Der1 promotes movement of misfolded proteins through the endoplasmic reticulum membrane

Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD ligase) and degraded by cytosolic 26S proteasomes. The movement of these proteins through the lipid bilayer is assumed to occur via a protein-conducting channel of unknown nature. We show that the integral membrane protein Der1 oligomerizes, which relies on its interaction with the scaffolding protein Usa1. Mutations in the transmembrane domains of Der1 block the passage of soluble proteins across the ER membrane. As determined by site-specific photocrosslinking, the ER-luminal exposed parts of Der1 are in spatial proximity to the substrate receptor Hrd3, whereas the membrane-embedded domains reside adjacent to the ubiquitin ligase Hrd1. Intriguingly, both regions also form crosslinks to client proteins. Our data imply that Der1 initiates the export of aberrant polypeptides from the ER lumen by threading such molecules into the ER membrane and routing them to Hrd1 for ubiquitylation

A Systematic Approach from a Comparison of Three Glucocorticoids

Solid lipid nanoparticles (SLNs) can enhance drug penetration into the skin, yet the mechanism of the improved transport is not known in full. To unravel the influence of the drug-particle interaction on penetration enhancement, 3 glucocorticoids (GCs), prednisolone (PD), the diester prednicarbate (PC) and the monoester betamethasone 17-valerate (BMV), varying in structure and lipophilicity, were loaded onto SLNs. Theoretical permeability coefficients (cm/s) of the agents rank BMV (–6.38) ≧ PC (–6.57) > PD (–7.30). GC-particle interaction, drug release and skin penetration were investigated including a conventional oil-in-water cream for reference. Both with SLN and cream, PD release was clearly superior to PC release which exceeded BMV release. With the cream, the rank order did not change when studying skin penetration, and skin penetration is thus predominantly influenced by drug release. Yet, the penetration profile for the GCs loaded onto SLNs completely changed, and differences between the steroids were almost lost. Thus, SLNs influence skin penetration by an intrinsic mechanism linked to a specific interaction of the drug-carrier complex and the skin surface, which becomes possible by the lipid nature and nanosize of the carrier and appears not to be derived by testing drug release. Interestingly, PC and PD uptake from SLN even resulted in epidermal targeting. Thus, SLNs are not only able to improve skin penetration of topically applied drugs, but may also be of particular interest when specifically aiming to influence epidermal dysfunction

Engineering with logic: Rigorous test-oracle specification and validation for TCP/IP and the Sockets API

