bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of motoneurons and neuropil using facial nerve axotomy in SOD1 mice
Authors: Haulcomb Melissa M , Mesnard Nichole A , Sanders Virginia M , Jones Kathryn J 
Published: June 2021

The facial nerve axotomy is used to study motoneuron (MN) survival and peripheral nerve regeneration. Using this model in wild‐type (WT) mice we have identified two populations of MN, regenerative MN in the ventromedial (VM) subnucleus and degenerative MN in the ventrolateral (VL) subnucleus. Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease caused by MN cell death. In the SOD1 transgenic mouse model of ALS, a recent study demonstrated the development of MN populations, one compensatory and one degenerative, following disease progression. To further examine these findings we propose to use facial nerve axotomy as a tool to characterize pre‐symptomatic SOD1 MN or neuropil compared to WT. RT‐PCR was conducted using laser microdissected control vs. axotomized facial nuclei and VM vs. VL subnuclei to determine axotomy‐induced mRNA expression changes in apoptotic‐related genes. We found axotomy‐induced expression of tumor necrosis factor‐á (TNFá) and upregulation of caspase 8 in WT mice. However, SOD1 mice have TNFá constitutively expressed in the facial nucleus, with axotomy‐induced upregulation of both TNFá and caspase 8. The colocalization of TNFá, caspase 8, caspase 3, or TNFR1, with microglia, astrocytes, or MN will be examined via immunocytochemistry.Supported by the Les Turner ALS Foundation (NAM & KJJ) and by NIH grant NS40433 (KJJ & VMS).Grant Funding SourceLes Turner ALS Foundation and NIH
Pre‐phrenic interneurons as an anatomical substrate for plasticity following cervical spinal cord injury (SCI) in the adult rat
Authors: Lane Michael Aron , Jones Alex L. , O'Steen Barbara E. , Hunsaker Forest L. , Vavrousek Jennie , Salazar Krystal , Fuller David D. , Reier Paul J. 
Published: June 2021

Injury in the cervical spinal cord frequently results in some respiratory dysfunction and contributes to secondary complications, which are the leading causes of morbidity and mortality amongst humans. However, a number of studies have shown potential for respiratory plasticity following SCI. In the present work, using combined anterograde and transneuronal retrograde tracing, we have identified a population of spinal interneurons with projections onto PhMNs in the adult rat that receive input from the ipsi‐ and contralateral VRC. Immunohistochemical studies have revealed that some of these pre‐phrenic interneurons are cholinergic. Following a C2HMx, quantitative analyses demonstrated a significant reduction in the number of interneurons around the central canal. In addition, the extent of ipsilateral VRC input to PhMNs and pre‐phrenic interneurons is dramatically reduced. However, the VRC contralateral to injury has projections to ipsilateral PhMNs and pre‐phrenic interneurons both ipsi‐ and contralateral to injury. As such some pre‐phrenic interneurons could represent an anatomical substrate that modulates the extent and onset of plasticity. Supported by NIH (PJR) and the Craig H. Neilsen Foundation. (MAL)Grant Funding SourceNational Institute of Health
Improved Method for Non‐Invasively Monitoring Peripheral Neuropathy
Authors: Tucker Kelsey , Latman Neal S 
Published: June 2021

Non‐invasive determination of peripheral neuropathy is crucial in diagnosing and monitoring a variety of diseases. Semmes‐Weinstein (SW) Monofilaments have been the standard in this area. Vibration perception threshold (VPT) instruments have been developed as an alternative method. Typically, VPT measurements at 120 Hz show a low correlation (r = 0.39) with SW. The sensitivity of touch receptors also varies with temperature. The purpose of this study was to determine if a novel VPT at 50 Hz, designed to stimulate the same receptors as the SW, provides a higher correlation, and determine if skin temperature improves the instrument's reliability and accuracy.100 subjects, 18 to 88 years of age, were examined using the SW, novel VPT, and the Surface Temperature Scanner. Each subject was tested for nerve sensitivity and temperature on their fingertips.The correlation coefficient between SW and the VPT measurements was 0.88 (p=0.0000). Combining the VPT, subject's age and finger temperature resulted in a 72% accurate assignment of subjects into their correct clinical category for loss of sensation.Therefore, this study shows that the novel VPT provides measurements of peripheral neuropathy that have a stronger correlation with SW than current VPT instruments. This study also shows that by adding skin temperature, the novel VPT instrument would be more accurate and reliable in monitoring peripheral neuropathy.
Long Patch Base Excision Repair proceeds via coordinated stimulation of the multienzyme repair complex
Authors: Balakrishnan Lata , Brandt Patrick , Lindsey‐Boltz Laura , Sancar Aziz , Bambara Robert 
Published: June 2021

