Tauopathy-Associated Tau Fragment Ending at Amino Acid 224 Is Generated by Calpain-2 Cleavage

BACKGROUND: Tau aggregation in neurons and glial cells characterizes tauopathies as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Tau proteolysis has been proposed as a trigger for tau aggregation and tau fragments have been observed in brain and cerebrospinal fluid (CSF). Our group identified a major tau cleavage at amino acid (aa) 224 in CSF; N-terminal tau fragments ending at aa 224 (N-224) were significantly increased in AD and lacked correlation to total tau (t-tau) and phosphorylated tau (p-tau) in PSP and CBD. OBJECTIVE: Previous studies have shown cleavage from calpain proteases at sites adjacent to aa 224. Our aim was to investigate if calpain-1 or -2 could be responsible for cleavage at aa 224. METHODS: Proteolytic activity of calpain-1, calpain-2, and brain protein extract was assessed on a custom tau peptide (aa 220-228), engineered with fluorescence resonance energy transfer (FRET) technology. Findings were confirmed with in-gel trypsination and mass spectrometry (MS) analysis of brain-derived bands with proteolytic activity on the FRET substrate. Finally, knock-down of the calpain-2 catalytic subunit gene (CAPN2) was performed in a neuroblastoma cell line (SH-SY5Y). RESULTS: Calpain-2 and brain protein extract, but not calpain-1, showed proteolytic activity on the FRET substrate. MS analysis of active gel bands revealed presence of calpain-2 subunits, but not calpain-1. Calpain-2 depletion and chemical inhibition suppressed proteolysis of the FRET substrate. CAPN2 knock-down caused a 76.4% reduction of N-224 tau in the cell-conditioned media. CONCLUSIONS: Further investigation of the calpain-2 pathway in the pathogenesis of tauopathies is encouraged

Increasing awareness and reputation of MERCK S.A. Portugal through employee advocacy

Merck KGaA is a global Science and Technology company, focusing on the Healthcare Industry, Life Science and Performance Materials. Merck in Portugal is a relatively small subsidiary with a midsized office employing approximately 114 employees. Merck S.A. Portugal focuses mainly on the Healthcare Market. As the company went through a major transformation in 2016, it created a need to communicate on a low cost basis and in an efficient way, using digital as its main channel. The current paper shows the implementation of an employee advocacy strategic plan as a solution to digital communication. Employee Advocacy is a new communications concept derived from employee social media usage and employee word of mouth. It has been noted that employee networks are wider than company’s own and employee word of mouth is more trustworthy. Before the tool could be implemented a research on what motivates employees to commit to Brand Citizenship Behavior was performed. Findings revealed that in the case of Merck S.A. Portugal employees individual internal drive and brand knowledge are factors affecting willingness to share on social media. Based on the results a series of internal strategies were applied on- and offline.info:eu-repo/semantics/acceptedVersio

Skp is a multivalent chaperone of outer membrane proteins

The trimeric chaperone Skp sequesters outer-membrane proteins (OMPs) within a hydrophobic cage, thereby preventing their aggregation during transport across the periplasm in Gram-negative bacteria. Here, we studied the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding revealed that higher Skp/OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry–mass spectrometry (IMS–MS) data, computer modeling and molecular dynamics simulations provided evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs that cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp in protecting a single substrate from aggregation

Fine tuning of the E. coli NusB:NusE complex affinity to BoxA RNA is required for processive antitermination

Phage λ propagation in Escherichia coli host cells requires transcription antitermination on the λ chromosome mediated by λN protein and four host Nus factors, NusA, B, E (ribosomal S10) and G. Interaction of E. coli NusB:NusE heterodimer with the single stranded BoxA motif of λnutL or λnutR RNA is crucial for this reaction. Similarly, binding of NusB:NusE to a BoxA motif is essential to suppress transcription termination in the ribosomal RNA (rrn) operons. We used fluorescence anisotropy to measure the binding properties of NusB and of NusB:NusE heterodimer to BoxA-containing RNAs differing in length and sequence. Our results demonstrate that BoxA is necessary and sufficient for binding. We also studied the gain-of-function D118N NusB mutant that allows λ growth in nusA1 or nusE71 mutants. In vivo λ burst-size determinations, CD thermal unfolding measurements and X-ray crystallography of this as well as various other NusB D118 mutants showed the importance of size and polarity of amino acid 118 for RNA binding and other interactions. Our work suggests that the affinity of the NusB:NusE complex to BoxA RNA is precisely tuned to maximize control of transcription termination

RfaH Suppresses Small RNA MicA Inhibition of fimB Expression in Escherichia coli K-12

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE , and hence MicA, in the absence of RfaH. Additional work con- firms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression

Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM

The β-barrel assembly machinery (BAM) is a ~203 kDa complex of five proteins (BamA-E) which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a ‘lateral gating’ motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex, and interactions between BamA, B, D, and E and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM

Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OFMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding

Strategisches Handeln von Startups im Kontext der Mediatisierung

Junge Gründer und Start-ups müssen sich in einer schnell wandelnden und mediatisierten Wettbewerbsumwelt behaupten. Ihr Handeln wird geprägt von sozialen Netzwerkmedien wie Facebook, LinkedIn oder Instagram. Um auf diesen Medien-plattformen erfolgreich zu sein, müssen Markenführung und Markenkommunikation strategisch verankert sein. Der Aufsatz präsentiert daher eine qualitative Analyse empirischer Daten aus dem Kontext des Start-up-Incubator neudeli der Bauhaus-Universität Weimar und verdeutlicht, dass die Mediatisierung grundlegend in die strategische Entwicklung der Marke von jungen Gründern und Start-ups eingreift. Die Studie verdeutlicht das Verständnis strategischer Markenführung in mediatisierten Kontexten und zeigt, dass drei idealtypische Praktiken zur Markenführung und strategischen Entwicklung beitragen: 1) Bürokratische Medienarbeit, 2) Mediale Kreativarbeit, 3) Netzwerkarbeit durch Medien

A brand within a brand: an integrated understanding of internal brand management and brand architecture in the public sector

Branding in the public sector is emerging as an interesting area of research, as diverse organisations find themselves using branding principles to promote a consistent, clear brand. However, very little is known how public organisations could, or should, manage their brands. The purpose of this research, therefore, is to explore brand management processes in the public sector, and its implication for brand architecture, from an employee perspective. With a qualitative approach, the study argues that branding is important not only for the organisation, but also for individual departments. Further, unlike branding in the private sector, public organisations may be more concerned with supporting a positive perception and organisational attractiveness rather than a unique and differentiated brand. This may have implications for brand architecture. By allowing individual departments to manage their brand with support from organisational structures that provide alignment and focus, organisations can form a brand architecture that supports a strong organisational brand and employee brand commitment