ResearchPad - molecular-biology-assays-and-analysis-techniques https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Crystal structure of <i>Thermus thermophilus</i> methylenetetrahydrofolate dehydrogenase and determinants of thermostability]]> https://www.researchpad.co/article/elastic_article_13865 The elucidation of mechanisms behind the thermostability of proteins is extremely important both from the theoretical and applied perspective. Here we report the crystal structure of methylenetetrahydrofolate dehydrogenase (MTHFD) from Thermus thermophilus HB8, a thermophilic model organism. Molecular dynamics trajectory analysis of this protein at different temperatures (303 K, 333 K and 363 K) was compared with homologous proteins from the less temperature resistant organism Thermoplasma acidophilum and the mesophilic organism Acinetobacter baumannii using several data reduction techniques like principal component analysis (PCA), residue interaction network (RIN) analysis and rotamer analysis. These methods enabled the determination of important residues for the thermostability of this enzyme. The description of rotamer distributions by Gini coefficients and Kullback–Leibler (KL) divergence both revealed significant correlations with temperature. The emerging view seems to indicate that a static salt bridge/charged residue network plays a fundamental role in the temperature resistance of Thermus thermophilus MTHFD by enhancing both electrostatic interactions and entropic energy dispersion. Furthermore, this analysis uncovered a relationship between residue mutations and evolutionary pressure acting on thermophilic organisms and thus could be of use for the design of future thermostable enzymes.

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<![CDATA[Host interactors of effector proteins of the lettuce downy mildew <i>Bremia lactucae</i> obtained by yeast two-hybrid screening]]> https://www.researchpad.co/article/elastic_article_13834 Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four protein-pairs upon their co-expression, supporting their interaction in planta.

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<![CDATA[Amino acids serve as an important energy source for adult flukes of <i>Clonorchis sinensis</i>]]> https://www.researchpad.co/article/elastic_article_13829 Clonorchiasis, closely related to cholangiocarcinoma and hepatocellular carcinoma, has led to a negative socioeconomic impact in global areas especially some Asian endemic regions. Owing to the emergence of drug resistance and hypersensitivity reactions after the massive and repeated use of praziquantel as well as the lack of effective vaccines, searching for new strategies that prevent and treat clonorchiasis has become an urgent matter. Clonorchis sinensis, the causative agent of clonorchiasis, long-term inhabits the microaerobic and limited-glucose environment of the bile ducts. Adequate nutrients are essential for adult flukes to resist the adverse condition and survive in the crowed habitat. Studies on energy metabolism of adult flukes are beneficial for further exploring host-parasite interactions and developing novel anti-parasitic drugs. Our results suggest that gluconeogenesis probably plays a vital role in energy metabolism of Clonorchis sinensis and exogenous amino acids might be an essential energy source for adult flukes to successfully survive in the host. Our foundational study opens a new avenue for explaining energy metabolism of Clonorchis sinensis and provides a valuable strategy that the gluconeogenesis pathway will be a potential and novel target for the prevention and treatment of clonorchiasis.

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<![CDATA[Medusa: Software to build and analyze ensembles of genome-scale metabolic network reconstructions]]> https://www.researchpad.co/article/elastic_article_7734 Uncertainty in the structure and parameters of networks is ubiquitous across computational biology. In constraint-based reconstruction and analysis of metabolic networks, this uncertainty is present both during the reconstruction of networks and in simulations performed with them. Here, we present Medusa, a Python package for the generation and analysis of ensembles of genome-scale metabolic network reconstructions. Medusa builds on the COBRApy package for constraint-based reconstruction and analysis by compressing a set of models into a compact ensemble object, providing functions for the generation of ensembles using experimental data, and extending constraint-based analyses to ensemble scale. We demonstrate how Medusa can be used to generate ensembles and perform ensemble simulations, and how machine learning can be used in conjunction with Medusa to guide the curation of genome-scale metabolic network reconstructions. Medusa is available under the permissive MIT license from the Python Packaging Index (https://pypi.org) and from github (https://github.com/opencobra/Medusa), and comprehensive documentation is available at https://medusa.readthedocs.io/en/latest.

