed in a variety of cancers. ZBP1 was first identified by its ability to promote the localization of beta‐actin mRNA to the leading edge of chicken embryo fibroblasts through its interaction with cis‐acting elements contained within the beta‐actin mRNA.

ZBP1 contains two RNA recognition motifs (RRM) followed by four hnRNP K homology (KH) domains. We have identified a fragment of ZBP1, which contains both KH3 and KH4, that binds to a 28 nucleotide region of the beta‐actin mRNA with high affinity. KH3 and KH4 function together as one domain and bind RNA as a monomer. Mutation of the amino acids that link KH3 and KH4 reduces binding affinity by greater than 100‐fold suggesting that the orientation of the RNA binding surfaces is important for RNA recognition.

While ZBP1 KH34 has remained recalcitrant towards crystallization, we have recently obtained crystals of IMP2 KH34, a paralog of ZBP1 with 82% sequence identity, that diffract to 2Å resolution. A molecular replacement solution has not yet been identified, so experimental phases are being determined.
Engineering artificial splicing factors with designed specificities
Authors: Wang Zefeng , Wang Yang , Cheong Cheom‐Gil , Hall Traci M
Published: June 2021

The majority of human genes undergo alternative splicing to produce multiple isoforms with distinct functions. This process is tightly regulated by trans‐acting splicing factors that bind specifically to exons or introns to promote or inhibit their splicing. Here we report the engineering of novel artificial splicing factors that can be designed to specifically modulate alternative splicing of target genes. Sequence‐specific RNA‐binding domains of human Pumilio1 were fused with splicing regulatory motifs to produce artificial splicing factors with diverse activities in regulating different types of alternative splicing in several reporter systems. Because disruption of splicing regulation is closely associated with various human diseases, a new method to modulate disease‐associated splicing events provides therapeutic potential. We demonstrated such potential by engineering a splicing factor to modulate alternative splicing of the endogenous Bcl‐x gene, a target of anti‐cancer therapy. This engineered splicing factor shifted splicing toward the pro‐apoptotic Bcl‐xS isoform, promoted apoptosis, and increased chemosensitivity of several cancer cell lines to common anti‐tumor drugs. The ability to design the RNA sequence specificity of Pumilio1 permits creation of artificial splicing factors that can target virtually any pre‐mRNA.
Small RNA, Big Impact: Probing miRNA pathways in living cells using single particle tracking
Authors: Pitchiaya Sethuramasundaram , Androsavich John R , Sobhy Mohamed A , Walter Nils G
Published: June 2021

MicroRNAs (miRNAs) are short non‐coding RNAs that control the expression of an estimated 30% of all human proteins post‐transcriptionally. Mature miRNA, typically 21 base pairs long, associates with several proteins to form a miRNA Induced Silencing Complex (miRISC). This complex associates with the Un‐Translated Region (UTR) of mRNAs in a sequence specific manner and forms an active miRISC that controls protein expression. While several different mechanisms have been proposed for miRNA mediated gene repression, they are highly debated. We are investigating the mechanism of miRNA action by observing the diffusive motions of miRISC‐mRNA complexes in living cells using single particle tracking microscopy (SPTM). To this end, we microinject fluorophore labeled miRNA probes into living cultured cells. The probe can bind to multiple sites on its target mRNA which results in an inherent fluorescent amplification and significantly aids in tracking the particle. Quantitative Real Time ‐ Polymerase Chain Reactions (qRT‐PCR) were performed to estimate the level of the HMGA2 mRNA in F9 cells and preliminary tests suggests that there are ~8 mRNA molecules/cell, which is a suitable target number for our SPT experiments. SPT experiments are being performed using which we can deduce whether a specific miRISC‐mRNA complex progresses through multiple mechanisms of repression.

