Fourier transform infrared spectroscopy provides a fingerprint for the tetramer and for the aggregates of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular beta-sheet structure followed by an increase in nonnative, probably antiparallel beta-sheet contacts (bands at 1,616 and 1,686 cm(-1)) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12 kbar), TTR loses only part of its beta-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of beta-sheets does not change much upon fibrillization; however, the alignment of beta-sheets is altered, resulting in the formation of distinct beta-sheet contacts (band at 1,625 cm(-1)). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the beta-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37 degrees C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.     

Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler''s diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K_m(NAD⁺) = 6 μm, K_m(actin) = 24 μm, and k_cat = 22 s⁻¹. VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC₅₀ = 20 μm). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.     

Bias in effect size of systemic lupus erythematosus susceptibility loci across Europe: a case-control study

Introduction

Prevalence of an abnormal Papanicolaou smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk human papillomavirus (HPV) infection. The nucleic acid-specific Toll-like receptors (TLRs) locate at the endolysosomal compartments and trigger the induction of cytokines for the innate immune response. This study evaluated whether abnormal host innate immune response in lupus patients may enhance HPV persistence.

Methods

Protein levels of TLRs 3, 7, 8 and 9 in cervical epithelial cells of lupus patients and controls with or without HPV infection were assessed using flow cytometry. Characteristics associated with the differential expression of TLRs in systemic lupus erythematosus (SLE) were elucidated. The effect and interferon-stimulated genes (ISGs) (ISG15 and Mx-1) gene expressions were then measured in oncogenic HeLa (HPV18), CaSki (HPV) and C33A (HPV negative) cell lines using flow cytometry and quantitative real-time PCR. Ex vivo productions of cytokines and interferon-gamma (IFN-??) upon TLR ligands stimulations were subsequently measured using cytometric bead array and ELISA.

Results

For subjects with HPV infection, levels of TLR3 and TLR7 were significantly lower in lupus patients compared with controls. Significantly decreased TLRs 7, 8 and 9 levels were observed in HPV-negative SLE compared to healthy controls. For SLE with and without HPV infection, TLR7 and 9 levels were significantly lower in infected SLE than those in HPV-negative patients. Independent explanatory variables associated with down-regulation of TLR7 level included HPV infection and a higher cumulative dose of prednisolone; while a higher cumulative dose of hydroxychloroquine and HPV infection were associated with down-regulation of TLR9 level. In cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral ISG15 and Mx-1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands (R837, ssRNA and ODN2395) was greatly impaired in CaSki and HeLa than C33A cells.

Conclusions

In conclusion, prednisolone and TLR antagonist (hydroxychloroquine) may down-regulate protein levels of TLR7 and TLR9 in lupus patients, thereby decreasing the innate immune response against HPV infection. Upon infection, HPV further down-regulate TLR7 and 9 levels for viral persistence. Furthermore, reduction of nucleic acid-sensing TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs (ISG15 and Mx-1) on a biologically relevant antiviral response.     

Bias in effect size of systemic lupus erythematosus susceptibility loci across Europe: a case-control study

Introduction

We aimed to investigate whether the effect size of the systemic lupus erythematosus (SLE) risk alleles varies across European subpopulations.

Methods

European SLE patients (n = 1,742) and ethnically matched healthy controls (n = 2,101) were recruited at 17 centres from 10 different countries. Only individuals with self-reported ancestry from the country of origin were included. In addition, participants were genotyped for top ancestry informative markers and for 25 SLE associated SNPs. The results were used to compare effect sizes between the Central Eureopan and Southern European subgroups.

Results

Twenty of the 25 SNPs showed independent association with SLE, These SNPs showed a significant bias to larger effect sizes in the Southern subgroup, with 15/20 showing this trend (P = 0.019) and a larger mean odds ratio of the 20 SNPs (1.46 vs. 1.34, P = 0.02) as well as a larger difference in the number of risk alleles (2.06 vs. 1.63, P = 0.027) between SLE patients and controls than for Central Europeans. This bias was reflected in a very significant difference in the cumulative genetic risk score (4.31 vs. 3.48, P = 1.8 × 10^-32). Effect size bias was accompanied by a lower number of SLE risk alleles in the Southern subjects, both patients and controls, the difference being more marked between the controls (P = 1.1 × 10^-8) than between the Southern and Central European patients (P = 0.016). Seven of these SNPs showed significant allele frequency clines.

