Immunoreactivity of the AAA plus chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals

Citation: Krajewska, J., Arent, Z., Wieckowski, D., Zolkiewski, M., & Kedzierska-Mieszkowska, S. (2016). Immunoreactivity of the AAA plus chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals. Bmc Microbiology, 16, 8. doi:10.1186/s12866-016-0774-8Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that L. interrogans ClpB (ClpB(Li)) is essential for bacterial survival under stressful conditions and also during infection. The aim of this study was to provide further insight into the role of ClpB in L. interrogans and answer the question whether ClpB(Li) as a potential virulence factor may be a target of the humoral immune response during leptospiral infections in mammals. Results: ClpB(Li) consists of 860 amino acid residues with a predicted molecular mass of 96.3 kDa and shows multi-domain organization similar to that of the well-characterized ClpB from Escherichia coli. The amino acid sequence identity between ClpB(Li) and E. coli ClpB is 52 %. The coding sequence of the clpB(Li) gene was cloned and expressed in E. coli BL21(DE3) strain. Immunoreactivity of the recombinant ClpB(Li) protein was assessed with the sera collected from Leptospira-infected animals and uninfected healthy controls. Western blotting and ELISA analysis demonstrated that ClpB(Li) activates the host immune system, as evidenced by an increased level of antibodies against ClpB(Li) in the sera from infected animals, as compared to the control group. Additionally, ClpB(Li) was found in kidney tissues of Leptospira-infected hamsters. Conclusions: ClpB(Li) is both synthesized and immunogenic during the infectious process, further supporting its involvement in the pathogenicity of Leptospira. In addition, the immunological properties of ClpB(Li) point to its potential value as a diagnostic antigen for the detection of leptospirosis

Identifying (territorial) tensions in the servitization value chain

Purpose: To understand the tensions that servitization activities create between actors within networks. Design/methodology/approach: Interviews were conducted with manufacturers, intermediaries and customers across a range of industrial sectors. Findings: Tensions relating to two key sets of capabilities are identified: in developing or acquiring (i) operant technical expertise and (ii) operand service infrastructure. The former tension concerns whom knowledge is co-created with and where expertise resides. The latter involves a territorial investment component; firms developing strategies to acquire greater access to, or ownership of, infrastructures closer to customers. Developing and acquiring these capabilities is a strategic decision on the part of managers of servitizing firms, in order to gain recognized power and control in a particular territory. Originality/value: This paper explores how firms’ servitization activities involve value appropriation (from the rest of the network), contrasting with the narrative norm for servitization: that it creates additional value. There is a need to understand the tensions that servitization activities create within networks. Some firms may be able to improve servitization performance through co-operation rather than competition, generating co-opetitive relationships. Others may need to become much more aggressive, if they are to take a greater share of the ‘value’ from the value chain

Identifying tensions in the servitized value chain

OVERVIEW: Servitization is recognized as an opportunity for manufacturing firms to harvest additional value by accessing new sources of revenue and expanding their reach up and down the value chain. It is a network activity, as it involves not just the servitizing firm but actors across the firm’s ecosystem. Most studies argue that servitization creates value for all network actors. However, service innovation activities may also result in the firm appropriating value from other actors, creating tensions in the network. Those tensions can undermine servitization efforts and destroy value for all participants. To avoid this outcome, firms must anticipate and manage tensions to create cooperative relationships with value chain partners. Through a series of semi-structured interviews with key actors at servitizing firms and their customers and intermediaries, we identified specific types and sources of tensions in the servitization process and explored how they might be mitigated or managed

Dealing with missing data for prognostic purposes

© 2016 IEEE. Centrifugal compressors are considered one of the most critical components in oil industry, making the minimisation of their downtime and the maximisation of their availability a major target. Maintenance is thought to be a key aspect towards achieving this goal, leading to various maintenance schemes being proposed over the years. Condition based maintenance and prognostics and health management (CBM/PHM), which is relying on the concepts of diagnostics and prognostics, has been gaining ground over the last years due to its ability of being able to plan the maintenance schedule in advance. The successful application of this policy is heavily dependent on the quality of data used and a major issue affecting it, is that of missing data. Missing data's presence may compromise the information contained within a set, thus having a significant effect on the conclusions that can be drawn from the data, as there might be bias or misleading results. Consequently, it is important to address this matter. A number of methodologies to recover the data, called imputation techniques, have been proposed. This paper reviews the most widely used techniques and presents a case study with the use of actual industrial centrifugal compressor data, in order to identify the most suitable ones

