bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth factor and cytokine regulation in ventral cervical spinal cord following C2 hemisection in rats
Authors: Windelborn James A , Mitchell Gordon S
Published: June 2021

Large shifts in growth factor and cytokine expression have been reported in several models of spinal injury. However, published reports seldom specify changes in spinal motor areas of the ventral horn, particularly in segments below the injury. Understanding injury‐induced changes in these molecules will help define abnormalities in the molecular environment that underlie spontaneous functional recovery and/or alter the response to therapeutic intervention. Thus, we studied changes in the expression of growth factors and inflammatory mediators in the spinal ventral horn after C2 hemisection (C2HS) or sham surgery in male Lewis rats 1‐28 days post‐injury. Relative changes in BDNF, VEGFA, TNF‐α and TrkB protein levels at the site of injury and near the phrenic motor nucleus (C4) were quantified by densitometry and expressed as a percentage of time‐matched sham rats. Surprisingly, no protein measured was changed 14 or 28 days post‐C2HS. BDNF levels were increased in C2 ventral horn 1 and 3 days post‐C2HS (p<0.01), but not in C4. TNF‐α increased 1 day post‐C2HS in both C2 and C4 (p<0.05), but was only marginally elevated 3 days post‐C2HS (p=0.058). Thus, despite traumatic spinal injury, BDNF and TNF‐α expression only transiently increase in the cervical ventral horn whereas other growth factors/receptors studied are unaffected by injury.

Support by NIH HL69064 and HL007654 (JW)

Grant Funding Source

NIH HL007654
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 Source

Les 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 Source

National Institute of Health
Beta secretase localization, amyloid deposition and aberrant synaptic and neuritic sprouting in transgenic mouse Alzheimer models: a role for BACE1 in synaptic remodeling and plaque formation?
Authors: Clough Rich W , Zhang Xue‐Mei , Cai Huaibin , Ebersole Jeremy , Yan Cai , Struble Robert G , Patrylo Peter R , Yan Xiao‐Xin
Published: June 2021

Amyloid plaques are a pathological hallmark of Alzheimer's disease (AD). Diffuse plaques consist largely of deposited beta amyloid peptides (Ab). Neuritic plaques also contain dystrophic neurites and reactive gliosis in the vicinity of Ab deposits. The origin of Ab deposits and process of plaque development remain unknown. We hypothesize that Ab deposition begins at sites of aberrant neuroplasticity. BACE immunolabeling, Ab accumulation and neuritic alterations were analyzed in transgenic mouse models of AD (with single and combined human APP, PS1 and tau mutations: Tg2576, 2XFAD, 5XFAD and 3XTg). BACE1 immunoreactivity occurred in neuritic‐like plaques in all strains of transgenics. BACE1‐labeled elements in the plaques co‐expressed synaptophysin and growth associated protein‐43 (GAP‐43), suggestive of sprouting axons. Of note, perisomatic BACE1 and Ab labeling coexisted with synaptophysin around principal neurons in younger transgenics. These findings suggest: 1) BACE1 elevation coincides with Ab overload in neurons, synaptic terminals and plaques in transgenic AD models; (2) axonal misrouting or aberrant sprouting may be an early and crucial event of plaque pathogenesis; (3) BACE1‐mediated Ab overproduction and release from sprouting or remodeling neurites play an active role in plaque formation. Supported by SIUSOM and the Illinois Department of Public 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
Thermodynamic and kinetic characterization of binding stoichiometry and mismatch repair activity by human thymine DNA glycosylase
Authors: Morgan Michael Thomas , Drohat Alexander C.
Published: June 2021

