bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional Regulation of Xenobiotic Detoxification in Drosophila
Authors: Misra Jyoti Ranjan , Horner Michael A , Lam Geanette , Thummel Carl S
Published: June 2021

Xenobiotic compounds pose a constant threat to the survival of all organisms and to overcome this, animals mount an elaborate transcriptional response, regulating a battery of enzymes that detoxify these compounds. Several transcription factors have been identified in vertebrates that regulate this response. In contrast, little is known about this pathway in insects. We show that the Drosophila Nrf2 ortholog, CncC, is a central regulator of xenobiotic detoxification responses. A binding site for CncC and its heterodimer partner Maf is sufficient and necessary for robust transcriptional responses to three xenobiotic compounds, phenobarbital (PB), chlorpromazine, and caffeine. Genetic manipulations that alter the levels of CncC, or its negative regulator Keap1, lead to predictable changes in xenobiotic‐inducible gene expression. Transcriptional profiling studies reveal that more than half of the genes regulated by PB are also controlled by CncC. Consistent with these effects on detoxification gene expression, activation of the CncC/Keap1 pathway in Drosophila is sufficient to confer resistance to the pesticide malathion. Further, in the two pesticide‐resistant strains of Drosophila, the pathway is constitutively active, leading to overexpression of several detoxification genes. Our current efforts are aimed at identifying the mutations that constitutively activate the pathway in these strains.
Identification of Protein‐Interacting Partners of USF2 in Erythroid Cells
Authors: Masannat Jude , Barrow Joeva , Bungert Jorg
Published: June 2021

Adult hemoglobin, comprised of α and β‐globin protein chains, is the major constituent of red blood cells that functions to transport oxygen around the body. Mutations of these genes lead to hemoglobinopathies such as sickle cell anemia. It has been previously demonstrated that the Upstream Stimulatory Factor (USF) activates globin gene expression. USF, composed of USF1 and USF2, is a ubiquitously expressed protein and likely interacts with erythroid‐specific activities to regulate globin gene expression. The goal of this project is to identify protein‐interacting partners of USF in order to derive a molecular pathway of adult β‐globin activation.

Using the murine erythroleukemia (MEL) and the human erythroleukemia (K562) cell lines as models for erythroid globin expression, we have successfully expressed a biotin‐flag‐tagged USF protein in these cells in an inducible manner. USF interacting proteins are identified by tandem affinity purification followed by mass spectrometry analyses. Here, we present data on the novel identification and characterization of the multiple protein‐interacting partners of USF. This knowledge provides insight into developing novel therapies for hemoglobinopathies.
Competitive titrations of SULT1A1:TNP‐AMP reveals biological nucleotide affinities for a sulfotransferase
Authors: Gaffney Kristen , Macdonald Jane , Martin Emily , Beckmann Joe D.
Published: June 2021

Cytosolic sulfotransferases (SULTs) sulfurylate xenobiotics, hormones, and neurotransmitters, with 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) as the required cosubstrate. We are investigating the regulation of recombinant bovine SULT1A1, which prefers simples phenol substrates, by biological nucleotides. SULT1A1 inhibition by PAP, ATP, and (acyl)CoAs is known; however, it is unclear whether regulation by other nucleotides might occur. Kinetic evaluation of the many such possibilities would be difficult, so we have investigated 3′‐trinitrophenylated‐adenylates as possible displaceable fluorescent probes. Fluorimetric titrations revealed TNP‐AMP to have the lowest SULT affinity of these probes, making it most readily displaceable by biological nucleotides. Therefore, bSULT1A1 is first titrated with TNP‐AMP (Kd = 0.50 μM), followed by competitive titration with a nucleotide of interest. The titrations were then numerically fit with an updated mathematical model for competition to obtain Kd values. Competition (and Kd values) observed to date are: PAP (0.22 μM), palmitoyl‐CoA (0.15 μM), octanoyl‐CoA (2.33 μM), malonyl‐CoA (38 μM), ATP (45 μM), GTP (61 μM), CTP (120 μM), UTP (164 μM), dATP (83 μM), dGTP (125 μM), and TTP (347 μM). Evaluation of additional nucleotides is ongoing.
The promise of CD44v6, a therapeutic target for colon cancer
Authors: Misra Suniti , Hascall Vincent C. , Markwald Roger R. , Ghatak Shibnath
Published: June 2021

Objective

The proposed study links CD44v6 and its cross‐talk with growth factor receptor signaling pathways in colon tumorigenesis, and the use of colon tumor specific delivery of CD44v6shRNA through nanoparticles will suppress tumorigenesis.

Methods

Mechanism of sustained production of CD44v6 in response to stimulation of hepatocyte growth factor (HGF) were studied in non metastatic primary intestinal epithelial cell line Apc10.1 isolated from Apc Min/+ mice. Involvement of CD44v6 in clonogenic growth, sphere formation, motility and in vitro angiogenesis were studied in human colon rectal cancer (CRC) cell lines. CD44v6shRNA through nanoparticles were delivered intraperitonially to immunodeficient mice carrying HT29 xenograft and the effect of CD44v6shRNA on tumorigenesis and CD44v6 signaling were studied.

Results

Mechanistically, CD44v6 acts as co‐receptor for c‐Met and VEGFR2 were observed. HGF effect on CD44v6 is inducible whereas CCD44v6 physically binds with VEGFR2 for its activation. CD44v6 forms a positive feed‐back loop with PI3K for sustained production of CD44v6. CD44v6shRNA reduces xenograft growth by 80–90% within ten treatments.

