bluuebunny/crossref_metadata_2025_split huggingface dataset:
XRCC4 Protein Interactions with XRCC4‐like Factor (XLF) Create an Extended Grooved Scaffold for DNA Ligation and Double Strand Break Repair
Authors: Hammel Michal , Rey Martial , Yu Yaping , Lees‐Miller Susan , Tainer John 
Published: June 2021

The XRCC4‐like factor (XLF)‐XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double strand breaks in human cells. Yet, how XLF bindsXRCC4 and impacts nonhomologous end joining functions has been enigmatic. Here, we report the XLF‐XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define the XLF‐XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain interfaces forming parallel superhelical filaments. XLF Leu‐115 (“Leu‐lock”) inserts into a hydrophobic pocket formed by XRCC4 Met 59, Met‐61, Lys‐65, Lys‐99, Phe‐106, and Leu‐108 in synergy with pseudo‐symmetric beta‐zipper hydrogen bonds to drive specificity. XLF C terminus and DNA enhance parallel filament formation. Super‐helical XLF‐XRCC4 filaments form a positively charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF‐XRCC4 stimulation of DNA ligation.
RNAseq analysis of embryonic and postnatal stages of mouse retina reveal transcriptome diversity and novel alternatively spliced isoforms
Authors: BANDAY ABDUL ROUF , Congdon Sean , Seesi Sahar Al , Mandoiu Ion , Kanadia Rahul N 
Published: June 2021

Mammalian retina exhibits a highly organized laminar structure consisting more than fifty cell types which are produced and eventually differentiated in specific manner during development. Several gene expression studies have been performed in retina. However, the landscape of alternatively spliced isoforms on global level at embryonic and postnatal stages of retinal development has not been done.Here we employed high throughput RNAseq from illumina HiSeq 2000 platform for retina samples of embryonic day, E16 and postnatal day, P0. The sequence reads were analyzed by using combination of tools such as Bowtie, IsoEM, Cuflinks and Seqmonk.We have identified 18021 and 17320 transcripts in E16 and P0 time points, respectively. Comparison of the E16 and P0 transcriptomes showed differential expression patterns and levels (as FPKM values) of transcripts. qRT‐PCR analysis of genes such as Pax6 further confirmed the results. Our results suggest that regulation of alternative spliced isoforms may be the key to the differentiation of retinal cell‐types during development. Our approach of global transcriptome characterization would advance the findings in cellular networks of proteins required for specific functions and transitions required in the developing retina. In all, this analysis will provide a database of alternatively spliced isoforms of genes expressed in the retina. Funding from NEI K99/ROO‐5ROOEYO1954704.
The role of NK1 receptor antagonist L‐703, 606 in the experimental traumatic brain injury
Authors: Duan Hubin , Hao Chunyan , Li Shuzhen , Fan Yimin 
Published: June 2021

Background and purposeL‐703, 606 is a NK1 receptor antagonist. the purpose of the research is to explore the the role of L‐703, 606 in the experimental traumatic brain injury£TBI£©.Methods45 male Wistar rats were randomly divided into control group, L‐703, 606 intervention group (group L) ,TBI trauma group (group T). L‐703, 606 was injected into the tail vein of rats in group L after TBI. HE staining were used to observe the pathological changes in each group. The gene and protein expression of substance P(SP), calcitonin gene related peptide (CGRP) and nuclear transcription factor‐kappa B (NF‐¦ÊB) were detected by immunohistochemical staining and RT‐PCR.ResultsThe gene and protein expression of SP, CGRP and NF‐¦ÊB in rat cerebral cortical lesions of group L and group T was increased compared with that of control group. Above indicators of group L was significantly reduced compared with that of group T. Only mild congestion, a small amount of inflammatory cell infiltration showed in group L. (P <0.05)ConclusionL‐703, 606 has a protective effect on the brain injury in rats. The possible mechanism maybe reducing the inflammatory reaction induced by SP and CGRP which released from cerebral cortical lesion.
Stem cells, pluripotency and nuclear reprogramming
Authors: Jaenisch Rudolf 
Published: June 2021

