Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.     

Eye Movement Patterns in Natural Reading: A Comparison of Monolingual and Bilingual Reading of a Novel

Introduction and Method

This paper presents a corpus of sentence level eye movement parameters for unbalanced bilingual first language (L1) and second-language (L2) reading and monolingual reading of a complete novel (56 000 words). We present important sentence-level basic eye movement parameters of both bilingual and monolingual natural reading extracted from this large data corpus.

Results and Conclusion

Bilingual L2 reading patterns show longer sentence reading times (20%), more fixations (21%), shorter saccades (12%) and less word skipping (4.6%), than L1 reading patterns. Regression rates are the same for L1 and L2 reading. These results could indicate, analogous to a previous simulation with the E-Z reader model in the literature, that it is primarily the speeding up of lexical access that drives both L1 and L2 reading development. Bilingual L1 reading does not differ in any major way from monolingual reading. This contrasts with predictions made by the weaker links account, which predicts a bilingual disadvantage in language processing caused by divided exposure between languages.     

Potential short-term earthquake forecasting by farm animal monitoring

Whether changes in animal behavior allow for short-term earthquake predictions has been debated for a long time. Before, during and after the 2016/2017 earthquake sequence in Italy, we deployed bio-logging tags to continuously observe the activity of farm animals (cows, dogs, and sheep) close to the epicenter of the devastating magnitude M6.6 Norcia earthquake (Oct–Nov 2016) and over a subsequent longer observation period (Jan–Apr 2017). Relating 5,304 (in 2016) and 12,948 (in 2017) earthquakes with a wide magnitude range (0.4 ≤ M ≤ 6.6) to continuously measured animal activity, we detected how the animals collectively reacted to earthquakes. We also found consistent anticipatory activity prior to earthquakes during times when the animals were in a building (stable), but not during their time on a pasture. We detected these anticipatory patterns not only in periods with high, but also in periods of low seismic activity. Earthquake anticipation times (1–20 hr) are negatively correlated with the distance between the farm and earthquake hypocenters. Our study suggests that continuous bio-logging of animal collectives has the potential to provide statistically reliable patterns of pre-seismic activity that could yield valuable insights for short-term earthquake forecasting. Based on a priori model parameters, we provide empirical threshold values for pre-seismic animal activities to be used in real-time observation stations.     

Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3)

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3.     

Characterization of a Discontinuous Neutralizing Epitope on Glycoprotein B of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal antibodies (MAbs), we have recently identified antigenic region 4 (AD-4) on gB as an important target for neutralizing antibodies. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 of gB of HCMV strain AD169. To map epitopes for human antibodies on this protein domain, we used a three-dimensional (3D) model of HCMV gB to identify surface-exposed amino acids on AD-4 and selected juxtaposed residues for alanine scans. A tyrosine (Y) at position 364 and a lysine (K) at position 379 (the YK epitope), which are immediate neighbors on the AD-4 surface, were found to be essential for binding of the human MAbs. Recognition of AD-4 by sera from HCMV-infected individuals also was largely dependent on these two residues, indicating a general importance for the antibody response against AD-4. A panel of AD-4 recombinant viruses harboring mutations at the crucial antibody binding sites was generated. The viruses showed significantly reduced susceptibility to neutralization by AD-4-specific MAbs or polyclonal AD-4-specific antibodies, indicating that the YK epitope is dominant for the AD-4-specific neutralizing antibody response during infection. To our knowledge, this is the first molecular identification of a functional discontinuous epitope on HCMV gB. Induction of antibodies specific for this epitope may be a desirable goal following vaccination with gB.     

B cell progenitors are arrested in maturation but have intact VDJ recombination in the absence of Ig-alpha and Ig-beta

Ig-alpha and Ig-beta mediate surface expression and signaling of diverse B cell receptor complexes on precursor, immature, and mature B cells. Their expression begins before that of the Ig chains in early progenitor B cells. In this study, we describe the generation of Ig-alpha-deficient mice and their comparative analysis to mice deficient for Ig-beta, the membrane-IgM, and recombination-activating gene 2 to determine the requirement of Ig-alpha and Ig-beta in survival and differentiation of pro-B cells. We find that in the absence of Ig-alpha, B cell development does not progress beyond the progenitor stage, similar to what is observed in humans lacking this molecule. However, neither in Ig-alpha- nor in Ig-beta-deficient mice are pro-B cells impaired in V(D)J recombination, in the expression of intracellular Ig micro-chains, or in surviving in the bone marrow microenvironment. Finally, Ig-alpha and Ig-beta are not redundant in their putative function, as pro-B cells from Ig-alpha and Ig-beta double-deficient mice are similar to those from single-deficient animals in every aspect analyzed.