bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lanthanide‐based FRET biosensors for time‐gated imaging and detection of protein‐protein interactions in live mammalian cells
Authors: Chen Ting , Pham Ha , Miller Lawrence 
Published: November 2023

Förster resonance energy transfer (FRET) using fluorescent proteins (FPs) as donors and acceptors is commonly used to microscopically image protein‐protein interactions in living cells. However, overlapping FP spectra lower signal‐to‐noise because the FRET‐sensitized acceptor emission signal is convoluted with donor bleedthrough and directly excited acceptor emission, or crosstalk. Quantitative FRET imaging is even more difficult when the local concentrations of donor‐labeled and acceptor labeled proteins are substantially different. We used the well‐studied interaction between FK binding protein 12 (FKBP12) and the rapamycin binding domain of m‐Tor (FRB) as a model system to show that lanthanide‐based biosensors can overcome the limitations of conventional FP‐FRET. The essential features of this approach include the use of luminescent Tb(III) complexes as FRET donors and the elimination of non‐specific fluorescence background that comes from time‐gated detection of long‐lived (~ms) Tb(III) and Tb(III)‐to‐FP emission signals. We prepared stably transformed NIH 3T3 fibroblast lines that express a single polypeptide consisting of the following elements (N to C): 1) FRB; 2) Escherichia coli dihydrofolate reductase (eDHFR); 3) a linker sequence; 4) GFP; and 5) FKBP12. A Tb(III) complex (TMP‐Lumi4) that selectively binds to eDHFR completes the sensor. The linker comprises alternating repeats of four glutamic acid residues followed by four arginine or lysine residues (ER/K) that form a rigid alpha helix in solution. Stochastic breaks in the helix permit close approach and binding of affinity elements positioned at each end. The effective concentration of each binding element depends on linker length and not solution concentration. Consequently, the fraction of two elements in the closed (high‐FRET) state depends on KD and linker length. We tested constructs with ER/K linker lengths of 10, 20 and 30 nm. Following addition of cell permeable TMP‐Lumi4 and rapamycin to culture medium, two‐color, time‐gated images showed significant, ~3‐fold increases in acceptor denominated FRET ratios relative to baseline (without rapamycin). The high sensor dynamic range permitted robust, time‐gated detection (Z'‐factor > 0.8) of the FKBP12/FRB interaction and its inhibition by ascomycin in cells grown in 96‐well plates.Support or Funding InformationNIGMS R01 081030
Effect of Zhibai Dihuang Granule on anti‐inflammatory proteins associated with Yin deficiency heat syndrome
Authors: Mao Liangen , Liu Changming , Jiang Tingting , Wang Chong , Chen Zhongliang , Li Jicheng 
Published: November 2023

This study aims to explore the effect of Zhibai Dihuang Granule (ZDG) on anti‐inflammatory proteins in the serum of Yin deficiency heat syndrome rats. The Yin deficiency syndrome rat model was established by triiodothyronine, and differentially expressed proteins in the serum of rats were analyzed by iTRAQ. The levels of anti‐inflammatory proteins such as Gelsolin (Gsn), Plasminogen (Plg), Fibrinogen alpha chain (Fga), and Thymosin beta‐4 (T‐β4) were significantly increased in the ZDG treated model rats, compared with the control rats. However, the level of Coagulation factor XIII A chain was significantly decreased in the ZDG treated group. Gsn has been found to remove the damaged tissue and release actin, thereby inhibiting cell apoptosis. Gsn was significantly increased in the ZDG treated group, indicating that ZDG may affect Gsn to inhibit cell apoptosis. Colla2 and Plg have been shown to regulate the blood rheology in the inflammatory response. Colla2 was significantly decreased and Plg wes significantly increased in the ZDG treated group, suggesting that ZDG can regulate the blood rheology of the inflammatory response by down‐regulating the Colla2 and up‐regulating Plg. Gsn and T‐β4 have been shown to promote the healing of inflammatory damage by regulating cytokines. Gsn and T‐β4 were significantly increased in the ZDG treated group, indicating that ZDG could inhibit apoptosis by regulating cytokines.ConclusionZDP can treat Yin deficiency heat syndrome by removing damaged tissue, releasing actin and lipopolysaccharide, inhibiting cell apoptosis, regulating inflammatory factors and the blood rheology of inflammation, and promoting the healing of inflammatory damage.Support or Funding InformationThis work was supported by grants from the National Basic Research Program of China (No.2014CB543002), the National Natural Science Foundation of China (No.81573709, No.81273882)
Screening and identification of novel serum potential biomarkers for pulmonary tuberculosis by iTRAQ‐2D LC‐MS/MS
Authors: Jiang Tingting , Shi Liying , Li Xiang , Wei Liliang , Yang Su , Wang Chong , Liu Changming , Chen Zhongliang , Tu Huihui , Li Zhongjie , Li Jicheng 
Published: November 2023