Conclusion

The data provide new insights into growth factor induced CD44v6 splicing mechanism and its role in CRC and suggest that CD44v6shRNA/nanoparticles delivery is a promising strategy for targeting CRC tumor growth and progression.
Comparative induction of p53‐regulated gene expression by replicative senescence, pharmacological senescence, and DNA damage
Authors: Gorjala Priyatham , Mohammedabdul Ataur-Rahman , Gary Ronald K.
Published: June 2021

The p53 transcription factor is activated rapidly in response to DNA damage. Replicative senescence also requires p53 activity, but the transition from a proliferative to a non‐dividing state occurs gradually over a period of weeks in culture. As cells advance towards senescence, p53‐regulated gene products accumulate slowly but steadily. Beryllium salt induces a senescence‐like state without causing DNA damage, so this reagent can be used to activate the senescence‐signaling functions of p53 more rapidly. Human fibroblasts were: (1) exposed to ionizing radiation to elicit DNA damage, (2) exposed to beryllium to elicit pharmacological senescence, or (3) cultured to attain normal senescence. The mRNA levels of senescence or DNA damage response genes were measured in each treatment group. Some p53‐regulated genes, such as the p21 cell cycle inhibitor, were induced to a similar extent by all three treatments. Other genes, such as BTG2 and GDF15, were overexpressed most strongly after pharmacological induction of senescence. These results suggest that the activity of p53 undergoes incremental adjustments when senescence occurs naturally, but that the senescence‐signaling pathway is capable of pronounced acute activation when provided with a suitable pharmacological stimulus. This work is part of a study of cellular radiation responses, supported by the Nevada NASA EPSCoR Program.
Ligand induced activation of VEGFR during angiogenesis
Authors: Li Xiaobo , Xu Ping , Toombs Jason E. , Parker Matthew W. , Brekken Rolf A. , Vander Kooi Craig W.
Published: June 2021

Vascular endothelial growth factor (VEGF) is one of the most potent pro‐angiogenic growth factors and a key regulator of both physiological and pathological angiogenesis. VEGF signals through VEGF receptor (VEGFR) tyrosine kinase family members. VEGFR‐2 is a main regulator of angiogenic effects of VEGF under both normal and abnormal conditions. VEGF binding to VEGFR‐2 induces specific dimerization and subsequent activation of angiogenesis. A model of VEGFR‐2 activation shows that ligand binding induces specific dimerization of the juxtamembrane D7 domain of VEGFR‐2. We report here the high‐resolution structure of the VEGFR‐2 Ig d7 dimer with a molecular interface centered around R724 that is primarily composed of salt‐bridges and hydrogen bonds. We further demonstrate a monoclonal antibody that binds to the D7 dimerization interface in a manner that is mutually exclusive with dimerization. We further show that antibody binding reduces VEGFR‐2 signaling. Taken together, these results indicate that ligand binding is necessary but not sufficient for VEGFR‐2 activation and define a new potential mechanism for therapeutic intervention.

Supported by NIH Grant GM094155.
In vivo microdialysis sampling of adipokines from rat mammary fat pad. Comparison of dialysates to whole tissue concentrations
Authors: Bajpai Geetika , Stenken Julie A
Published: June 2021

Objective

Microdialysis sampling was used to collect adipokines, CCL2 (MCP‐1) and IL‐6, from mammary fat pads of female rats (Figure 1). A critical issue with microdialysis sampling is the calibration and determining actual tissue concentrations. Tissue concentrations of the adipokines were obtained and compared with dialysates.

Methods

CMA 20 polyethersulfone, 100 kDa MWCO, probes were implanted in the right abdominal mammary fat pad of female Sprague‐Dawley rats (220–250g). Tissue around the probe was harvested for protein extraction. CCL2 and IL‐6 were quantified using Luminex and ELISA kits.

Results and discussion

Concentrations of IL‐6 and CCL2 increased after probe implantation (Table 1). Interestingly, IL‐6 levels were not different, but CCL2 levels were between the implanted vs. control tissue. This may suggest that IL‐6 levels are not influenced by the probe insertion trauma.

Conclusions

Adipokines can be collected from mammary fat pad using microdialysis sampling. Up to ~50 fold differences exist between tissue and dialysate levels.

Funding: COBRE 1P30RR031154‐01 and Arkansas Biosciences Institute.
Diversity of Heterotrimeric G proteins in Plant Innate Immunity – Evidence of a G protein Code
Authors: Lawrence Stacey Alicia , Clay Nicole K.
Published: June 2021

Heterotrimeric G proteins are one of the most conserved eukaryotic signaling components that are responsible for transducing ligand‐recognition signals from transmembrane receptors. Upon ligand recognition, the heterotrimeric G protein complex divides into two functional components: the GTP‐bound Gα‐subunit and the Gβγ dimer. However, it remains unclear how G proteins decode receptor signals and activate appropriate effectors. This is particularly true for innate immunity, where G proteins have long been recognized to play a role. By contrast to the 40 subunits characterized in humans, the model plant Arabidopsis thaliana (A. thaliana) has only one Gα, one Gβ (AGB1), and three Gγ subunits characterized to date. This small repertoire of subunits offers a simplified system to study the role of G proteins in innate immunity. Using sequence homology to guide our functional genomic screens, we identified a number of putative Gβ subunits involved in the activation of different immune responses (e.g. cell wall reinforcement) upon the recognition of specific microbe‐associated ligands, known collectively as MAMPs. We will present a case that one of the candidates, AGB2, is a novel Gβ subunit, and that like AGB1, the known Gβ subunit, is involved in transducing receptor signals for fungal vs. bacterial pathogens. More specifically, both AGB1 and AGB2 are involved in activating the bacterial MAMP‐elicited lignin deposition response as well as activating the fungal MAMP‐elicited callose deposition response. Furthermore, we will describe the mechanism for their antagonistic and additive interactions for each defense response as well as present a model on how the variability of subunits would allow G proteins to be assigned to different signal transduction effector function (defense response) for the same ligand‐receptor pair, and to couple different ligand‐receptor pairs to the same defense response.
The CC‐type chemokine receptor 1 (CCR1) is constitutively associated with βarrestin2: Role of receptor domains
Authors: Gilliland Christian Taylor , Salanga Catherina , Trejo JoAnn , Handel Tracy
Published: June 2021

Careful regulation of the signaling activity of chemokine receptors is crucial to prevent inappropriate inflammatory responses, and arrestins play a key role in this process. The purpose of this study was to determine how arrestins associate with CCR1 and to identify the receptor's structural features that mediate this interaction. Using the bioluminescence resonance energy transfer (BRET) assay, it was observed that CCR1 is constitutively associated with βarrestin2 and that this association is dependent upon a series of Ser/Thr residues in its distal C‐terminal tail. This ligand‐independent interaction was confirmed by co‐immunoprecipitation and fluorescence microscopy assays in endogenous and transient receptor expression systems. This can be explained by the observation that CCR1 is basally phosphorylated. Mutation of the Ser/Thr clusters inhibited receptor desensitization and internalization as well. Agonist binding to CCR1 was shown to primarily induce a conformational change in a pre‐formed CCR1/βarrestin2 complex, as demonstrated in a BRET saturation assay. This represents the first report of ligand‐independent association of βarrestin2 with CCR1 and could play an important role in developing CCR1 ligands that selectively modulate this interaction for therapeutic benefit. This work was supported by the NIH Pharmacological Sciences Training Grant and NIH grant R01AI037113‐14A1.
In Silico Modelling of Novel Analgesic peptides and Expression in Suitable host organisms
Authors: Joginapally Venkateshwara Rao , Pasha Chand , Banothu Venkanna , Shravan P , kiran Jayasree
Published: June 2021

Two hybrid peptide were constructed by using three active peptides reported in the peptide literature linked with polyglycine links. Peptide 1 consists of Met enkephalins, four glycine residues, Endomorphin 1, again four glycine residues and Leu enkephalins. In same way Peptide 2 hybrid was constructed using Met enkephalins, PDB structures for all the sequences were developed by the MODELLOR. For these structures, docking was done for opioid peptides binding with opioid receptors. This docking study was done by using the FlexX Software. Docking studies were carried out using the FlexX program interfaced with SYBYL6.7. All the molecules for docking were sketched in the SYBYL and minimized using Gasteiger‐Huckel charges and all the charges were removed. The 3D coordinates of the active sites were taken from the X‐ray crystal structure. The active site was defined as the area within 6.5 A°. The wild and selected mutant sequences of all the five peptides were codon optimized using DNA 2.0 software for maximum expression of peptides in Saccharomyces cerevisiae and E.coli. Codon optimization was carried out at above 15% threshold and as per the codon usage frequency of standard Saccharomyces and E.coli table. After conforming the mutants cloning strategies were adapted and expressed them in E.coli and Yeast Cells.
Lipid metabolism alterations in U87 glioma cells deficient in very long‐chain acyl‐CoA synthetase 3 are associated with a less malignant phenotype
Authors: Watkins Paul A. , Pei Zhengtong , Kolar Elizabeth A. , Clay Emily M. , Shi Xiaohai , Laterra John
Published: June 2021

Expression of ACSVL3, an acyl‐CoA synthetase that activates long‐ to very long‐chain fatty acids (FA), is elevated in malignant gliomas and in glioma cells. Knocking down ACSVL3 expression in U87 glioma cells by RNA interference decreased their in vitro and in vivo malignant properties. To determine whether the beneficial effects of ACSVL3 depletion were mediated by changes in downstream lipid metabolism, we investigated the metabolic fate of FAs in control and ACSVL3 knockdown (KD) U87 cells. Beta‐oxidation of both C16:0 and C24:0 was decreased in KD cells. There was decreased incorporation of radiolabeled FAs (C16:0, C18:0, C18:1, C18:2n‐6, C20:4n‐6, C22:6n‐3) into phospholipids PI, PS, and PE, but not into PC. ACSVL3 KD had little effect on incorporation of several FAs into most neutral lipids. However, increased incorporation of C20:4n‐6 and C22:6n‐ 3 into triacylglycerol and cholesterol esters was seen in KD cells. ACSVL3 KD altered the total FA profile of U87 cells, and KD cells had decreased rates of de novo FA synthesis from acetate. Thus, depletion of ACSVL3 had multiple effects on U87 glioma cell lipid metabolism. Further study is needed to determine whether any of these changes are critical for switching these cells from a malignant to a more normal phenotype. Supported by NS062043.
Structural properties of functional HDL and variants of apoA‐I
Authors: Petrlova Jitka , Zhu Lin , Mörgelin Matthias , Hebert Hans , Jegerschöld Caroline , Voss John C. , Lagerstedt Jens O.
Published: June 2021

Apolipoprotein A‐I (apoA‐I) is the main protein involved in the formation of high density lipoprotein (HDL). HDL is the principal mediator of the reverse cholesterol transfer (RCT) pathway and provides cardio‐protection. The RCT process involves several sub‐species of HDL where structural plasticity of apoA‐I is critical. This property, and the general high hydrophobicity of apoA‐I, has made structural analyses of lipid‐free and lipid‐bound apoA‐I challenging. We have previously used electron paramagnetic resonance spectroscopy to analyze the structure of apoA‐I [J Biol Chem 282:9143; 286:2966]. We here extend those studies by use of Cryo‐Electron Microscopy (Cryo‐EM). Cryo‐EM is a stain‐free 3D‐structure determination technique and thus highly suitable for structural analysis of the different species of HDL protein‐lipid complexes. In addition to functional apoA‐I, several variants of apoA‐I have been shown to form tissue‐specific amyloid deposits in vivo. We previously characterized G26Rvariant [Biochemistry 46:9693] and L178H variant, which possesses decreased stability. Currently, we are analyzing the structural and functional properties of the C‐terminal variant R173C that is associated with low level of HDL and in contrast, with low prevalence of developing atherosclerosis, and longevity.

This work was funding by grants from the Swedish Research Council (522‐2008‐3724,7480).
Human apoA‐I lysine residues promote association with lipopolysaccharides and phosphatidylglycerol bilayers
Authors: Biglang-awa Ivan , Beck Wendy Hyun Jung , Adams Chris , Vincent Heather , Haas-Stapleton Eric J. , Weers Paul Michiel
Published: June 2021

Human apolipoprotein A‐I (apoA‐I) is a 28 kDa protein and a major component of high‐density lipoproteins, mediating several essential metabolic functions related to heart disease. Recently, a novel protective role against bacterial pathogens has emerged. ApoA‐I suppressed bacterial growth of log phase Escherichia coli and Klebsiella pneumoniae, resulting in a reduced colony count. We suggest that apoA‐I interacts with gram‐negative bacteria in two ways: 1) by associating with lipopolysaccharides (LPS) which are the major constituent of the outer bacterial membrane, thereby reducing endotoxic effects, and 2) destabilizing the negatively charged phospholipid bilayer of the inner bacterial membrane, resulting in cell lysis. To analyze the role of lysine in LPS and phospholipid binding, lysine side chains were acetylated using acetic anhydride in saturated sodium acetate. Electrophoresis analysis and 1‐anilino‐naphthalene‐8‐sulfonate fluorescence of apoA‐I in the absence or presence of LPS indicated decreased binding upon acetylation. ApoA‐I was able to lyse phosphatidylglycerol vesicles; however, acetylated apoA‐I showed a marked decrease in activity. These results indicate the potential for apoA‐I to function as an antimicrobial protein and the importance of lysine residues.
DISSECTION OF THE NECK: A WAX ANATOMICAL MODEL FROM THE SPITZNER COLLECTION IN THE ORFILA MUSEUM OF ANATOMY: AN OBSERVATION AND PHOTOGRAPHIC TRI‐DIMENSIONAL RECONSTRUCTION
Authors: Le Floch-Prigent Patrice Pierre , MARTINELLO Karen
Published: June 2021

An anatomical model of the dissection of the human neck with its supra‐clavicular and sub‐mandibular regions was studied in order to determine its anatomical accuracy. the model was made of brightly colored wax and was of female appearance. Formerly it belonged to the collection of Dr Spitzner (1833–1896), and was labelled as #23. It was 36cm wide and 27cm high. The model's primary views were photographed at 5° increments in alignment with a 270 degree vertical axis, in order to obtain a tri‐dimensional photographic reconstruction. The arterial and venous vessels were enhanced as well as the thyroid and sub‐mandibular glands. The musculo‐cutaneous frames were limited to five main regions of dissection. The nerves and the lymphatics were not represented.
Adding New Properties to Proteins via Reassigning AGA Codon to Encode for Unnatural Amino Acids in E. coli
Authors: Wang Yiyan , Tsao Meng-Lin
Published: June 2021

The ability to introduce unnatural amino acids (uaas) with novel chemical, physical and biological properties at specific sites into proteins adds a new dimension to study protein structure and function. The UAG amber stop codon has been utilized as the top choice to encode for uaas. To provide more powerful codons to further expand the genetic code, here we report our study in reassignment of AGA codon, one of the rare codons, from arginine to uaas in E. coli. This was achieved through a two‐step approach. Firstly, the anticodon recognizing region of Methanococcus Jannaschii tyrosyl‐tRNA synthetase (MjTyrRS) was altered to host tRNAUCU instead of original tRNAGUA by using a mutant GFPuv as the reporter. Secondly, subsequently modifications on amino acid binding pocket of the mutated MjTyrRSuaa allowed the incorporation of target unnatural amino acids into proteins in response to AGA codon. Our results showed that in E.coli the suppression rate of AGA codon by the evolved MjTyrRSuaa‐MjtRNAUCU pair was generally above 90% and the uaa‐decorated protein yield reached about 100 mg/L. This research provided a powerful new approach to add new building blocks to the genetic code and to the structural/functional study of protein with novel properties. The project was sponsored by UC Merced start‐up fund.
Chaperoning activity of peroxiredoxins protects brain‐type creatine kinase under various stresses through specific interaction
Authors: Rakhmetov Anar , Lee Sang Pil , Kim Kanghwa , Chae Ho Zoon
Published: June 2021

Peroxiredoxins (Prxs) are a family of peroxidase proteins which, although, exhibit chaperone activity due to the structural transition from dimer to decamer or high‐molecular weight complex. We have identified brain‐type creatine kinase (CKBB) as a Prx I binding protein from the heat‐stressed rat brain extracts. Co‐IP results from the transfected cells demonstrated that the interaction of CKBB with PrxII in addition to PrxI. PrxII showed much higher binding affinity to CKBB than PrxI under normal physiological condition as well as heat‐stressed condition. Prx II effectively protected the kinase activity of CKBB from oxidative inactivation and heat inactivation. This protection activity is not depends on the structural transition but depends on the site‐specific interaction. Thus, we suggest this chaperoning activity of Prx through the sitespecific interaction as “client specific chaperoning”, and that through structural transition as “general chaperoning”. For specific clients, Prx binds to their native state and provides the protection for maintain their native state. The beneficiary of chaperoning through structural transition (general chaperoning) opens to wide variety of clients, however, that through site‐specific interaction (client specific chaperoning) opens to very limited number of clients.
Chronic treatment with Azidothymidine (AZT) alters cytoskeletal proteins responsible for cardiac function
Authors: McKee Edward E. , Kamath Vasudeva , Donahue Deborah L , Tan John , Zeng Erliang
Published: June 2021

Metabolic complications resulting in cardiomyopathy are associated with long‐term treatment with the antiviral thymidine analog, AZT (3′‐azido‐3′ deoxythymidine; zidovudine). While mitochondrial toxicity is thought to be a major underlying cause, the molecular mechanism(s) are still ambiguous. We identified molecular and cellular changes associated with cardiac toxicity that occur after long‐term treatment. Adult male rats were chronically exposed to AZT in the drinking water for a period of six months. Mild changes in echocardiogram data suggested impaired cardiac function in AZT treated rats. Respiratory control rates and electron transport chain capacity as measured in isolated heart mitochondria by a high resolution respirometer were unchanged in the treated rats. There was also no change in the mitochondrial DNA copy numbers in treated animals. Interestingly, microarray analysis of total mRNA from heart samples presented with dramatic alterations in expression of mRNA in rats treated with AZT. Among these mRNAs, cytoskeletal protein mRNA alterations were evident in a number of pathways. Cytoskeletal/ contractile proteins such as actin, cofilin, profilin, myosin II, myosin light chain and myosin heavy chain were significantly altered contributing to contractile dysfunction. The role of mitochondrial toxicity in this model is unclear.
EXPRESSION OF BIOMARKERS OF OSTEOARTHRITIS IN MICE WITH AND WITHOUT RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE)
Authors: Kartchner Jeffrey Zane , Peterson D. M. , Larkin D. J. , Doxey A. S. , Hollis W. R. , Rees J. L. , Ingersoll C. , Jackson G. G. , Wilhelm S. K. , Haynie S. S. , Seegmiller R. E. , Stogdill J. A. , Reynolds P. R.
Published: June 2021

HtrA1 and Mmp‐13 are reliable biomarkers of osteoarthritis (OA) and are upregulated at days post knee destabilization surgery (Polur et al., 2010). While expression of RAGE and its ligands are suggested, the exact mechanism for HtrA1 upregulation is not known (Cecil et al., 2005; Loeser et al., 2005). Our objective was to examine biomarkers of OA in the presence (control mice) and absence (RAGE knock‐out) of RAGE. We performed right knee destabilization and sham surgery on 8 week old control and RAGE knock‐out (KO) mice. We assayed for HtrA1, Mmp‐13, RAGE and S‐100 at 3, 7, 14 and 28 days post‐surgery by immunohistochemistry on both knee joints. We did not detect biomarkers of OA including Mmp‐13 in any sham surgery mice. In RAGE KO mice, we observed negligible staining of HtrA1 and faint staining of S‐100 at 28 days post‐surgery but not Mmp‐13. In contrast, we detected the presence of S‐100, HtrA1 and Mmp‐13 at focal points 3 days post‐surgery in control mice. By 28 days post‐surgery HtrA1 and Mmp‐13 expression is uniformly expressed in articular cartilage in control mice. RAGE KO does not entirely prevent HtrA1 upregulation but does protect against Mmp‐13 activation. Since the expression of Mmp‐13 is a terminal event resulting in OA, we conclude that RAGE KO protects against the most vital component of articular cartilage degradation and subsequently, OA.
Can fluorescently labeled cytokinins become a new approach for receptor domain mapping?
Authors: Szucova Lucie , Plihal Ondrej , Zahajska Lenka , Mik Vaclav , Dolezal Karel , Spichal Lukas , Strnad Miroslav
Published: June 2021

Cytokinins belong to a class of plant hormones structurally based on adenine. They play important roles in many aspects of plant growth and development. Isoprenoid cytokinin N6‐ isopentenyladenine was accompanied with various spacers in C2, C8 and N9 position of adenine and fluorescently labeled with NBD, FITC, rhodamine B and many other common fluorescent labels. The activation of signaling pathway was assessed with Arabidopsis cytokinin receptors AHK3 and AHK4 and maize cytokinin receptors ZmHK1 and ZmHK3a in a bacterial receptor test where specific activation of the cytokinin signaling pathway (YojN> RcsB > cps::lacZ) was quantified. Although the structural changes led to the loss of the ability to activate signaling pathway through the receptors, some derivatives were still able to interact with the receptor binding site as was evident from competitive ligand receptor testing. One of the N9 substituted iP derivatives was promising in staining procedures: it was shown that the derivative was transported to the cell cytosol and the signal was associated with subcellular structures, especially with the endoplasmic reticulum, where these receptors are probably localized.

The work was supported by GACR P501/10/1450.
PHOSPHORYLATION OF THE C‐TERMINAL DOMAIN OF MUC1 IN INFLAMMATION
Authors: Nguyen Lynne , Van Eugene , Tsai Jeffrey
Published: June 2021

Mucins are protective glycoproteins in the respiratory, digestive, and urogenital tracts. MUC1 is a large O‐glycosylated transmembrane protein expressed on the apical surface of epithelial tissues. It exhibits structural features of receptors for cytokines and growth factors. Its intracellular cytoplasmic tail contains multiple amino acid sequence motifs that, once phosphorylated, serve as docking sites for protein kinases for signal transduction. Most studies have focused on breast and pancreatic cancer cells in related to overexpression of MUC1. Few have examined phosphorylation in normal epithelial cells in the oral cavity. MUC1 plays a role in the pathogenesis of cancer and innate immunity. The present study was to determine whether MUC1 expressed in oral epithelial cells is phosphorylated in response to pro‐inflammatory mediators of inflammation. EGF, IL‐1‐beta, IFN‐gamma, and LPS was treated in KB and TR146 cells and examined the pro‐inflammatory mediators on the C‐terminal domain of MUC1. The results showed that the C‐terminal domain of MUC1 is phosphorylated in response to treatments of pro‐inflammatory cytokines. It suggests that phosphorylation of MUC1 may function to initiate inflammatory signaling cascades during the early stages of inflammation in response to bacterial infection.
Inducible nitric acid synthase/Src axis mediates TLR3 Tyr759 phosphorylation, nuclear translocation of IRF3 and IRF7 that leads to interferon‐beta synthesis in double‐stranded RNA‐stimulated macrophage
Authors: Maa Ming-Chei , Chang Miao Ying , Leu Tzeng-Horng
Published: June 2021

As a frequent byproduct of viral infection, double‐stranded RNA is recognized by Toll‐like receptor 3 (TLR3) and triggers signaling pathways that result in production of interferon‐β (IFN‐β). Despite mounting evidence reveals the involvement of protein tyrosine kinases (PTKs) in this process, the identity of the responsive PTKs is still elusive. In this study, we demonstrated Src attenuation impaired TLR3 Pi‐Tyr759, nuclear accumulation of transcription factors IRF3 and IRF7, and the increment of ifn‐β transcript and IFN‐β production in polyinosinic‐polycytidylic acid (polyI:C)‐stimulated macrophages. Remarkably, re‐introduction of siRNA‐resistant avian Src reversed all these defects. Consistent with the expression of Src was inducible‐nitric oxide synthase (iNOS)‐dependent, polyI:C‐elicited TLR3 Pi‐Tyr759, nuclear translocation of IRF3 and IRF7, as well as IFN‐β generation were inhibited in iNOS‐null macrophages, which could be rescued by SNAP (a NO donor). Notably, Lyn and FAK did not contribute to polyI:C‐evoked IFN‐β secretion. This was the first time an essential role of iNOS/Src axis in IFN‐β production in TLR3‐engaged macrophages was corroborated. This work was supported by the National Science Council (NSC98‐2311‐B‐039‐002‐MY3).
Identification of cAMP‐responsive substrates of SIK2 in adipocytes
Authors: Henriksson Emma , Säll Johanna , Göransson Olga
Published: June 2021

Salt‐inducible kinase (SIK) 2 is an AMP‐activated protein kinase (AMPK)‐related kinase and is highly expressed in adipose tissue. SIK2 activity is regulated by LKB1‐phosphorylation of a conserved residue in the activation (T)‐loop. In adipocytes, SIK2 is phosphorylated at Ser358 upon cAMP‐induction, leading to its binding to 14‐3‐3 proteins and re‐localisation into the cytosol. This regulation may restrict its ability to interact with potential substrates and the aim of our study was to investigate if histone deacetylase ‐4 (HDAC4), CREB regulated transcription co‐activator 2 (CRTC2) and ‐3, all regulated by cAMP, are substrates of SIK2 in adipocytes. Adenovirus‐mediated over‐expression of wild type SIK2 in adipocytes increased the phosphorylation of all proposed substrates, compared to kinase inactive SIK2 and GFP‐transduced cells. In addition, HDAC4, CRTC2 and ‐3 were found to co‐immunoprecipitate with HA‐SIK2, with a reduction in binding upon cAMP‐elevation for all but HDAC4. After siRNA‐mediated silencing of SIK2, the phosphorylation of CRTCs was reduced. Ongoing experiments address the role of Ser358 phosphorylation of SIK2 in the regulation of HDAC4, CRTC2 and ‐3 in adipocytes. These proteins, which we suggest are substrates of SIK2 in adipocytes, are known to be involved in the control of metabolic gene expression, supporting a role for SIK2 in adipocyte metabolism. This work was mainly supported by the Swedish Research Council, The Craaford Foundation and the Novo Nordisk Foundation.
N‐acetylcysteine potentiates doxorubicin‐induced ATM and p53 activation in ovarian cancer cells
Authors: Brum Gabriella Maria , Carbone Thomas , Still Eric , Correia Vendita , Ji Fang , Di Wen , Wan Yinsheng
Published: June 2021

Doxorubicin has been used clinically to treat various types of cancer, and yet the mode of actions of doxorubicin remains to be fully unraveled and the cell signaling pathways to be uncovered. In this study, we investigated the effect of doxorubicin on cultured ovarian cancer cells (CaOV3). MTT assay data showed that doxorubicin inhibits cell proliferation in a time and dose dependent manner. Phagokinetic cell motility assay data indicated that doxorubicin inhibits both basal level and EGF‐induced cell migration in CaOV3 cells. Confocal microscopic data revealed that doxorubicin induces reorganization of cytoskeletal proteins including actin, tubulin and vimentin. Western blot analysis showed that doxorubicin induces phosphorylation of p53 at ser 15 and 20, acetylation of p53, and ATM activation. Doxorubicin also induces phosphorylation of histone H2AX at ser 139. Interestingly, Doxorubicin inhibits mTOR activity, measured by phosphorylation of S6 ribosomal protein. Contrary to the previous report on other cells, pretreatment of CaOV3 cells with antioxidant N‐acetylcysteine or NAC but not PDTC potentiates doxorubicin‐induced phosphorylation of p53. Collectively, we conclude that doxorubicin induces ATM/p53 activation leading to reorganization of cytoskeletal networks, inhibition of mTOR activity, and inhibition of cell proliferation and migration. Our data also suggest that removal of oxidants may enhance the efficacy of doxorubicin in vivo.
Natural ethanol availability directed the evolution of short‐chain alcohol dehydrogenase in animals
Authors: Julián-Sánchez Adriana , Hernández-Tobías Aída , Piña Enrique , Riveros-Rosas Héctor
Published: June 2021

The presence of alcohol dehydrogenase (ADH) in animals has been assumed to be a consequence of chronic exposure to ethanol. Therefore, in this work we investigate if natural ethanol availability directed the evolution of type II or short‐chain ADHs evolution. In animals, 15‐hydroxyProstaglandin dehydrogenase (PGDH) is the closest homologous to type II ADHs. In contrast, bona fide type II ADHs and ADH‐like proteins appear to be restricted to Diptera (flies). Thus, it can be assumed that the origin of PGDH predates the origin of type II ADHs in Diptera. Adh locus in Drosophila has undergone at least five duplication events, and all them occurred within the Diptera. Because point mutations in Adh can abolish ADH activity in this fly, it can be considered the unique fly enzyme involved in ethanol metabolism.

