Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90αN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90αN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding

A Typical Case Presentation with Spontaneous Visual Recovery in Patient Diagnosed with Leber Hereditary Optic Neuropathy Due to Rare Point Mutation in <i>MT-ND4</i> Gene (<i>m.11253T>C</i>) and Literature Review

Leber hereditary optic neuropathy (LHON) is one of the most common inherited mitochondrial optic neuropathies, caused by mitochondrial DNA (mtDNA) mutations. Three most common mutations, namely m.11778G>A, m.14484T>G and m.3460G>A, account for the majority of LHON cases. These mutations lead to mitochondrial respiratory chain complex I damage. Typically, LHON presents at the 15–35 years of age with male predominance. LHON is associated with severe, subacute, painless bilateral vision loss and account for one of the most common causes of legal blindness in young individuals. Spontaneous visual acuity recovery is rare and has been reported in patients harbouring m.14484T>C mutation. Up to date LHON treatment is limited. Idebenone has been approved by European Medicines Agency (EMA) to treat LHON. However better understanding of disease mechanisms and ongoing treatment trials are promising and brings hope for patients. In this article we report on a patient diagnosed with LHON harbouring rare m.11253T>C mutation in MT-ND4 gene, who experienced spontaneous visual recovery. In addition, we summarise clinical presentation, diagnostic features, and treatment

S100A9 Alters the Pathway of Alpha-Synuclein Amyloid Aggregation

The formation of amyloid fibril plaques in the brain creates inflammation and neuron death. This process is observed in neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases. Alpha-synuclein is the main protein found in neuronal inclusions of patients who have suffered from Parkinson’s disease. S100A9 is a calcium-binding, pro-inflammation protein, which is also found in such amyloid plaques. To understand the influence of S100A9 on the aggregation of α-synuclein, we analyzed their co-aggregation kinetics and the resulting amyloid fibril structure by Fourier-transform infrared spectroscopy and atomic force microscopy. We found that the presence of S100A9 alters the aggregation kinetics of α-synuclein and stabilizes the formation of a particular amyloid fibril structure. We also show that the solution’s ionic strength influences the interplay between S100A9 and α-synuclein, stabilizing a different structure of α-synuclein fibrils

Cadmium Exposure and Risk of Breast Cancer by Histological and Tumor Receptor Subtype in White Caucasian Women: A Hospital-Based Case-Control Study

As the majority of experimental studies suggest cadmium being metalloestrogen, we examined cadmium/breast cancer (BC) association by histological and tumor receptor subtype in 509 invasive BC patients and 1170 controls. Urinary cadmium was determined by atomic absorption spectrometry, and categorized using tertiles of its distribution in the controls: &lt;0.18, 0.18&ndash;0.33, &gt;0.33 kg &times; 10&minus;9/kg &times; 10&minus;3 creatinine. Relative to the lowest category of urinary cadmium adjusted odds ratio (OR) of ductal BC was 1.18 (95% confidence interval (CI): 0.89&ndash;1.58) in the intermediate and 1.53 (95% CI: 1.15&ndash;2.04) in the highest category. There was a significant association for hormone receptor-positive ductal BC: ORs per category increase were 1.34 (95% CI: 1.14&ndash;1.59) for estrogen receptor-positive (ER+), 1.33 (95% CI: 1.09&ndash;1.61) for progesterone receptor-positive (PR+) and 1.35 (95% CI: 1.11&ndash;1.65) for ER+/PR+ BC. We found a significant association between cadmium and human epidermal growth factor receptor 2-negative (HER2&minus;) ductal BC. The strongest association with cadmium was for ER+/PR+/HER2&minus; ductal BC. The associations between cadmium and lobular BC with hormone receptor-positive and HER2&minus; were positive but insignificant. There was no evidence that the associations with cadmium differed for cancers with different tumor histology (p-heterogeneity &gt; 0.05). This study provides evidence that urinary cadmium is associated with the risk of hormone receptor-positive and HER2&minus; breast cancer independent of tumor histology

Phenotypic characterization of <i>Gardnerella vaginalis</i> subgroups suggests differences in their virulence potential

