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The work presents results of the in vitro and in silico study of formation of amyloid-like structures under harsh denaturing conditions by non-specific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with β-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with acidic pH at elevated temperature followed by long-term incubation at room temperature is necessary. After heating at 95°C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of tertiary and secondary structure of the protein, which are accompanied by the increase in the content of total β-structure and sharp decrease in the value of characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to the Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of the changes in intrinsic fluorescence during protein aggregation suggest that during formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only the areas with an internally disordered structure corresponding to the external loops of the porin, but also main framework of the molecule, which has a rigid spatial structure inherent to β-barrel.

Tumour-infiltrating lymphocytes are associated with a survival benefit in several tumour types and with the response to immunotherapy 1 – 8 . However, the reason some tumours have high CD8 T cell infiltration while others do not remains unclear. Here we investigate the requirements for maintaining a CD8 T cell response against human cancer. We find that CD8 T cells within tumours consist of distinct populations of terminally differentiated and stem-like cells. On proliferation, stem-like CD8 T cells give rise to more terminally differentiated, effector-molecule-expressing daughter cells. For many T cells to infiltrate the tumour, it is critical that this effector differentiation process occur. In addition, we show that these stem-like T cells reside in dense antigen-presenting-cell niches within the tumour, and that tumours that fail to form these structures are not extensively infiltrated by T cells. Patients with progressive disease lack these immune niches, suggesting that niche breakdown may be a key mechanism of immune escape. The authors examine the immune cell infiltrates of human tumours and provide evidence for a population of CD8 T cells with stem-cell characteristics and proliferative capacity that reside in an antigen-presenting niche within tumours.

Fibroblasts isolated and expanded from ReLEx SMILE lenticules can be a source of human keratocytes. Since corneal keratocytes are quiescent cells, it is difficult to expand them in vitro in suitable numbers for clinical and experimental use. In the present study, this problem was solved by isolating and growing corneal fibroblasts (CFs) with a high proliferative potential and their reversion to keratocytes in a selective serum-free medium. Fibroblasts reversed into keratocytes (rCFs) had a dendritic morphology and ultrastructural signs of activation of protein synthesis and metabolism. The cultivation of CFs in a medium with 10% FCS and their reversion into keratocytes was not accompanied by the induction of myofibroblasts. After reversion, the cells spontaneously formed spheroids and expressed keratocan and lumican markers, but not mesenchymal ones. The rCFs had low proliferative and migratory activity, and their conditioned medium contained a low level of VEGF. CF reversion was not accompanied by a change with the levels of IGF-1, TNF-alpha, SDF-1a, and sICAM-1. In the present study, it has been demonstrated that fibroblasts from ReLEx SMILE lenticules reverse into keratocytes in serum-free KGM, maintaining the morphology and functional properties of primary keratocytes. These keratocytes have a potential for tissue engineering and cell therapy of various corneal pathologies.

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn’s disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death. The authors define molecular mechanisms by which distinct domains of the ubiquitin editing enzyme A20 contribute to the regulation of inflammation and cell death. Anti-apoptotic/anti-inflammatory actions of protein A20 Compromised function of the ubiquitin-editing enzyme A20 contributes to various inflammatory syndromes and autoimmune diseases including rheumatoid arthritis, multiple sclerosis, Crohn's disease and psoriasis. Here Ingrid Wertz et al . define the molecular mechanisms by which distinct domains of A20 contribute to the regulation of inflammation and cell death.

Background Our preclinical work revealed tumour hypoxia induces homologous recombination deficiency (HRD), increasing sensitivity to Poly (ADP-ribose) polymerase inhibitors. We aimed to induce tumour hypoxia with ramucirumab thereby sensitising tumours to olaparib. Patients and methods This multi-institution single-arm Phase 1/2 trial enrolled patients with metastatic gastroesophageal adenocarcinoma refractory to ≥1 systemic treatment. In dose escalation, olaparib was evaluated at escalating dose levels with ramucirumab 8 mg/kg day 1 in 14-day cycles. The primary endpoint of Phase 1 was the recommended Phase 2 dose (RP2D), and in Phase 2 the primary endpoint was the overall response rate (ORR). Results Fifty-one patients received ramucirumab and olaparib. The RP2D was olaparib 300 mg twice daily with ramucirumab 8 mg/kg. In evaluable patients at the RP2D the ORR was 6/43 (14%) (95% CI 4.7–25.6). The median progression-free survival (PFS) was 2.8 months (95% CI 2.3–4.2) and median overall survival (OS) was 7.3 months (95% CI 5.7–13.0). Non-statistically significant improvements in PFS and OS were observed for patients with tumours with mutations in HRD genes. Conclusions Olaparib and ramucirumab is well-tolerated with efficacy that exceeds historical controls with ramucirumab single agent for gastric cancer in a heavily pre-treated patient population.

Nature 528, 370–375 (2015); doi: 10.1038/nature16165. In this Article, owing to a typesetter error the ‘received date’ was incorrectly shown as ‘5 November 2015’ instead of ‘5 November 2013’; this has been corrected in the online versions of the paper.