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Cold stress is a key climatic factor that limits grape productivity and quality. Although β-amylase (BAM) is known to play an important role as a mediator of starch degradation under conditions of cold stress, the mechanism by which BAM regulates cold tolerance in grape remains unclear. Here, we identified VaBAM1 from Vitis amurensis and characterized its interactive regulating mechanism under cold stress in Arabidopsis thaliana and grape. VaBAM1 -overexpressing A. thaliana plants (OEs) exhibited high freezing tolerance. Soluble sugar content and amylase activity were increased in OEs and VaBAM1 -overexpressing grape calli (VaBAM1-OEs) under cold stress; however, they were decreased in grape calli in which VaBAM1 was edited using CRISPR/Cas9. The results of yeast two-hybrid, bimolecular fluorescence complementation, and pull-down experiments showed that serine/arginine-rich splicing factor 1 (VaSR1) interacted with VaBAM1. Furthermore, the expression of VaSR1 was opposite that of VaBAM1 in phloem tissue of Vitis amurensis during winter dormancy. In VaSR1 -overexpressing grape calli (VaSR1-OEs), BAM activity and the expression levels of C-repeat binding transcription factor and cold response genes were all significantly lower than that in untransformed calli subjected to cold stress. Moreover, VvBAM1 was downregulated in VaSR1-OEs under cold stress. Overall, we identified that VaSR1 interacts with VaBAM1, negatively regulating BAM activity and resulting in decreased plant cold tolerance. • VaBAM1 overexpression enhanced cold tolerance in Arabidopsis leaves and grape calli. • CRISPR/Cas9 editing of VaBAM1 resulted in decreased cold tolerance in grape calli. • VaSR1 and VaBAM1 interaction inhibited BAM activity, decreasing cold tolerance in grape calli. [ABSTRACT FROM AUTHOR]

• Much refrigerant in the F-evaporator causes the starvation of R-evaporator at RC. • Liquefied refrigerant held-up in the bottom of F-evaporator affect FC performance. • The multi-parameters optimization experimental methods are performed. • R-cabinet temperature drop rate decreased with the increase of ambient temperature. • F-cabinet temperature drop rate was almost unaffected by the ambient temperature. This paper presents an experimental research on configuration optimization of a frost-free refrigerator-freezer with the parallel circuit cycle based on the analysis of circuit switching characteristics. Through combination optimization of R-fan voltage, F-fan voltage, R-capillary tube flow capacity, refrigerant charge, structure of suction pipe, F-compressor speed and R-compressor speed, the system could save 9.27% energy consumption compared with the initial condition. The experimental results also showed that the larger the R-capillary tube flow capacity, the larger the flow rate of the RC, resulting in the quickly temperature drop in R-cabinet and slowly temperature drop in R-cabinet based on "R-cabinet temperature priority" control strategy. Additionally, the R-cabinet temperature drop rate decreased with the increase of ambient temperature but the F-cabinet temperature drop rate was almost unaffected by the ambient temperature. [ABSTRACT FROM AUTHOR]

Abstract The interactions between the four Dengue virus (DENV) serotypes and plasma proteins are crucial in the initial steps of viral infection to humans. Affinity purification combined with quantitative mass spectrometry analysis, has become one of the most powerful tools for the investigation on novel protein-protein interactions. Using this approach, we report here that a significant number of bait-interacting proteins do not dissociate under standard elution conditions, i.e. acid pH and chaotropic agents, and that this problem can be circumvented by using the "on-matrix" digestion procedure described here. This procedure enabled the identification of 16 human plasma proteins interacting with domain III from the envelope protein of DENV serotypes 1, 3 and 4 that would have not been detected otherwise and increased the known DIIIE interactors in human plasma to 59 proteins. Selected Reaction Monitoring analysis evidenced DENV interactome in human plasma is rather conserved although significant differences on the reactivity of viral serotypes with specific proteins do exist. A comparison between the serotype-dependent profile of reactivity and the conservation pattern of amino acid residues suggests an evolutionary selection of highly conserved interactions with the host and other interactions mediated for surface regions of higher variability. Significance False negative results on the identification of interacting proteins in pull-down experiments compromise the subsequent interpretation of results and the formulation of a working hypothesis for the derived future work. In this study we demonstrate the presence of bait-interacting proteins reluctant to dissociate under elution conditions of acid pH and presence of chaotropics. We propose the direct proteolytic digestion of proteins while still bound to the affinity matrix ("on-matrix" digestion) and evaluate the impact of this methodology in the comparative study of the interactome of the four serotypes of Dengue virus mediated by the domain III of the viral envelope glycoprotein. Fifty nine proteins were identified as putative interaction partners of Dengue virus (IPs) either due to direct binding or by co-isolation with interacting proteins. Collectively the IPs identified from the pull-down with the recombinant domain III proteins representing the four viral serotypes, 29% were identified only after "on-matrix" digestion which demonstrate the usefulness of this method of recovering bait-bound proteins. Results highlight a particular importance of "on-matrix" digestion procedure for comparative studies where a stronger interaction with one of the interest baits could prevent a bound protein to elute under standard conditions thus leading to misinterpretation as absent in the interactome of this particular bait. The analysis of the Interaction Network indicates that Dengue virus interactome mediated by the domain III of the envelope protein is rather conserved in the viral complex suggesting a key role of these interactions for viral infection thus making candidates to explore for potential biomarkers of clinical outcome in DENV-caused disease. Interestingly, some particular IPs exhibit significant differences in the strength of the interaction with the viral serotypes representing interactions that involve more variable regions in the surface of the domain III. Since such variable regions are the consequence of the interaction with antibodies generated by human immune response; this result relates the interaction with proteins from human plasma with the interplay of the virus and the human immune system. Graphical abstract Image 2 Highlights • The presence of proteins reluctant to elute with acid pH and chaotropes in pull-down experiments is demonstrated. • Digestion of proteins that remained bound to the affinity matrix proved to be useful to avoid false negatives in AP-MS. • Dengue virus interactome with human plasma proteins is highly conserved for the four viral serotypes • Human plasma proteins with significant differences in reactivity with the dengue virus serotypes are identified [ABSTRACT FROM AUTHOR]

Abstract In China, the high-oleic acid rapeseed has an oil content of ∼42% and oleic acid (18:1) content of ∼80%. Compared to ordinary rapeseed, high-oleic acid rapeseed has higher levels of monounsaturated fatty acids and lower levels of saturated fatty acid and polyunsaturated fatty acids, and thus is of high nutritional and health value. In addition, high-oleic acid rapeseed oil imparts cardiovascular protective effects. Based on these properties, high-oleic acid oil crops have been extensively investigated and cultivated. We previously identified a CCCH-type transcription factor (BnZFP1, GenBank accession number XM_013796508) that is associated with high oleic acid traits from a Brassica napus subtractive hybridization library. In the present study, we overexpressed and silenced the BnZFP1 gene of B. napus. BnZFP1 -overexpressing plants exhibited an 18.8% increase in oleic acid levels and a 3.8% increase in oil content. However, BNZFP1 -silenced plants showed a 4.5% decrease in oleic acid levels, whereas no significant change in oil content was observed. Microarray and pull-down assays indicated that BnZFP1 has a total of thirty potential target genes. Further analysis and validation of one of the potential target genes, namely, diacylglycerol O-acyltransferases 1 (DGAT1) gene, indicated that it is positively regulated by BnZFP1. We also observed a correlation between elevated DGAT1 gene expression levels and higher oil content and oleic acid levels in rapeseed. Highlights • BnZFP1 -overexpressing plants exhibited an 18.8% increase in oleic acid levels and a 3.8% increase in oil content. • BNZFP1 -silenced plants exhibited a 4.5% decrease in oleic acid levels. • Pull-down assays and microarray studies have shown that DGAT1-1 is a candidate target gene of transcription factor BnZFP1. • BnZFP1 overexpression enhances the positive regulation of the transcription factor BnZFP1 on the DGAT1-1 gene. [ABSTRACT FROM AUTHOR]

Summary Condensins load onto DNA to organize chromosomes. Smc-ScpAB clearly loads onto the parS sites bound by Spo0J, but other loading site(s) must operate independently of parS . In this study, we asked where and how Smc-ScpAB normally selects its loading site. Our results suggest that rDNA is also a loading site. A pull-down assay revealed that Smc-ScpAB preferentially loads onto rDNA in the wild-type cell and even in a Δ spo0J mutant but not in a Δ smc mutant. Moreover, we showed that deletion mutants of rDNAs cause a defect in nucleoid separation, and at least two rDNAs near oriC are essential for separation. Full-length rDNA, including promoters, is required for loading and nucleoid separation. A synthetic defect by deletions of both rDNA and spo0J resulted in more aberrant nucleoid separation. We propose that a single-stranded segment of DNA that is exposed at highly transcribed rRNA operons would become a target for Smc-ScpAB loading. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E. Open reading frame 2 (ORF2) encodes the capsid protein of HEV, which is the main structural protein and may participate, together with the host factors, in viral entry and egress. However, it is still not clear which host proteins are involved in the interaction with ORF2 and what the functions of these ORF2-interacting proteins are. In this study, we have applied a split-ubiquitin yeast two-hybrid screening approach in combination with the pull-down and coimmunoprecipitation assays, identified and validated multiple interacting partners of ORF2 of genotype 1 and 4, which have diverse biological functions. Among these novel candidates that have not been previously reported, we have found that 20 of them are located in endoplasmic reticulum. TMEM134, which interacts and co-localizes with ORF2 in the endoplasmic reticulum, negatively regulates ORF2-mediated inhibition of the NF-κB signaling pathway. Our study for the first time has systematically mapped the ORF2 interactome in two genotypes of HEV, providing a new insight of understanding the virus-host interaction during the pathogenesis of HEV, and may offer potential therapeutic targets to intervene the HEV life cycle. [ABSTRACT FROM AUTHOR]