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<![CDATA[Host factors that promote retrotransposon integration are similar in distantly related eukaryotes]]> https://www.researchpad.co/article/5ab4e87b463d7e0cbd0422e6

Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.

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<![CDATA[Physicochemical and biological evaluation of JR-131 as a biosimilar to a long-acting erythropoiesis-stimulating agent darbepoetin alfa]]> https://www.researchpad.co/article/Na789b0ff-1b14-409c-afa6-0c7a70fc7c42

Renal anemia is predominantly caused by a relative deficiency in erythropoietin (EPO). Conventional treatment for renal anemia includes the use of recombinant human EPO (rhEPO) or a long-acting erythropoiesis-activating agent named darbepoetin alfa, which is a modified rhEPO with a carbohydrate chain structure that differs from native hEPO. We have developed a biosimilar to darbepoetin alfa designated JR-131. Here, we comprehensively compare the physicochemical and biological characteristics of JR-131 to darbepoetin alfa. JR-131 demonstrated similar protein structure to the originator, darbepoetin alfa, by peptide mapping and circular dichroism spectroscopy. Additionally, mass spectroscopic analyses and capillary zone electrophoresis revealed similar glycosylation patterns between the two products. Human bone marrow-derived erythroblasts differentiated and proliferated to form colonies with JR-131 to a similar degree as darbepoetin alfa. Finally, JR-131 stimulated erythropoiesis and improved anemia in rats similarly to darbepoetin alfa. Our data show the similarity in physicochemical and biological properties of JR-131 to those of darbepoetin alfa, and JR-131 therefore represents a biosimilar for use in the treatment of renal anemia.

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<![CDATA[Sugar, amino acid and inorganic ion profiling of the honeydew from different hemipteran species feeding on Abies alba and Picea abies]]> https://www.researchpad.co/article/Neb889307-c28f-4dcd-8ba6-6ff5a5f28e34

Several hemipteran species feed on the phloem sap of plants and produce large amounts of honeydew that is collected by bees to produce honeydew honey. Therefore, it is important to know whether it is predominantly the hemipteran species or the host plant to influence the honeydew composition. This is particularly relevant for those botanical and zoological species from which the majority of honeydew honey originates. To investigate this issue, honeydew from two Cinara species located on Abies alba as well as from two Cinara and two Physokermes species located on Picea abies were collected. Phloem exudates of the host plants were also analyzed. Honeydew of all species contained different proportions of hexoses, sucrose, melezitose, erlose, and further di- and trisaccharides, whereas the phloem exudates of the host trees contained no trisaccharides. Moreover, the proportions of sugars differed significantly between hemipteran species feeding on the same tree species. Sucrose hydrolysis and oligosaccharide formation was shown in whole-body homogenates of aphids. The type of the produced oligosaccharides in the aphid-extracts correlated with the oligosaccharide composition in the honeydew of the different aphid species. The total contents of amino acids and inorganic ions in the honeydew were much lower than the sugar content. Glutamine and glutamate were predominant amino acids in the honeydew of all six hemipteran species and also in the phloem exudates of both tree species. Potassium was the dominant inorganic ion in all honeydew samples and also in the phloem exudate. Statistical analyses reveal that the sugar composition of honeydew is determined more by the hemipteran species than by the host plant. Consequently, it can be assumed that the sugar composition of honeydew honey is also more influenced by the hemipteran species than by the host tree.

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<![CDATA[Increased 14-3-3β and γ protein isoform expressions in parasitic eosinophilic meningitis caused by Angiostrongylus cantonensis infection in mice]]> https://www.researchpad.co/article/5c8acccdd5eed0c484990014

The 14-3-3 proteins are cerebrospinal fluid (CSF) markers of neuronal damage during infectious meningitis and Creutzfeldt-Jakob disease. Little is known about dynamic changes in the individual isoforms in response to parasitic eosinophilic meningitis. The purposes of this study were to determine the 14-3-3 protein isoform patterns, examine the kinetics and correlate the severity of blood brain barrier (BBB) damage with the expressions of these markers in mice with eosinophilic meningitis.