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA damage and maintaining genomic integrity. Recent reports show that the Rad 9‐Rad1‐Hus1 complex (9‐1‐1) stimulates enzymes proposed to perform long patch base excision repair (LP‐BER) including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA Polymerase β(Polβ), flap endonuclease 1 (FEN‐1) and DNA Ligase 1 (Lig 1). Since these stimulatory effects were studied in incomplete enzyme systems, we reconstituted the entire LP‐BER pathway in vitro using purified human proteins to characterize the functional interactions in the multi‐enzyme repair complex. We found that Pol βstimulated the flap cleavage activity of FEN‐1, the highest value, 12‐fold, in connection with strand displacement synthesis. Even in the absence of dNTPs or using a Pol βsynthesis‐deficient mutant, Pol βstimulated FEN‐1 nearly five‐fold. We also observed a two‐fold stimulation of Lig1 activity by Polβ. Stimulation of FEN‐1 and Lig 1 activities by Pol βare likely responsible for the diminished responsiveness of these proteins to the 9‐1‐1 complex in reconstituted LP‐BER. Observed stimulations suggest a significant role for Pol βactivity in base excision repair and provide further evidence for the existence of LP‐BER in vivo.This work was supported by National Institutes of Health Grant GM024441
Structural basis of G‐tract recognition by hnRNP F: implication for alternative splicing
Authors: Dominguez Cyril , Fisette Jean‐François , Chabot Benoit , Allain Frédéric 
Published: June 2021

HnRNP F regulates alternative splicing of many pre‐mRNAs, among which the Bcl‐x pre‐mRNA. HnRNP F contains three quasi RNA recognition motifs (qRRMs) that specifically recognizes Guanosine tract (G‐tract) RNA sequences that are crucial for splice site recognition.We solved the structure of the three qRRMs of HnRNP F in complex with a G‐tract RNA (AGGGAU) by NMR. The structures explain how qRRMs specifically recognize three consecutive guanosines. The RNA binding surface of the qRRM is very different than that of the classical RRM and consists of residues located in three loops and involves aromatic residues stacking the RNA and hydrogen bonds between main‐chain and positively charged side‐chains with the three guanosines. Mutagenesis experiments confirmed the importance of these residues in RNA binding. These structures define a novel RNA recognition mechanism.Our data show that G‐tract RNAs form stable G‐quadruplex structures that are destabilized by hnRNP F. We propose that hnRNP F regulates alternative splicing by modifying the RNA structure. Consistently, we show that single qRRMs of hnRNP F regulate splicing of the Bcl‐x pre‐mRNA with almost the same efficiency as the full‐length protein.
Increased enzymatic glycosylation of mitochondrial proteins impairs mitochondrial function in cardiomyocytes
Authors: Suarez Jorge , Hu Yong , Fricovsky Eduardo , Wang Hong , Scott Brian T , Han Wenlong , Dillmann Wolfgang H 
Published: June 2021

Increased nuclear protein O‐linked β‐N‐acetylglucosamine glycosylation (O‐GlcNAcylation) mediated by hyperglycemia contributes to cardiomyocyte dysfunction. However, whether mitochondrial (MITO) proteins are submitted to O‐GlcNAcylation or excessive O‐GlcNAcylation alters MITO function is unknown. Here, we investigated if MITO proteins are O‐GlcNAcylated and if increased O‐GlcNAcylation is linked to HG‐induced MITO dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that MITO proteins, members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core1 and core2 of complex III, and the MITO DNA encoded subunit I of complex IV (COX I) are O‐GlcNAcylated. By Mass Spectrometry, we identified that Serine 156 on NDUFA9 is O‐GlcNAcylated. HG treatment (30mM glucose) increases MITO protein O‐GlcNAcylation, including those of COX I and NDUFA9 which is reduced by expression of O‐GlcNAcase (GCA). Increased MITO O‐GlcNAcylation is associated with impaired activity of complex I, III and IV, and lower MITO calcium and cellular ATP content. Reducing O‐GlcNAcylation increased the activity of complex I, III and IV and MITO calcium and cellular ATP content returned to control levels. In conclusion, specific MITO proteins of cardiac myocytes are O‐GlcNAcylated and HG increases O‐GlcNAcylation of MITO proteins impairing MITO function.NIH Grant HL‐66917.
Increased Glucose Transport Prevents the Development of Contractile Dysfunctions in Diabetic Hearts
Authors: Bian Fang , Li Wei , Jia Zhong , Chaudhuri Priyanajana , Kerr Douglas , Tian Rong , Yu Xin 
Published: June 2021

