A large neighbourhood based heuristic for two-echelon routing problems

In this paper, we address two optimisation problems arising in the context of city logistics and two-level transportation systems. The two-echelon vehicle routing problem and the two-echelon location routing problem seek to produce vehicle itineraries to deliver goods to customers, with transits through intermediate facilities. To efficiently solve these problems, we propose a hybrid metaheuristic which combines enumerative local searches with destroy-and-repair principles, as well as some tailored operators to optimise the selections of intermediate facilities. We conduct extensive computational experiments to investigate the contribution of these operators to the search performance, and measure the performance of the method on both problem classes. The proposed algorithm finds the current best known solutions, or better ones, for 95% of the two-echelon vehicle routing problem benchmark instances. Overall, for both problems, it achieves high-quality solutions within short computing times. Finally, for future reference, we resolve inconsistencies between different versions of benchmark instances, document their differences, and provide them all online in a unified format

CD40, an extracellular receptor for binding and uptake of Hsp70–peptide complexes

Tumor and viral antigens elicit a potent immune response by heat shock protein–dependent uptake of antigenic peptide with subsequent presentation by MHC I. Receptors on antigen-presenting cells that specifically bind and internalize a heat shock protein–peptide complex have not yet been identified. Here, we show that cells expressing CD40, a cell surface protein crucial for B cell function and autoimmunity, specifically bind and internalize human Hsp70 with bound peptide. Binding of Hsp70–peptide complex to the exoplasmic domain of CD40 is mediated by the NH2-terminal nucleotide–binding domain of Hsp70 in its ADP state. The Hsp70 cochaperone Hip, but not the bacterial Hsp70 homologue DnaK, competes formation of the Hsp70–CD40 complex. Binding of Hsp70-ADP to CD40 is strongly increased in the presence of Hsp70 peptide substrate, and induces signaling via p38. We suggest that CD40 is a cochaperone-like receptor mediating the uptake of exogenous Hsp70–peptide complexes by macrophages and dendritic cells

Hsp90: a specialized but essential protein-folding tool

Hsp90 is unique among molecular chaperones. The majority of its known substrates are signal transduction proteins, and recent work indicates that it uses a novel protein-folding strategy

Formation of toxic oligomers of polyQ-expanded Huntingtin by prion-mediated cross-seeding

Manifestation of aggregate pathology in Huntington's disease is thought to be facilitated by a preferential vulnerability of affected brain cells to age-dependent proteostatic decline. To understand how specific cellular backgrounds may facilitate pathologic aggregation, we utilized the yeast model in which polyQ-expanded Huntingtin forms aggregates only when the endogenous prion-forming protein Rnq1 is in its amyloid-like prion [PIN+] conformation. We employed optogenetic clustering of polyQ protein as an orthog-onal method to induce polyQ aggregation in prion-free [pin-] cells. Optogenetic aggregation circumvented the prion requirement for the formation of detergent-resistant polyQ inclusions but bypassed the formation of toxic polyQ oligomers, which accumulated specifically in [PIN+] cells. Reconstitution of aggregation in vitro suggested that these polyQ oligomers formed through direct templating on Rnq1 prions. These findings shed light on the mechanism of prion-mediated formation of oligomers, which may play a role in triggering polyQ pathology in the patient brain

Multiple pathways of toxicity induced by C9orf72 dipeptide repeat aggregates and G4C2 RNA in a cellular model

The most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia is a G4C2 repeat expansion in the C9orf72 gene. This expansion gives rise to translation of aggregating dipeptide repeat (DPR) proteins, including poly-GA as the most abundant species. However, gain of toxic function effects have been attributed to either the DPRs or the pathological G4C2 RNA. Here, we analyzed in a cellular model the relative toxicity of DPRs and RNA. Cytoplasmic poly-GA aggregates, generated in the absence of G4C2 RNA, interfered with nucleocytoplasmic protein transport, but had little effect on cell viability. In contrast, nuclear poly-GA was more toxic, impairing nucleolar protein quality control and protein biosynthesis. Production of the G4C2 RNA strongly reduced viability independent of DPR translation and caused pronounced inhibition of nuclear mRNA export and protein biogenesis. Thus, while the toxic effects of G4C2 RNA predominate in the cellular model used, DPRs exert additive effects that may contribute to pathology

Sis1 potentiates the stress response to protein aggregation and elevated temperature

Cells adapt to conditions that compromise protein conformational stability by activating various stress response pathways, but the mechanisms used in sensing misfolded proteins remain unclear. Moreover, aggregates of disease proteins often fail to induce a productive stress response. Here, using a yeast model of polyQ protein aggregation, we identified Sis1, an essential Hsp40 co-chaperone of Hsp70, as a critical sensor of proteotoxic stress. At elevated levels, Sis1 prevented the formation of dense polyQ inclusions and directed soluble polyQ oligomers towards the formation of permeable condensates. Hsp70 accumulated in a liquid-like state within this polyQ meshwork, resulting in a potent activation of the HSF1 dependent stress response. Sis1, and the homologous DnaJB6 in mammalian cells, also regulated the magnitude of the cellular heat stress response, suggesting a general role in sensing protein misfolding. Sis1/DnaJB6 functions as a limiting regulator to enable a dynamic stress response and avoid hypersensitivity to environmental changes. Identifying factors that enable cells to induce a potent stress response to amyloid-like aggregation can provide further insight into the mechanism of stress regulation. Here, the authors express polyglutamine-expanded Huntingtin as a model disease protein in yeast cells and perform a genetic screen for chaperone factors that allow yeast cells to activate a potent stress response. They identify Sis1, an essential Hsp40 co-chaperone of Hsp70, as a critical sensor of proteotoxic stress and further show that both Sis1 and its mammalian homolog DnaJB6 regulate the magnitude of the cellular heat stress response, indicating that this mechanism is conserved

The Native 3D Organization of Bacterial Polysomes

SummaryRecent advances have led to insights into the structure of the bacterial ribosome, but little is known about the 3D organization of ribosomes in the context of translating polysomes. We employed cryoelectron tomography and a template-matching approach to map 70S ribosomes in vitrified bacterial translation extracts and in lysates of active E. coli spheroplasts. In these preparations, polysomal arrangements were observed in which neighboring ribosomes are densely packed and exhibit preferred orientations. Analysis of characteristic examples of polysomes reveals a staggered or pseudohelical organization of ribosomes along the mRNA trace, with the transcript being sequestered on the inside, the tRNA entrance sites being accessible, and the polypeptide exit sites facing the cytosol. Modeling of elongating nascent polypeptide chains suggests that this arrangement maximizes the distance between nascent chains on adjacent ribosomes, thereby reducing the probability of intermolecular interactions that would give rise to aggregation and limit productive folding