<div><p>The well-known genotypic and phenotypic diversity of <i>G</i>. <i>vaginalis</i> resulted in its classification into at least four subgroups (clades) with diverse genomic properties. To evaluate the virulence potential of <i>G</i>. <i>vaginalis</i> subgroups, we analyzed the virulence-related phenotypic characteristics of 14 isolates of clade 1, 12 isolates of clade 2, 8 isolates of clade 4 assessing their <i>in vitro</i> ability to grow as a biofilm, produce the toxin vaginolysin, and express sialidase activity. Significant differences in VLY production were found (<i>p</i> = 0.023), but further analysis of clade pairs did not confirm this finding. The amount of biofim did not differ significantly among the clades. Analysis of sialidase activity indicated statistically significant differences among the clades (<i>p</i> < 0.001). Production of active recombinant <i>G</i>. <i>vaginalis</i> sialidase demonstrated the link between the <i>sld</i> gene and enzymatic activity, which may be differentially regulated at the transcriptional level. Statistical classification analysis (random forests algorithm) showed that <i>G</i>. <i>vaginalis</i> clades could be best defined by the profiles of two phenotypic characteristics: sialidase activity and vaginolysin production. The results of principal component analysis and hierarchical clustering suggested that all isolates can be subgrouped into three clusters, the structures of which are determined based on phenotypic characteristics of the isolates. Clade 4 was the most homogenous group, as all isolates were found in the same cluster, which is characterized by low production of all studied virulence factors. Clade 2 isolates were mainly distributed between two clusters, whereas clade 1 isolates were found in all three clusters that were characterized by a distinct profile of phenotypic characteristics. Our findings suggest that <i>G</i>. <i>vaginalis</i> subgroups with different virulence potential might play distinct roles in vaginal microbiota.</p></div

Assessment of phenotypic characteristics of <i>G</i>. <i>vaginalis</i> clinical isolates cultivated <i>in vitro</i>.

<p>(A) The ELISA-determined VLY concentration (ng/mL) was adjusted to the OD of the cultures and mapped on a log scale. (B) The OD₄₉₂ of bacterial films after a 24-h incubation is expressed as log OD. (C) Calculated sialidase activity normalized to the OD of cultures was mapped on a log scale. Individual values are indicated by colored circles (randomly moved in the direction parallel to the X-axis to avoid overlapping) and summarized as Tukey-style boxplots [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200625#pone.0200625.ref037" target="_blank">37</a>]. Boxes restrict the interquartile range from the 25th to the 75th percentiles. Horizontal lines in the boxes indicate the median, and whiskers show the range from minimum to maximum non-outlying values. Possible outlier values are indicated by black dots. The CLD letters (where indicated) above the boxplots summarize the results of pairwise comparisons using the Games-Howell test (A) and the Conover-Iman test (C).</p

Results of hierarchical clustering and principal component analysis based on four characteristics: Sialidase activity, VLY production, and biofilm amount after 24 h and 48 h incubation.

<p>(A) Dendogram. The colors of the branches represent the three largest clusters. (B) PCA biplot. Axes represent the first and the second principal components (PCs). Percentages on the axes indicate the percentage of total variance explained by each PC. Arrows in the biplot represent the phenotypic characteristics. Dots indicate isolates, and the colors of the dots/isolate numbers correspond to particular clades. Closed lines indicate the areas in the space of PCs restricted by the isolates of the same clade. Shaded fields indicate areas restricted by the clusters determined in the dendogram.</p

Results of hierarchical clustering and principal component analysis based on two characteristics: Sialidase activity and biofilm amount after 24 h incubation.

<p>(A) Dendogram that shows similarity between the <i>G</i>. <i>vaginalis</i> isolates. The colors of the branches represent the three largest clusters. (B) PCA biplot. Axes represent the first and the second principal components (PCs). Percentages on the axes indicate the percentage of the total variance explained by each PC. Arrows in the biplot represent the phenotypic characteristics. Dots indicate isolates, and the colors of the dots/isolate numbers correspond to particular clades. Closed lines indicate the areas in the space of PCs restricted by the isolates of the same clade. Shaded fields indicate areas restricted by the clusters determined in the dendogram.</p