The Mechanism of ER to Cytosol Retrotranslocation of Proteins Targeted to ERAD (ER Associated Degradation)

Retro-translocation from the endoplasmic reticulum (ER) to the cytosol of secretory and membrane proteins takes place on misfolded molecules targeted for proteasomal degradation, in a process called ER associated degradation (ERAD). Because of the difficulties in clearly discriminating the fraction of molecules already retro-translocated from the ones in the ER, we took advantage of the E. coli biotin-ligase (BirA) expressed in the cytosol of mammalian cells, to specifically biotin-label proteins that undergo retro-translocation. The method was validated using four different model proteins, known to undergo retro-translocation upon different conditions: the MHC-1α chain, the non-secretory null-Hong Kong mutant of α1 antitrypsin, the immunoglobulin γH chain and calreticulin. The specific mono-biotinylation of only cytosolically dislocated molecules resulted in a novel quantitative method to determine the extent of retro-translocation. The method was used to study dislocation of CD4 and BST-2/Tetherin, two membrane proteins targeted to degradation by the HIV-1 protein Vpu. It was found that CD4 retro-translocates with oxidised intra-chain disulphide bridges that only upon proteasomal inhibition accumulates in the cytosol in reduced and de-glycosylated form. Similarly, BST-2/Tetherin is first exposed to the cytosol as a dimeric-oxidised complex, which then becomes de-glycosylated and reduced to monomers. Experiments with the non-secreted NS1 Ig-κ light chain showed that also this ERAD model protein is retro-translocated with oxidised cysteines. The role of VCP/p97-ATPase in retro-translocation was investigated. In contrast to what previously reported, it was found that it is not required to dislocate ERAD substrates from the ER lumen to the cytosol, while it is required for efficient de-glycosylation and proteasomal degradation of ERAD substrates. The results obtained indicate that complete cysteine reduction and unfolding is not strictly required for retro-translocation, suggesting alternative mechanisms of the ERAD pathway. In addition, the role of VCP/p97-ATPase was found associated to stages downstream of membrane crossing

A Possible Role of Copernicus Sentinel-2 Data to Support Common Agricultural Policy Controls in Agriculture

Farmers that intend to access Common Agricultural Policy (CAP) contributions must submit an application to the territorially competent Paying Agencies (PA). Agencies are called to verify consistency of CAP contributions requirements through ground campaigns. Recently, EU regulation (N. 746/2018) proposed an alternative methodology to control CAP applications based on Earth Observation data. Accordingly, this work was aimed at designing and implementing a prototype of service based on Copernicus Sentinel-2 (S2) data for the classification of soybean, corn, wheat, rice, and meadow crops. The approach relies on the classification of S2 NDVI time-series (TS) by “user-friendly” supervised classification algorithms: Minimum Distance (MD) and Random Forest (RF). The study area was located in the Vercelli province (NW Italy), which represents a strategic agricultural area in the Piemonte region. Crop classes separability proved to be a key factor during the classification process. Confusion matrices were generated with respect to ground checks (GCs); they showed a high Overall Accuracy (>80%) for both MD and RF approaches. With respect to MD and RF, a new raster layer was generated (hereinafter called Controls Map layer), mapping four levels of classification occurrences, useful for administrative procedures required by PA. The Control Map layer highlighted that only the eight percent of CAP 2019 applications appeared to be critical in terms of consistency between farmers’ declarations and classification results. Only for these ones, a GC was warmly suggested, while the 12% must be desirable and the 80% was not required. This information alone suggested that the proposed methodology is able to optimize GCs, making possible to focus ground checks on a limited number of fields, thus determining an economic saving for PA and/or a more effective strategy of controls

The Importance of Agronomic Knowledge for Crop Detection by Sentinel-2 in the CAP Controls Framework: A Possible Rule-Based Classification Approach