Conclusion

Our findings showed a bias to larger effect sizes of SLE loci in the Southern Europeans relative to the Central Europeans together with clines of SLE risk allele frequencies. These results indicate the need to study risk allele clines and the implications of the polygenic model of inheritance in SLE.     

Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition

Actin-based stress fiber formation is coupled to microtubule depolymerization through the local activation of RhoA. While the RhoGEF Lfc has been implicated in this cytoskeleton coupling process, it has remained elusive how Lfc is recruited to microtubules and how microtubule recruitment moderates Lfc activity. Here, we demonstrate that the dynein light chain protein Tctex-1 is required for localization of Lfc to microtubules. Lfc residues 139-161 interact with Tctex-1 at a site distinct from the cleft that binds dynein intermediate chain. An NMR-based GEF assay revealed that interaction with Tctex-1 represses Lfc nucleotide exchange activity in an indirect manner that requires both polymerized microtubules and phosphorylation of S885 by PKA. We show that inhibition of Lfc by Tctex-1 is dynein dependent. These studies demonstrate a pivotal role of Tctex-1 as a negative regulator of actin filament organization through its control of Lfc in the crosstalk between microtubule and actin cytoskeletons.     

Nothofagus dombeyi regeneration in declining Austrocedrus chilensis forests: Effects of overstory mortality and climatic events

This research examines the regeneration dynamics of Nothofagus dombeyi and Austrocedrus chilensis in A. chilensis-dominated forests growing near the eastern limit of N. dombeyi where precipitation is limiting. In these forests the widespread decline and mortality of overstory A. chilensis trees, known as ‘mal del ciprés’ (cypress sickness), generates large canopy gaps in which new individuals establish. Our objective was to study the population dynamics of N. dombeyi and A. chilensis in these forests to investigate the influences of overstory tree death and climatic variation on establishment. We sampled 6 symptomatic A. chilensis stands and used dendrochronological techniques to reconstruct basal area development and regeneration establishment over time. Bivariate event analysis was performed to examine the temporal relationships between tree establishment and mortality events and climatic variation. Overstory A. chilensis trees established as post-fire cohorts, with subsequent establishment of A. chilensis and N. dombeyi during the past 50–60 years. Regeneration in the past two decades was primarily N. dombeyi. The establishment of both A. chilensis and N. dombeyi was synchronous with overstory tree mortality events, but it was more consistent among stands and prolonged for N. dombeyi. Establishment of A. chilensis was not associated with climatic events but N. dombeyi establishment was synchronous with droughts, possibly related to climate-driven mortality creating canopy gaps or reducing competition within gaps. We have demonstrated that N. dombeyi has the ability to establish in post-fire A. chilensis-dominated forests resulting in mixed-species, uneven-aged forests. The ongoing increase in the abundance of N. dombeyi relative to A. chilensis represents a shift in composition and increased complexity in stand structure driven by ‘mal del ciprés’ and climatic variation.     

Cognitive Functions in Middle Aged Individuals Are Related to Metabolic Disturbances and Aerobic Capacity: A Cross-Sectional Study

Aims

Metabolic disturbances may contribute to cognitive dysfunction in patients with type 2 diabetes. We investigated the relation between cognitive impairment and metabolic deteriorations, low physical fitness, low-grade inflammation and abdominal obesity in middle aged individuals.

Methods

We conducted a cross-sectional study including 40 to 65 year-old patients with type 2 diabetes and limited co morbidity (N = 56), age-matched individuals with impaired glucose tolerance (N = 56) as well as age-matched controls with normal glucose tolerance (N = 72). Specific cognitive functions were assessed with focus on verbal memory, processing speed, executive functions, and a composite overall mean score. Oral glucose tolerance test, VO₂max test, systemic inflammation, DXA scanning and abdominal MRI were measured.

Results

Multiple linear regression analyses adjusting for age, gender and verbal intelligence demonstrated that a low score in processing speed, executive functions and overall cognitive function were related to high fasting C-peptide, as well as low insulin sensitivity, beta-cell function and VO₂max. Measurements of blood glucose, obesity and inflammation were not associated with cognitive function.

Conclusion

Low cognitive scores are seen in middle aged individuals with hyperinsulinemia, low insulin sensitivity, beta-cell function and low aerobic capacity. These findings emphasize the importance of appropriate lifestyle and not only blood glucose control in prevention of cognitive disability.