A Tobacco Homolog of DCN1 is Involved in Cellular Reprogramming and in Developmental Transitions

Plant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein destruction regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called defective in cullin neddylation 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8, and associates with cullin. When knocked down by RNAi, tobacco pollen formation stopped and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1, however, accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is specifically required for transitions during pollen development and embryogenesis, and for pollen tube tip growth

Stability of Lysozyme in Aqueous Extremolyte Solutions during Heat Shock and Accelerated Thermal Conditions

The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin) under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectroscopy and a bioactivity assay. During heat shock (10 min at 70uC), betaine, trehalose, ectoin and firoin protected lysozyme against inactivation while hydroxyectoine, did not have a significant effect. During accelerated thermal conditions (4 weeks at 55uC), firoin also acted as a stabilizer. In contrast, betaine, hydroxyectoine, trehalose and ectoine destabilized lysozyme under this condition. These findings surprisingly indicate that some extremolytes can stabilize a protein under certain stress conditions but destabilize the same protein under other stress conditions. Therefore it is suggested that for the screening extremolytes to be used for protein stabilization, an appropriate storage conditions should also be taken into account

A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles

<p>Abstract</p> <p>Background</p> <p>Engineered nanoparticles are becoming increasingly ubiquitous and their toxicological effects on human health, as well as on the ecosystem, have become a concern. Since initial contact with nanoparticles occurs at the epithelium in the lungs (or skin, or eyes), <it>in vitro </it>cell studies with nanoparticles require dose-controlled systems for delivery of nanoparticles to epithelial cells cultured at the air-liquid interface.</p> <p>Results</p> <p>A novel air-liquid interface cell exposure system (ALICE) for nanoparticles in liquids is presented and validated. The ALICE generates a dense cloud of droplets with a vibrating membrane nebulizer and utilizes combined cloud settling and single particle sedimentation for fast (~10 min; entire exposure), repeatable (<12%), low-stress and efficient delivery of nanoparticles, or dissolved substances, to cells cultured at the air-liquid interface. Validation with various types of nanoparticles (Au, ZnO and carbon black nanoparticles) and solutes (such as NaCl) showed that the ALICE provided spatially uniform deposition (<1.6% variability) and had no adverse effect on the viability of a widely used alveolar human epithelial-like cell line (A549). The cell deposited dose can be controlled with a quartz crystal microbalance (QCM) over a dynamic range of at least 0.02-200 μg/cm². The cell-specific deposition efficiency is currently limited to 0.072 (7.2% for two commercially available 6-er transwell plates), but a deposition efficiency of up to 0.57 (57%) is possible for better cell coverage of the exposure chamber.</p> <p>Dose-response measurements with ZnO nanoparticles (0.3-8.5 μg/cm²) showed significant differences in mRNA expression of pro-inflammatory (IL-8) and oxidative stress (HO-1) markers when comparing submerged and air-liquid interface exposures. Both exposure methods showed no cellular response below 1 μg/cm²ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels.</p> <p>Conclusion</p> <p>The ALICE is a useful tool for dose-controlled nanoparticle (or solute) exposure of cells at the air-liquid interface. Significant differences between cellular response after ZnO nanoparticle exposure under submerged and air-liquid interface conditions suggest that pharmaceutical and toxicological studies with inhaled (nano-)particles should be performed under the more realistic air-liquid interface, rather than submerged cell conditions.</p

