Epigenomic Profiling Advises Therapeutic Potential of Leukotriene Receptor Inhibitors for a Subset of Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype, with a poor survival rate compared to others subtypes. For a long time, chemotherapy was the only systemic treatment for TNBC, and the identification of actionable molecular targets might ultimately improve the prognosis for TNBC patients. We performed a genome-wide analysis of DNA methylation at CpG islands on a collection of one hundred ten breast carcinoma samples and six normal breast tissue samples using reduced representation bisulfite sequencing with the XmaI restriction enzyme (XmaI-RRBS) and identified a subset of TNBC samples with significant hypomethylation at the LTB4R/LTB4R2 genes' CpG islands, including CpG dinucleotides covered with cg12853742 and cg21886367 HumanMethylation 450K microarray probes. Abnormal DNA hypomethylation of this region in TNBC compared to normal samples was confirmed by bisulfite Sanger sequencing. Gene expression generally anticorrelates with promoter methylation, and thus, the promoter hypomethylation detected and confirmed in our study might be revealed as an indirect marker of high LTB4R/LTB4R2 expression using a simple methylation-sensitive PCR test. Analysis of RNA-seq expression and DNA methylation data from the TCGA dataset demonstrates that the expression of the LTB4R and LTB4R2 genes significantly negatively correlates with DNA methylation at both CpG sites cg12853742 (R = -0.4, p = 2.6 × 10-6; R = -0.21, p = 0.015) and cg21886367 (R = -0.45, p = 7.3 × 10-8; R = -0.24, p = 0.005), suggesting the upregulation of these genes in tumors with abnormal hypomethylation of their CpG island. Kaplan-Meier analysis using the TCGA-BRCA gene expression and clinical data revealed poorer overall survival for TNBC patients with an upregulated LTB4R. To this day, only the leukotriene inhibitor LY255283 has been tested on an MCF-7/DOX cell line, which is a luminal A breast cancer molecular subtype. Other studies compare the effects of Montelukast and Zafirlukast (inhibitors of the cysteinyl leukotriene receptor, which is different from LTB4R/LTB4R2) on the MDA-MB-231 (TNBC) cell line, with high methylation and low expression levels of LTB4R. In our study, we assess the therapeutic effects of various drugs (including leukotriene receptor inhibitors) with the DepMap gene effect and drug sensitivity data for TNBC cell lines with hypomethylated and upregulated LTB4R/LTB4R2 genes. LY255283, Minocycline, Silibinin, Piceatannol, Mitiglinide, 1-Azakenpaullone, Carbetocin, and Pim-1-inhibitor-2 can be considered as candidates for the additional treatment of TNBC patients with tumors demonstrating LTB4R/LTB4R2 hypomethylation/upregulation. Finally, our results suggest that the epigenetic status of leukotriene B4 receptors is a novel, potential, predictive, and prognostic biomarker for TNBC. These findings might improve individualized therapy for TNBC patients by introducing new therapeutic adjuncts as anticancer agents.

DNA Methylation and Prospects for Predicting the Therapeutic Effect of Neoadjuvant Chemotherapy for Triple-Negative and Luminal B Breast Cancer.

Despite advances in the diagnosis and treatment of breast cancer (BC), the main cause of deaths is resistance to existing therapies. An approach to improve the effectiveness of therapy in patients with aggressive BC subtypes is neoadjuvant chemotherapy (NACT). Yet, the response to NACT for aggressive subtypes is less than 65% according to large clinical trials. An obvious fact is the lack of biomarkers predicting the therapeutic effect of NACT. In a search for epigenetic markers, we performed genome-wide differential methylation screening by XmaI-RRBS in cohorts of NACT responders and nonresponders, for triple-negative (TN) and luminal B tumors. The predictive potential of the most discriminative loci was further assessed in independent cohorts by methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR), a promising method for the implementation of DNA methylation markers in diagnostic laboratories. The selected most informative individual markers were combined into panels demonstrating cvAUC = 0.83 (TMEM132D and MYO15B markers panel) for TN tumors and cvAUC = 0.76 (TTC34, LTBR and CLEC14A) for luminal B tumors. The combination of methylation markers with clinical features that correlate with NACT effect (clinical stage for TN and lymph node status for luminal B tumors) produces better classifiers, with cvAUC = 0.87 for TN tumors and cvAUC = 0.83 for luminal B tumors. Thus, clinical characteristics predictive of NACT response are independently additive to the epigenetic classifier and in combination improve prediction.

Photobase-Triggered Formation of 3D Epitaxially Fused Quantum Dot Superlattices with High Uniformity and Low Bulk Defect Densities.