Increased fatty acid oxidation and decreased glucose utilization are the metabolic characteristics of diabetic hearts. However, whether this shift in substrate utilization is causally related to the development of diabetic cardiomyopathy is not fully understood. We investigated the functional impact of increased glucose transport/utilization on diabetic hearts. We hypothesized that increased glucose oxidation can prevent diabetic cardiomyopathy.Type I diabetes was induced by steptozotocin injection in mice with cardiac‐specific overexpression of GLUT1 (TG) and their wildtype littermates (WT). Saline injected mice were used as controls. Cardiac function, substrate utilization, pyruvate dehydrogenase (PDH) activity, and mortality rate were determined after four months.Mortality rate was 26% for WT and 16% for TG (P<0.05). In diabetic hearts, fatty acid oxidation increased significantly from 58±1% to 64±2% in WT, and from 39±2% to 48±4% in TG (P<0.05). Due to increased glucose uptake and PDH activity, diabetic TG hearts showed increased glucose oxidation than WT: 24±1% vs 7±1% (P<0.05). MRI study showed significant decrease in ventricular function in WT diabetic mice (P<0.05). However, ventricular function in diabetic TG mice was preserved.We concluded that enhanced glucose oxidation lead to improved survival rate and prevented the functional deterioration in diabetic hearts.
Lectin Binding Studies of Buccal Cell Glycoproteins
Authors: Vanderpuye Oluseyi Adewale , Jones Roger , Ivey Frederick 
Published: June 2021

Oral cavity buccal cells are models for oral cancers and drug delivery systems. There is limited knowledge of buccal cell membrane protein composition in relation to interactions with toxins such as ricin and pathogens. Our objective is to better understand buccal cell polymorphisms and receptors for the toxin ricin and other lectins. Buccal surface glycoproteins were studied by fluorescence microscopy, lectin blots of nitrocellulose transfers of SDS gels and flow cytometry.The lectins ConA, WGA, peanut agglutinin (PNA), Ricinus communis agglutinin‐I, Ricinus communis agglutinin ‐II (ricin), Mackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin bound to different proteins in the range 60kDA to 250kDaIn fluorescence microscopy, buccal cells bound strongly to PNA, ConA, and WGA. Lesser binding was observed for RCA‐I and ricin, SNA, MAA , Tomato and Jacalin lectins. Ulex europeus‐I, Vicia villosa, Solanum tuberosum and Griffonia simplicifolia II lectins stained cells from some donors more strongly than others.Flow cytometry showed that WGA and RCA‐I bound to all buccal cells but antibodies to the complement regulators, CD59, CD55 and CD46 bound to only 50% of cells.We conclude lectins can be used to study glycoprotein polymorphisms in buccal cells, proteins of 200 kDa and 80 kDa are potential major ricin receptors in buccal cells, complement regulators expression differ on buccal cells.
Bioinformatics in a High School Research Curriculum; Phage Hunting and Soil Metagenomics at The Pingry School
Authors: Hata Tommie Shintaro , Conti Brooke , Lashley Danielle , Gatof Max 
Published: June 2021

The Pingry School offers a lab‐based research course that provides high school students with the opportunity to explore the concepts, methods, and applications of molecular biology. The Pingry School is a coeducational, independent, college‐preparatory country day school in Martinsville, New Jersey.To incorporate an authentic research experience into the class, we have partnered with labs at local universities to screen soil samples collected around The Pingry School. Since the 2005‐2006 academic year, numerous bacteriophage have been successfully isolated and used to construct libraries. These libraries are used to screen for functional genes such as lytic enzymes. The libraries also allow for genome analysis and comparison to other known phage genomes. "Phage hunting" and the analysis of phage genomes involve methods and concepts that are exciting and accessible to high school students. Students take part in a scientifically valid and interesting project while having the opportunity to interact with scientists working on bacteriophage technology and applications.
Optimization of SPR‐based assay for monitoring SV40 large T‐antigen helicase activity
Authors: Howard Rhonda , Plyler Jason R , Williams Steven , Araki Naoko , David Wendi M. 
Published: June 2021

Replication of the SV40 virus is widely used as a model system for cellular DNA replication. The SV40 large T‐antigen (T‐ag), a member of the SF3 family of helicases, is essential for SV40 DNA replication. We have recently reported a real‐time assay for monitoring T‐ag helicase activity using surface plasmon resonance (SPR). In this current study, optimization of the duplex and quadruplex DNA unwinding assay for T‐ag has been carried out. The influence of varying contact time between helicase and DNA substrates, T‐ag and ATP concentrations, and salt/buffer compositions on DNA unwinding were determined. Higher ionic strength decreased the efficiency of unwinding. Optimal contact time depended on the length of DNA substrate to be unwound and the sequence of DNA; DNA substrates containing the SV40 origin sequence were bound much more tightly than random sequence oligonucleotides, but unwound much more slowly, if at all, under the assay conditions. Of particular importance is the improved direct observation of quadruplex DNA substrate unwinding, in which no secondary reporter of unwinding is required. Future studies will investigate the real‐time inhibition of this quadruplex DNA helicase activity, with potential implications for small‐molecule regulation of gene function.Support of the Welch Foundation (Grant AI 0045) is gratefully acknowledged.
Purification of MBP‐ and Strep‐tag II proteins
Authors: Heijbel Anna , Carlsson Marianne , Karlsson Anneli 
Published: June 2021

Recombinant tagged proteins can be purified to high purity in one step using affinity chromatography. The tag can either be a peptide or a protein, and the choice of tag depends on target protein characteristics and the aim for purifying the protein. Two new chromatography media are presented, StrepTactinTM SepharoseTM High Performance that utilizes that the small 8 amino acid peptide, Strep(II)‐tag, has high affinity for the Strep‐tactin ligand. Dextrin Sepharose High Performance, on the other hand is based on the protein tag, Maltose binding protein (MBP, Mr: ~42 000), which binds specific to carbohydrates (e.g. dextrin). The specificity of the two media towards their respective tag gives high purity of the target proteins.In this study, simple protein purifications on ÄKTAdesignTM chromatography systems using MBPTrapTM HP (prepacked with Dextrin Sepharose High Performance) and StrepTrapTM HP, (prepacked with StrepTactin Sepharose High Performance) are presented. Purifications were also scaled‐up giving equivalent results. Robustness was tested by running six repeated purifications of a Strep(II)‐tagged protein expressed in E. coli on the same StrepTrap HP column including regeneration with 0.5 M NaOH between each run. No tendencies in reduction of final purity and yield of the target protein were observed, demonstrating the high reproducibility during repeated usage of StrepTrap HP.In summary, the results show the ease of using prepacked StrepTrap HP and MBPTrap HP columns together with chromatography systems. Equivalent results are obtained when scaling up showing the simplicity to purify larger quantities of pure target protein. Results also show that StrepTrap HP can be efficiently regenerated repeated times with 0.5 M NaOH without losing binding capacity.
Lysosomal sorting of ABC transporter TAP‐like (ABCB9)
Authors: Fujimoto Yasuyuki , Maeda Masatomo 
Published: June 2021

TAP‐like (TAPL, ABCB9) is an ATP‐binding cassette (ABC) transporter with similarity to TAP, a transporter associated with antigen processing. Both are half‐type transporters of ABCB subfamily and they localize distinctly in lysosome and endoplasmic reticulum, respectively. Here, we identified a region necessary for lysosomal targeting of TAPL.PROCEDURESVarious truncated forms of TAPL tagged with GFP were stably expressed in CHO‐K1 cells. Subcellular distributions of the tagged proteins were analyzed by confocal microscopy.RESULTSTAPL has ten transmembrane helices followed by conserved ABC region. Amino‐terminal four helices are unique to TAPL. The remaining six helices and the ABC region form the core structure of ABC transporters. The amino‐terminal four helices were revealed to be sufficient for the lysosomal targeting. A truncated protein lacking this region failed in sorting to lysosomes. The latter construct could dimerize, which is essential for half‐type transporters, while the former could not (BBRC, in press). The results demonstrate that TAPL can be dissected into two parts, each required for subcellular localization and transporter machinery.Supported by JSPS grants (13142206, 18590072).
Effects of Triglyceride‐Rich Lipoproteins on the Inflammatory Potential of Endothelial Cells
Authors: Wang Ying , Wong Maelene Ling , Raymond Nadine , Passerini Anthony , Simon Scott Irwin 
Published: June 2021