Mycobacterium avium subsp. paratuberculosis Proteome Changes Profoundly in Milk

Mycobacterium avium subspecies paratuberculosis (MAP) are detectable viable in milk and other dairy products. The molecular mechanisms allowing the adaptation of MAP in these products are still poorly understood. To obtain information about respective adaptation of MAP in milk, we differentially analyzed the proteomes of MAP cultivated for 48 h in either milk at 37 °C or 4 °C or Middlebrook 7H9 broth as a control. From a total of 2197 MAP proteins identified, 242 proteins were at least fivefold higher in abundance in milk. MAP responded to the nutritional shortage in milk with upregulation of 32% of proteins with function in metabolism and 17% in fatty acid metabolism/synthesis. Additionally, MAP upregulated clusters of 19% proteins with roles in stress responses and immune evasion, 19% in transcription/translation, and 13% in bacterial cell wall synthesis. Dut, MmpL4_1, and RecA were only detected in MAP incubated in milk, pointing to very important roles of these proteins for MAP coping with a stressful environment. Dut is essential and plays an exclusive role for growth, MmpL4_1 for virulence through secretion of specific lipids, and RecA for SOS response of mycobacteria. Further, 35 candidates with stable expression in all conditions were detected, which could serve as targets for detection. Data are available via ProteomeXchange with identifier PXD027444

Applications of Machine Learning in Palliative Care: A Systematic Review

Objective: To summarize the available literature on using machine learning (ML) for palliative care practice as well as research and to assess the adherence of the published studies to the most important ML best practices. Methods: The MEDLINE database was searched for the use of ML in palliative care practice or research, and the records were screened according to PRISMA guidelines. Results: In total, 22 publications using machine learning for mortality prediction (n = 15), data annotation (n = 5), predicting morbidity under palliative therapy (n = 1), and predicting response to palliative therapy (n = 1) were included. Publications used a variety of supervised or unsupervised models, but mostly tree-based classifiers and neural networks. Two publications had code uploaded to a public repository, and one publication uploaded the dataset. Conclusions: Machine learning in palliative care is mainly used to predict mortality. Similarly to other applications of ML, external test sets and prospective validations are the exception

Applications of Machine Learning in Palliative Care: A Systematic Review.

Objective: To summarize the available literature on using machine learning (ML) for palliative care practice as well as research and to assess the adherence of the published studies to the most important ML best practices. Methods: The MEDLINE database was searched for the use of ML in palliative care practice or research, and the records were screened according to PRISMA guidelines. Results: In total, 22 publications using machine learning for mortality prediction (n = 15), data annotation (n = 5), predicting morbidity under palliative therapy (n = 1), and predicting response to palliative therapy (n = 1) were included. Publications used a variety of supervised or unsupervised models, but mostly tree-based classifiers and neural networks. Two publications had code uploaded to a public repository, and one publication uploaded the dataset. Conclusions: Machine learning in palliative care is mainly used to predict mortality. Similarly to other applications of ML, external test sets and prospective validations are the exception

Probiotic Bacillus cereus Strains, a Potential Risk for Public Health in China

Bacillus cereus is an important cause of foodborne infectious disease and food poisoning. However, B. cereus has also been used as a probiotic in human medicine and livestock production, with low standards of safety assessment. In this study, we evaluated the safety of 15 commercial probiotic B. cereus preparations from China in terms of mislabeling, toxin production, and transferable antimicrobial resistance. Most preparations were incorrectly labeled, as they contained additional bacterial species;one product did not contain viable B. cereus at all. In total, 18 B. cereus group strains-specifically B. cereus and Bacillus thuringiensis were isolated. Enterotoxin genes nhe, hbl, and cytK1, as well as the ces-gene were assessed by PCR. Enterotoxin production and cytotoxicity were confirmed by ELISA and cell culture assays, respectively. All isolated B. cereus group strains produced the enterotoxin Nhe;15 strains additionally produced Hbl. Antimicrobial resistance was assessed by microdilution;resistance genes were detected by PCR and further characterized by sequencing, transformation and conjugation assays. Nearly half of the strains harbored the antimicrobial resistance gene tet(45). In one strain, tet(45) was situated on a mobile genetic element-encoding a site-specific recombination mechanism and was transferable to Staphylococcus aureus and Bacillus subtilis by electro-transformation. In view of the wide and uncontrolled use of these products, stricter regulations for safety assessment, including determination of virulence factors and transferable antimicrobial resistance genes, are urgently needed

Thyroid Receptor Antibody and the Development of Graves’ Orbitopathy: Clinical Experience of using Radioiodine Ablation in the Management of Graves’ Orbitopathy in post-iodine ablation hypothyroid patient