Human thymine DNA glycosylase (TDG) is involved in efficiently repairing G:T and other mismatches through the base excision repair pathway. Our recent crystal structure revealed the catalytic core of TDG bound to abasic DNA in a 2:1 (protein:DNA) complex. The N‐terminal domain of TDG promotes the 2:1 complex and enhances the activity for G:T lesions. Here we investigate the relationship between 2:1 binding and T removal. Our work seeks to understand the mechanisms of this dimerization event, the substrate characteristics that favor 1:1 or 2:1 binding, the role of the N‐terminus in mediating such an event, and how the second subunit affects activity of the productively bound subunit. We find that, as compared with U, T removal is more sharply diminished when the substrate is only capable of binding a single copy of TDG. Furthermore, we find that both subunits are catalytically active, and the activity depends on the number of bases between two mispairs. ITC experiments show that TDG binding is significantly less favorable for DNA that forms a 1:1 versus a 2:1 complex , and this effect is greater for G:T versus G:U substrates. Supported by NIH grant to ACD.
The Genomics Education Partnership
Authors: Hauser Charles , Bailey C , Elgin S CR , Lopatto D
Published: June 2021

The advances in genome sequencing technology have led to the generation of massive amounts of publicly available data creating new opportunities for student‐scientist research partnerships. With appropriate training, undergraduate students can provide the labor‐intensive sequence improvement and annotation required for effective use of sequence data. The Genomics Education Partnership (GEP) brings together faculty from over 40 colleges and universities in a collaborative project to provide their students with a research experience in genomics. The first project undertaken by the GEP is the sequence improvement and annotation of the dot chromosomes of several Drosophila species. This unique domain has properties of both euchromatin, including normal gene density, and heterochromatin. Undergraduates improved the posted sequence data to an error rate of less than 1:1000 bp, and generate carefully annotated gene models; the pooled data is being used for analysis of the chromosome as a whole, including comparisons among different Drosophila species. Students appreciate the opportunity to make novel contributions to the scientific database, and that their work will result in a scientific publication. We find that genomics research is a rewarding way for us to teach, and a rewarding way for our students to learn. We are looking for additional schools to join us, and additional partners from the scientific community to pose meaningful annotation problems for the students to tackle. Funded by grants from HHMI and NIH.
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.
Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA
Authors: Graham William Darnell , Scheunemann Ann Elizabeth , Agris Paul
Published: June 2021

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome's subunits by binding to Helix 69 (H69) of 23S rRNA. Aminoglycoside binding to the chemically synthesized terminal domains of the E. coli and human H69 was detected and quantified using UV‐monitored thermal denaturation, circular dichroism spectrapolarimetry, and isothermal titration calorimetry. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin and tobramycin than for paromomycin, while the binding of streptomycin was too weak to assess. The three conserved pseudouridine modifications (Ψ1911, Ψ1915, Ψ1917) occurring in the loop of the E. coli H69 did not significantly effect the dissociation constant or stoichiometry for the binding of paromomycin. In contrast, the human 28S rRNA H69 had a considerably decreased affinity for the antibiotics. The higher affinity of the bacterial 23S rRNA H69 for aminoglycosides is an important validation of the antibiotic target. The higher affinity of the E. coli H69 for neomycin and tobramycin as compared to paromomycin and streptomycin indicates differences in the efficacy of the aminoglycosides as anti‐microbial agents.
Mnk1/2 kinase activity mediates integrin (α6β4) dependent translation of cancer related mRNAs
Authors: Korneeva Nadejda , Kim HongIm , Giordano Anthony , Chung Jun
Published: June 2021