Reciprocating compressor prognostics

Reciprocating compressors are vital components in oil and gas industry though their maintenance cost can be high. The valves are considered the most frequent failing part accounting for almost half the maintenance cost. Condition Based Maintenance and Prognostics and Health Management which is based on diagnostics and prognostics principles can assist towards reducing cost and downtime while increasing safety and availability by offering a proactive means for scheduling maintenance. Although diagnostics is an established field for reciprocating compressors, there is limited information regarding prognostics, particularly given the nature of failures can be instantaneous. This paper compares several prognostics methods (multiple liner regression, polynomial regression, K-Nearest Neighbours Regression (KNNR)) using valve failure data from an operating industrial compressor. Moreover, a variation about Remaining Useful Life (RUL) estimation process based on KNNR is proposed along with an ensemble method combining the output of all aforementioned algorithms. In conclusion it is showed that even when RUL is relatively short given the instantaneous failure mode, good estimates are feasible using the proposed methods

Reciprocating compressor prognostics of an instantaneous failure mode utilising temperature only measurements

Reciprocating compressors are critical components in the oil and gas sector, though their maintenance cost is known to be relatively high. Compressor valves are the weakest component, being the most frequent failure mode, accounting for almost half the maintenance cost. One of the major targets in industry is minimisation of downtime and cost, while maximising availability and safety of a machine, with maintenance considered a key aspect in achieving this objective. The concept of Condition Based Maintenance and Prognostics and Health Management (CBM/PHM) which is founded on the diagnostics and prognostics principles, is a step towards this direction as it offers a proactive means for scheduling maintenance. Despite the fact that diagnostics is an established area for reciprocating compressors, to date there is limited information in the open literature regarding prognostics, especially given the nature of failures can be instantaneous. This work presents an analysis of prognostic performance of several methods (multiple linear regression, polynomial regression, K-Nearest Neighbours Regression (KNNR)), in relation to their accuracy and variability, using actual temperature only valve failure data, an instantaneous failure mode, from an operating industrial compressor. Furthermore, a variation for Remaining Useful Life (RUL) estimation based on KNNR, along with an ensemble technique merging the results of all aforementioned methods are proposed. Prior to analysis, principal components analysis and statistical process control were employed to create !! and ! metrics, which were proposed to be used as health indicators reflecting degradation process of the valve failure mode and are proposed to be used for direct RUL estimation for the first time. Results demonstrated that even when RUL is relatively short due to instantaneous nature of failure mode, it is feasible to perform good RUL estimates using the proposed techniques

Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and β-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration

The Chaperone ClpX Stimulates Expression of Staphylococcus aureus Protein A by Rot Dependent and Independent Pathways

The Clp ATPases (Hsp100) constitute a family of closely related proteins that have protein reactivating and remodelling activities typical of molecular chaperones. In Staphylococcus aureus the ClpX chaperone is essential for virulence and for transcription of spa encoding Protein A. The present study was undertaken to elucidate the mechanism by which ClpX stimulates expression of Protein A. For this purpose, we prepared antibodies directed against Rot, an activator of spa transcription, and demonstrated that cells devoid of ClpX contain three-fold less Rot than wild-type cells. By varying Rot expression from an inducible promoter we showed that expression of Protein A requires a threshold level of Rot. In the absence of ClpX the Rot content is reduced below this threshold level, hence, explaining the substantially reduced Protein A expression in the clpX mutant. Experiments addressed at pinpointing the role of ClpX in Rot synthesis revealed that ClpX is required for translation of Rot. Interestingly, translation of the spa mRNA was, like the rot mRNA, enhanced by ClpX. These data demonstrate that ClpX performs dual roles in regulating Protein A expression, as ClpX stimulates transcription of spa by enhancing translation of Rot, and that ClpX additionally is required for full translation of the spa mRNA. The current findings emphasize that ClpX has a central role in fine-tuning virulence regulation in S. aureus

Heat Resistance Mediated by a New Plasmid Encoded Clp ATPase, ClpK, as a Possible Novel Mechanism for Nosocomial Persistence of Klebsiella pneumoniae

Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably thermotolerant providing a conceivable explanation of its persistence in the hospital environment. This marked phenotype is mediated by a novel type of Clp ATPase, designated ClpK. The clpK gene is encoded by a conjugative plasmid and we find that the clpK gene alone renders an otherwise sensitive E. coli strain resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance of Clp ATPases in acquired environmental fitness and highlights the challenges of mobile genetic elements in fighting nosocomial infections