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) poses great threat to human health. Delay in the diagnosis of TB may increase the severity and mortality, as well as increase the risk of transmission of the disease. Therefore, early diagnosis and treatment are important for the control of TB spread. The aim of this study was to discover novel biomarkers for TB. Differentially expressed proteins in the serum of patients with TB were screened and identified by iTRAQ‐2DLC‐MS/MS analysis. A total of 47 abnormal proteins were discovered in TB patients, compared with healthy controls, pneumonia and chronic obstructive pulmonary disease (COPD) patients. The levels of serum amyloid A (SAA), vitamin K‐dependent protein Z (PROZ), and C4b‐binding protein β chain (C4BPB) were significantly higher in TB patients (n=136) than healthy controls (n=66) (P<0.0001, P<0.0001, and P<0.0001, respectively), whereas, significantly lower levels of SAA, PROZ, and C4BPB were observed in TB patients compared with pneumonia patients (n=72) (P<0.0001, P<0.0001, and P<0.0001, respectively) and COPD patients (n=72) (P<0.0001, P<0.0001, and P=0.0016, respectively). After 6 months of treatment, the levels of SAA and PROZ were significantly increased (P=0.022 and P<0.0001, respectively), while the level of C4BPB was significantly decreased (P=0.0038) in treated TB cases (n=72). Significantly increased SAA and PROZ levels were observed in TB patients with cavitary lung lesions (P=0.006 and P=0.03, respectively), compared with TB patients with non‐cavitary lung lesions. Meanwhile, the serum concentrations of SAA were higher in TB patients with double lung lesions than TB patients with single lung lesions (P=0.003). The serum SAA levels were also found to be increased in smear positive TB patients than smear negative TB patients. Decision trees of TB were constructed, and receiver operating characteristic curve analysis revealed that the area under the curve value (AUC) of the diagnostic model combined by SAA, PROZ, and C4BPB was more accurate than the single serum protein. The AUC was 0.972, 0.928, 0.957, and 0.969, to discriminate the TB group from the healthy controls, pneumonia, COPD, and cured TB groups, respectively. Therefore, the results suggested that SAA, PROZ, and C4BPB may serve as potential biomarkers for TB. Our study could provide experimental data for the differential diagnosis of TB.Support or Funding InformationThis work was supported by grants from the National Basic Research Program of China (No.2014CB543002), the National Natural Science Foundation of China (No.81573709, No.81273882), and the scientific research fund project of South China University of Technology School of Medicine (NO. yxy2016006).
Investigation of Induced Obstructive Sleep Apnea (OSA) in Rat Atria using Mass Spectrometry based Proteomics
Authors: Channaveerappa Devika , Lux Jacob , Wormwood Kelly L. , McLerie Meredith , Panama Brian K. , Darie Costel C. 
Published: November 2023

Obstructive sleep apnea (OSA) affects up to 24% of the adult population and is associated with several atrial diseases. It is associated with daytime sleepiness, headache, depression, hypertension, obesity, arthritis, type 2 diabetes, and heart disease. OSA is characterized by transient cessations in respiration lasting >10 seconds as a result of narrowing or occlusion of the upper airway during sleep. OSA severity is assessed by the apnea‐hypopnea index (AHI), or the average number of apneas (complete obstructions) or hypopneas (partial obstructions) per hour. An estimated 20% of adults have mild OSA (AHI 5–15) and 7% of adults have moderate (AHI 15–30) to severe (AHI>30) OSA, with 85% of patients remaining undiagnosed. Although clinical evidence linking OSA to proarrhythmaic atrial changes is well known, the specific molecular mechanisms by which OSA causes atrial disease remain elusive. To study the OSA‐induced cardiac changes, we have initiated a recently developed rat model which closely recapitulates the characteristics of OSA.Male Sprague Dawley rats, aged 50–70 days, received surgically implanted tracheal balloons which were inflated to cause transient airway obstructions. Apnea groups experienced 60 apneas per hour of either 13 seconds (moderate apnea) or 23 seconds (severe apnea) for 2 weeks and 8 hours per day. Control rats received surgeries but no inflations. Proteomics analysis was done on the rat atria homogenates to identify dysregulated proteins in moderate and severe apnea when compared to control. SDS‐PAGE was performed on the homogenates to separate the proteins and the gel bands were trypsin digested to obtain the peptide mixtures. The peptides were analyzed by a Nano Acquity UPLC coupled with Xevo G2 Mass Spectrometer. Data analysis was done using ProteinLynx Global Server (PLGS 2.4), Mascot server and Scaffold 4.1 software.The proteomics analysis revealed that 3 of the 9 enzymes in glycolysis and 2 proteins related to oxidative phosphorylation were down‐regulated in the severe apnea group. In contrast, several structural and pro‐hypertrophic proteins were up‐regulated with chronic OSA. The data suggests the chronic OSA causes proteins changes which lead to cessation of glycolysis, a diminished capacity to generate reducing equivalents (i.e. NADH) as well as promotion of cardiac hypertrophy.Support or Funding InformationClarkson University
Temporal quantitative proteome analysis reveals dynamic change of cellular and secreted protein profiles of Clostridium cellulovorans depending on carbon sources
Authors: Shunsuke Aburaya , Wataru Aoki , Kouichi Kuroda , Mitsuyoshi Ueda 
Published: November 2023

