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N-terminal extensions ("tags") have proven valuable for producing peptides using high throughput recombinant expression technologies. However, the applicability is hampered by the limited options for specific and efficient proteases to release the fully native sequence without additional amino acids in the N-terminal. Here we describe the Escherichia coli (E. coli) expression, purification and characterization of engineered variants of Xaa-Pro dipeptidyl aminopeptidase (Xaa-Pro-DAP) derived from Lactococcus lactis for cleavage of Gly-Pro dipeptide extension in the N-terminal of glucagon and glucagon-like peptide 1 (GLP-1(7-37)). By single amino acid substitution in the Xaa-Pro-DAP protease, significantly higher product yields were achieved. The combination of HRV14 3C protease and engineered Xaa-Pro-DAP is suggested for obtaining native N-terminal of peptides., Competing Interests: Declaration of competing interest M.Ø.P and A.C.S. are employees of Novo Nordisk A/S. E.V. and H.T were employees of Novo Nordisk A/S at the time of this study. E.V. is an employee of Novo Nordisk Research Center Seattle Inc., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We investigated the effect of aging on the multi-dimensional characteristics and heterogeneity of human peripheral CD8 + T cells defined by the expression of a set of molecules at the single cell level using the recently developed mass cytometry or Cytometry by Time-Of-Flight (CyTOF) and computational algorithms. CD8 + T cells of young and older adults had differential expression of molecules, especially those related to cell activation and migration, permitting the clustering of young and older adults through an unbiased approach. The changes in the expression of individual molecules were collectively reflected in the altered high-dimensional profiles of CD8 + T cells in older adults as visualized by the dimensionality reduction analysis tools principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE). A combination of PhenoGraph clustering and t-SNE analysis revealed heterogeneous subsets of CD8 + T cells that altered with aging. Furthermore, intermolecular quantitative relationships in CD8 + T cells appeared to change with age as determined by the computational algorithm conditional-Density Resampled Estimate of Mutual Information (DREMI). The results of our study showed that heterogeneity, multidimensional characteristics, and intermolecular quantitative relationships in human CD8 + T cells altered with age, distinctively clustering young and older adults through an unbiased approach., (Copyright © 2019 Elsevier Inc. All rights reserved.)

IL-15 is involved in regulating host defense and inflammation. Monocytes produce the biologically active cell surface IL-15 in response to IFN-γ. Although aging can alter the immune system, little is known about whether and how aging affects IFN-γ-mediated IL-15 production in human monocytes. We showed that monocytes of healthy older adults (age ≥ 65) had increased cell surface IL-15 expression in response to IFN-γ compared to those of healthy young adults (age ≤ 40). This finding stems in part from increased IFN-γ receptor (R)1/2 expression on monocytes in older adults, leading to enhanced STAT1 activation and interferon regulatory factor 1 synthesis with increased IL15 gene expression. Our study suggests that with aging the IFN-γ-mediated IL-15 production pathway in human monocytes is uncompromised, but rather augmented, and could be considered as a therapeutic target point to modulate host defense and inflammation in older adults., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Alterations in T cell immunity occur with aging. Influenza causes significant morbidity and mortality in the elderly. We investigated the relationship of serum IgG responses with hemagglutinin inhibition (HI) antibody titers and the frequency of distinct T cell subsets in young and elderly people who received the inactivated influenza vaccine. Influenza vaccine-specific IgG responses correlated with the increase of HI antibody titers and the frequency of CD4(+) T cells producing IFN-γ and IL-17 in young, but not elderly, people. Also, only in young people, such IgG responses correlated with the frequency of memory T cells, especially central memory cells, CD45RA(-) effector memory CD8(+) T cells and IL-7 receptor alpha high effector memory CD8(+) T cells with potent survival and proliferative capacity. These findings suggest that aging alters the association of influenza-vaccine specific IgG responses with HI antibody titers, cytokine-producing capacity and proportions of memory T cells in humans., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In larval lamprey, partial lesions were made in the rostral spinal cord to determine which spinal tracts are important for descending activation of locomotion and to identify descending brain neurons that project in these tracts. In whole animals and in vitro brain/spinal cord preparations, brain-initiated spinal locomotor activity was present when the lateral or intermediate spinal tracts were spared but usually was abolished when the medial tracts were spared. We previously showed that descending brain neurons are located in eleven cell groups, including reticulospinal (RS) neurons in the mesenecephalic reticular nucleus (MRN) as well as the anterior (ARRN), middle (MRRN), and posterior (PRRN) rhombencephalic reticular nuclei. Other descending brain neurons are located in the diencephalic (Di) as well as the anterolateral (ALV), dorsolateral (DLV), and posterolateral (PLV) vagal groups. In the present study, the Mauthner and auxillary Mauthner cells, most neurons in the Di, ALV, DLV, and PLV cell groups, and some neurons in the ARRN and PRRN had crossed descending axons. The majority of neurons projecting in medial spinal tracts included large identified Müller cells and neurons in the Di, MRN, ALV, and DLV. Axons of individual descending brain neurons usually did not switch spinal tracts, have branches in multiple tracts, or cross the midline within the rostral cord. Most neurons that projected in the lateral/intermediate spinal tracts were in the ARRN, MRRN, and PRRN. Thus, output neurons of the locomotor command system are distributed in several reticular nuclei, whose neurons project in relatively wide areas of the cord., ((c) 2010 Elsevier Inc. All rights reserved.)