Erasing marks: Functions of plant deubiquitylating enzymes in modulating the ubiquitin code.

Plant cells need to respond to environmental stimuli and developmental signals accurately and promptly. Ubiquitylation is a reversible posttranslational modification that enables the adaptation of cellular proteostasis to internal or external factors. The different topologies of ubiquitin linkages serve as the structural basis for the ubiquitin code, which can be interpreted by ubiquitin-binding proteins or readers in specific processes. The ubiquitylation status of target proteins is regulated by ubiquitylating enzymes or writers, and deubiquitylating enzymes (DUBs) or erasers. DUBs can remove ubiquitin molecules from target proteins. Arabidopsis (A. thaliana) DUBs belong to seven protein families and exhibit a wide range of functions and play an important role in regulating selective protein degradation processes, including proteasomal-, endocytic-, and autophagic protein degradation. DUBs also shape the epigenetic landscape and modulate DNA damage repair processes. In this review, we summarize the current knowledge on DUBs in plants, their cellular functions, and the regulatory mechanisms involved in the spatiotemporal regulation of plant DUBs.

Deubiquitylating enzymes in Arabidopsis thaliana endocytic protein degradation.

The regulation of ubiquitylation is key for plant growth and development, in which the activities of ubiquitylating enzymes as well as deubiquitylating enzymes (DUBs) determine the stability or function of the modified proteins. In contrast with ubiquitylating enzymes, there are less numbers of DUBs. DUBs can be classified into seven protein families according to the amino acid sequence of their catalytic domains. The catalytic domains of animal and plant DUB families show high homology, whereas the regions outside of the catalytic site can vary a lot. By hydrolyzing the ubiquitin molecules from ubiquitylated proteins, DUBs control ubiquitin-dependent selective protein degradation pathways such as the proteasomal-, autophagic-, and endocytic degradation pathways. In the endocytic degradation pathway, DUBs can modulate the endocytic trafficking and thus the stability of plasma membrane proteins including receptors and transporters. To date, three DUB families were shown to control the endocytic degradation pathway namely associated molecule with the SH3 domain of STAM (AMSH) 3, ubiquitin-specific protease (UBP) 12 and UBP13, and ovarian tumor protease (OTU) 11 and OTU12. In this review we will summarize the activity, molecular functions, and target protein of these DUBs and how they contribute to the environmental response of plants.

Home-based (virtual) rehabilitation improves motor and cognitive function for stroke patients: a randomized controlled trial of the Elements (EDNA-22) system.

BACKGROUND: Home-based rehabilitation of arm function is a significant gap in service provision for adult stroke. The EDNA-22 tablet is a portable virtual rehabilitation-based system that provides a viable option for home-based rehabilitation using a suite of tailored movement tasks, and performance monitoring via cloud computing data storage. The study reported here aimed to compare use of the EDNA system with an active control (Graded Repetitive Arm Supplementary Program-GRASP training) group using a parallel RCT design. METHODS: Of 19 originally randomized, 17 acute-care patients with upper-extremity dysfunction following unilateral stroke completed training in either the treatment (n = 10) or active control groups (n = 7), each receiving 8-weeks of in-home training involving 30-min sessions scheduled 3-4 times weekly. Performance was assessed across motor, cognitive and functional behaviour in the home. Primary motor measures, collected by a blinded assessor, were the Box and Blocks Task (BBT) and 9-Hole Pegboard Test (9HPT), and for cognition the Montreal Cognitive Assessment (MoCA). Functional behaviour was assessed using the Stroke Impact Scale (SIS) and Neurobehavioural Functioning Inventory (NFI). RESULTS: One participant from each group withdrew for personal reasons. No adverse events were reported. Results showed a significant and large improvement in performance on the BBT for the more-affected hand in the EDNA training group, only (g = 0.90). There was a mild-to-moderate effect of training on the 9HPT for EDNA (g = 0.55) and control (g = 0.42) groups, again for the more affected hand. In relation to cognition, performance on the MoCA improved for the EDNA group (g = 0.70). Finally, the EDNA group showed moderate (but non-significant) improvement in functional behaviour on the SIS (g = 0.57) and NFI (g = 0.49). CONCLUSION: A short course of home-based training using the EDNA-22 system can yield significant gains in motor and cognitive performance, over and above an active control training that also targets upper-limb function. Intriguingly, these changes in performance were corroborated only tentatively in the reports of caregivers. We suggest that future research consider how the implementation of home-based rehabilitation technology can be optimized. We contend that self-administered digitally-enhanced training needs to become part of the health literacy of all stakeholders who are impacted by stroke and other acquired brain injuries. Trial registration Australian New Zealand Clinical Trials Registry (ANZCTR) Number: ACTRN12619001557123. Registered 12 November 2019, http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=378298&isReview=true.

Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3.

Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I-binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta, indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.

Upper limb motor training using a Saebo™ orthosis is feasible for increasing task-specific practice in hospital after stroke.

BACKGROUND/AIM: Assistive technologies have the potential to increase the amount of movement practice provided during inpatient stroke rehabilitation. The primary aim of this study was to investigate the feasibility of using the Saebo-Flex™ device in a subacute stroke setting to increase task-specific practice for people with little or no active hand movement. The secondary aim was to collect preliminary data comparing hand/upper limb function between a control group that received usual rehabilitation and an intervention group that used, in addition, the Saebo-Flex™ device. METHODS: Nine inpatients (mean three months (median six weeks) post-stroke) participated in this feasibility study conducted in an Australian rehabilitation setting, using a randomised pre-test and post-test design with concealed allocation and blinded outcome assessment. In addition to usual rehabilitation, the intervention group received eight weeks of daily motor training using the Saebo-Flex™ device. The control group received usual rehabilitation (task-specific motor training) only. Participants were assessed at baseline (pre-randomisation) and at the end of the eight-week study period. Feasibility was assessed with respect to ease of recruitment, application of the device, compliance with the treatment programme and safety. Secondary outcome measures included the Motor Assessment Scale (upper limb items), Box and Block Test, grip strength and the Stroke Impact Scale. RESULTS: Recruitment to the study was very slow because of the low number of patients with little or no active hand movement. Otherwise, the study was feasible in terms of being able to apply the Saebo-Flex™ device and compliance with the treatment programme. There were no adverse events, and a greater amount of upper limb rehabilitation was provided to the intervention group. While there were trends in favour of the intervention group, particularly for dexterity, no between-group differences were seen for any of the secondary outcomes. CONCLUSIONS: This pilot feasibility study showed that the use of assistive technology, specifically the Saebo-Flex™ device, could be successfully used in a sample of stroke patients with little or no active hand movement. However, recruitment to the trial was very slow. The use of the Saebo-FlexTM device had variable results on outcomes, with some positive trends seen in hand function, particularly dexterity.

Combined cytotoxic activity of an infectious, but non-replicative herpes simplex virus type 1 and plasmacytoid dendritic cells against tumour cells.

Malignant melanoma is an aggressive tumour of the skin with increasing incidence, frequent metastasis and poor prognosis. At the same time, it is an immunogenic type of cancer with spontaneous regressions. Most recently, the tumoricidal effect of plasmacytoid dendritic cells (pDC) and their capacity to overcome the immunosuppressive tumour microenvironment are being investigated. In this respect, we studied the effect of the infectious, but replication-deficient, herpes simplex virus 1 (HSV-1) d106S vaccine strain, which lacks essential immediate early genes, in pDC co-cultures with 11 melanoma cell lines. We observed a strong cytotoxic activity, inducing apoptotic and necrotic cell death in most melanoma cell lines. The cytotoxic activity of HSV-1 d106S plus pDC was comparable to the levels of cytotoxicity induced by natural killer cells, but required only a fraction of cells with effector : target ratios of 1 : 20 (P < 0·05). The suppressive activity of cell-free supernatants derived from virus-stimulated pDC was significantly neutralized using antibodies against the interferon-α receptor (P < 0·05). In addition to type I interferons, TRAIL and granzyme B contributed to the inhibitory effect of HSV-1 d106S plus pDC to a minor extent. UV-irradiated viral stocks were significantly less active than infectious particles, both in the absence and presence of pDC (P < 0·05), indicating that residual activity of HSV-1 d106S is a major component and sensitizes the tumour cells to interferon-producing pDC. Three leukaemic cell lines were also susceptible to this treatment, suggesting a general anti-tumour effect. In conclusion, the potential of HSV-1 d106S for therapeutic vaccination should be further evaluated in patients suffering from different malignancies.

Both plasmacytoid dendritic cells and monocytes stimulate natural killer cells early during human herpes simplex virus type 1 infections.