Atherosclerosis, a chronic inflammatory arterial disease featured by focal accumulation of fat‐laden deposits, is exacerbated by prolonged circulation of elevated levels of triglyceride‐rich lipoproteins (TGRL). This study aims to examine the effects of TGRL on the inflammatory potential of endothelial cells and the mechanisms for enhanced recruitment of mononuclear cells using vascular mimetics. We have developed an ex vivo atherogenesis model, in which human aortic endothelial cells (HAEC) in culture are perfused with postprandial TGRL freshly isolated from high‐fat diet fed individuals under varied shear rates. The expressions of cell adhesion molecules (CAM) are examined by flow cytometry, while the binding and internalization of TGRL are visualized by immunofluorescence. The results show an enhanced CAM upregulation upon TNF‐stimulation in TGRL perfused HAECs. Moreover, a member of low density lipoprotein receptors, LR11 is found involved in TGRL binding and upregulated in TNF‐α stimulated HAECs. We conclude that excessive exposure of endothelium to TGRL primes them for enhanced inflammatory response to cytokines or pro‐athero shear flow profiles. This elevated inflammatory state further enhances the recruitment of TGRL, thus revealing a positive feedback loop for metabolically driven development of atherosclerosis.This study is supported by NIH RO1 HL 082689 to S.I.S.
Large‐scale Detection of O‐GlcNAc Modification Sites using Lectin Chromatography and Electron Transfer Dissociation Mass Spectrometry
Authors: Chalkley Robert , Thalhammer Agnes , Schoepfer Ralf , Burlingame A L 
Published: June 2021

O‐GlcNAc modification is a transient, regulatory post‐translational modification of serine and threonine residues in nuclear and cytoplasmic proteins. It has been shown to have roles in cell regulation in response to nutritional state and cellular stress. The modification is poorly understood, partly due to the difficulty in detecting and identifying sites of modification by proteomic approaches.Levels of O‐GlcNAc modification are known to be high in the brain. In this study we demonstrate how the combination of lectin weak‐affinity chromatography and subsequent analysis by electron transfer dissociation mass spectrometry has allowed identification of large numbers of O‐GlcNAc modification sites in post‐synaptic density preparations, doubling the number of known sites in the literature. Several of the identified sites of O‐GlcNAc modification have also been observed in a phosphorylated state, further supporting the proposition that these two modifications may regulate each other.This work is supported by NIH NCRR grant RR001614, the Wellcome Trust and the Biotechnology and Biological Sciences Research Council.
GIV is a Non‐Receptor GEF for Gαi with a Unique Motif that Regulates Akt Signaling
Authors: Garcia‐Marcos Mikel , Ghosh Pradipta , Ear Jason , Farquhar Marilyn Gist 
Published: June 2021

Heterotrimeric G proteins are molecular switches that control signal transduction. Ligand‐occupied G‐protein coupled receptors serve are the canonical guanine nucleotide exchange factors (GEFs) that activate heterotrimeric G proteins. A few unrelated non‐receptor GEFs have also been described, but little or nothing is known about their structure, mechanism of action or cellular functions in mammals. We have discovered that GIV/Girdin serves as a non‐receptor GEF for Gαi through an evolutionarily conserved motif that forms a unique interface with the Gα‐subunit. Using insights from the 3‐dimensional structure of this interface, we demonstrate that this GEF motif enhances Akt signaling via the G ??‐PI3K pathway and that mutational disruption of the Gαi‐GIV interface inhibits Akt enhancement and cell migration in cancer cells. Recently we also found that GIV is a metastasis‐related protein in carcinomas by virtue of its ability to promote unrestricted Akt activation. Thus, the novel regulatory motif described here provides the structural and biochemical basis for the pro‐metastatic features of GIV, making the functional disruption of the Gαi‐GIV interface a promising target for therapy against cancer metastasis.Supported by NIH grants CA100768 and DKI7780 to M.G.F, by Basque Government Postdoctoral fellowship BFI06.300 and Susan G. Komen Postdoctoral fellowship KG080079 to MG‐M and by training grant NIH/T32 DK07202 and a Research Scholar Award from American Gastroenterology Association (AGA) to PG.