The natural breeding substrates of Drosophila are fermenting fruits. The Drosophila genus is among the most diverse of the Diptera, encompassing >2,000 species. The efficient exploitation of such niches is the reasons for their successful worldwide spreading. Thus, induction of type II Adh by ethanol exposure, a positive correlation between ADH activity and ethanol resistance, ethanol utilization as a nutrient, and the fact that flies and type II Adh diversification occurred in concert with angiosperm diversification, strongly suggest that type II ADHs were recruited to allow larval flies to exploit new restricted niches with high ethanol content. Supported by DGAPA‐UNAM grant IN208510.
Innervation of the Extensor Carpi Radialis Brevis muscle by the Median nerve is extremely rare
Authors: Farkas Gabrielle A , Anyah Anwuli , Assefa Mahaletwork , Borman William , Fisher Cara , Olinger Anthony
Published: June 2021

Compartmentalization of skeletal musculature into distinct neuromuscular components is an important clinical consideration due to the regular use of tendon transfer surgeries. Traumatic injury can result in loss of function of the forearm musculature. The extensor carpi radialis muscles are often used in tendon transfer procedures to restore function after a traumatic injury. A recent study described a previously unreported variation whereby a portion of the extensor carpi radialis brevis (ECRB) muscle was innervated by a branch of the median nerve. This finding is of particular interest given the potential to then use that portion of the muscle to restore function via tendon transfer using a portion of an extensor muscle not innervated by the radial nerve. The purpose of the current study was to determine the frequency by which the ECRB was innervated by a branch of the median nerve. Upper limbs from 208 cadavers were dissected and examined to determine the innervation distribution of the ECRB. In all cases the ECRB was innervated entirely by branches of the radial nerve. The median nerve was never seen sending any branches to the ECRB. These findings suggest that the observation made in that study, whereby the median nerve innervated a part of the ECRB, is extremely rare and that this is not a frequent enough variation to rely on using a portion of the ECRB to restore function when the radial nerve has been compromised.
Construction of synthetic protein‐based library by combinatorial approach
Authors: Su Yue , Parameswaran Hariharan , Guan Lan
Published: June 2021

Protein‐capture reagents have been shown to be a useful tool for scientific studies and clinical applications. We have constructed a repeat protein‐based combinatorial library for further structural and functional study of membrane proteins. This library was constructed by an ordered assembly of repeat DNA fragments containing random amino acid codons at certain positions. Theoretically, it should contain a diversity of ~2 × 1022. We obtained 45 % error‐free full‐length proteins, reducing the theoretic diversity to ~1 × 1022. Although feasible diversity size is limited by a screening method, a fraction of such library is still sufficient for many applications. By DNA sequencing analysis, 48 out of 49 correct clones are unique in protein sequence; among them, 90 % individuals have >10 positions that are different with one and another. Biophysical analyses of purified proteins from four randomly selected clones suggested correct Mr values and protein folding. Thermostability study using circular dichroism spectroscopy revealed that the melting temperatures for all four proteins are > 95 °C. Taken together, the diversity and stability of this library provide a pool of candidates for further screening of capture reagents that specifically bind to a protein of interest with high affinity.

This work was supported by National Institutes of Health Grants R01 GM095538 to LG.
Molecular Design and functional analysis of Starch Binding Domain of Rizopus oryzae Glucoamylase
Authors: Ng Sim-Kun , Fang Tsai-Wei , Lee Yuan-Chuan , Huang Han-Tsz , Jiang Ting-Ying , Ci Yuan-Pei , Chou Wei-I , Chang Dah-Tsyr Margaret
Published: June 2021

Starch‐binding domain (SBD) is a functional domain which effectively adsorbs onto granular starch and other soluble oligosaccharides. The 11.7 kDa N‐terminal SBD of Rhizopus oryzae glucoamylase (RoSBD) belongs to carbohydrate binding module (CBM) family 21. RoSBD has been developed as a purification tag that successfully overexpress and purify recombinant proteins in bacteria and yeast. Here RoSBD‐eGFP expression in mammalian cell line HEK293FT is also successfully expressed, and recovered with high purity (95%) and fair yield (30 mg/L). Since tandem repeat of CBMs is known to promote ligand binding affinity, multivalent protein binders including mouse IgG1 Fc and collagen‐like peptide (CLP) are engineered to generate respectively dimeric and trimeric RoSBD. The polymeric RoSBDs are expressed stably with high quantity in E. coli and form desired dimer and trimer. Improvement in binding activity of polymeric RoSBDs to granular starch and soluble oligosaccharides is observed employing depletion isotherm and isothermal titration calorimetry (ITC). Although natural CBM‐glycan interaction is usually too weak to be precisely detected, the improvement of binding affinity between RoSBD and several glycans may enable us to investigate novel binding partners of RoSBD. These results may also facilitate development of novel protein purification systems vitally important for biotechnological applications.
Increasing telomerase in mouse brain by novel compound protects neurons from various insults
Authors: Eitan Erez , Tichon Ailon , Gitler Daniel , Priel Esther
Published: June 2021