Mice were orally infected with 50 A. cantonensis L3 via an oro-gastric tube and sacrificed every week for 3 consecutive weeks after infection. The Evans blue method and BBB junctional protein expressions were used to measure changes in the BBB. Hematoxylin and eosin staining was used to analyze pathological changes in the mice brains following 1–3 weeks of infection with A. cantonensis. The levels of 14-3-3 protein isoforms in serum/CSF and brain homogenates were analyzed by Western blot, and immunohistochemistry (IHC) was used to explore the different isoform distributions of 14-3-3 proteins and changes in BBB junctional proteins in the mice brain meninges. Dexamethasone was injected intraperitoneally from the seventh day post infection (dpi) until the end of the study (21 dpi) to study the changes in BBB junctional proteins. The amounts of Evans blue, tight junction and 14-3-3 protein isoforms in the different groups of mice were compared using the nonparametric Kruskal-Wallis test.

There were significant increases in 14-3-3 protein isoforms β and γ in the CSF in the second and third weeks after infection compared to the controls and first week of infection, which were correlated with the severity of BBB damage in brain histology, and Evans blue extravasation. Using IHC to assess the distribution of 14-3-3 protein isoforms and changes in BBB junctional proteins in the mice brain meninges, the expressions of isoforms β, γ, ε, and θ and junctional proteins occludin and claudin-5 in the brain meninges increased over a 3-week period after infection compared to the controls and 1 week after infection. The administration of dexamethasone decreased the expressions of BBB junctional proteins occludin and claudin-5 in the mice brain meninges.

Our findings support that 14-3-3 proteins β and γ can potentially be used as a CSF marker of neuronal damage in parasitic eosinophilic meningitis caused by A. cantonensis.

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<![CDATA[Swift Large-scale Examination of Directed Genome Editing]]> https://www.researchpad.co/article/5c8823fad5eed0c484639487

In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, the handling-time and material costs per sample remain major limiting factors. Here we present a robust method for low-cost DIY-pipet tips addressing these needs; i.e. using a cellulose filter disc inserted into a regular pipet tip. These filter-in-tips allow for a rapid, stand-alone four-step genotyping workflow by simply binding the DNA contained in the primary lysate to the cellulose filter, washing it in water and eluting it directly into the buffer for the downstream application (e.g. PCR). This drastically cuts down processing time to maximum 30 seconds per sample, with the potential for parallelizing and automation. We show the ease and sensitivity of our procedure by genotyping genetically modified medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos (targeted by CRISPR/Cas9 knock-out and knock-in) in a 96-well plate format. The robust isolation and detection of multiple alleles of various abundancies in a mosaic genetic background allows phenotype-genotype correlation already in the injected generation, demonstrating the reliability and sensitivity of the protocol. Our method is applicable across kingdoms to samples ranging from cells to tissues i. e. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips.

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<![CDATA[Molecular features of steroid-binding antidins and their use for assaying serum progesterone]]> https://www.researchpad.co/article/5c76fe5fd5eed0c484e5b998

Chicken avidin (Avd) and streptavidin from Streptomyces avidinii are extensively used in bionanotechnology due to their extremely tight binding to biotin (Kd ~ 10−15 M for chicken Avd). We previously reported engineered Avds known as antidins, which have micro- to nanomolar affinities for steroids, non-natural ligands of Avd. Here, we report the 2.8 Å X-ray structure of the sbAvd-2 (I117Y) antidin co-crystallized with progesterone. We describe the creation of new synthetic phage display libraries and report the experimental as well as computational binding analysis of progesterone-binding antidins. We introduce a next-generation antidin with 5 nM binding affinity for progesterone, and demonstrate the use of antidins for measuring progesterone in serum samples. Our data give insights on how to engineer and alter the binding preferences of Avds and to develop better molecular tools for modern bionanotechnological applications.