An anaerobic bacterium Clostridium cellulovorans secretes a large cell‐surface enzyme complex called cellulosome and this can degrade and metabolize cellulose, hemicellulose, and pectin which constitute plant cell wall. Furthermore, C. cellulovorans optimizes profiles of cellulosomal enzymes and non‐cellulosomal enzymes, which are secreted to media, depending on carbon sources. [1] Under the degradation of each carbon source, dynamical changes of composition of metabolic proteins occurred. [2] C. cellulovorans recognizes carbon sources in media and optimizes cell surface, secreted, and metabolic proteins to adapt to environment.It is very interesting to reveal a dynamic adaptation mechanism of cellular and secreted protein profiles depending on carbon sources. In addition, temporal microbial profiling of cellular and secreted protein derived from the one‐pot culture has not been reported. In this research, we cultured C. cellulovorans in 5 carbon sources and took cellular and secreted samples temporally. After that, quantitative proteome analysis using tandem mass tag was carried out. From this analysis, we attempted to clarify temporal dynamic changes of protein profiles depending on carbon sources and culture times.After the cultivation and sampling of each time point in glucose, cellulose, xylan, pectin, and galactomannan, the cellular proteins were extracted and the secreted proteins were concentrated. Each protein was alkylated after reduction, and digested by trypsin. Peptides were labeled with the stable isotope label and subjected to the LC‐MS/MS system equipped with unique super‐long monolithic silica capillary column. [2]We could totally identify about 1500 proteins of C. cellulovorans in one‐shot proteome analysis, and these profiles were significantly dependent on each substrate and time point. Obtained data were analyzed statistically and changing proteins depending on substrates and time points was significantly identified. Focusing on central metabolic enzymes, we could clarify dynamic changes of these enzymes with time‐ and substrate‐depending manners. Cellulosomal and non‐cellulosomal proteins derived from each fraction are also dynamically changed as carbon sources were degraded. From these results, we considered adaptation and degradation mechanisms of C. cellulovorans with various carbon sources. [3]
Proteomic research on the therapeutic mechanism of Zhibai Dihuang Granule for treating hyperthyroidism yin‐deficiency rats
Authors: Liu Changming , Mao Liangen , Yang Su , Jiang Tingting , Wang Chong , Chen Zhongliang , Tu Huihui , Li Zhongjie , Li Jicheng 
Published: November 2023

Zhibai Dihuang Granule (ZDG) is a classical traditional Chinese medicine that has been used to treat yin‐deficiency‐heat (YDH) syndrome. However, the therapeutic mechanism of ZDG is still unclear. The aim of this study was to observe the therapeutic effect and mechanism of ZDG in hyperthyroidism yin‐deficiency rats by detecting the serum proteome expression. Triiodothyronine (T3) was used to induce the hyperthyroidism yin‐deficiency model of rats, and the rats were treated daily with ZDG by gastrogavage. In the proteomics experiment, a total of 93 differentially expressed serum proteins (49 up‐regulated proteins and 44 down‐regulated proteins) were identified by iTRAQ labeling coupled with two‐dimensional liquid chromatography‐tandem mass spectrometry (2D LC‐MS/MS) analysis (fold change >1.50 or <0.60, p < 0.05) between model rats and control rats. After ZDG treatment, 16 proteins among up‐regulated proteins showed obvious downward trend, and 10 proteins among down‐regulated proteins showed obvious upward trend. Bioinformatics analysis of the proteins which returned to normal level revealed that 10 of these proteins were involved in immune response, especially in complement and coagulation cascades. After ZDG treatment, L‐selectin, C‐reactive protein (CRP), and Zinc‐alpha‐2‐glycoprotein (Azgp1) levels were significantly increased (p=0.0002, p=0.0003, p=0.0051, respectively). Therefore, we concluded that ZDG may treat YDH syndrome by regulating immune response, especially complement and coagulation cascades, and L‐selectin, CRP and Azgp1 may act as potential biomarkers to evaluate the therapeutic efficacy of ZDG. Our work provides important experimental basis to understand the mechanism of ZDG for treating YDH syndrome, and provide potential serum biomarkers for evaluating therapeutic efficacy of ZDG.Support or Funding InformationThis work was supported by grants from National Basic Research Program of China (No.2014CB543002), National Natural Science Foundation of China (No.81573709, No.81273882).
Alcohol's Effect on the Metabolome and CCL2
Authors: Berndt Calvin Wayne , Schumann Peter , Dunn Alyssa , Bray Jennifer , Lawrence Jim 
Published: November 2023