The recent demonstration of in vitro reprogramming using transduction of 4 transcription factors by Yamanaka and colleagues represents a major advance in the field. However, major questions regarding the mechanism of in vitro reprogramming need to be understood and will be one focus of the talk.During cellular reprogramming only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells and impeded identification of events at the single‐cell level. We utilized two different gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. We show that Esrrb, Utf1, Lin28, and Dppa2 have a better potential to predict cells that will become iPSCs, compared to Fbxo15, Fgf4, and Oct4, which were previously suggested to be markers of reprogramming. Our data suggest that stochastic gene expression early in reprogramming is followed by a late sequential phase with Sox2 activation upstream in a gene expression hierarchy. Finally, we demonstrate that subsets of downstream factors derived from the sequential phase can activate the pluripotency circuitry.A major impediment in realizing the potential of ES and iPS cells to study human diseases is the inefficiency of gene targeting. Using Zn finger or TALEN mediated genome editing we have established efficient protocols to target expressed and silent genes in human ES and iPS cells. Finally, our progress in using iPS cells for therapy and for the study of complex human diseases will be summarized.
Effect of glutathione on the phospholipase A2 activity of peroxiredoxin 6
Authors: Zhou Suiping , Dodia Chandra , Shuvaeva Tea , Sorokina Elena , Harper Sandra , Speicher David W , Feinstein Sheldon I , Fisher Aron B 
Published: June 2021

Peroxiredoxin 6 (Prdx6) is a unique non‐seleno 1‐Cys peroxidase with both peroxidase and phospholipase A2 (PLA2) activities. We have shown that phosphorylation of Prdx6 at Thr177 increases its PLA2 activity and broadens its pH‐activity spectrum (Biochem. J. 419:669, 2009). Our recent study indicates that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhanced enzymatic activity at pH 7.4 (Biochemistry. 51:5521–30. 2012). Here, we studied the effect of glutathione (GSH) on the PLA2 activity of Prdx6 using biochemical and biophysical approaches. With 3H‐DPPC as substrate, basal Prdx6 PLA2 activity was 0.17 ± 0.1 nmole/min/mg and increased to 49 ± 1 nmole/min/mg upon addition of 5 mM GSH. Addition of other thiols (GSSG, DTT) had no effect. The enhancing effect of GSH on the PLA2 activity of Prdx6 depended on the redox status of substrates and phosphorylation status of the enzyme. GSH has no effect on PLA2 activity if the substrate is oxidized by Cu(2+)‐ ascorbate or if the enzyme is phosphorylated. By isothermal titration calorimetry (ITC), GSH interacts with Prdx6. Circular dichroism (CD) indicates that GSH induces a change of Prdx6 conformation. Thus, a change in the conformation of Prdx6 in the presence of GSH is the basis for enhancing effect of GSH on PLA2 activity of non‐phosphorylated Prdx6 in the presence of reduced substrate.This work was supported by NIH grants HL102016 and HL19737
Zhx2 and the balance between cardiovascular and hepatic health
Authors: Creasy Kate Townsend , Clinkenbeard Erica , Spear Brett T 
Published: June 2021

BackgroundThe Zinc fingers and homeoboxes 2(Zhx2) gene has been identified as being responsible for the mouse Hyplip2 trait characterized by hyperlipidemia and atherosclerosis. This finding is supported by a recent human GWAS study identifying ZHX2 as an important risk factor for carotid intima media thickness, a measure of subclinical atherosclerosis. Mice with mutated Zhx2 incur less cardiovascular damage from a high‐fat diet than wildtype mice.ObjectiveTo explore the role of Zhx2 on lipid metabolism and homeostasis, as well as its impact on cardiovascular and hepatic health.MethodsWild‐type and Zhx2‐mutant mice were fed either a control or high‐fat (15%) diet for 8 weeks. Body weight was measured and serum was collected during this period, and tissues were collected post‐sacrifice. Quantitative RT‐PCR analysis was performed to assess gene expression.ResultsMice with low Zhx2 levels have elevated serum ALT levels, increased hepatic AFP mRNA, and elevated liver pathology and lipid accumulation. Additionally, Lpl, Elovl3, and Cyp8B1, genes involved in cholesterol/lipid metabolism, were identified as Zhx2 targets.ConclusionsThese data indicate that reduced Zhx2 levels may be cardioprotective but at the same time lead to increased liver damage in mice on a high‐fat diet. Further studies will investigate the role of Zhx2 in human cardiovascular and liver disease.This research is supported by PHS Grants DK074816 and RR021954 from the NIH.
Preliminary Investigation of The Nutritional Qualities, Phytochemical Properties and Antioxidant Activities of Aqueous Extract of Mistletoe Leaves (Tapinanthus Bangwensis) Grown on Orange Trees.
Authors: Omojufehinsi Moyosore , Nwoke Stella C , Imaga Ngozi A , Magbagdeola Abiola O , Ebuehi Albert O. , Okafor Uzoma A 
Published: June 2021

