Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

Asparaginyl endopeptidases (AEPs) are ideal for peptide and protein labeling. However, because of the reaction reversibility, a large excess of labels or backbone modified substrates are needed. In turn, simple and cheap reagents can be used to label N-terminal cysteine, but its availability inherently limits the potential applications. Aiming to address these issues, we have created a chemo-enzymatic labeling system that exploits the substrate promiscuity of AEP with the facile chemical reaction between N-terminal cysteine and 2-formyl phenylboronic acid (FPBA). In this approach, AEP is used to ligate polypeptides with a Asn–Cys–Leu recognition sequence with counterparts possessing an N-terminal Gly–Leu. Instead of being a labeling reagent, the commercially available FPBA serves as a scavenger converting the byproduct Cys–Leu into an inert thiazolidine derivative. This consequently drives the AEP labeling reaction forward to product formation with a lower ratio of label to protein substrate. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP-ligation/FPBA-coupling system was further demonstrated by site-specifically labeling the N- or C-termini of various proteins

Castration causes an increase in lysosomal size and upregulation of cathepsin D expression in principal cells along with increased secretion of procathepsin D and prosaposin oligomers in adult rat epididymis

In the epididymis, lysosomal proteins of the epithelial cells are normally targeted from the Golgi apparatus to lysosomes for degradation, although their secretion into the epididymal lumen has been documented and associated with sperm maturation. In this study, cathepsin D (CatD) and prosaposin (PSAP) were examined in adult epididymis of control, and 2-day castrated rats without (Ct) and with testosterone replacement (Ct+T) to evaluate their expression and regulation within epididymal epithelial cells. By light microscope-immunocytochemistry, a quantitative increase in size of lysosomes in principal cells of Ct animals was noted from the distal initial segment to the proximal cauda. Androgen replacement did not restore the size of lysosomes to control levels. Western blot analysis revealed a significant increase in CatD expression in the epididymis of Ct animals, which suggested an upregulation of its expression in principal cells; androgens restored levels of CatD to that of controls. In contrast, PSAP expression in Ct animals was not altered from controls. Additionally, an increase in procathepsin D levels was noted from samples of the epididymal fluid of Ct compared to control animals, accompanied by an increased complex formation with PSAP. Moreover, an increased oligomerization of prosaposin was observed in the epididymal lumen of Ct rats, with changes reverted to controls in Ct+T animals. Taken together these data suggest castration causes an increased uptake of substrates that are acted upon by CatD in lysosomes of principal cells and in the lumen by procathepsin D. These substrates may be derived from apoptotic cells noted in the lumen of proximal regions and possibly by degenerating sperm in distal regions of the epididymis of Ct animals. Exploring the mechanisms by which lysosomal enzymes are synthesized and secreted by the epididymis may help resolve some of the issues originating from epididymal dysfunctions with relevance to sperm maturation.Fil: Carvelli, Flavia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Aguilera, Andrea Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Zyla, Leila Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Pereyra, Laura Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Morales, Carlos R.. McGill University; CanadáFil: Hermo, Louis. McGill University; CanadáFil: Sosa Escudero, Miguel Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentin

The Quasi-Verbal Dispute Between Kripke and 'Frege-Russell'

Traditional descriptivism and Kripkean causalism are standardly interpreted as rival theories on a single topic. I argue that there is no such shared topic, i.e. that there is no question that they can be interpreted as giving rival answers to. The only way to make sense of the commitment to epistemic transparency that characterizes traditional descriptivism is to interpret Russell and Frege as proposing rival accounts of how to characterize a subject’s beliefs about what names refer to. My argument relies on a development of the distinction between speaker’s reference and semantic reference

Application of texture analysis to muscle MRI: 1-What kind of information should be expected from texture analysis?

Several previous clinical or preclinical studies using computerized texture analysis of MR Images have demonstrated much more clinical discrimination than visual image analysis by the radiologist. In muscular dystrophy, a discriminating power has been already demonstrated with various methods of texture analysis of magnetic resonance images (MRI-TA). Unfortunately, a scale gap exists between the spatial resolutions of histological and MR images making a direct correlation impossible. Furthermore, the effect of the various histological modifications on the gray level of each pixel is complex and cannot be easily analyzed. Consequently, clinicians will not accept the use of MRI-TA in routine practice if TA remains a “black box” without clinical correspondence at a tissue level. A goal therefore of the multicenter European COST action MYO-MRI is to optimize MRI-TA methods in muscular dystrophy and to elucidate the histological meaning of MRI textures.info:eu-repo/semantics/publishedVersio

Autocrine regulation of human prostate carcinoma cell proliferation by somatostatin through the modulation of the SH2 domain containing protein tyrosine phosphatase (SHP)-1