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<![CDATA[Subunits of the mechano-electrical transduction channel, Tmc1/2b, require Tmie to localize in zebrafish sensory hair cells]]> https://www.researchpad.co/article/5c648d15d5eed0c484c81f40

Mutations in transmembrane inner ear (TMIE) cause deafness in humans; previous studies suggest involvement in the mechano-electrical transduction (MET) complex in sensory hair cells, but TMIE’s precise role is unclear. In tmie zebrafish mutants, we observed that GFP-tagged Tmc1 and Tmc2b, which are subunits of the MET channel, fail to target to the hair bundle. In contrast, overexpression of Tmie strongly enhances the targeting of Tmc1-GFP and Tmc2b-GFP to stereocilia. To identify the motifs of Tmie underlying the regulation of the Tmcs, we systematically deleted or replaced peptide segments. We then assessed localization and functional rescue of each mutated/chimeric form of Tmie in tmie mutants. We determined that the first putative helix was dispensable and identified a novel critical region of Tmie, the extracellular region and transmembrane domain, which is required for both mechanosensitivity and Tmc2b-GFP expression in bundles. Collectively, our results suggest that Tmie’s role in sensory hair cells is to target and stabilize Tmc channel subunits to the site of MET.

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<![CDATA[Sexual dimorphism of acute doxorubicin-induced nephrotoxicity in C57Bl/6 mice]]> https://www.researchpad.co/article/5c76fe23d5eed0c484e5b5ad

Doxorubicin (DOX) is a chemotherapeutic agent that has been reported to cause nephrotoxicity in rodent models and to a lesser degree in cancer patients. Female rodents have been shown to be protected against several features of DOX-induced nephrotoxicity. Nevertheless, the underlying mechanisms of this sexual dimorphism are not fully elucidated. Therefore, in the current study, we investigated the sex and time-dependent changes in pathological lesions as well as apoptotic and fibrotic markers in response to acute DOX-induced nephrotoxicity. We also determined the effect of acute DOX treatment on the renal expression of the sexually dimorphic enzyme, soluble epoxide hydrolase (sEH), since inhibition of sEH has been shown to protect against DOX-induced nephrotoxicity. Acute DOX-induced nephrotoxicity was induced by a single intra-peritoneal injection of 20 mg/kg DOX to male and female adult C57Bl/6 mice. The kidneys were isolated 1, 3 and 6 days after DOX administration. Histopathology assessment, gene expression of the apoptotic marker, BAX, protein expression of the fibrotic marker, transforming growth factor-β (TGF-β), and gene and protein expression of sEH were assessed. DOX administration caused more severe pathological lesions as well as higher induction of the apoptotic and fibrotic markers in kidneys of male than in female mice. Intriguingly, DOX inhibited sEH protein expression in kidneys of male mice sacrificed at 3 and 6 days following administration, suggesting that induction of sEH is not necessary for acute DOX-induced nephrotoxicity. However, DOX-induced inhibition of renal sEH in male mice may protect the kidney from further DOX-induced injury in a negative feedback mechanism. We also observed lower constitutive expressions of TGF-β and sEH in the kidney of female mice which may contribute, at least in part, to sexual dimorphism of DOX-induced nephrotoxicity.

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<![CDATA[Comparative analysis of mutational robustness of the intrinsically disordered viral protein VPg and of its interactor eIF4E]]> https://www.researchpad.co/article/5c6f148fd5eed0c48467a2e5

Conformational intrinsic disorder is a feature present in many virus proteins. Intrinsically disordered regions (IDRs) have weaker structural requirement than ordered regions and mutations in IDRs could have a lower impact on the virus fitness. This could favor its exploration of adaptive solutions. The potyviral protein VPg contains IDRs with determinants for adaptation to its host plant. To experimentally assess whether IDRs are more resistant to mutations than ordered regions, the biologically relevant interaction between mutant libraries of both VPg and the eukaryotic translation initiation factor 4E (eIF4E) and their respective wild type partner was examined using yeast two hybrid assay. Our data shows that VPg is significantly more robust to mutations than eIF4E and as such belongs to a particular class of intrinsically disordered proteins. This result is discussed from the standpoint of IDRs involvement in the virus adaptive processes.