Herpes simplex virus type 1 (HSV-1), a member of the herpes virus family, is characterized by a short replication cycle, high cytopathogenicity and distinct neurotropism. Primary infection and reactivation may cause severe diseases in immunocompetent and immunosuppressed individuals. This study investigated the role of human plasmacytoid dendritic cells (pDC) in the activation of natural killer (NK) cells for the control of herpesviral infections. Within peripheral blood mononuclear cells, UV-inactivated HSV-1 and CpG-A induced CD69 up-regulation on NK cells, whereas infectious HSV-1 was particularly active in inducing NK cell effector functions interferon-γ (IFN-γ) secretion and degranulation. The pDC-derived IFN-α significantly contributed to NK cell activation, as evident from neutralization and cell depletion experiments. In addition, monocyte-derived tumour necrosis factor-α (TNF-α) induced after exposure to infectious HSV-1 was found to stimulate IFN-γ secretion. A minority of monocytes was shown to be non-productively infected in experiments using fluorescently labelled viruses and quantitative PCR analyses. HSV-1-exposed monocytes up-regulated classical HLA-ABC and non-classical HLA-E molecules at the cell surface in an IFN-α-dependent manner, whereas stress molecules MICA/B were not induced. Notably, depletion of monocytes reduced NK cell effector functions induced by infectious HSV-1 (P < 0.05). Altogether, our data suggest a model in which HSV-1-stimulated pDC and monocytes activate NK cells via secretion of IFN-α and TNF-α. In addition, infection of monocytes induces NK cell effector functions via TNF-α-dependent and TNF-α-independent mechanisms. Hence, pDC and monocytes, which are among the first cells infiltrating herpetic lesions, appear to have important bystander functions for NK cells to control these viral infections.

Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation.

Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca2+-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.

Measuring the DUB Activity of Arabidopsis Deubiquitylating Enzymes.

Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here we show methods to analyze DUB activity using immunodetection, Coomassie brilliant blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

Lipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking.

The abundance of plasma membrane-resident receptors and transporters has to be tightly regulated by ubiquitin-mediated endosomal degradation for the proper coordination of environmental stimuli and intracellular signaling. Arabidopsis OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 are plasma membrane-localized deubiquitylating enzymes (DUBs) that bind to phospholipids through a polybasic motif in the OTU domain. Here we show that the DUB activity of OTU11 and OTU12 towards K63-linked ubiquitin is stimulated by binding to lipid membranes containing anionic lipids. In addition, we show that the DUB activity of OTU11 against K6- and K11-linkages is also stimulated by anionic lipids, and that OTU11 and OTU12 can modulate the endosomal degradation of a model cargo and the auxin efflux transporter PIN2-GFP in vivo. Our results suggest that the catalytic activity of OTU11 and OTU12 is tightly connected to their ability to bind membranes and that OTU11 and OTU12 are involved in the fine-tuning of plasma membrane proteins in Arabidopsis.

Detection of Phosphorylation on Immunoprecipitates from Total Protein Extracts of Arabidopsis thaliana Seedlings.

Phosphorylation is a versatile posttranslational modification that can regulate the localization, stability, and conformation of proteins; protein-protein interactions; and enzyme activities. Phosphorylation of plasma membrane proteins, for example, can serve as recognition signals for ubiquitin ligases and hence can trigger its endocytic degradation. Key determinants of protein phosphorylation are kinases and phosphatases that are spatiotemporally regulated to phosphorylate or dephosphorylate specific target proteins. To understand the dynamics and regulatory mechanisms of protein phosphorylation, it is essential to analyze the phosphorylation status of the proteins and identify phosphorylation sites as well as the modifying enzymes. In this chapter, we describe methods that can be used for the detection of phosphoproteins that are immunoprecipitated from Arabidopsis total extracts.

Transient Expression of ESCRT Components in Arabidopsis Root Cell Suspension Culture-Derived Protoplasts.

Localization studies are important to understand the function of diverse proteins. The endosomal trafficking pathway is very complex, and a lot of proteins function in this pathway, primarily the endosomal sorting complexes required for transport (ESCRTs). Some of the ESCRT-related proteins or mutant variants cannot be stably expressed in planta due to the toxicity of their expression. Therefore, a transient expression system is necessary to study their function. Transient expression in protoplasts from Arabidopsis root cell-derived culture serves as a fast and reliable method for the expression and cell biological and biochemical analyses of otherwise toxic constructs.

Model-based magnetization retrieval from holographic phase images.

The phase shift of the electron wave is a useful measure for the projected magnetic flux density of magnetic objects at the nanometer scale. More important for materials science, however, is the knowledge about the magnetization in a magnetic nano-structure. As demonstrated here, a dominating presence of stray fields prohibits a direct interpretation of the phase in terms of magnetization modulus and direction. We therefore present a model-based approach for retrieving the magnetization by considering the projected shape of the nano-structure and assuming a homogeneous magnetization therein. We apply this method to FePt nano-islands epitaxially grown on a SrTiO3 substrate, which indicates an inclination of their magnetization direction relative to the structural easy magnetic [001] axis. By means of this real-world example, we discuss prospects and limits of this approach.

Effect of finishing and polishing procedures on surface roughness, gloss and color of resin-based composites.