Farmers are supported by European Union (EU) through contributions related to the common agricultural policy (CAP). To obtain grants, farmers have to apply every year according to the national/regional procedure that, presently, relies on the Geo-Spatial Aid Application (GSAA). To ensure the properness of applications, national/regional payment agencies (PA) operate random controls through in-field surveys. EU regulation n. 809/2014 has introduced a new approach to CAP controls based on Copernicus Sentinel-2 (S2) data. These are expected to better address PA checks on the field, suggesting eventual inconsistencies between satellite-based deductions and farmers’ declarations. Within this framework, this work proposed a hierarchical (HI) approach to the classification of crops (soya, corn, wheat, rice, and meadow) explicitly aimed at supporting CAP controls in agriculture, with special concerns about the Piemonte Region (NW Italy) agricultural situation. To demonstrate the effectiveness of the proposed approach, a comparison is made between HI and other, more ordinary approaches. In particular, two algorithms were considered as references: the minimum distance (MD) and the random forest (RF). Tests were operated in a study area located in the southern part of the Vercelli province (Piemonte), which is mainly devoted to agriculture. Training and validation steps were performed for all the classification approaches (HI, MD, RF) using the same ground data. MD and RF were based on S2-derived NDVI image time series (TS) for the 2020 year. Differently, HI was built according to a rule-based approach developing according to the following steps: (a) TS standard deviation analysis in the time domain for meadows mapping; (b) MD classification of winter part of TS in the time domain for wheat detection; (c) MD classification of summer part of TS in the time domain for corn classification; (d) selection of a proper summer multi-spectral image (SMSI) useful for separating rice from soya with MD operated in the spectral domain. To separate crops of interest from other classes, MD-based classifications belonging to HI were thresholded by Otsu’s method. Overall accuracy for MD, RF, and HI were found to be 63%, 80%, and 89%, respectively. It is worth remarking that thanks to the SMSI-based approach of HI, a significant improvement was obtained in soya and rice classification

Piezoelectric energy harvesting solutions

This paper reviews the state of the art in piezoelectric energy harvesting. It presents the basics of piezoelectricity and discusses materials choice. The work places emphasis on material operating modes and device configurations, from resonant to non-resonant devices and also to rotational solutions. The reviewed literature is compared based on power density and bandwidth. Lastly, the question of power conversion is addressed by reviewing various circuit solutions

Enabling personalised medical support for chronic disease management through a hybrid robot-cloud approach

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Mechanism-based traps enable protease and hydrolase substrate discovery.

Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes

Mechanism-based traps enable protease and hydrolase substrate discovery.

Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes

VSV-G-Enveloped Vesicles for Traceless Delivery of CRISPR-Cas9

The method of delivery of CRISPR-Cas9 into target cells is a strong determinant of efficacy and specificity in genome editing. Even though high efficiency of Cas9 delivery is necessary for optimal editing, its long-term and high levels of expression correlate with increased off-target activity. We developed vesicles (VEsiCas) carrying CRISPR-SpCas9 ribonucleoprotein complexes (RNPs) that are efficiently delivered into target cells through the fusogenic glycoprotein of the vesicular stomatitis virus (VSV-G). A crucial step for VEsiCas production is the synthesis of the single guide RNA (sgRNA) mediated by the T7 RNA polymerase in the cytoplasm of producing cells as opposed to canonical U6-driven Pol III nuclear transcription. In VEsiCas, the absence of DNA encoding SpCas9 and sgRNA allows rapid clearance of the nuclease components in target cells, which correlates with reduced genome-wide off-target cleavages. Compared with SpCas9 RNPs electroporation, which is currently the method of choice to obtain transient SpCas9 activity, VEsiCas deliver the nuclease with higher efficiency and lower toxicity. We show that a wide variety of cells can be edited through VEsiCas, including a variety of transformed cells, induced pluripotent stem cells (iPSCs), and cardiomyocytes, in vivo. VEsiCas is a traceless CRISPR-Cas9 delivery tool for efficient and safe genome editing that represents a further advancement toward the therapeutic use of the CRISPR-Cas9 technology

Mechanism-based traps enable protease and hydrolase substrate discovery.

Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes