Tyrosine kinase inhibitors can activate the NLRP3 inflammasome in myeloid cells through lysosomal damage and cell lysis.

Inflammasomes are intracellular protein complexes that promote an inflammatory host defense in response to pathogens and damaged or neoplastic tissues and are implicated in inflammatory disorders and therapeutic-induced toxicity. We investigated the mechanisms of activation for inflammasomes nucleated by NOD-like receptor (NLR) protiens. A screen of a small-molecule library revealed that several tyrosine kinase inhibitors (TKIs)-including those that are clinically approved (such as imatinib and crizotinib) or are in clinical trials (such as masitinib)-activated the NLRP3 inflammasome. Furthermore, imatinib and masitinib caused lysosomal swelling and damage independently of their kinase target, leading to cathepsin-mediated destabilization of myeloid cell membranes and, ultimately, cell lysis that was accompanied by potassium (K+) efflux, which activated NLRP3. This effect was specific to primary myeloid cells (such as peripheral blood mononuclear cells and mouse bone marrow-derived dendritic cells) and did not occur in other primary cell types or various cell lines. TKI-induced lytic cell death and NLRP3 activation, but not lysosomal damage, were prevented by stabilizing cell membranes. Our findings reveal a potential immunological off-target of some TKIs that may contribute to their clinical efficacy or to their adverse effects.

Can adjunctive perioperative radiation improve range of motion after total knee revision for arthrofibrosis?

BACKGROUND: Total knee arthroplasty revision (TKRev) can be performed to treat chronic arthrofibrosis. Low-dose irradiation may decrease fibro-osseous proliferation of soft tissue; therefore, it may be effective at increasing range of motion (ROM) after TKRev. Our hypothesis is that low-dose radiation administered in the immediate postoperative period leads to increased ROM after TKRev for arthrofibrosis. METHODS: A retrospective analysis was conducted from 2008-2015 on 26 patients who underwent TKRev for treating chronic arthrofibrosis. Fifteen patients (XRT group) received 800 cGy radiation within 48 hours after TKRev and 11 patients (CTL group) did not. Measurements of extension, flexion, and total arc of ROM were performed preoperatively and at one, six, and 12 months postoperatively. RESULTS: ROM improved from 14.3° extension, 69.0° flexion, and 54.7° total ROM preoperatively, to 3.3° extension, 94.0° flexion, and 90.7° total ROM postoperatively in the XRT group. ROM improved from 18.6° extension, 85.9° flexion, and 67.3° total ROM preoperatively to 4.1° extension, 102.5° flexion, and 98.5° total ROM postoperatively in the CTL group. The 1-year overall improvement in extension (12.5°), flexion (21.4°), and total ROM (33.9°) vs preoperative ROM was significant for all measurements (p < 0.001). The 8.4° improvement in flexion (25.0° vs 16.6°, p = 0.10) in the XRT group vs the CTL group approached, but did not reach significance. CONCLUSIONS: TKRev for arthrofibrosis showed significant improvement in extension, flexion, and total ROM at one year. The use of low dose irradiation showed promise with improved flexion, but the result did not reach statistical significance in this small sample of patients.

Emerging drugs for the treatment of acromegaly.

INTRODUCTION: Acromegaly is a disease characterized by elevated growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels. Surgery is the only curative treatment, while medical therapies are administered life-long. To date, almost 30% of patients treated with the currently available medical therapies do not achieve biochemical control. AREAS COVERED: This review focuses on new drugs in development for acromegaly. In detail, we provide an overview of the new molecules designed to improve disease control rate (such as novel somotostatin receptor ligands and antisense oligonucleotides), as well as the new formulations of existing medications aiming to improve patients' compliance (e.g. oral or long-acting subcutaneous octreotide). EXPERT OPINION: The constant progresses in the medical treatment of acromegaly could lead to an individualized therapy based on tumor, as well as patient's characteristics. Besides disease control, patient's need represents a major target of medical treatment in chronic diseases such as acromegaly, in order to improve compliance to therapy and patients' quality of life.

Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis.

Thiol-based redox-regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin-dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione-mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo-lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (EGSH ) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal EGSH dynamics, we show that stromal EGSH is highly reducing in wild-type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.

Translation, adaptation, and reliability of the Stanford Integrated Psychosocial Assessment for Transplantation in the Spanish population.

BACKGROUND: Psychosocial risk factors influence the course of transplantation. Psychosocial evaluation is an important part of pre-transplantation evaluation processes, yet there are no standardized instruments in Spanish. OBJECTIVE: To translate, adapt, and test the reliability of the Stanford Integrated Psychosocial Assessment for Transplantation (SIPAT) in organ and cell transplantation patients in the Spanish context. METHOD: A Spanish version was developed and adapted using WHO's guidelines. The first 30 candidates' SIPAT interviews were recorded and scored by four independent examiners to test the inter-rater reliability. The internal consistency of the SIPAT items was calculated with a sample of 150 heart, liver, and allogeneic haematopoietic stem cell transplant candidates. Evaluations were conducted by SIPAT-trained and transplantation-experienced clinical psychologists and psychiatrists. RESULTS: Stanford Integrated Psychosocial Assessment for Transplantation achieved excellent intra-class correlation reliability coefficients between investigators (ICC = 0.93 for the general score and from 0.77 to 0.94 for domain scores). Good internal consistency was found with Cronbach's alpha of 0.84 (from 0.69-0.71 for domains). DISCUSSION: This study presents the translated and adapted version of SIPAT. It has been found to have strong inter-rater reliability and good internal consistency. Further research is needed to confirm reliability (eg, test-retest) and establish its validity (eg, concurrent, predictive).

