Electrochemical Investigations into Kinase-Catalyzed Transformations of Tau Protein

The formation of neurofibrillary tangles by hyperphosphorylated tau is a well-recognized hallmark of Alzheimer’s disease. Resulting from malfunctioning protein kinases, hyperphosphorylated tau is unable to bind microtubules properly, causing it to self-associate and aggregate. The effects of tau phosphorylation on tau conformation and aggregation are still largely unexplored. The conformational analysis of tau and its hyperphosphorylated forms is usually performed by a variety of spectroscopic techniques, all of which require ample sample concentrations and/or volumes. Here we report on the use of surface based electrochemical techniques that allow for detection of conformational changes and orientation of tau protein as a function of tau phosphorylation by tyrosine and serine/threonine kinases. The electrochemical methods utilize 5′-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative as a cosubstrate and tau immobilized on gold surface to probe the role of the following protein kinases: Sarcoma related kinase (Src), Abelson tyrosine kinase (Abl), tau-tubulin kinase (TTBK), proto-oncogene tyrosine protein kinase Fyn (Fyn), and glycogen synthase kinase 3-β (Gsk-3β). The single kinase and sequential kinase-catalyzed Fc-phosphorylations modulate the electrochemical signal, pointing to the dramatic changes around the Fc group in the Fc-phosphorylated tau films. The location and orientation of the Fc-group in Fc-tau film was investigated by the surface plasmon resonance based on antiferrocene antibodies. Additional surface characterization of the Fc-tau films by time-of-flight secondary ion-mass spectrometry and X-ray photoelectron spectroscopy revealed that Fc-phosphorylations influence the tau orientation and conformation on surfaces. When Fc-phosphorylations were performed in solution, the subsequently immobilized Fc-tau exhibited similar trends. This study illustrates the validity and the utility of the labeled electrochemical approach for probing the changes in protein film properties, conformation, and orientation as a function of the enzymatically catalyzed modifications

Descriptive statistics for the six dimensions of psychotic experiences as measured by SPEQ and PLIKS-Q.

<p>Note. SD, standard deviation.</p

Summary of results for schizophrenia PRS as a predictor of SPEQ subscales at p_T = 0.5.

<p>Note. β, unstandardized beta value; C.I., confidence intervals; t, t-statistic; N, sample size; df, degrees of freedom; SPEQ Paranoia subscale was log transformed; 8 PCs were included as covariates.</p

Mean sex differences on psychotic experiences.

<p>Note. M = Mean; SD = standard deviations; df = degrees of freedom. Raw scores provided. Paranoia, Hallucinations, Grandiosity and Parent-rated Negative Symptoms were transformed prior to statistical testing.</p

Results of Allelic & Genotypic Association Analyses for PLIKS-Q Transformed Data (adjusted for sex and age)

<p>Note: Chr, chromosome; Allele, risk allele; Beta, regression coefficient; ADD, additive linear regression model; GENO_2DF, two degrees of freedom joint test of additivity and dominance deviation (it does not assume a linear relationship); * proxies for rs17512836.</p

Results of regression analysis with the schizophrenia composite SNP score as a predictor of the SPEQ measures (adjusted for sex and age and performed on the transformed data).

<p>Note: β, Beta; SE, standard error; C.I., confidence interval.</p

Summary results of allelic & genotypic association analyses for transformed SPEQ data with <i>p</i>-values <0.05 (adjusted for sex and age).

<p>Note: Chr, chromosome; Position, positions based on Human Genome Build 36; Allele, risk allele; Beta, regression coefficient; ADD, additive linear regression model; GENO_2DF, two degrees of freedom joint test of additivity and dominance deviation (it does not assume a linear relationship); Neg. Sympt., Parent-rated Negative Symptoms</p

Summary of results for bipolar disorder PRS as a predictor of SPEQ subscales at p_T = 0.5.

<p>Note. β, unstandardized beta value; C.I., confidence intervals; t, t-statistic; N, sample size; df, degrees of freedom; 8 PCs were included as covariates.</p

Image_5_Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies.tif

Hepato-pancreatico-biliary (HPB) malignancies are difficult-to-treat and continue to to have a high mortality and significant therapeutic resistance to standard therapies. Immune oncology (IO) therapies have demonstrated efficacy in several solid malignancies when combined with chemotherapy, whereas response rates in pancreatic ductal adenocarcinoma (PDA) are poor. While promising in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), there remains an unmet need to fully leverage IO therapies to treat HPB tumors. We therefore defined T cell subsets in the tumor microenvironment of HPB patients utilizing a novel, multiparameter flow cytometry and bioinformatics analysis. Our findings quantify the T cell phenotypic states in relation to checkpoint receptor expression. We demonstrate the presence of CD103+ tissue resident memory T cells (TRM), CCR7+ central memory T cells, and CD57+ terminally differentiated effector cells across all HPB cancers, while the anti-tumor function was dampened by expression of multiple co-inhibitory checkpoint receptors. Terminally exhausted T cells lacking co-stimulatory receptors were more prevalent in PDA, whereas partially exhausted T cells expressing both co-inhibitory and co-stimulatory receptors were most prevalent in HCC, especially in early stage. HCC patients had significantly higher TRM with a phenotype that could confer restored activation in response to immune checkpoint therapies. Further, we found a lack of robust alteration in T cell activation state or checkpoint expression in response to chemotherapy in PDA patients. These results support that HCC patients might benefit most from combined checkpoint therapies, whereas efforts other than cytotoxic chemotherapy will likely be necessary to increase overall T cell activation in CCA and PDA for future clinical development.</p

Image_3_Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies.tif

Hepato-pancreatico-biliary (HPB) malignancies are difficult-to-treat and continue to to have a high mortality and significant therapeutic resistance to standard therapies. Immune oncology (IO) therapies have demonstrated efficacy in several solid malignancies when combined with chemotherapy, whereas response rates in pancreatic ductal adenocarcinoma (PDA) are poor. While promising in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), there remains an unmet need to fully leverage IO therapies to treat HPB tumors. We therefore defined T cell subsets in the tumor microenvironment of HPB patients utilizing a novel, multiparameter flow cytometry and bioinformatics analysis. Our findings quantify the T cell phenotypic states in relation to checkpoint receptor expression. We demonstrate the presence of CD103+ tissue resident memory T cells (TRM), CCR7+ central memory T cells, and CD57+ terminally differentiated effector cells across all HPB cancers, while the anti-tumor function was dampened by expression of multiple co-inhibitory checkpoint receptors. Terminally exhausted T cells lacking co-stimulatory receptors were more prevalent in PDA, whereas partially exhausted T cells expressing both co-inhibitory and co-stimulatory receptors were most prevalent in HCC, especially in early stage. HCC patients had significantly higher TRM with a phenotype that could confer restored activation in response to immune checkpoint therapies. Further, we found a lack of robust alteration in T cell activation state or checkpoint expression in response to chemotherapy in PDA patients. These results support that HCC patients might benefit most from combined checkpoint therapies, whereas efforts other than cytotoxic chemotherapy will likely be necessary to increase overall T cell activation in CCA and PDA for future clinical development.</p