Conventional computer engineering relies on test-and-debug development processes, with the behavior of common interfaces described (at best) with prose specification documents. But prose specifications cannot be used in test-and-debug development in any automated way, and prose is a poor medium for expressing complex (and loose) specifications. The TCP/IP protocols and Sockets API are a good example of this: they play a vital role in modern communication and computation, and interoperability between implementations is essential. But what exactly they are is surprisingly obscure: their original development focused on “rough consensus and running code,” augmented by prose RFC specifications that do not precisely define what it means for an implementation to be correct. Ultimately, the actual standard is the de facto one of the common implementations, including, for example, the 15 000 to 20 000 lines of the BSD implementation—optimized and multithreaded C code, time dependent, with asynchronous event handlers, intertwined with the operating system, and security critical. This article reports on work done in the Netsem project to develop lightweight mathematically rigorous techniques that can be applied to such systems: to specify their behavior precisely (but loosely enough to permit the required implementation variation) and to test whether these specifications and the implementations correspond with specifications that are executable as test oracles . We developed post hoc specifications of TCP, UDP, and the Sockets API, both of the service that they provide to applications (in terms of TCP bidirectional stream connections) and of the internal operation of the protocol (in terms of TCP segments and UDP datagrams), together with a testable abstraction function relating the two. These specifications are rigorous, detailed, readable, with broad coverage, and rather accurate. Working within a general-purpose proof assistant (HOL4), we developed language idioms (within higher-order logic) in which to write the specifications: operational semantics with nondeterminism, time, system calls, monadic relational programming, and so forth. We followed an experimental semantics approach, validating the specifications against several thousand traces captured from three implementations (FreeBSD, Linux, and WinXP). Many differences between these were identified, as were a number of bugs. Validation was done using a special-purpose symbolic model checker programmed above HOL4. Having demonstrated that our logic-based engineering techniques suffice for handling real-world protocols, we argue that similar techniques could be applied to future critical software infrastructure at design time, leading to cleaner designs and (via specification-based testing) more robust and predictable implementations. In cases where specification looseness can be controlled, this should be possible with lightweight techniques, without the need for a general-purpose proof assistant, at relatively little cost.EPSRC Programme Grant EP/K008528/1 REMS: Rigorous Engineering for Mainstream Systems EPSRC Leadership Fellowship EP/H005633 (Sewell) Royal Society University Research Fellowship (Sewell) St Catharine's College Heller Research Fellowship (Wansbrough), EPSRC grant GR/N24872 Wide-area programming: Language, Semantics and Infrastructure Design EPSRC grant EP/C510712 NETSEM: Rigorous Semantics for Real Systems EC FET-GC project IST-2001-33234 PEPITO Peer-to-Peer Computing: Implementation and Theory CMI UROP internship support (Smith) EC Thematic Network IST-2001-38957 APPSEM 2 NICTA was funded by the Australian Government's Backing Australia's Ability initiative, in part through the Australian Research Council

The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase) and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR, which is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70 type chaperone Kar2 and the Hsp40 type cochaperone Scj1 bind to Hrd3KR. Noteworthy, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream acting components like Der1

Formalized Verification of Snapshotable Trees: Separation and Sharing

Abstract. We use separation logic to specify and verify a Java program that implements snapshotable search trees, fully formalizing the specification and verification in the Coq proof assistant. We achieve local and modular reasoning about a tree and its snapshots and their iterators, although the implementation involves shared mutable heap data structures with no separation or ownership relation between the various data. The paper also introduces a series of four increasingly sophisticated implementations and verifies the first one. The others are included as future work and as a set of challenge problems for full functional specification and verification, whether by separation logic or by other formalisms.

Urodynamic investigations in patients with spinal cord injury: should the ice water test follow or precede the standard filling cystometry?

OBJECTIVES To evaluate whether the ice water test (IWT) should be performed before or after the standard urodynamic investigation (UDI). PATIENTS AND METHODS Two cohorts of patients suffering from neurogenic lower urinary tract dysfunction (NLUTD) due to spinal cord injury (SCI) were matched by lesion level and age. The patients of cohort A (n=55, retrospective cohort) underwent the IWT before and the patients of cohort B (n=110, prospective cohort) after standard UDI. The IWT effect on urodynamic parameters has been compared between the two groups using the Mann-Whitney U-test for independent samples. UDI was performed according to good urodynamic practices recommended by the International Continence Society. RESULTS The mean age of both cohorts was 49 years. Performing the IWT before versus after standard UDI resulted in a significantly lower maximum cystometric bladder capacity (P=0.01), lower incidence of detrusor overactivity (P=0.017) and lower maximum detrusor pressure during IWT (P=0.04). All other urodynamic parameters assessed demonstrated no significant difference (P>0.05). CONCLUSIONS Our results are in line with findings from animal studies demonstrating a bladder cooling-induced gating effect on the micturition reflex volume threshold on the level of sacral interneurons. Since the IWT is an unphysiological investigation that might significantly bias subsequent urodynamics, we suggest that the IWT should not precede more physiological standard UDI.Spinal Cord advance online publication, 22 September 2015; doi:10.1038/sc.2015.152

Hydroxyapatite coating does not improve uncemented stem survival after total hip arthroplasty!: An analysis of 116,069 THAs in the Nordic Arthroplasty Register Association (NARA) database