The present study was intended to gain additional information on the growth regulation of prostate by somatostatin\ud (SRIF) and the intracellular events involved. The humanprostate adenocarcinoma cell lines PC-3 and LNCaP produce SRIF and express subtypes 2 and 5 of SRIF receptors. The secretion\ud of SRIF is related to the proliferative status of these cells; an inverse relationship exists between cell proliferation and the amount of secreted SRIF. Moreover, the growth of PC-3 cells\ud is inhibited by SRIF overexpression and increased by blockage of endogenous SRIF. Coincident with the increase in SRIF\ud secretion, the activity and levels of theSH2domain containing protein tyrosine phosphatase (SHP)-1, present in PC-3 cells are augmented, but the effect can be partially prevented by neutralization of secreted endogenously SRIF. The activity of SHP-1 is also stimulated by the SRIF analog RC160. Overexpression of SHP-1 induces inhibition of PC-3 cell growth.\ud SHP-1 is also present in normal prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and well differentiated adenocarcinoma. In contrast, no signal is detected in poorly differentiated prostate cancer. These findings demonstrate that SRIF inhibits PC-3 and LNCaP cell proliferation through an autocrine/paracrine SRIF loop. This effect could be mediated by activation of the tyrosine phosphatase SHP-1 detected in these cells as well as in human prostate and prostate cancer.Fundación para la Investigación en UrologíaMinisterio de Asuntos Exteriore

Exploring the chemoselectivity towards cysteine arylation by cyclometalated Au(III) compounds: new mechanistic insights

To gain more insight into the factors controlling the efficient cysteine arylation by cyclometalated Au(III) complexes, the reaction between selected gold compounds and different peptides was investigated by high‐resolution liquid chromatography electrospray ionization mass spectrometry (HR‐LC‐ESI‐MS). The deducted mechanisms of C–S cross‐coupling, also supported by density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations, evidenced the key role of secondary peptidic gold binding sites in favouring the process of reductive elimination

The Pandora multi-algorithm approach to automated pattern recognition of cosmic-ray muon and neutrino events in the MicroBooNE detector.

The development and operation of liquid-argon time-projection chambers for neutrino physics has created a need for new approaches to pattern recognition in order to fully exploit the imaging capabilities offered by this technology. Whereas the human brain can excel at identifying features in the recorded events, it is a significant challenge to develop an automated, algorithmic solution. The Pandora Software Development Kit provides functionality to aid the design and implementation of pattern-recognition algorithms. It promotes the use of a multi-algorithm approach to pattern recognition, in which individual algorithms each address a specific task in a particular topology. Many tens of algorithms then carefully build up a picture of the event and, together, provide a robust automated pattern-recognition solution. This paper describes details of the chain of over one hundred Pandora algorithms and tools used to reconstruct cosmic-ray muon and neutrino events in the MicroBooNE detector. Metrics that assess the current pattern-recognition performance are presented for simulated MicroBooNE events, using a selection of final-state event topologies

Convolutional Neural Networks Applied to Neutrino Events in a Liquid Argon Time Projection Chamber

We present several studies of convolutional neural networks applied to data coming from the MicroBooNE detector, a liquid argon time projection chamber (LArTPC). The algorithms studied include the classification of single particle images, the localization of single particle and neutrino interactions in an image, and the detection of a simulated neutrino event overlaid with cosmic ray backgrounds taken from real detector data. These studies demonstrate the potential of convolutional neural networks for particle identification or event detection on simulated neutrino interactions. We also address technical issues that arise when applying this technique to data from a large LArTPC at or near ground level

Noise Characterization and Filtering in the MicroBooNE Liquid Argon TPC

The low-noise operation of readout electronics in a liquid argon time projection chamber (LArTPC) is critical to properly extract the distribution of ionization charge deposited on the wire planes of the TPC, especially for the induction planes. This paper describes the characteristics and mitigation of the observed noise in the MicroBooNE detector. The MicroBooNE's single-phase LArTPC comprises two induction planes and one collection sense wire plane with a total of 8256 wires. Current induced on each TPC wire is amplified and shaped by custom low-power, low-noise ASICs immersed in the liquid argon. The digitization of the signal waveform occurs outside the cryostat. Using data from the first year of MicroBooNE operations, several excess noise sources in the TPC were identified and mitigated. The residual equivalent noise charge (ENC) after noise filtering varies with wire length and is found to be below 400 electrons for the longest wires (4.7 m). The response is consistent with the cold electronics design expectations and is found to be stable with time and uniform over the functioning channels. This noise level is significantly lower than previous experiments utilizing warm front-end electronics.Comment: 36 pages, 20 figure

The Pandora multi-algorithm approach to automated pattern recognition of cosmic-ray muon and neutrino events in the MicroBooNE detector

The development and operation of Liquid-Argon Time-Projection Chambers for neutrino physics has created a need for new approaches to pattern recognition in order to fully exploit the imaging capabilities offered by this technology. Whereas the human brain can excel at identifying features in the recorded events, it is a significant challenge to develop an automated, algorithmic solution. The Pandora Software Development Kit provides functionality to aid the design and implementation of pattern-recognition algorithms. It promotes the use of a multi-algorithm approach to pattern recognition, in which individual algorithms each address a specific task in a particular topology. Many tens of algorithms then carefully build up a picture of the event and, together, provide a robust automated pattern-recognition solution. This paper describes details of the chain of over one hundred Pandora algorithms and tools used to reconstruct cosmic-ray muon and neutrino events in the MicroBooNE detector. Metrics that assess the current pattern-recognition performance are presented for simulated MicroBooNE events, using a selection of final-state event topologies.Comment: Preprint to be submitted to The European Physical Journal