When alcohol enters the central nervous system it disrupts the physiological balance, producing a plethora of negative effects on the nervous system, specifically learning and memory. It has been shown that the neuroimmune protein CCL2 perturbs the negative effects alcohol has on the hippocampus, the region of the brain associated with learning and memory. Although the signaling cascade initiated by CCL2 is currently unknown, elucidation of the pathway is possible through the use of a quadrupole time‐of‐flight mass spectrometer (Q‐TOF LC/MS). Using the previously specified instrument a molecular fingerprint can be produced by unambiguous and untargeted identification of metabolites located in hippocampus of CCL2‐transgenic mice. By comparing the fingerprint of non‐transgenic mice with that of CCL2‐transgenic mice one can identify dysregulated metabolites.Over a time course of ten days mice of varying ages consumed 6g/kg of 20% ethanol every day. After the binge the left‐hemisphere hippocampus was extracted for metabolite analysis and the right‐hemisphere hippocampus was used for quantification of CCL2 concentration. This ensured identical biological samples for cross‐comparison between protein expression and the effects this had on the metabolome of the hippocampus. ELISA was used to quantify CCL2 concentration while the Q‐TOF LC/MS was used to identify metabolites. Statistical analysis (PCA) of mass spectra was done through XCMS Online, a cloud‐based metabolic data processor. Tentative assignment of m/z to compound names was done through built‐in Agilent software (MassHunter Qualitative and ProFinder) using METLIN databases.Metabolites and their regulation tell a story about the physiology of the cell. Metabolic pathways can become temporarily dysregulated by external factors, such as alcohol consumption. By using transgenic mice, we can study the differing effects of alcohol on the metabolome by controlling a single molecular variable, such as CCL2 levels. By altering conditions of transgenic versus non‐transgenic mice, we can develop a broader knowledge base about the molecular fingerprint for alcoholism in the mammalian brain and understand the mechanisms of certain neurological disorders, such as alcoholism.Support or Funding InformationUniversity of Wisconsin ‐ Stevens Point URSCA Grant ($1,000)ASBMB Undergraduate Research Grant ($1,000)
O‐GlcNAcylation of the Human Kinome
Authors: Liu Xin , Han Guanghui , Pandey Akhilesh , Hart Gerald 
Published: November 2023

O‐GlcNAc, first characterized more than 30 years ago, is an O‐linked β‐N‐acetylglucosamine moiety attached to the side chain hydroxyl of a serine or threonine residue. O‐GlcNAc has been found on over 4,000 cytoplasmic and nuclear proteins. The addition of O‐GlcNAc to proteins is catalyzed by O‐GlcNAc transferase (OGT) while its removal is catalyzed by O‐linked N‐acetyl‐β‐D‐glucosaminidase (OGA). This dynamic and reversible modification is emerging as a key regulator of various cellular processes, such as signal transduction, transcription, cell cycle progression and protein‐protein interactions. O‐GlcNAc plays important roles in human diseases, such as cancer, diabetes and neurological disorders.Protein phosphorylation is a post‐translational modification that serves as a rapid and reversible means to modulate protein activity and transduce signals, the regulation of phosphorylation is a central mechanism in cell health and disease. The addition and removal of phosphorylation is through kinases and phosphates respectively, but little is known about O‐GlcNAcylation of kinases.We have done kinase microarray which indicate that 297 kinases are substrates for OGT and then we used home‐made kinase inhibitor beads to pull down the kinases in Hela cells to further confirm the results in vivo. More than 200 kinases were identified by mass spectrometry. Currently we are using click chemistry followed by O‐GlcNAc enrichment to map the sites for all the O‐GlcNAcylated kinases we have pulled down.Support or Funding InformationSupported by NIH P01HL107153, R01DK61671 and R01GM116891. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.
Cancer‐Related Mutations of ERK3 Promote Cancer Cell Invasiveness
Authors: Alsaran Hadel , Elkhadragy Lobna , Long Weiwen 
Published: November 2023