Mnk1 and Mnk2 kinases are targets of both the ERK and p38 MAPK pathways in response to a broad spectrum of extracellular stimuli. Mnk1/2 phosphorylate eIF4E and eIF4G. Modification of eIF4E and eIF4G as members of eIF4F complex can affect the spectrum of mRNAs translated. It was shown that integrin α6β4 is involved in activation of Ras pathway leading to translational activation of cancer specific mRNA and eIF4E phosphorylation in breast carcinoma cells. We investigated the impact of Mnk inhibition on α6β4 dependent functions in cells that stably express α6β4 integrin by using a Mnk inhibitor, CGP57380. Our data suggest that Mnk contributes to the α6β4 integrin‐dependent proliferation and survival mechanisms of breast carcinomas. To further investigate whether Mnk inhibition of α6β4 functions is related to α6β4 dependent mRNA translation, we performed polysomal ultracentrifugation followed by real‐time PCR analyses in β4 transfectants. Expression of β4 integrin in cells caused a shift of cancer‐related mRNAs into actively‐translated polysomal complexes. However, the addition of CGP57380 blocked this shift and caused a polysomal redistribution of cancer related mRNAs into poorly‐translated complexes. In summary, our results demonstrate that modulation of Mnk activity affects integrin α6β4 dependent translation of mRNAs with highly structured 5'UTR, which are dependent on the level and activity of eIF4F.
TRIM5α as an E3 ubiquitin ligase
Authors: Tanaka Makoto , Yamauchi Keiko , Kamitani Tetsu
Published: June 2021

Human immunodeficiency virus type1 (HIV‐1) efficiently infects susceptible cells and causes acquired immunodeficiency syndrome (AIDS) in humans. Although HIV can also enter the cells of Old World monkeys, it encounters a block before reverse transcription. Data have shown that this species‐specific restriction is mediated at the post‐entry stage by a RING‐finger protein TRIM5α. The function of TRIM5α, however, is unknown. Since many RING‐finger proteins, such as TRIM21 (Ro52), have been identified as E3 ubiquitin ligases, we hypothesized that TRIM5α functions as an E3 ubiquitin ligase. To test this possibility, we examined the E3 ligase activity of TRIM5α in vitro. Its enzymatic activity was also tested by overexpressing TRIM5α in human embryonic kidney 293T cells. Our results indicated that TRIM5α ubiquitinates itself (self‐ubiquitination) in cooperation with the E2 ubiquitin‐conjugating enzyme UbcH5B; this showed that TRIM5α is a RING‐finger‐type E3 ubiquitin ligase. Interestingly, in HEK293T cells, TRIM5α was clearly conjugated with a monoubiquitin, as well as polyubiquitin chain, which does not target to the proteasome for degradation. Thus, we identified TRIM5α as an E3 ubiquitin ligase.
Functional Analysis of Arabidopsis BRIZ Proteins
Authors: Hsia Mandy
Published: June 2021

Eukaryotes have adopted a multi‐enzymatic Ub/26S proteasome system to regulate protein degradation, which consists E1, E2 and E3. E3, the ubiquitin ligase, is often the key component for substrate specificity and a major type of E3 ligase uses a RING domain as the E2 docking site. Previous analysis has shown that there are more than 400 RING‐domain containing proteins in the Arabidopsis predicted proteome and some have been shown to catalyze polyubiquitination in vitro. However, most of them do not have defined in vivo functions. My project focuses on functional analysis of two related Arabidopsis RING‐domain containing proteins, BRIZ1 and 2. Both of these proteins have multiple predicted domains; a BRAP2, RING, ZnF‐UBP, and coiled‐coil domain from the N‐terminus to C‐terminus. Despite their sequence similarity and shared predicted domains, no homozygous T‐DNA insertion lines can be isolated from insertions in either loci, indicating that they are not functionally redundant. Heterozygous plants from insertions in BRIZ1 or BRIZ2 produce 3:1 viable:inviable seed. Homozygous mutant lines are recovered from both the BRIZ1 and 2 T‐DNA insertion lines after introduction of a plasmid containing coding sequences, indicating that disruption of BRIZ1 or 2 is responsible for the lethality. These observations suggest that both proteins together might be essential for proper germination through complex formation. BRIZ1 and BRIZ2 interacted after synthesis in in vitro transcription and translation reactions. In addition, GST‐tagged BRIZ1 and 6His‐tagged BRIZ2 preferentially interacted. These observations strongly suggest that hetero‐dimer formation is favored over homo‐dimer formation.
Nitric oxide regulates skeletal muscle performance by compartmentalization of nNOS splice variants
Authors: Percival Justin Mark , Anderson Kendra N , Huang Paul L , Froehner Stanley C
Published: June 2021

Regulation of the subcellular localization of nitric oxide synthases is important for conferring specificity to intracrine nitric oxide signaling. We report that two splice variants of nNOS, nNOSμ and nNOSβ, are differentially targeted to the sarcolemma and the Golgi complex in skeletal muscle, respectively. Sarcolemmal nNOSμ and Golgi nNOSβ?exhibit independent and overlapping functions in modulating the muscle phenotype. First, nNOSμ and NOSβ?differentially regulate skeletal muscle mass through sex‐specific mechanisms. Second, nNOSμ‐deficient skeletal muscles display increased susceptibility to contraction‐induced fatigue, independently of structural defects. However, muscles lacking both nNOSμ and nNOSβ display markedly exacerbated fatigue associated with specific force deficits, an aberrant microtubule cytoskeleton, fewer fatigue‐resistant type IIa fibers and greater numbers of fatigable type IIb fibers. Thus, Golgi nNOSβ appears to modulate susceptibility to contraction‐induced fatigue through alterations in muscle structural integrity and shifts in fiber composition. These data suggest that one function of alternative splicing of the NOS1 gene is the creation of isozyme‐specific signaling compartments. We conclude that sarcolemmal nNOSμ and Golgi nNOSβ? are critical regulators of skeletal muscle performance and that the spatial confinement of nNOS isozymes plays an important role in imparting specificity to NO signaling.
Expression and characterization of triosephosphate isomerase from Epulopiscium sp. Type B
Authors: Deal‐Laroda Nikolas Antonio , Piefer Andrew John
Published: June 2021

The main goals of this project are to express and characterize the putative Triosephosphate isomerase gene (TPI) from the bacterium Epulopiscium sp. Type B. This large bacterium (~250 µm in length) has never been cultured, grows in the gut of Naso tonganus (a surgeonfish), and may assist in the fish's algae digestion. TPI is responsible for the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3‐phosphate. This catalytic step bridges the initial ATP priming stage and the energy production stage of glycolysis. PCR was performed to amplify TPI for subsequent cloning and expression in Escherichia coli. The Epulopiscium TPI protein contains approximately 247 amino acids and its theoretical molecular weight is 26,841 Daltons. Most characterized TPIs from other organisms are homodimeric in structure, but this remains to be determined for Epulopiscium TPI. Epulopiscium TPI was aligned to TPI from Clostridium botulinum, E. coli, and Homo sapiens, showing 91.5% amino acid conservation among these diverse organisms. Furthermore, the alignment shows that Epulopiscium TPI has 63.7 % identity with Clostridium botulinum, a 41.4 % identity with E. coli, and a 42.9 % identity with Homo sapiens. These studies may provide a glimpse into this unique organism's physiology through examination of a highly conserved enzymatic process. This project was funded by the Hartwick College Department of Chemistry.
Cloning, Expression and Purification of Yeast Aminotransferase
Authors: Chooback Lilian , Bobyk Kostyantyn D. , Aktas Deniz , Karsten William E. , Cook Paul F.
Published: June 2021

Lysine is synthesized in Saccharomyces cerevisiae via the α‐aminoadipate pathway. α‐aminoadipate is made from α‐ketoadipate via the action of PLP‐dependent aminotransferase that has not been identified. Sequence alignment and a homology search for conserved amino acid residues were used to identify six aminotransferase genes in S. cerevisiae. On the basis of sequence homology the identified genes code for two aromatic aminotransferases, two aspartate aminotransferases, one kynurenine aminotransferase and one putative amiontransferase of unknown function. The cloned genes were obtained from the Harvard Institute of Proteomics Plasmid Repository. The genes were subcloned into expression vector pET16b. The expressed his‐tagged proteins have been purified using a Ni‐NTA affinity column. Kinetic characterization of these enzymes is at the early stages.
PPL‐240 modulates VEGF‐A secretion by HMC‐1 after ultraviolet radiation (UVR)
Authors: Reyes Eduardo , San Antonio Esther , Guerrero Antonio , Brieva Aurora , Valdes‐Hevia Marta D , Gonzalez Salvador , Alvarez‐Mon Melchor
Published: June 2021

Mast cells may be recruited by tumor‐derived chemoattractants and selectively secrete molecules such as VEGF that permit the formation of new blood vessels and metastases. We analyze the VEGF‐A and C secretion by HMC‐1 after UVR and their modulation by Polypodium leucotomos extract (PPL‐240). HMC‐1 cells were incubated for 2h in presence or absence of PPL‐240 and exposed to UVR (HB406). Finally, cells were culture in presence or absence of PMA during 24h in order to induce the secretion of soluble mediators. VEGF‐A and ‐C levels were determined by ELISA. UVR increased VEGF‐A levels by 65% (70±13 to 116±24 pg/ml) (p<0.018), levels that stay at baseline in PPL‐240‐treated cells. When UVR exposed cells were treated with PMA, VEGF‐A levels were amplified (over 278%, 324±136 pg/ml) (p<0.001) in comparison to irradiated and PMA untreated cells (p<0.001) and to irradiated and treated with PMA (74±6 pg/ml) (p<0.002). PPL‐240 reduced its release reaching to control levels in PMA untreated HMC‐1 (65±3 pg/ml) and in PMA treated HMC‐1 (79±11 pg/ml) (p<0.012) after UVR exposure. Regarding VEGF‐C secretion, UVR did not altered its levels with and without PPL‐240 treatment. PMA addition reduced the levels of VEGF‐C induced by UV‐exposed cells (118±17 to 95±10 pg/ml). We conclude that UVR increases VEGF‐A by HMC‐1 and PPL‐240 treatment normalizes these levels, which may be relevant for reducing tumour growth and metastasis.
Down‐aging effect of melatonin by long‐term treatment in ovariectomized rats
Authors: Lee Sunghoon , Lee Sang‐kil , Kim Joo‐heon , Hong Yonggeun
Published: June 2021

The aging theory postulates that this process may be due to the accumulation of oxidative damage in cells and molecules. The present study has investigated the effect of aging and ovariectomy on various markers related to aging and anticancer in muscles obtained from ovariectomized rat, and the influence of long‐term administration of melatonin. Female Sprague‐Dawley rats(n=15) of 2‐month of age were used. Animals were divided into five experimental groups: Intact groups(group‐C) that were untreated and four ovariectomized groups that group‐A, B and D treated with melatonin (1 mg/kg/day). At 9:00 am, 9:00 pm, 9:00 am plus 9:00 pm, animal were introduced to melatonin(i.p.). Group E treated with vehicle(0.9% saline) for 12 weeks. We measured in rats for 12 weeks after surgery. After 12 weeks of treatment, rats were sacrificed by decapitation, and skeletal muscle was collected and extracted RNA. We confirmed the differential expression of aging and anticancer‐related genes such as p53 and SMP30 in muscle tissue. The expression of SMP30 increased in melatonin treated group compared to non‐treated group but no differences of gene expression depend on introducing time points. Our results indicate that decreased expression of aging and anticancer related genes with melatonin introduction. Keywords: Autophagy, renal ischemia, renal artery occlusion, ovariectomy. Supported by BioGreen 21 Program (#20070401034006) from Rural Development Administration, South Korea.
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.
In PDE3B KO mice, White Adipose Tissue (WAT) Exhibits Characteristics of “Good Fat”, i.e., Brown Adipose Tissue (BAT): I. Alterations in Mitochondrial Biogenesis and Function
Authors: Chung Youn Wook , Lagranha Claudia , Zmuda‐Trzebiatowska Emilia , Khan Faiyaz A , Murphy Elizabeth
Published: June 2021