Graves’ disease (GD) is the commonest cause of hyperthyroidism, accounted for 70-80% in iodine sufficient countries and up to 50% in iodine deficient countries. Combination of genetic predisposition and environmental factors influences the development of GD. Graves’ orbitopathy (GO) represents the most common extra-thyroidal manifestation of GD with substantial impact on morbidity and quality of life. Expression of thyroid stimulating hormone receptor (TSHR) mRNA and protein in orbital tissues infiltrated by the activated lymphocytes produced by thyroid cells (Thyroid Receptor Antibody) results in the secretion of inflammatory cytokines that leads to the development of histological and clinical characteristics of GO. A subdivision of TRAb, thyroid stimulating antibody (TSAb), was found to have a close relationship with the activity and severity of GO, and suggested to be considered as a direct parameter of GO. Here, we present a 75-year-old female with a history of GD that has successfully been treated with radioiodine treatment, who developed GO 13 months after therapy while being hypothyroid with high TRAb level. The patient was given a second dose of radioiodine ablation to maintain GO with successful result

Temporal variability of lagoon–sea water exchange and seawater circulation through a Mediterranean barrier beach

The subterranean flow of water through sand barriers between coastal lagoons and the sea, driven by a positive hydraulic gradient, is a net new pathway for solute transfer to the sea. On the sea side of sand barriers, seawater circulation in the swash-zone generates a flux of recycled and new solutes. The significance and temporal variability of these vectors to the French Mediterranean Sea is unknown, despite lagoons constituting ~ 50% of the coastline. A one-dimensional 224Raex/223Ra reactive-transport model was used to quantify water flow between a coastal lagoon (La Palme) and the sea over a 6-month period. Horizontal flow between the lagoon and sea decreased from ~ 85 cm d−1 during May 2017 (0.3 m3 d−1 m−1 of shoreline) to ~ 20 cm d−1 in July and was negligible in the summer months thereafter due to a decreasing hydraulic gradient. Seawater circulation in the swash-zone varied from 10 to 52 cm d−1 (0.4–2.1 m3 d−1 m−1), driven by short-term changes in the prevailing wind and wave regimes. Both flow paths supply minor dissolved silica fluxes on the order of ~ 3–10 mmol Si d−1 m−1. Lagoon–sea water exchange supplies a net dissolved inorganic carbon (DIC) flux (320–1100 mmol C d−1 m−1) two orders of magnitude greater than seawater circulation and may impact coastal ocean acidification. The subterranean flow of water through sand barriers represents a significant source of new DIC, and potentially other solutes, to the Mediterranean Sea during high lagoon water-level periods and should be considered in seasonal element budgets

Essential role of Notch signaling in effector memory CD8+ T cell–mediated airway hyperresponsiveness and inflammation

Adoptive transfer of in vivo–primed CD8+ T cells or in vitro–generated effector memory CD8+ T (TEFF) cells restores airway hyperresponsiveness (AHR) and airway inflammation in CD8-deficient (CD8−/−) mice. Examining transcription levels, there was a strong induction of Notch1 in TEFF cells compared with central memory CD8+ T cells. Treatment of TEFF cells with a γ-secretase inhibitor (GSI) strongly inhibited Notch signaling in these cells, and after adoptive transfer, GSI-treated TEFF cells failed to restore AHR and airway inflammation in sensitized and challenged recipient CD8−/− mice, or to enhance these responses in recipient wild-type (WT) mice. These effects of GSI were also associated with increased expression of the Notch ligand Delta1 in TEFF cells. Treatment of sensitized and challenged WT mice with Delta1-Fc resulted in decreased AHR and airway inflammation accompanied by higher levels of interferon γ in bronchoalveolar lavage fluid. These results demonstrate a role for Notch in skewing the T cell response from a T helper (Th)2 to a Th1 phenotype as a consequence of the inhibition of Notch receptor activation and the up-regulation of the Notch ligand Delta1. These data are the first to show a functional role for Notch in the challenge phase of CD8+ T cell–mediated development of AHR and airway inflammation, and identify Delta1 as an important regulator of allergic airway inflammation