Telomerase was shown to protect developing neurons from apoptosis induced by DNA damage or oxidative stress. Hence, we and other hypothesized that increasing telomerase expression by pharmaceutical compounds may protect brain cells. This study demonstrates the ability of a novel compound, AGS, to significantly increase telomerase activity and expression (3 fold) in the mouse brain and to exert neuroprotective effects. Treatment with AGS, prior to NMDA systemic injection, increased the survival and decreased the seizures of NMDA injected mice. AGS treatment also enhanced DNA repair in purkinje neurons following brain irradiation. AGS protective effect is mediated by telomerase expression, since it did not protect motor neuron‐ like cell line (NSC‐34) from oxidative stress when TERT expression was diminished by shRNA or overexpressed. AGS treatment significantly delayed the onset of Amyotrophic lateral sclerosis in SOD1 Tg mouse model and prolongs their life span (18 day) in a dose dependent manner. Moreover, TERT expression is reduced with the disease progression in the brainstem and spinal cord. We suggest that TERT serves as a surviving enzyme in the CNS both by its canonical telomere stabilization activity and by its non‐canonical anti‐apoptotic function. Therefore transient increase of telomerase expression and activity by AGS may have a therapeutic effect in neurodegenerative diseases
Di‐n‐Butyl 2‐Chlorophenyl Phosphate: A Potential Therapeutic for Alzheimer's Disease
Authors: Hartman Lauren K , Del Cid Joselyn S , Acey Roger A
Published: June 2021

Alzheimer's Disease (AD) is characterized by decreased levels of acetylcholine and formation of neurotoxic β‐amyloid plaque. The result is a loss of cognitive function. Both effects correlate with elevated levels of butyrylcholinesterase (BuChE) activity. Reversible cholinesterase inhibitors have been shown to decrease β‐amyloid peptide formation in animal models. We have shown that aryl dialkyl phosphates are potent and highly selective irreversible inhibitors of BuChE. In this study, the toxicity of di‐n‐butyl 2‐chlorophenyl phosphate (DB2ClPP) was determined in cultured human SK‐N‐SH neuroblastoma cells. Cells were exposed to increasing concentrations of DB2ClPP for 24 hrs and then assayed for changes in cell proliferation, membrane integrity, and induction of apoptosis. Concentrations of DB2ClPP as high as 10 μM had no effect on any of these parameters. Incubation of whole blood with DB2ClPP revealed that the compound is present at detectable levels in plasma after 1 hr. These studies suggest that there is a significant potential for this class of compounds as a therapeutic to treat the symptoms of AD.
Is a calorie a calorie? Predictions of a validated computational model of human macronutrient metabolism
Authors: Hall Kevin D
Published: June 2021

A common assumption regarding weight loss is that “a calorie is a calorie” and therefore all equivalently energy‐reduced diets should lead to identical body fat loss regardless of their macronutrient composition. However, this assumption is not a necessary consequence of the first law of thermodynamics since the macronutrient balance concept allows for an effect of diet composition while also obeying the law of energy conservation. Here, I used a validated computational model of human macronutrient metabolism to predict the metabolic consequences and body composition changes resulting from a selective isocaloric reduction of dietary carbohydrate versus fat in a variety of obese ‘virtual study subjects’. The model predicts that, while keeping dietary fat and protein at baseline, selective reduction of carbohydrate by ~800 kcal/d results in augmented rates of lipolysis and fat oxidation with concomitant reductions of body fat and 24 hr RQ. In contrast, selective reduction of ~800 kcal/d of dietary fat results in no change in the 24 hr RQ or fat oxidation rate (Figure). While enhanced fat oxidation with the reduced carbohydrate diet might be expected to lead to greater body fat loss, the net cumulative fat imbalance was predicted to be more negative with the reduced fat diet. These model predictions are now being tested in a clinical research study at the NIH (NCT00846040).
Use of the Medifast meal replacement program for weight loss in overweight and obese clients: A retrospective chart review of three Medifast Weight Control Centers (MWCC)
Authors: Coleman Christopher David , Kiel Jessica R. , Davis Lisa M. , Mitola Andrea , Sonzone Carmine M.
Published: June 2021

Objective

A chart review was performed to evaluate the effectiveness of the Medifast meal replacement plan (MD) on body weight and body composition at 4, 12, 24 weeks (wk), and final weight loss visit (19.6 ± 13.5 wk) (FV) at 3 MWCCs for the years 2007–10.

Methods

Charts included adults aged 18–70 following MD with a BMI ≥ 25 that signed a patient health information consent form. 453 charts (males n=60 (13.5%); females n= 386 (86.5%)) were included. Data were collected electronically and included weight (Wt), systolic and diastolic blood pressure (SBP, DBP), lean muscle mass (LMM), body fat mass (BFM), % body fat (%BF), and abdominal circumference (AC). Additional data collected included: demographics, side effects, and compliance.

Results

Significant improvements (p<0.0001) were seen at all time‐points: wk 4, 12, 24, and FV. Wt loss: −12.7, −26.3, −38.2, −25.7 lbs; % Wt loss: −6.0, −12.1, −17.0, −12.3%; %BF: −2.2, −5.2, −8.5, −6.6; BFM: −10.1, −20.1, −31.6, −23.5 lbs; AC: −2.2, −4.0, −6.0, −4.7 in; SBP: −8.0, −8.6, −15.6, −8.8 mmHG; DBP: −5.3, −6.4, −9.2, −6.0 mmHG. Percent total body weight that was LMM improved by 3.5, 9.8, 16.0, and 13.9% (P<0.0001) respectively.

Conclusion

Medifast's meal replacement plan, along with the support available from MWCC, is an efficacious model that promotes significant weight loss and improvements in body composition. This research was funded by Medifast, Inc., Owings Mills, MD.