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<![CDATA[Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1]]> https://www.researchpad.co/article/5c6dc9e1d5eed0c48452a3da

Cholesterol oxidase is a bifunctional bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This valuable enzyme has attracted a great deal of attention because of its wide application in the clinical laboratory, synthesis of steroid derived drugs, food industries, and its potentially insecticidal activity. Therefore, development of an efficient protocol for overproduction of cholesterol oxidase could be valuable and beneficial in this regard. The present study examined the role of various parameters (host strain, culture media, induction time, isopropyl ß-D-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature) on over-expression of cholesterol oxidase from Chromobacterium sp. DS1. Applying the optimized protocol, the yield of recombinant cholesterol oxidase significantly increased from 92 U/L to 2115 U/L. Under the optimized conditions, the enzyme was produced on a large-scale, and overexpressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver-Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were determined. This study reports a straightforward protocol for cholesterol oxidase production which can be performed in any laboratory.

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<![CDATA[Metabolomic profiling reveals correlations between spermiogram parameters and the metabolites present in human spermatozoa and seminal plasma]]> https://www.researchpad.co/article/5c76fe4fd5eed0c484e5b889

In 50% of all infertility cases, the male is subfertile or infertile, however, the underlying mechanisms are often unknown. Even when assisted reproductive procedures such as in vitro fertilization and intracytoplasmic sperm injection are performed, the causes of male factor infertility frequently remain elusive. Since the overall activity of cells is closely linked to their metabolic capacity, we analyzed a panel of 180 metabolites in human sperm and seminal plasma and elucidated their associations with spermiogram parameters. Therefore, metabolites from a group of 20 healthy donors were investigated using a targeted LC-MS/MS approach. The correlation analyses of the amino acids, biogenic amines, acylcarnitines, lysophosphatidylcholines, phosphatidylcholines, sphingomyelins and sugars from sperm and seminal plasma with standard spermiogram parameters revealed that metabolites in sperm are closely related to sperm motility, whereas those in seminal plasma are closely related to sperm concentration and morphology. This study provides essential insights into the metabolome of human sperm and seminal plasma and its associations with sperm functions. This metabolomics technique could be a promising screening tool to detect the factors of male infertility in cases where the cause of infertility is unclear.

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<![CDATA[Inherent versus induced protein flexibility: Comparisons within and between apo and holo structures]]> https://www.researchpad.co/article/5c5b52c9d5eed0c4842bd003

Understanding how ligand binding influences protein flexibility is important, especially in rational drug design. Protein flexibility upon ligand binding is analyzed herein using 305 proteins with 2369 crystal structures with ligands (holo) and 1679 without (apo). Each protein has at least two apo and two holo structures for analysis. The inherent variation in structures with and without ligands is first established as a baseline. This baseline is then compared to the change in conformation in going from the apo to holo states to probe induced flexibility. The inherent backbone flexibility across the apo structures is roughly the same as the variation across holo structures. The induced backbone flexibility across apo-holo pairs is larger than that of the apo or holo states, but the increase in RMSD is less than 0.5 Å. Analysis of χ1 angles revealed a distinctly different pattern with significant influences seen for ligand binding on side-chain conformations in the binding site. Within the apo and holo states themselves, the variation of the χ1 angles is the same. However, the data combining both apo and holo states show significant displacements. Upon ligand binding, χ1 angles are frequently pushed to new orientations outside the range seen in the apo states. Influences on binding-site variation could not be easily attributed to features such as ligand size or x-ray structure resolution. By combining these findings, we find that most binding site flexibility is compatible with the common practice in flexible docking, where backbones are kept rigid and side chains are allowed some degree of flexibility.

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<![CDATA[ARR22 overexpression can suppress plant Two-Component Regulatory Systems]]> https://www.researchpad.co/article/5c6b2616d5eed0c4842892c3

In plants, several developmental processes are co-coordinated by cytokinins via phosphorylation dependent processes of the Two-Component System (TCS). An outstanding challenge is to track phosphorelay flow from cytokinin perception to its molecular outputs, of which gene activation plays a major role. To address this issue, a kinetic-based reporter system was expounded to track TCS phosphorelay activity in vivo that can distinguish between basal and cytokinin dependent effects of overexpressed TCS members. The TCS phosphorelay can be positively activated by cytokinin and inhibited by pharmaceuticals or naturally interfering components. In this case we took advantage of the phosphohistidine-phosphatase Arabidopsis Response Regulator (ARR) 22 and investigated its phosphocompetition with other TCS members in regulating promoters of ARR5 and WUS in Arabidopsis thaliana cell culture protoplasts. In congruency with the proposed function of ARR22, overexpression of ARR22 blocked the activation of all B-type ARRs in this study in a TCS dependent manner. Furthermore, this effect could not be mimicked by A-type response regulator overexpression or compensated by AHP overexpression. Compared to other reporter assays, ours mimicked effects previously observed only in transgenic plants for all of the TCS proteins studied, suggesting that it is possible to expose phosphocompetition. Thus, our approach can be used to investigate gene signaling networks involving the TCS by leveraging ARR22 as a TCS inhibitor along with B-type ARR overexpression.