Mistletoe (Tapinanthus bangwensis) plant has been used traditionally in Nigeria and other parts of Africa for the treatment and management of ailments such as diabetics, female reproductive disorders, arthritis, rheumatism, hypertension, asthma, epilepsy and diabetes. In view of the traditional usage of Mistletoe, this has no detailed scientific data; we investigated the nutritional potentials of aqueous crude extract of Tapinanthus bangwensis (Mistletoe) and determination of in vitro antioxidant activity, qualitative and quantitative physiochemical properties.The proximate analysis revealed the crude protein to be 6.8 ± 0.4%, crude fat (9.65 ± 0.3%), total carbohydrate (12.72 ± 0.1%), moisture (67.2 ± 0.1%), ash (3.5 ± 0.1%), crude fibre (0.13±0.2%) and the energy value is 164.94KJ/mole. The mineral evaluation in mg/100g dry weight showed Potassium (147.3±0.01) >; Calcium (90.70±0.025) >; Phosphorus (62.9±0.025) >; Sodium (54.6±0.03) >; Magnesium (43.05±0.02) >; Manganese (34.00±0.01) >; Zinc (24.90±0.015) and Iodine (2.05±0.01). Further tests revealed that heavy metals such as lead, chromium and cadmium were not detected. The qualitative phytochemical analysis revealed the presence of tannins, steroidal glycosides, phenols, saponin, terpenoids and flavonoids while the quantitative (in mg/100g) phytochemical analysis revealed tannin (108.65±0.04 ) >;phenol (66.7±0.01)>; flavonoid (44.58±0.03)>; Cynogenic glycoside (24.53±0.02)>; saponin (2.40±0.02). Reducing sugar was revealed to be 33.07±0.05.
Multiple cycles of acetyl‐CoA enolization catalyzed by citrate synthase (CS) and ATP‐citrate lyase (ACL)
Authors: Li Qingling , Anderson Vernon E. , Brunengraber Henri , Zhang Guo‐Fang 
Published: June 2021

Enolization of acetyl‐CoA on CS had been identified in Eggerer¡s experiments with [3H]acetyl‐CoA (Biochem. Z. 343: 111, 1965). To test whether CS catalyzes sequential cycles of acetyl‐CoA enolization, we incubated [2H3, 13C2]acetyl‐CoA with pig heart CS + oxaloacetate (OAA) (added as either a bolus, a bolus of OAA/malate, or malate + NAD + malate dehydrogenase). We measured the mass isotopomer distribution (MID) of acetyl‐CoA and citrate. The productions of M4 + M3 + M2 acetyl‐CoA, and of M4 + M3 + M2 citrate revealed up to 3 cycles of acetyl‐CoA enolization. This was confirmed when condensation of unlabeled acetyl‐CoA + OAA was conducted in 50% D2O. Acetyl‐CoA was up to M3 labeled and citrate was M1 labeled. Thus, up to 3 cycles of acetyl‐CoA enolization were catalyzed by CS.We hypothesized that ACL could also catalyze enolization of acetyl‐CoA. We incubated [2,2,4,4‐2H4]citrate + Mg++, CoA, ATP and rat liver ACL. The production of M2 + M1 + M acetyl‐ CoA, and the progressive de‐labeling of acetyl‐CoA demonstrated multiple cycles of enolization of acetyl‐CoA on ACL. This was confirmed when cleavage of unlabeled citrate was conducted in 50% D2O, by the accumulation of M1 + M2 + M3 acetyl‐CoA. Thus, multiple cycles of acetyl‐CoA enolization were catalyzed by ACL.(Supported by the NIDDK Metabolomic Initiative grant R33DK070291).
Crosslinking mass spectrometry with novel aryl‐dialdehyde reagents reveal extended conformational dynamics of RNA polymerase II
Authors: Trnka Michael J , Burlingame A. L. 
Published: June 2021