Funded by NIGMS.
Transcriptome‐wide Identification of the mRNA target sites of the Fragile‐X Mental Retardation Proteins
Authors: Ascano Manuel , Landthaler Markus , Khorshid Mohsen , Burger Lukas , Hafner Markus , Zavolan Mihaela , Tuschl Thomas
Published: June 2021

A leading cause of human mental retardation is the functional loss of the Fragile X mental retardation protein (FMRP). FMRP is the founding member of an evolutionarily conserved gene family that encodes RNA‐binding proteins (RBP) critical for numerous post‐transcriptional regulatory networks illustrated by their importance in neurological and muscular development. Despite their requisite nature, their in vivo targets remain largely unknown. Towards this end, we have improved upon existing methods for RBP‐target identification. We report the transcriptome‐wide discovery of RNA targets for FMRP and its two homologs Fragile‐X related proteins 1 and 2. The exact nucleic acid sequences of the messenger RNAs they bind are identified. Ribonucleoprotein particles (RNPs) are predicted to bring discrete mRNAs into functional groups, or regulons, to elicit specific biological processes, conceptually similar to prokaryotic operons. We find a number of genes targeted by the FMR protein family that function within the same cellular process such as: neuromuscular development, mRNA transport and various signal transduction pathways.
Structural investigation of the NTF2‐like RNA binding domain of C. elegans NXF‐2 in complex with NXT‐1
Authors: Kerkow Donald E , Carmel Andrew B , Williamson James R
Published: June 2021

The first step governing mRNA export and metabolism is the sorting of processed transcripts into specific nuclear export pathways. mRNA nuclear export and regulation is orchestrated by the nuclear export factor (NXF) family. The NXF NTF2‐like domain, required for nuclear export, is an important interaction domain that heterodimerizes with NXT1 (also an NTF2‐like protein) and directly binds nuclear pore FG repeats. We found that the NTF2‐like domain of both C. elegans NXF‐2 (CeNXF‐2) and human NXF1 possesses novel high affinity RNA binding activity. Here, we present the 1.8Å crystal structure of the NTF2‐like domain from CeNXF‐2 bound to its protein partner CeNXT‐1. Structural analysis reveals a putative RNA binding site on CeNXF2, not present on CeNXT1, and also shows a clear FG binding pocket suggesting possible nuclear pore binding and shuttling activity. Additionally, both CeNXF‐2 and CeNXT‐1 appear to be fairly flexible/dynamic outside of their dimer interface. The presence of additional binding pockets in this structure suggests that the list of functions ascribed to the NTF2 fold is not yet complete. Furthermore, RNA binding activity of the NTF2‐like domain implies that its role in NXF mediated RNA export/regulation is not completely understood. Structural characterization of the putative RNA binding site and of the additional binding pockets is currently underway.
A switch from BAG1 to BAG3 during ageing triggers the enhanced use of the autophagic‐lysosomal system for the degradation of polyubiquitinated proteins
Authors: Gamerdinger Martin , Hajieva Parvana , Behl Christian
Published: June 2021

BAG1 and BAG3 are members of the Bcl‐2‐associated athanogene (BAG) family, a conserved group of Hsp70 co‐regulators. We show that both, BAG1 and BAG3, are involved in protein quality control: whereas BAG1 regulates the removal of polyubiquitinated proteins by the proteasomal system, BAG3 acts in concert with the ubiquitin‐binding protein p62/SQSTM1 to stimulate the turnover of polyubiquitinated proteins by the autophagic‐lysosomal pathway. Interestingly, a switch from BAG1 to BAG3 occurs during ageing of human cells as well as in neurons of the aged rodent brain. Similar to the ageing models, a shift from BAG1 to BAG3 is observed in HEK cells under protein cross‐linking conditions provoked by oxidative stress. Moreover, we show that, triggered by the increased BAG3 to BAG1 ratio, aged cells use increasingly the autophagic‐lysosomal pathway for the turnover of polyubiquitinated proteins. We conclude that the BAG3‐mediated recruitment of the autophagy pathway is an important adaptation of the cellular protein quality control system to maintain protein homeostasis in the presence of an enhanced pro‐oxidant and aggregation‐prone milieu characteristic of ageing.

This work was supported by grants from the Fritz und Hildegard Berg‐Stiftung
Changes in β‐secretase activity in down syndrome and aging
Authors: Webb Robin L. , Holler Christopher J. , Beckett Tina L. , Head Elizabeth , Murphy M. Paul
Published: June 2021

Background

Down Syndrome (DS) or trisomy 21, leads to Alzheimer's disease (AD) pathology by 40 years of age. Chromosome 21 harbors several genes implicated in AD, including the amyloid precursor protein (APP), and one isoform of the β‐site APP cleaving enzyme (BACE2).

Objective

Our laboratory is interested in β‐secretase and its regulation in different disease states. DS provides a unique model for evaluating changes in regulatory factors that influence the proportion of DS patients with AD.