Semi-automatic determination of cell surface areas used in systems biology.

Quantitative biology requires high precision measurement of cellular parameters such as surface areas or volumes. Here, we have developed an integrated approach in which the data from 3D confocal microscopy and 2D high-resolution transmission electron microscopy were combined. The volumes and diameters of the cells within one population were automatically measured from the confocal data sets. The perimeter of the cell slices was measured in the TEM images using a semi-automated segmentation into background, cytoplasm and nucleus. These data in conjunction with approaches from stereology allowed for an unbiased estimate of surface areas with high accuracy. We have determined the volumes and surface areas of the cells and nuclei of six different immune cell types. In mast cells for example, the resulting cell surface was 3.5 times larger than the theoretical surface assuming the cell was a sphere with the same volume. Thus, our accurate data can now serve as inputs in modeling approaches in systems immunology.

Formation of duodenal atresias in fibroblast growth factor receptor 2IIIb-/- mouse embryos occurs in the absence of an endodermal plug.

PURPOSE: Duodenal atresia in humans has been hypothesized to arise from a failure of the duodenal lumen to recanalize after formation of an endodermal plug. Recently, mutations in the fibroblast growth factor receptor 2 gene (Fgfr2IIIb) have been shown to cause atretic defects of the duodenum in mice. However, work in rats suggests that murine species do not form an endodermal plug during normal duodenal development. These lines of data led us to hypothesize that mice are able to form a duodenal atresia in the absence of an endodermal plug. To test this hypothesis, we examined duodenal development in wild-type and Fgfr2IIIb-/- embryos. METHODS: Paraffin sections were generated for H&E, E-cadherin, or terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling staining from Fgfr2IIIb-/- and wild-type embryos between embryonic days (E) 10.5 and E14.5. Sections were photographed and reconstructed into 3-dimensional display using Adobe Photoshop and Amira Visage software. RESULTS: Normal mouse duodenum does not form an endodermal plug, although a plug does form in the pyloric region of the stomach at E14.5. Fgfr2IIIb-/- embryos experience significant apoptosis in the duodenal region at E10.5, followed by the disappearance of the endoderm in the atretic precursor by E11.5. Thereafter, the mesoderm of the atretic precursor involutes over the next 2 days in the absence of further apoptosis. Interestingly, an endodermal plug was not observed at any point during the formation of a duodenal atresia. CONCLUSIONS: These results suggest that duodenal atresia in the Fgfr2IIIb-/- model does not arise from persistence of an epithelial plug. Rather it appears to result from the loss of the endoderm because of apoptosis very early in development.

Use of Physcomitrella patens actin 5' regions for high transgene expression: importance of 5' introns.

We have isolated four actin (Act) genes from Physcomitrella patens and used their corresponding 5' regions for recombinant expression of the human vascular endothelial growth factor (rhVEGF121) in transiently transformed Physcomitrella protoplasts and in stable transformed lines. In the transient system, we found up to 11-fold activity of the corresponding 5' regions as compared with that of the plant constitutive 35S promoter. Moreover, the use of an optimised expression vector in which the human VEGF signal peptide was exchanged with a plant signal peptide resulted in an additional 7-fold increase in secreted rhVEGF. We found that the 5' introns of PpAct1, PpAct5 and PpAct7 are essential for high expression. The enhancing mechanisms of the introns, however, seem to be different since in the case of PpAct1, the expression level is stimulated only in the presence of the endogenous promoter, whereas the 5' introns of PpAct5 and PpAct7 stimulate expression also in combination with the 35S promoter. Beyond this, the isolated 5' regions are shown to be useful for high expression levels in transgenic moss lines with values of secreted rhVEGF up to 96 microg g(-1) dry weight.

Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine.

Molecular chaperones and proteases monitor the folded state of other proteins. In addition to recognizing non-native conformations, these quality control factors distinguish substrates that can be refolded from those that need to be degraded. To investigate the molecular basis of this process, we have solved the crystal structure of DegP (also known as HtrA), a widely conserved heat shock protein that combines refolding and proteolytic activities. The DegP hexamer is formed by staggered association of trimeric rings. The proteolytic sites are located in a central cavity that is only accessible laterally. The mobile side-walls are constructed by twelve PDZ domains, which mediate the opening and closing of the particle and probably the initial binding of substrate. The inner cavity is lined by several hydrophobic patches that may act as docking sites for unfolded polypeptides. In the chaperone conformation, the protease domain of DegP exists in an inactive state, in which substrate binding in addition to catalysis is abolished.