Class I small heat shock proteins (CI sHSPs), OsHsp16.9A and OsHsp18.0, share 74% identity in amino acid sequences and accumulate in response to heat shock treatments. Individual rice transformants overexpressing OsHsp16.9A and OsHsp18.0 exhibit distinct thermoprotection/thermotolerance modes. Under high temperature stress, OsHsp16.9A -overexpressing lines showed higher seed germination rate, seedling survival, and pollen germination than wild-type controls, while OsHsp18.0 overexpression provided higher thermoprotection/thermotolerance for seedling survival. To elucidate the functional roles of OsHsp16.9A, mass spectrometry was used to identify OsHsp16.9A-interacting proteins. OsHsp101 was consistently identified in the OsHsp16.9A protein complex in several mass spectrometry analyses of seed proteins from OsHsp16.9A -overexpressing lines. Both OsHsp16.9A and OsHsp101 proteins accumulated during similar developmental stages of rice seeds and formed a heat-stable complex under high temperature treatments in in vitro assays. Co-localization of OsHsp16.9A and OsHsp101 was observed via ratiometric bimolecular fluorescence complementation analyses. Amino acid mutation studies revealed that OsHsp16.9A glutamate residue 74 and amino acid residues 23–36 were essential for OsHsp16.9A–OsHsp101 interaction. Moreover, overexpressing OsHsp16.9A in OsHsp101 knockdown mutants did not increase the seed germination rate under heat stress, which further confirmed the functional roles of OsHsp16.9A–OsHsp101 interaction in conferring thermotolerance to rice plants. • Development-activated OsHsp16.9A was closely related thermotolerance of rice seeds. • The interaction with OsHsp101 was necessary for OsHsp16.9A to confer thermoprotection. • Glutamate 74 of OsHsp16.9A was responsible for the OsHsp16.9A–OsHsp101 interaction. [ABSTRACT FROM AUTHOR]

This report presents the results of a study on the performance of thin-walled cold-formed steel wall frames with different sheathing systems affected by angular distortions simulating ground differential settlements due to land subsidence. The wall frames are sheathed with different systems: expanded polystyrene, OSB, calcium silicate and gypsum board. Using experimental testing and numerical simulation, moment–rotation curves were calculated for the studs. A non-linear static pull-down analysis was performed producing several degrees of angular distortion simulating ground settlements. Lastly, fragility curves were calculated based on three levels of damage for wall frames with different sheathing systems. [ABSTRACT FROM AUTHOR]

Abstract: HSP70 protein is involved in Leishmania differentiation, apoptosis, antimony-resistance and host-immune response. Therefore, this protein and the regulatory mechanisms of HSP70 gene expression are promising targets for therapeutic intervention against leishmaniasis. The regulation of mRNA expression in trypanosomatids operates mostly through the interaction of trans-acting proteins, and elements located in the untranslated regions of mRNAs. The aim of this work was to identify protein factors interacting specifically with the Leishmania braziliensis HSP70 mRNAs. Thus, the 5′ UTR and the two types of 3′ UTRs (UTR-I and UTR-II) from L. braziliensis HSP70 genes were used as baits in pull down assays using total protein extracts from parasites cultured at 26 or 35°C. The captured proteins were resolved by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS) analysis. As a result, 52 different proteins were identified based on their binding to the L. braziliensis HSP70-mRNAs. As expected, several of the identified proteins were related to RNA metabolism (27%) and translation process (7%). In addition, five hypothetical conserved proteins having motifs related with RNA interaction were also identified (9.6%). Nevertheless, unexpected proteins, apparently unrelated to the mRNA expression, were also identified. The biological significance of these and others L. braziliensis detected proteins, including the HSP70 itself, is discussed. Biological significance: For the first time, a riboproteomic analysis of the proteins interacting with the untranslated regions of the heat shock protein 70 (HSP70) mRNA from Leishmania braziliensis was carried out. This work provides new insights related to protein factors putatively involved in the regulation of HSP70 gene expression in L. braziliensis, and thereby, contributes to a better understanding of the parasite biology, and ultimately to the development of novel therapeutic interventions for controlling the important diseases caused by this parasite. [Copyright &y& Elsevier]