To study effects of cyclic nucleotides on energy homeostatic mechanisms, mice were generated by targeted inactivation of the Pde3b gene, which encodes Cyclic Nucleotide Phosphodiesterase 3B (PDE3B), an enzyme that catalyzes hydrolysis of cAMP and cGMP. In PDE3B‐KO mice, adipose tissue mass is reduced and epididymal white adipose tissue (EWAT) exhibits characteristics of BAT, including changes in morphology, and increased expression of genes related to BAT differentiation, mitochondrial biogenesis, and energy dissipation, including PPARγ coactivator‐1 alpha (PGC‐1α? and uncoupling protein 1 (UCP1). Mitochondria were isolated and studied by electron microscopic and proteomics techniques and functional assays. In KO EWAT, mitochondria are larger than WT mitochondria and uncoupled, with dysfunction of mitochondrial complex I, consuming less oxygen (using glutamate and malate as substrate) and generating less ATP than WT mitochondria. Proteomic patterns in KO mitochondria predict increased fatty acid β‐oxidation and decreased oxidative damage. In WAT to BAT differentiation, PDE3B is a critical molecular switch, inducing UCP1 to promote mitochondria uncoupling and inhibiting complex I to generate less oxidative stress. In KO mice, the presence of 'good fat' may compensate for hepatic insulin resistance.
A New Mechanism of Action for Metformin
Authors: Parakhia Rahulkumar A. , Ouyang Jiangyong , Ochs Raymond S.
Published: June 2021

Metformin activates AMP Kinase (AMPK) through an unknown mechanism. Using L6 muscle cells, we replicated the stimulation of AMPK phosphorylation by metformin, as well as the stimulation of glucose transport and fatty acid oxidation. We examined the leading hypothesis for metformin action, inhibition of mitochondrial respiration. It is difficult to rationalize this hypothesis with the established metformin stimulation of fatty acid oxidation. We conducted rotenone titrations on L6 muscle cell cultures for both glucose transport and fatty acid oxidation and compared this with metformin. Rotenone stimulated glucose transport, but inhibited fatty acid oxidation at all levels of the inhibitor. A similar divergence was found using agonists for peroxynitrite. Knockout experiments using siRNA suggested that the upstream kinase for AMPK was not involved. We formed a novel hypothesis: inhibition of the AMP destruction through the inhibition of AMP deaminase. We found metformin (and phenformin) inhibited purified AMP deaminase. Moreover, a known inhibitor of AMP deaminase stimulated glucose uptake and fatty acid oxidation. We have recently used several inhibitors of AMP deaminase, and found that they replicate metformin action. We conclude that AMP deaminase inhibition may be the link between metformin and AMPK activation.
The role of PEPCK‐C in the mammary gland
Authors: Hsieh Chang‐Wen , Millward Carrie , Croniger Colleen
Published: June 2021

PEPCK‐C has been proven that plays an important role in both carbohydrate and lipid metabolism, and is involved in type II diabetes. Recently, we found the milk from lactating PPARE‐/‐ mice which PEPCK‐C gene expression is ablated in the WAT and the mammary gland exhibited significantly lower triglyceride than from wild type mice. We hypothesized that the expression of PEPCK‐C might be regulated in lactating mammary gland and contribute to triglyceride synthesis. To determine the role of PEPCK‐C expression in mammary epithelial cells, we investigated the PEPCK‐C expression by transient transfection. By the deletion analysis of PEPCK‐C promoter, we found the region from ‐402 to ‐206 is required for the expression of PEPCK‐C in mammary epithelial cell line. Besides, we also found that the PPAR£^2 binding site and a glucocorticoid response element, GR1, were also required. According to the less milk triglyceride in PPARE‐/‐ lactating mice, we suggested PEPCK‐C expression is regulated by prolactin in mammary epithelium. To determine the effect of prolactin on PEPCK‐C expression in mammary epithelium, mRNA was measured by RT‐PCR in HC‐11 cells. We found prolactin decreased PEPCK‐C expression in mammary epithelial cells. Moreover, by transient transfection, we also found PEPCK‐C promoter activity was repressed after prolactin treatment. Thus, we concluded that the expression of PEPCK‐C is regulated by prolactin.
Low catalase activity is associated with diabetes caused by alloxan
Authors: Takemoto Kazunori , Tanaka Miho , Wang Da‐Hong , Ogino Keiki , Taniuchi Koji , Masuoka Noriyoshi
Published: June 2021