Methods

We used a fluorometric assay developed in our lab to evaluate both BACE1 and BACE2 activity from a set of 24 DS (0.42‐62 years) and 16 control (0.45‐67 years) brains (Brodman area 9). Results and

Conclusions

BACE1 significantly decreased (p<0.04) with age, a finding differing from the situation seen in both AD and with normal aging in rodents and non‐human primates. Surprisingly, BACE2 was unchanged, even though the gene for BACE2 is located within the DS obligate region of chromosome 21. Finally, BACE1 and BACE2 activity levels were highly correlated in this series (r2 = 0.95), indicating that there may be a higher degree of shared regulation than previously believed. Additional studies will examine the relationship between amyloid pathology and enzymatic activity. Insight gathered from these studies will help understand the nature of the shared pathology in both diseases. Supported by NIH NS058382.
Dynein light chain facilitates dimerization of its primarily monomeric disordered binding partners
Authors: Barbar Elisar , Hall Justin , Nyarko Afua
Published: June 2021

Dynein light chain LC8 was first discovered as an essential component of the microtubule‐based molecular motor dynein. LC8 also interacts with non‐dynein proteins in a broad array of systems, including apoptosis, viral pathogenesis, enzyme regulation, and kidney development. The many diverse interactions of LC8 led to the widely held view that these proteins are dynein cargo, and that LC8 serves as an adaptor for attaching cargo to dynein. Our recent structural and binding studies suggest that LC8 cannot simultaneously assemble with dynein and bind to other proteins. We propose that LC8 functions to facilitate dimerization of its monomeric disordered binding partners. We test this hypothesis by probing the interactions of LC8 with dynein intermediate chain and a non dynein protein Swallow. Both free IC and Swallow are disordered and monomeric at physiological temperature. Using a combination of techniques including fluorescence resonance energy transfer, isothermal titration calorimetry, circular dichroism, NMR and X‐ray crystallography we show that for IC, LC8 binding is coupled to IC self‐association at a dimerization domain distant from the LC8 site, while the rest of IC remains disordered. Likewise, for Swallow, LC8 binding promotes self‐association of the Swa dimerization domain to a stable coiled‐coil.
Rocking Motion of the Equatorial Domains of a Group II Chaperonin between Two Biochemical States Revealed by Single‐Particle Cryo‐EM at Near‐atomic and Subnanometer Resolutions
Authors: Zhang Junjie , Baker Matthew , Schroeder Gunnar , Douglas Nick , Jakana Joanita , Fu Caroline , Levitt Michael , Ludtke Steven , Frydman Judith , Chiu Wah
Published: June 2021

Chaperonins consist of two rings which are stacked back‐to‐back, forming folding cavities where substrates are encapsulated. The closure of the chaperonin cavity is triggered by ATP binding or hydrolysis. We report four single‐particle cryo‐EM structures of both wildtype and mutant Mm‐cpn, an Archaeal chaperonin in the nucleotide free and nucleotide bound states. The 4.3‐4.8Å resolution maps of the Mm‐cpn closed, nucleotide‐bound state, clearly show pitches of α‐helices and separation of β‐strands. More than 56% non‐small sidechain densities are visible in the 4.3Å resolution map of the wildtype Mm‐cpn. Protein backbones are modeled for both of the closed conformations. Models are built with geometry‐constrained flexible fitting to the 8‐10Å maps of the nucleotide free Mm‐cpn which exhibits an open conformation. The open and closed structures reveal a rocking motion in the equatorial domain of all subunits which results in different molecular contacts between the two rings in these two states. We observe a ~40° tilting of the region containing the N and C termini, as mm‐cpn switches from the closed to open state. This swinging movement of the N and C termini induced by the nucleotide hydrolysis may suggest their role in the substrate folding and/or release.