Chemical crosslinking mass spectrometry is a means of probing the structures of protein machines in their native states. Bifunctional reagents covalently label stable complexes, reflecting their topology. Identification of the modified sites generates distance restraints, which together with crystallography of individual subunits and cryoEM visualization of entire complexes, can be used to model large three‐dimensional structures. Furthermore, solid‐state protein structures are conformationally constrained but crosslinking can reveal the extent of their dynamics. Here, we probe RNA polymerase II (pol II) with aryl‐dialdehyde (ADA) crosslinking reagents. ADA reagents preserve positive charge at the site of crosslinking, which facilitates the enrichment of crosslinked peptides by cation exchange chromatography relative to conventional reagents. Furthermore, these highly charged precursors are well suited to analysis by electron transfer dissociation (ETD) mass spectrometry, which provides more comprehensive product ion coverage than collisional dissociation and facilitates identification of crosslinked peptides in complex searches. The data indicate that the pol II clamp domain is more dynamic than indicated by crystallography.Funding was provided by the Biomedical Technology Research Centers program of the NIH National Institute of General Medical Sciences NIH NIGMS 8P41GM103481
Human melanoma metabolic network analysis with combined 13C NMR/bioreactor technique: testing the Warburg effect
Authors: Shestov Alexander , Mancuso Anthony , Locasale Jason W , Glickson Jerry D 
Published: June 2021

The bioreactor techniques are becoming an important tool to study cancer cell metabolism(1). The goal of the present work was to develop a 13C metabolic “bonded cumomer” modeling approach adapted for fitting bioreactor data obtained with 13C glucose as substrate and to calculate the extent of the Warburg effect and how much energy comes from aerobic glycolysis vs oxidative phosphorylation.ResultsFigure 1 shows experimental time courses for labeled glutamate obtained during [1,6‐13C2] glucose perfusion of melanoma cells cultured in a CST bioreactor. Using extracted fluxes we estimated that 54% of the energy coming from oxidative phosphorylation (from which 44% are from glucose) and 46% from aerobic glycolysis and the extent of the Warburg effect (aerobic glycolysis) was 87%.ConclusionsUnder aerobic conditions glycolysis provides nearly as much ATP as oxidative phosphorylation. The model is validated by excellent agreement between model predicted and experimentally measured values of CMRO2 and cytosolic glutamate pool size. Dynamic high‐resolution MR spectra is very sensitive to changing/adding biochemical pathways and flux values, and bonded cumomer modeling allows one to check precisely the feasibility of assumed general bionetworks and particular metabolic pathways.This work was supported by NIH grants 2U24‐CA083105 and 5R01CA129544–02
The molecular mechanism of histone deacetylase inhibitor (HDACi) PCI‐24781 mediating anti‐tumor activity in neuroblastoma cells
Authors: Zhan Qing Lei , Ngai Sai Ming 
Published: June 2021

Neuroblastoma accounts for 7–10% of all childhood cancers, and it is the second most common solid tumor diagnosed during infancy. The survival rate among children with high‐risk neuroblastoma is less than 40%, despite intensive multi‐modality treatment, highlighting the urgent needs for new treatment strategies. Epigenetic changes are known to be important for tumorigenesis and a number of drugs targeting epigenetic regulation have been developed for anticancer therapies. Among those drugs, the most effective ones are histone deacetylase inhibitors (HDACi). Numerous HDACi are currently in phase I/II clinical trials for various tumor types. However, little is known about the role of PCI‐24781, a novel HDACi in nueroblastoma. In the present study, we investigated the underlying mechanisms that mediate anti‐tumor activity of PCI‐24781 in neuroblastoma cell line SK‐N‐DZ. PCI‐24781 treatment inhibited tumor growth at very low doses in SK‐N‐DZ, while normal cell line HS‐68 shown a relatively resistance to treatment. PCI‐24781 caused the accumulation of acetylated histone H3 and cell cycle arrest in G2/M phase in SK‐N‐ DZ. Treatment of SK‐N‐DZ with PCI‐24781 also induced apoptosis signaling pathways via up‐regulating some genes associated with apoptosis, including death receptor DR4, caspase3, p21 and p53. Furthermore, the 2D electrophoresis‐based proteomic analysis revealed different protein expression profile in SK‐N‐DZ cells treated with PCI‐24781 for 24 h and 36 h. The identified proteins involved in many biological processes, including energy metabolism, DNA repair, transcription regulation, cell cycle and proliferation. Immunoblotting analysis further confirmed that PCI‐24781 could increase the expression of prohibitin, hHR23a and RuvB2.These studies will enable a better understanding of molecular mechanisms underlying PCI‐24781‐mediated anti‐tumor effects in neuroblastoma cells SK‐N‐DZ.
Differential Protein Analysis of Developing Mouse Cochlea using Label‐Free Quantitative Mass Spectrometry
Authors: Darville Lancia , Sokolowski Bernd 
Published: June 2021