Catalase has a predominant role in protecting oxidative damage caused with hydrogen peroxide. No catalase activity in blood is acatalasemia and low activity is hypocatalasemia. As a higher incidence of diabetes mellitus was shown in Hangarian acatalasemic and hypocatalasemic patients, we examined whether low catalase activity is associated with diabetes. When the blood glucose concentration of acatalasemic mice (3% activity of normal mice) was compared with normal mice, no significant difference was seen. To normal and acatalasemic mice, alloxan (200 mg/kg of body weight) was intraperitoneally administrated. Blood glucose concentration of the acatalasemic mice became less stable than that of normal mice. With a glucose tolerance test (2.0 g/kg), blood glucose concentration of the acatalasemic mice is significantly higher than that of normal mice. And then, these mice were killed and the pancreatic tissues were stained with hematoxylin and eosin. No significant difference of numbers of β‐cells in islet of langerhans of the normal mice was seen between administration of alloxan and that of saline. However, numbers of β‐cells in the acatalasemic mice decreased by about 20% (P<0.005). As alloxan generates hydrogen peroxide, these results indicate that catalase protects pancreatic β‐cells from the damage with hydrogen peroxide and suggest that lower catalase activity develops higher incidence of diabetes.
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.
Self –peptidomic repertoire of the human pre‐nodal lymph
Authors: Clement Cristina Corina , Maitra Radhashree , Sahu Ranjit , Santambrogio Laura
Published: June 2021

The reticular network within the lymph node is a conduit system forming the infrastructure for the fast delivery of soluble substances from the afferent lymph to the lumen of high endothelial venules (HEVs). Conduit associated dendritic cells (DC) are capable of taking up and processing soluble antigens by directly fishing into the nanometer size conduits transporting the afferent lymph. No data are currently available on the antigenic composition of the human lymph available to the resident dendritic cells, which constantly sample the self‐antigenic repertoire. In this study we are reporting important sequence information of peptides from the human lymph of healthy donors. Using advanced proteomics approaches, such as high resolution electrospray ionization mass spectroscopy sequencing (LTQ‐MS/MS) together with proteins MS/MS database search using Mascot and SEQUEST algorithms we were able to sequence more than three hundred peptides present in the pre‐nodal lymph. A significant majority of these peptides derived from the "in vivo" processing and turnover of extracellular matrix proteins such as collagens, laminins, fibronectins, mucins. Sequenced peptides were then screened for their potential affinity for HLA‐DR1 and HLA‐DR4 MHC class II proteins using the Syf‐P9 algorithm. The best hit candidates were further screened and found to have high binding affinity for either HLA‐DR1 or HLA‐DR4.
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 250kDa

In 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.
Proteomic comparisons of differentiating embryonic stem cells, an in vitro model of embryogenesis
Authors: Orlando Ron , Angel Peggi , Weatherly Daniel Brent , Atwood James Alexander
Published: June 2021

Investigation of the genetic mechanisms driving embryogenetic differentiation has produced notable advances in disease research. However, reports on protein expression have been limited to comparisons between the undifferentiated and differentiated cellular states. Here, we use progressively differentiated embryonic stem cells as an in vitro model of embryogenesis. Using a quantitative proteomic approach, we explored protein regulation from mouse embryonic stem cells (ES), early primitive ectoderm‐like cells (EPL), and embryoid bodies (EB). A total of 2911 proteins were identified for the ES proteome, 1852 proteins for the EPL proteome, and 3328 proteins were identified in the embryoid body proteome. A total of 1509 proteins were shared across all three cell states and could be accuratly quantified. Differentially regulated proteins were then mapped to functional categories and correlated to embryogenetic events. This analysis represents a comprehensive investigation of protein expression during early embryogenesis.
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.
Retinoblastoma associated binding protein 46: a potential prognostic marker for distal metastasis in non‐small cell lung cancer
Authors: Wang Chih‐Liang , Wang Chun‐I , Liao Pao‐Chi , Liang Ying , Wu Chih‐Ching , Chuang Wen‐Yu , Tsai Ying‐Huang , Chen Hua‐Chien , Yu Jau‐Song , Yu Chia‐Jung
Published: June 2021