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<![CDATA[Deregulation of LRSAM1 expression impairs the levels of TSG101, UBE2N, VPS28, MDM2 and EGFR]]> https://www.researchpad.co/article/5c648d54d5eed0c484c825ab

CMT is the most common hereditary neuromuscular disorder of the peripheral nervous system with a prevalence of 1/2500 individuals and it is caused by mutations in more than 80 genes. LRSAM1, a RING finger ubiquitin ligase also known as TSG101-associated ligase (TAL), has been associated with Charcot-Marie-Tooth disease type 2P (CMT2P) and to date eight causative mutations have been identified. Little is currently known on the pathogenetic mechanisms that lead to the disease. We investigated the effect of LRSAM1 deregulation on possible LRSAM1 interacting molecules in cell based models. Possible LRSAM1 interacting molecules were identified using protein-protein interaction databases and literature data. Expression analysis of these molecules was performed in both CMT2P patient and control lymphoblastoid cell lines as well as in LRSAM1 and TSG101 downregulated SH-SY5Y cells.TSG101, UBE2N, VPS28, EGFR and MDM2 levels were significantly decreased in the CMT2P patient lymphoblastoid cell line as well as in LRSAM1 downregulated cells. TSG101 downregulation had a significant effect only on the expression of VPS28 and MDM2 and it did not affect the levels of LRSAM1. This study confirms that LRSAM1 is a regulator of TSG101 expression. Furthermore, deregulation of LRSAM1 significantly affects the levels of UBE2N, VPS28, EGFR and MDM2.

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<![CDATA[Expression, purification and characterization of the dimeric protruding domain of Macrobrachium rosenbergii nodavirus capsid protein expressed in Escherichia coli]]> https://www.researchpad.co/article/5c5df342d5eed0c484581038

Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1–252 and 253–371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.

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<![CDATA[Equilin in conjugated equine estrogen increases monocyte-endothelial adhesion via NF-κB signaling]]> https://www.researchpad.co/article/5c5b5254d5eed0c4842bc66d

The adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules, plays a crucial role in the onset of atherosclerosis. Conjugated equine estrogen, which is widely used for estrogen-replacement therapy, contains both estrone sulfate and various nonhuman estrogens, including equilin. To investigate the association between various estrogen types and atherosclerosis risk, we examined their effect on adhesion-molecule expression in human umbilical vein endothelial cells (HUVECs). In estrogen-treated HUVECs, the mRNA and protein expression levels of adhesion molecules were quantified by real-time polymerase chain reaction and enzyme immunoassay. Additionally, a flow-chamber system was used to assess the effects of estrogens on the adherence of U937 monocytoid cells to HUVECs. Equilin, but not 17β-estradiol (E2) or other types of estrogen, significantly increased the mRNA (P < 0.01) and protein (P < 0.05) expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 as compared with levels in controls. Equilin treatment increased the adherence of U937 monocytoid cells to HUVECs relative to the that in the control (P < 0.05), decreased estrogen receptor (ER)β expression, and increased the expression of proteins involved in nuclear factor kappa-B (NF-κB) activation relative to levels in controls. Furthermore, the accumulation of NF-κB subunit p65 in HUVEC nuclei was promoted by equilin treatment. By contrast, E2 treatment neither increased the number of adhered monocytoid cells to HUVECs nor altered the expression of ERβ or NF-κB-activating proteins. Our findings suggest that in terms of the adhesion of monocytes at the onset of atherosclerosis, E2 may be preferable for estrogen-replacement therapy. Further studies comparing equilin treatment with that of E2 are needed to investigate their differential impacts on atherosclerosis.

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