Abstract: Described is the synthesis of two biotinylated derivatives of a cytotoxic macrocycle. Pull-down assays indicate that this macrocycle targets the N-middle domain of Hsp90. Untagged compound can effectively compete away tagged compound–Hsp90 protein complexes, confirming the binding specificity of the macrocycle for Hsp90. The macrocycle is similar in potency to other structurally-related analogs of Sansalvamide A (San A) and induces apoptosis via a caspase 3 mechanism. Unlike other San A derivatives, we show that the macrocycle does not inhibit binding between C-terminal client proteins and co-chaperones and Hsp90, suggesting that it has a unique mechanism of action. [Copyright &y& Elsevier]

Summary: Objective: No standard regimen has been established for the management of postoperative voiding dysfunction following TVT insertion. This study was designed to compare two different strategies for management of this problem. Methods: This is a retrospective study of women operated on within a tertiary referral unit. Two cohorts were identified: those managed by clean intermittent self catheterization (CISC) during 2006, and those managed by ‘pull-down’ of tape in the immediate post-operative period during 2007. Specific measurements include number of patients with resumption of normal voiding and time taken for resolution of symptoms. Incidence of tape division, recurrence of stress incontinence and development of overactive bladder (OAB) were also measured. Results: The incidence of voiding difficulty following TVT insertion remained the same in 2006 and in 2007. 45.5% of the CISC group resumed normal voiding compared to 100% of the ‘pull-down’ group. The incidence of tape division and reappearance of stress incontinence was 54.5% and 27% respectively in the CISC group compared to none in the ‘pull-down’ group. The CISC group also had higher incidence of OAB. Conclusions: Immediate “pull-down” of tape is an effective way of managing voiding dysfunction following TVT with an excellent rate of symptom resolution without any need of further intervention. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we identified the multifunctional protein GC1q-R as a novel vasopressin V2 receptor (V2R) interacting protein. For this purpose, we have developed a proteomic approach combining pull-down assays using a cyclic peptide mimicking the third intracellular loop of V2R as a bait and mass spectrometry analyses of proteins isolated from either rat or human kidney tissues or the HEK 293 cell line. Co-immunoprecipitation of GC1q-R with the c-Myc-tagged h-V2R expressed in a HEK cell line confirmed the existence of a specific interaction between GC1q-R and the V2 receptor. Then, construction of a mutant receptor in i3 loop allowed us to identify the i3 loop arginine cluster of the vasopressin V2 receptor as the interacting determinant for GC1q-R interaction. Using purified receptor as a bait and recombinant (74–282) GC1q-R, we demonstrated a direct and specific interaction between these two proteins via the arginine cluster. [Copyright &y& Elsevier]

Protein–protein interactions, and the factors affecting them, are of fundamental importance to all biological systems. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITR) are powerful methods for assaying such interactions, but are expensive to implement. In contrast, bead-based pull-down assays using affinity tags such as glutathione-S-transferase (GST), require no specialist equipment. As a result, such assays are the most popular method for analysing protein–protein interactions, despite being time-consuming and prone to variability. In respect of these problems, we have modified this form of binding assay, using glutathione-coated 96-well plates rather than glutathione-Sepharose beads to bind the primary bait protein. Quantitation of bound protein utilises ELISA for purified proteins and scintillation counting for in vitro translated proteins, rather than the SDS-PAGE-based detection methods used in traditional bead-based assays. These modifications result in an approximately 10-fold increase in the number of samples that can be assayed daily, and allow results to be obtained within hours as opposed to days. We validate the modified assay by analysing the equilibrium binding of Munc18 and syntaxin, and also demonstrate that association and dissociation kinetics may be measured using this approach. The method we describe is generally applicable to any protein–protein interaction assay based on affinity tags and is amenable to automation, and so should benefit a wide range of biochemical research. [Copyright &y& Elsevier]

As an important marine fish pathogen, Edwardsiella piscicida infects a broad range of fish species and causes substantial economic losses. The EsrA-EsrB two-component system is essential for the expression of type III and type VI secretion systems (T3/T6SSs), the key virulence determinants in the bacterium. In this study, a pull-down assay with the esrB promoter as bait was performed to identify the upstream regulators of esrB. As a result, PepA, a leucyl aminopeptidase, was identified as a repressor of EsrB and T3/T6SS expression. PepA bound to the esrB promoter region and negatively regulated the production of T3/T6SS proteins in early stages. Moreover, PepA was found to affect the in vivo colonization of E. piscicida in turbot livers through the regulation of EsrB expression. Collectively, our results enhance the understanding of the virulence regulatory network and in vivo colonization mechanism of E. piscicida. One sentence summary: PepA regulates EsrB expression in Edwardsiella piscicida. [ABSTRACT FROM AUTHOR]