Previously, RbAp46 was identified as a novel body fluid protein from malignant plural effusions of lung adenocarcinoma by proteomic approach. In this report, we study the clinicopathologic significance of RbAp46 in non‐small cell lung cancer (NSCLC) and validate its application to be prognostic serum biomarker. We found RbAp46 mRNA was up‐regulated significantly in lung cancer tissues comparing to those in normal tissues. Strong nuclear stain of RbAp46 was observed in 75.3 % of lung cancer cells and 3.2 % of adjacent normal cells. RbAp46 was secreted from lung cancer cell lines and detected in conditioned media. Serum levels of RbAp46 were significantly higher in lung cancer patients versus healthy controls. The combination of serum RbAp46 and CEA levels could outperform CEA alone in discriminating patients from healthy controls in this case‐control study. Moreover, serum RbAp46 levels were positively correlated with distal metastasis of NSCLC. The migration ability of NSCLC cell line could be significantly inhibited by RbAp46 knockdown. Our studies suggest RbAp46 may be a novel biomarker and prognosticator for NSCLC and is involved in migration of lung cancer cells.
Growth Hormone Alters the Proteome of Differentiated MAC‐T Cells
Authors: Johnson Tasha Lynn , Peterson Daniel Gunnar
Published: June 2021

Growth Hormone (GH) improves growth and lactation in many species. Mammary epithelium is responsive to GH though the mechanism of these effects has not yet been fully characterized. Bovine Mammary Alveolar Cell‐T (MAC‐T) cells uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin and prolactin (DIP) and are a widely used ruminant mammary cell model. We have found significant changes in triacyglycerol, cholesterol and phospholipid distribution in MAC ‐T cells stimulated with 10 ng/ml GH. To characterize these direct effects of GH on a proteomic level, total protein was isolated from MAC‐T cells after differentiation for seven days with DIP and either 0 or 10 ng/ml GH. Proteins were separated by isoelectric point and molecular weight using two‐dimensional gel electrophoresis. We have observed a change in the abundance of multiple proteins when MAC‐T cells are stimulated with GH and the identification of these proteins is being pursued using MALDI‐TOF mass spectrometry.
Quantification of phosphoprotein variants in stem cells by mass spectrometry and a novel, digital high‐resolution isoelectric focusing approach.
Authors: Biringer Roger Gregory , Rosenblatt Michael , Rampalli Krystal , Hughes Douglas , Rogers John , Hühmer Andreas
Published: June 2021

Isoelectric focusing (IEF) is often used as a high resolution fractionation technique to separate proteins of interest from complex cellular background. We have combined a digital IEF approach with a multiplexed quantitation strategy based on isobaric mass labels to resolve and quantify protein expression and phosphoprotein changes in mesenchymal stem cells during differentiation into neurons. Proteins were labeled with Tandem Mass Tags (TMT), separated by digital IEF and quantified by complimentary approaches. Accurate relative quantities of phosphoprotein variants were determined using TMT labeling and LTQ‐Orbitrap‐based LCMS methodologies confirming phosphorylation events previously observed. Alternatively, Western blots and high content analysis with phospho‐specific and pan‐specific antibodies verified these phosphorylation changes in Erk1/2, Stat1, and Pak2 kinase, and several other proteins relevant to neural differentiation. The flexibility of the digital IEF approach enabled comparison of both methods. However, isobaric labeling and high resolution mass spectrometry provided resolution, throughput, and accuracy for phosphoprotein variant quantification superior to traditional methods.