Research is supported by NIH Nanomedicine Roadmap grant (PN2EY016525)
The crystal structure of the human NDRG2 protein, a potential tumor suppressor
Authors: Kim Yoonjeong , Oh Tae‐Kwang , Kim Myung Hee
Published: June 2021

The human N‐Myc downstream‐regulated gene (NDRG) family is composed of NDRG1, NDRG2, NDRG3 and NDRG4, which are important for cell proliferation and differentiation. The NDRG family members show 57‐65% amino acid sequence similarity, and are predicted that their N‐terminal domain contains an alpha/beta hydrolase fold. Current study identified NDRG2 as a potential meningioma‐associated tumor suppressor gene. There are two types of NDRG2 isoforms, NDRG2a and NDRG2b. The latter encodes a 357 amino acid protein while the former contains an additional sequence of 14 amino acids in its internal region. In the present study we crystallized the NDRG2b protein (residues 23‐304) through introduction of surface entropy reduction method and solved structure to 2.0 Åof resolution. We describe the three‐dimensional structure of NDRG2. The protein exhibits distinct structural similarities to alpha/beta hydrolase superfamily. Structural inspection of NDRG2 reveals that the protein comprises two domains, a large alpha/beta hydrolase domain and a small cap domain covering a small pocket located at the interface between two domains. Although the NDRG2 structure can be classified into alpha/beta hydrolase superfamily, the catalytic signature residues in hydrolase are missing. This work was supported by funding from the 21C Frontier Microbial Genomics and Applications Center Program, MEST, Republic of Korea.
Role of estrogen and leptin in the estrous cycle of ob/ob and ovariectomized mice
Authors: Ng Ka Yan , Yong Jennifer , Chakraborty Tandra R.
Published: June 2021

There is a known impact of gonadal hormones like estrogen and leptin in the regulation of the estrous cycle. Analyzes of estrous cycle is an important tool as female mice are considered to be an excellent model for the study of neuroendocrine mechanism underlying a wide variety of normal functions and pathophysiological conditions in human. Estrogen, a hormone secreted by the ovaries regulates reproductive cycle and has a negative effect on food intake. Leptin, a hormone secreted by the adipose tissue has an inhibitory effect on food intake. In this study we tried to analyze the estrous cycle in relation with leptin and estrogen levels in two mouse lines ob/ob and ovariectomized (OVX) female mice, both of which are infertile but with contrasting leptin levels. To assess the progression of estrous cycle, vaginal smears was performed on female WT, ob/ob and OVX (n=4) mice. We found that ob/ob mice were acyclic and were at a persistent estrous phase with cornified cells. OVX mice showed an increase in leukocytes and were also acyclic. Quantitization of beta‐estradiol showed reduced levels in ob/ob as compared to WT and no beta‐estradiol in OVX mice. Thus, this study leads us to believe the estrous cycle of these mouse models is dependent on estrogen levels and quantitation of the cells in the various stages of the estrous cycle could be an useful tool to determine the stage and use of the animal as a model.
Molecular Basis of D‐AKAP2 Binding to PKA Regulatory Subunits
Authors: Sarma Ganapathy , Kinderman Francis , Kim Choel , von Daake Sventja , Taylor Susan
Published: June 2021

cAMP‐dependent protein kinase (PKA) regulates a variety of biological activities by phosphorylating a broad range of target proteins. Spatial and temporal specificity of PKA activity is, in turn, achieved by a family of scaffolding proteins called as the A‐kinase anchoring proteins (AKAPs). One of these, dual‐specific AKAP 2 (D‐AKAP2) binds to both the Regulatory (R) isoforms (RI and RII) of PKA. Specifically, the dimerization and docking (D/D) domain at the N‐terminus of the R subunits forms a four‐helix bundle that provides an interface for AKAP binding.

In order to understand the molecular basis of D‐AKAP2 binding to the RIα subunit and understand the molecular determinants that make D‐AKAP2 dual‐specific, we have solved the structures of the RIα D/D domain with and without the D‐AKAP2 peptide. The structure of the apo crystal form was solved using the anomalous signal from inherently present sulfur atoms. Together with the existing peptide array data and the RIIα D/D structure, the present structures reveal atomic‐level information on the dual‐specific nature of D‐AKAP2. The structures also provide insights into the role of a pair of highly conserved disulfides that might function as a redox sensor in cardiac myocytes. This work was supported by an American Heart Association Postdoctoral Fellowship to G.N.S, NIH grant to DK54441 to S.T and the Howard Hughes Medical Institute.
Pdx1 promotes ESC differentiation into insulin producing cells
Authors: Raikwar Sudhanshu P. , Zavazava Nicholas
Published: June 2021

Type 1 diabetes can be treated by islet transplantation. However, acute shortage of organ donors, lifelong immunosuppression and chronic graft rejection are the current major limitations. Embryonic stem cells (ESC) offer a novel approach towards developing a treatment for type 1 diabetes. We hypothesized that reprogramming of the ESC using a pancreatic β cell specific transcription factor Pdx1 will lead to their enhanced differentiation into insulin producing cells (IPCs). A double stable ESC line R1 Pdx1AcGFP RIP Luc simultaneously co‐expressing EF1α promoter driven Pdx1AcGFP and rat insulin promoter (RIP) driven luciferase was generated. Undifferentiated R1 Pdx1AcGFP RIP Luc ESC line expressed Oct4, Nanog, Pdx1 and AcGFP. The R1 Pdx1AcGFP RIP Luc ESC line was subjected to Activin A treatment to generate definitive endoderm (DE) cells which were characterized by CXCR4 expression on the cell surface. The selectively enriched CXCR4+ DE cells were directly infused into streptozotocin induced diabetic mice which led to their in vivo homing into the pancreas, differentiation into IPCs and sustained correction of hyperglycemia. Real‐time non‐invasive bioluminescence imaging was used to monitor the long term in vivo fate of the transplanted IPCs. In conclusion, R1 Pdx1AcGFP RIP Luc‐derived IPCs offer a novel approach for the treatment of type 1 diabetes.
Effect of pentachlorophenol on MAP kinases in human natural killer cells
Authors: Whalen Margaret M
Published: June 2021

Pentachlorophenol (PCP) is an organochlorine compound that has been used as an insecticide and a fungicide. PCP use has been restricted in the USA, it is mainly used now as a wood preservative for power‐line poles and fence poles. PCP has been detected in human tissues through inhalation and consumption of contaminated food and water. Human natural killer (NK) lymphocytes play a central role in immune defense against viral infection and tumor development by killing (lysing) virally infected and tumor cells. The lytic function of NK cells depends in part upon the activation of mitogen activated protein kinases (MAPK). In previous studies we have shown that exposure to 10‐0.5µM PCP decreases the lytic function of NK cells. In this study we evaluated the effect of these same concentrations of PCP on the phosphorylation state and total levels of p44/42, p38, and JNK ,MAPKs. Preliminary studies indicate that PCP exposure is able to increase the phosphorylation (activation) of MAPKs. This PCP‐induced phosphorylation of MAPKs may leave them unavailable for activation by virally infected or tumor target cells and thus contribute to the observed decreases in lytic function seen in NK cells exposed to PCP.

Supported by NIH grant S06 GM008092‐33.
Plum polyphenolics decrease oxidative stress and inflammation in vascular endothelial cells
Authors: Townsley Emily D. , Noratto Giuliana , Angel Gabriela , Del Follo Armando , Byrne David , Mertens‐Talcott Susanne
Published: June 2021

Previous studies have demonstrated that plums are rich in antioxidant polyphenolics which may have anti‐inflammatory effects. In this study we determined the anti‐oxidant and anti‐inflammatory effects of polyphenolics extracted from ″Black Splendor″ (BS), a commercial variety of plum (Prunus salicina). BS concentrations of up to 10 mg gallic acid equiv./L did not significantly alter cell proliferation and therefore were selected for the determination of antioxidant and anti‐inflammatory effects in human umbilical vein endothelial cells (HUVEC). Reactive oxygen species (ROS) were induced with 10mM glucose for 30 min at 37°C and BS (≥2 mg/L) decreased the induced oxidative stress by up to 70% upon treatment . Treatment of HUVEC cells with 10mM glucose significantly increased messenger‐RNA expression of inflammation biomarkers IL‐6, IL‐8, and NF‐κB. An increase in transcription factor NF‐κB up‐regulates other pro‐inflammatory cytokines leading to increased vascular inflammation. Upon treatment with BS (4 mg/L), IL‐6, IL‐8, and NF‐κB were decreased by 4.4, 7.4, and 3.1 fold, respectively. This data suggests that antioxidant polyphenolics found in plum extracts help control inflammation and inhibit oxidative stress in human umbilical vein endothelial cells.

Grant Funding Source

Texas A&M University Vegetable and Fruit Improvment Center
Trans‐10, cis‐12 conjugated linoleic acid (CLA)‐mediated inflammatory signaling: Essential role of human adipocytes in initiating cross‐talk with preadipocytes
Authors: Martinez Kristina Brooke , Chung Soonkyu , LaPoint Kathleen , Kennedy Arion , Walker‐West Tiffany , McIntosh Michael
Published: June 2021

We have previously shown that both lipopolysaccharide (LPS) and CLA, a popular weight loss supplement, activate inflammatory signaling that promote insulin resistance in primary cultures of human (pre)adipocytes. Because our published data demonstrated that preadipocytes were the primary instigators of inflammatory signaling in LPS‐treated cultures, we hypothesized that they played the same role in CLA‐mediated inflammation. To test this hypothesis, we employed two experimental models; Model 1‐ a Co‐Culture Insert Model in which inserts containing ~50% adipocytes (AD50) or ~100% preadipocytes (AD0) were suspended over wells containing AD50 or AD0 cultures, and Model 2‐ a Conditioned Media (CM) Model in which naïve AD0 and AD50 cultures were treated with CM from CLA‐treated AD50 or AD0 cultures. In Model 1, CLA‐induced interleukin (IL)‐8 and IL‐1β mRNA levels were highest in AD50 cultures when co‐cultured with AD0 inserts, whereas no increase in these genes were detected in AD0 cultures. In Model 2, IL‐8 and monocyte chemoattractant protein (MCP)‐1 mRNA levels were increased in cultures treated with CM from CLA‐treated AD50, but not AD0, cultures. These data suggest that, in contrast to LPS, CLA instigates release of inflammatory signals from adipocytes that subsequently activate adjacent preadipocytes in primary cultures of human (pre)adipocytes. Supported by NIH‐NIDDK/ODS 2R01‐DK063070.

Grant Funding Source

NIH‐NIDDK/ODS 2R01‐DK063070 and NCARS 06771
Sub‐vasoactive concentrations of extracts from Amazonian palm berry (Euterpe oleraceae Martius) protect coronary arteries from damage following external exposure to superoxide
Authors: Bell David R , Moon Haley J
Published: June 2021

In chemical assays, extracts from the Amazonian palm berry ACAI (Euterpe oleraceae Martius) exhibit the highest ORACFL activity of any food yet reported with exceptional ability to scavenge O2−. However, it is not known whether these chemical properties translate into an ability of ACAI to protect living tissues from O2− mediated damage. Therefore we examined whether extracts from ACAI protected porcine coronary arteries from O2− mediated damage. Arterial rings from 6 pigs were incubated in sterile tissue culture media overnight for use in in vitro isometric vascular reactivity studies. Maximum relaxation and sensitivity to A23187 was impaired in rings exposed externally to O2− (400uM pyrogallol (Py) for 15 m) but markedly improved when rings were co‐exposed with 0.5mg/L of ACAI pulp extracted in either phosphate buffer (ACAIaq) or DMSO (ACAIDMSO). % Maximum relaxation and ‐logED50, mean+sem, were C, 84±12%, 7.82±0.2; Py, 63±12%, 7.60±0.06; Py+ACAIaq, 88±6%, 7.88+0.13; Py+ACAIDMSO, 73± 13%, 7.75± 0.13, n=4). At 10x its oxidant protective concentration, ACAI extracted with either phosphate buffer, DMSO, DDH2O or EtOH caused <10% total relaxation of rings. Our results demonstrate that ACAI extracts at concentrations too low to directly alter vasoreactivity protect coronary arteries from damage following external exposure to oxygen radicals. Thus the oxygen radical neutralizing effect of ACAI rather than direct modulation of vascular relaxation is responsible for the protective effects of ACAI in arteries exposed to reactive oxygen species.
Bench top fermentation system for food and dietary science
Authors: Thatcher Chad , Lin Bae
Published: June 2021

A multi‐purpose, modular programmable fermentation system has been developed for research applications involving bacteria, fungal and other food science research agents. Manufactured by Major Science, the fermentation system offers great versatility with vessel ranges from 0.5L to 10L. The MS‐FS series offers a modular option. This allows the researcher to choose only the necessary components with full ability to upgrade later. The controller allows constant monitoring of the media conditions in the various applications relevant to food science, where fermentation is involved. A pH probe enables automatic control of either acid or base media condition via peristaltic pumps and the DO probe controls the aerator. Additional ports are available to extend and customize control of the media environment. The small footprint, modular and upgradeable design, and proven high reproducibility demonstrated by this system make it an ideal choice for various food science research applications.