Background and purpose — It is still being debated whether HA coating of uncemented stems used in total hip arthroplasty (THA) improves implant survival. We therefore investigated different uncemented stem brands, with and without HA coating, regarding early and long-term survival. Patients and methods — We identified 152,410 THA procedures using uncemented stems that were performed between 1995 and 2011 and registered in the Nordic Arthroplasty Register Association (NARA) database. We excluded 19,446 procedures that used stem brands less than 500 times in each country, procedures performed due to diagnoses other than osteoarthritis or pediatric hip disease, and procedures with missing information on the type of coating. 22 stem brands remained (which were used in 116,069 procedures) for analysis of revision of any component. 79,192 procedures from Denmark, Norway, and Sweden were analyzed for the endpoint stem revision. Unadjusted survival rates were calculated according to Kaplan-Meier, and Cox proportional hazards models were fitted in order to calculate hazard ratios (HRs) for the risk of revision with 95% confidence intervals (CIs). Results — Unadjusted 10-year survival with the endpoint revision of any component for any reason was 92.1% (CI: 91.8–92.4). Unadjusted 10-year survival with the endpoint stem revision due to aseptic loosening varied between the stem brands investigated and ranged from 96.7% (CI: 94.4–99.0) to 99.9% (CI: 99.6–100). Of the stem brands with the best survival, stems with and without HA coating were found. The presence of HA coating was not associated with statistically significant effects on the adjusted risk of stem revision due to aseptic loosening, with an HR of 0.8 (CI: 0.5–1.3; p = 0.4). The adjusted risk of revision due to infection was similar in the groups of THAs using HA-coated and non-HA-coated stems, with an HR of 0.9 (CI: 0.8–1.1; p = 0.6) for the presence of HA coating. The commonly used Bimetric stem (n = 25,329) was available both with and without HA coating, and the adjusted risk of stem revision due to aseptic loosening was similar for the 2 variants, with an HR of 0.9 (CI: 0.5–1.4; p = 0.5) for the HA-coated Bimetric stem. Interpretation — Uncemented HA-coated stems had similar results to those of uncemented stems with porous coating or rough sand-blasted stems. The use of HA coating on stems available both with and without this surface treatment had no clinically relevant effect on their outcome, and we thus question whether HA coating adds any value to well-functioning stem designs.publishedVersio

Expression of Drug Targets in Patients Treated with Sorafenib, Carboplatin and Paclitaxel

Introduction: Sorafenib, a multitarget kinase inhibitor, targets members of the mitogen-activated protein kinase (MAPK) pathway and VEGFR kinases. Here we assessed the association between expression of sorafenib targets and biomarkers of taxane sensitivity and response to therapy in pre-treatment tumors from patients enrolled in ECOG 2603, a phase III comparing sorafenib, carboplatin and paclitaxel (SCP) to carboplatin, paclitaxel and placebo (CP). Methods: Using a method of automated quantitative analysis (AQUA) of in situ protein expression, we quantified expression of VEGF-R2, VEGF-R1, VEGF-R3, FGF-R1, PDGF-Rβ, c-Kit, B-Raf, C-Raf, MEK1, ERK1/2, STMN1, MAP2, EB1 and Bcl-2 in pretreatment specimens from 263 patients. Results: An association was found between high FGF-R1 and VEGF-R1 and increased progression-free survival (PFS) and overall survival (OS) in our combined cohort (SCP and CP arms). Expression of FGF-R1 and VEGF-R1 was higher in patients who responded to therapy ((CR+PR) vs. (SD+PD+ un-evaluable)). Conclusions: In light of the absence of treatment effect associated with sorafenib, the association found between FGF-R1 and VEGF-R1 expression and OS, PFS and response might reflect a predictive biomarker signature for carboplatin/paclitaxel-based therapy. Seeing that carboplatin and pacitaxel are now widely used for this disease, corroboration in another cohort might enable us to improve the therapeutic ratio of this regimen. © 2013 Jilaveanu et al