The sensory epithelium of the inner ear contains the receptor or hair cells responsible for hearing. To obtain insights into the proteome of these epithelial cells during development, we quantitatively compared proteins expressed in postnatal day (P) 14 versus P30 mice using label free shotgun proteomics with mass spectrometry. Proteins from the cochlear sensory epithelia were extracted, enzymatically digested, separated by strong cation exchange (SCX), and analyzed using nano LC‐MS/MS on an Orbitrap. Proteins were quantified and compared for P14 and P30 cochleae using spectral counts calculated by MaxQuant. Results show that proteins unique to the P14 and P30 proteome number 368 and 255, respectively, with 746 proteins common to both. An initial comparison of up‐regulated proteins, as defined by at least a 10‐fold increase in spectral count, revealed an increase in 69 and 52 proteins, respectively, on P14 and P30. Analyses, using gene ontology annotations, showed that oxidoreductase proteins had the greatest decrease in expression on P30 relative to P14. In comparison, cytoskeletal and transfer proteins were up‐regulated on P30 compared to P14. The use of label free quantitation to investigate the cochlear proteome revealed protein identifications that may have biological significance to hearing maturation.Supported by NIDCD grant DC004295 to BS.
Thermodynamics of cellulose solvation in novel solvent mixtures
Authors: Das Ritankar , Chu Jhih‐Wei 
Published: June 2021

Biomass contains abundant amounts of cellulose as crystalline microfibrils. A limiting step to using cellulose as an alternative energy source, however, is the hydrolysis of the biomass and subsequent transformation into fuels. Cellulose is insoluble in most solvents including organic solvents and water, but it is soluble in some ionic liquids like BMIM‐Cl. This project aims to find alternative solvents that are less expensive and are more environmentally benign than the ionic liquids.All‐atom molecular dynamics simulations were performed on dissociated glucan chains separated by multiple (4–5) solvation shells, in the presence of several novel solvents and solvent mixtures. The solubility of the chains in each solvent was indicated by contacts calculations after the equilibration of the molecular dynamics. It was discovered that pyridine and imidazole acted as the best solvents because their aromatic electronic structure was able to effectively disrupt the inter‐sheet interactions among the glucan chains in the axial direction, and because perturbation of the solvent interactions in the presence of glucan chains was minimal.
The pro‐cysteine drug, L‐2‐Oxothiazolidine‐4‐carboxylic acid (OTC) is a novel inducer of nSMase2 mRNA and protein expression in the liver
Authors: Deevska Gergana , Karakashian Claudia , Sunkara Manjula , Peppers Benjamin , Morris Andrew , Nikolova‐Karakashian Mariana 
Published: June 2021

Neutral sphingomyelinase 2 (nSMase2) is a signaling enzyme that mediates hepatic response to IL‐1β. It has been suggested that nSMase2 is activated by oxidative stress and during aging leading to hyperresponsiveness to inflammation. These changes in nSMase2 activity are not caused by increased protein, but are due to aging‐associated decline in the levels of hepatic GSH, a known nSMase2 inhibitor. In this study we sought to investigate the effect of a cysteine pro‐drug, L‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) on nMSase2 activity in vivo in young and aged mice (5 and 23 months old). OTC has been shown to replenish hepatic GSH and to improve the overall morbidity in elderly. C57Bl6 mice were fed three different diets for 4 weeks: a standard chow (Std), sulfur amino acid deficient control (SAA Def) and supplemented with 0.5% OTC diet (OTC). As anticipated, aging was associated with significant increases in hepatic nSMase activity. Mass‐Spec analyses showed significant accumulation of C16 and C24:1 ceramides in the aging liver. OTC diet lead to increase in hepatic GSH and abolished any aging‐associated differences. Surprisingly, the mice fed the OTC diet had significantly elevated nSMase activity than mice fed the other two diets. These changes were accompanied by corresponding increases in nSMase2 mRNA and protein levels suggesting that OTC has GSH‐independent effect. The effects were seemingly specific for nSMase2 and liver. Direct addition of OTC to HepG2 cells recapitulated the effects observed in vivo and lead to elevated nSMase2 mRNA and protein expression. Together, these studies provide evidence for a specific regulation of nSMase2 mRNA transcription and protein expression by a drug involved in the control of cellular red‐ox homeostasis.Support:R01AG19223,R01AG026711to MNK, AHA11POST7650060 to GD.