PURPOSE: To evaluate the effects of different finishing and polishing procedures on surface roughness, gloss and color of five resin composites: two experimental microhybrid composites - FZ-Dentin (FZD) and FZ-Enamel (FZE), one commercial microhybrid composite - Esthet-X (EX), and two microfilled composites - Heliomolar (HM) and Renamel Microfill (RM). METHODS: Surface roughness, gloss and color of the disc-shaped specimens (10 mm in diameter and 2-mm thick) were measured as Mylar (baseline), 16-fluted carbide bur and polishing were completed. Sixteen specimens of each composite were randomized to four groups of four. After finishing with a 16-fluted finishing bur, each group was polished by a different system: 1. Astropol (A), 2. Sof-lex disc (S), 3. Po-Go (P), 4. Enhance (E). Average surface roughness (Ra) was measured with a profilometer. Gloss measurements were performed using small-area glossmeter, while color coordinate values were recorded using a spectrophotometer. A deltaE*ab< or =1 was considered to be the limit of perceptibility. RESULTS: The order of surface roughness ranked according to polishing system (for all five composites together) was: P < S < E < A. The order of surface roughness ranked according to composites was: RM < FZD < FZ < HM < EX. The order of gloss ranked according to polishing system (for all five composites together) was: P > E > A > S. The order of gloss values for the polished composites (for each of four polishing systems) was: RM > FZD > FZE > HM > EX. Fisher's PLSD intervals at the 0.05 level of significance for comparisons of means of surface roughness among five composites and four polishing systems were 0.01 and 0.01 microm, respectively. Fisher's PLSD intervals at the 0.05 level of significance for comparisons of means of gloss among five composites and four polishing systems were 6 and 5 GU, respectively. Color differences (deltaE*ab) among five composites and four polishing methods were found to range from 0.2 to 1.1.

Electron holography for fields in solids: problems and progress.

Electron holography initially was invented by Dennis Gabor for solving the problems raised by the aberrations of electron lenses in Transmission Electron Microscopy. Nowadays, after hardware correction of aberrations allows true atomic resolution of the structure, for comprehensive understanding of solids, determination of electric and magnetic nanofields is the most challenging task. Since fields are phase objects in the TEM, electron holography is the unrivaled method of choice. After more than 40 years of experimental realization and steady improvement, holography is increasingly contributing to these highly sophisticated and essential questions in materials science, as well to the understanding of electron waves and their interaction with matter.

Effect of resin-composite filler particle size and shape on shrinkage-stress.

OBJECTIVES: The aim of this study was to investigate the effect of variations in filler particle size and shape on the polymerization shrinkage-stress kinetics of resin-composites. METHODS: A model series of 12 VLC resin-composites were studied. The particulate dispersed phase volume fraction was 56.7%: these filler particles were systematically graded in size, and further were either spherical or irregular. A Bioman instrument (cantilever beam method) was employed to determine the shrinkage-stress kinetics following 40s irradiation (600 mW/cm(2)) at 23°C (n=3). All data were captured for 60 min and the final shrinkage-stress calculated. RESULTS: Shrinkage-stress varied between 3.86 MPa (SD 0.14) for S3 (spherical filler particles of 500 nm) and 8.44 MPa (SD 0.41) for I1 (irregular filler particles of 450 nm). The shrinkage-stress values were generally lower for those composites with spherical filler particles than those with irregular filler particles. The differences in shrinkage-stress with filler particle size and shape were statistically significant (p<0.001). SIGNIFICANCE: Composites with spherical filler particles exhibit lower shrinkage-stress values compared to those with irregular filler particles. Shrinkage-stress and shrinkage-stress rate vary in a complex manner with variations in the size of the dispersed phase particles: a hypothesized explanation for the effect of filler particle size and shape is presented.

Effect of resin-composite filler particle size and shape on shrinkage-strain.

OBJECTIVES: The aim of this study was to investigate the effect of variations in filler particle size and shape on the polymerization shrinkage-strain kinetics of resin-composites. METHODS: A model series of 12 VLC resin-composites were studied. The particulate dispersed phase volume fraction was 56.7%: these filler particles were systematically graded in size, and further were either spherical or irregular. The bonded disk method was used to determine shrinkage-strain kinetics. Displacement was recorded following 40s irradiation (600mW/cm(2)) at 23 degrees C (n=3). All data were captured for 60min and the final shrinkage-strain calculated. RESULTS: For materials with spherical filler, shrinkage-strain was 2.66% (SD 0.18) for those with irregular filler it was 2.89% (SD 0.11). These differences were statistically significant (p<0.001): the Scheffé test identified two subsets, those with irregular filler (including materials with a multimodal mix) and those with spherical filler (including materials with a multimodal mix). Additionally, there was a trend for higher shrinkage-strain values with decreasing filler particle size which was apparent for both those composites with spherical filler particles and those with irregular filler particles. For irregular filler particles, linear regression gave a high correlation (r(2)=0.99). SIGNIFICANCE: Statistically significant differences were identified in the shrinkage behavior of resin-composites with differing filler size and shape.