Extracellular signal‐Regulated Kinase 3 (ERK3) is an atypical member of the mitogen‐activated protein kinase (MAPK) family. Recent studies have shown that ERK3 is highly upregulated in multiple cancers, such as lung cancer and colon cancer. In addition, ERK3 promotes cancer cell migration and invasion, at least partly by upregulating matrix metalloproteinase genes' expression. Gene mutation is a major cause of human cancers. However, little is known about ERK3 mutations in cancer development and progression. By reviewing COSMIC database of gene mutations in cancers, we found two ERK3 mutations L290P and L290V which exist in cancers of lung, large intestine and skin. Notably, ERK3 L290 residue is located in the kinase domain of ERK3 and is conserved in ERK1/2/3/4. In order to characterize these mutations, we generated plasmids for ERK3 with each of these point mutations, and overexpressed them in H1299 and A549 lung cancer cells. We found that in comparison with wild type (WT) ERK3, both of these cancer‐related mutants had greatly increased ability in promoting cancer cell migration and invasion. To elucidate the underlying mechanism by which ERK3 L290 mutants increased cancer cell invasiveness, we examined the kinase activity of ERK3 mutants by in vitro kinase assay. We found that ERK3 L290P and L290V mutants have similar kinase activity to WT ERK3. ERK3 protein is known to shuttle between the nucleus and the cytoplasm, which may alter its function. Interestingly, we found that both L290P and L290V mutations greatly increased the cytoplasmic localization of ERK3 proteins, whereas WT ERK3 is mostly nuclear. We are currently trying to elucidate the molecular mechanisms by which L290P (or V) mutations increase the cytoplasmic localization of ERK3 and its ability in increasing cancer cell migration and invasion. In conclusion, our present study has identified previously unstudied cancer‐related ERK3 mutations and their importance in cancer cell invasiveness.Support or Funding InformationNCI 1R01 CA193264‐01 to Weiwen Long and “Saudi Culture mission” support to Hadel Alsaran.
The Phosphatase PRL‐3 as a Novel Drug Target in T‐Cell Acute Lymphoblastic Leukemia
Authors: Wei Min , Jing Huang , Liu Jinpeng , Wang Chi , Blackburn Jessica 
Published: November 2023

PRL‐3, phosphatase of regenerating liver 3, is a member of a subclass of dual specific phosphatases. The up‐regulation of PRL‐3 is associated with progression of gastric, breast, liver, ovarian carcinomas, oral, and cervical caners and correlates survival in patients with colorectal, gastric, and breast cancers. However, it has not been associated with T cell acute lymphoblastic leukemia (T‐ALL). We have found that PRL‐3 was a top hit among genomic amplifications acquired in T‐ALL, concurrently with increased relapse potential, in an unbiased large‐scale cell transplantation experiment using zebrafish Myc‐induced T‐ALL model. Zebrafish injected at the one‐cell stage with rag2:Myc;rag2:GFP;rag2:PRL‐3 develop T‐ALL with a shorter latency and higher penetrance, compared with the fish injected with rag2:Myc; rag2:GFP alone. Growth after transplantation at low cell number was also significantly faster in the PRL3 expressing T‐ALL. Analysis of gene expression microarray data of 119 patient samples reveals that PRL‐3 was highly expressed in 58% of the patients. In addition, PRL‐3 had the highest expression in the most aggressive and treatment resistant subgroups of T‐ALL, TAL/LMO and Immature subtypes. PRL‐3 mRNA and protein expression is also highly expressed in human T‐ALL cell lines using quantitative RT‐PCR and immunoblotting, respectively. The treatment of T‐ALL cells using a small molecule 2‐cyano‐2‐ene‐ester compound (Analog 3), resulted in growth inhibition of T‐ALL cells. Annexin V staining revealed that Analog 3 treatment led to strong apoptosis in a subset of T‐ALL cells. Cell cycle analysis using DAPI staining showed that cell cycle distribution of the cells treated with Analog3 is not affected. In summary, our study provides evidence for a role of PRL‐3 in T‐ALL malignancy, and suggests that PRL3 may be a useful drug target in the treatment of this disease. Our future studies are focused on identifying the mechanisms by which PRL3 promotes T‐ALL progression and survival.Support or Funding InformationNCI 4R00CA181500NIGMS P30GM110787The V Foundation for Cancer Research