Highly ordered epitaxially fused colloidal quantum dot (QD) superlattices (epi-SLs) promise to combine the size-tunable photophysics of QDs with the efficient charge transport of bulk semiconductors. However, current epi-SL fabrication methods are crude and result in structurally and chemically inhomogeneous samples with high concentrations of extended defects that localize carriers and prevent the emergence of electronic mini-bands. Needed fabrication improvements are hampered by inadequate understanding of the ligand chemistry that causes epi-SL conversion from the unfused parent SL. Here we show that epi-SL formation by the conventional method of amine injection into an ethylene glycol subphase under a floating QD film occurs by deprotonation of glycol by the amine and subsequent exchange of oleate by glycoxide on the QD surface. By replacing the amine with hydroxide ion, we demonstrate that any Brønsted-Lowry base that creates a sufficient dose of glycoxide can produce the epi-SL. We then introduce an epi-SL fabrication method that replaces point injection of a base with contactless and uniform illumination of a dissolved photobase. Quantitative mapping of multilayer (3D) films shows that our photobase-made epi-SLs are chemically and structurally uniform and have much lower concentrations of bulk defects compared to the highly inhomogeneous and defect-rich epi-SLs produced by amine point injection. The structural-chemical uniformity and structural perfection of photobase-made epi-SLs make them leading candidates for achieving emergent mini-band charge transport in a self-assembled mesoscale solid.

Free energy difference to create the M-OH* intermediate of the oxygen evolution reaction by time-resolved optical spectroscopy.

Theoretical descriptors differentiate the catalytic activity of materials for the oxygen evolution reaction by the strength of oxygen binding in the reactive intermediate created upon electron transfer. Recently, time-resolved spectroscopy of a photo-electrochemically driven oxygen evolution reaction followed the vibrational and optical spectra of this intermediate, denoted M-OH*. However, these inherently kinetic experiments have not been connected to the relevant thermodynamic quantities. Here we discover that picosecond optical spectra of the Ti-OH* population on lightly doped SrTiO3 are ordered by the surface hydroxylation. A Langmuir isotherm as a function of pH extracts an effective equilibrium constant relatable to the free energy difference of the first oxygen evolution reaction step. Thus, time-resolved spectroscopy of the catalytic surface reveals both kinetic and energetic information of elementary reaction steps, which provides a critical new connection between theory and experiment by which to tailor the pathway of water oxidation and other surface reactions.

Coherent Acoustic Interferometry during the Photodriven Oxygen Evolution Reaction Associates Strain Fields with the Reactive Oxygen Intermediate (Ti-OH*).

The oxygen evolution reaction (OER) from water requires the formation of metastable, reactive oxygen intermediates to enable oxygen-oxygen bond formation. Conversely, such reactive intermediates could also structurally modify the catalyst. A descriptor for the overall catalytic activity, the first electron and proton transfer OER intermediate from water, (M-OH*), has been associated with significant distortions of the metal-oxygen bonds upon charge-trapping. Time-resolved spectroscopy of in situ, photodriven OER on transition metal oxide surfaces has characterized M-OH* for the charge trapping and the symmetry of the lattice distortions by optical and vibrational transitions, respectively, but had yet to detect an interfacial strain field arising from a surface coverage M-OH*. Here, we utilize picosecond, coherent acoustic interferometry to detect the uniaxial strain normal to the SrTiO3/aqueous interface directly caused by Ti-OH*. The spectral analysis applies a fairly general methodology for detecting a combination of the spatial extent, magnitude, and generation time of the interfacial strain through the coherent oscillations' phase. For lightly n-doped SrTiO3, we identify the strain generation time (1.31 ps), which occurs simultaneously with Ti-OH* formation, and a tensile strain of 0.06% (upper limit 0.6%). In addition to fully characterizing this intermediate across visible, mid-infrared, and now GHz-THz probes on SrTiO3, we show that strain fields occur with the creation of some M-OH*, which modifies design strategies for tuning catalytic activity and provides insight into photo-induced degradation so prevalent for OER. To that end, the work put forth here provides a unique methodology to characterize intermediate-induced interfacial strain across OER catalysts.

The electron-transfer intermediates of the oxygen evolution reaction (OER) as polarons by in situ spectroscopy.

The conversion of diffusive forms of energy (electrical and light) into short, compact chemical bonds by catalytic reactions regularly involves moving a carrier from an environment that favors delocalization to one that favors localization. While delocalization lowers the energy of the carrier through its kinetic energy, localization creates a polarization around the carrier that traps it in a potential energy minimum. The trapped carrier and its local distortion-termed a polaron in solids-can play a role as a highly reactive intermediate within energy-storing catalytic reactions but is rarely discussed as such. Here, we present this perspective of the polaron as a catalytic intermediate through recent in situ and time-resolved spectroscopic investigations of photo-triggered electrochemical reactions at material surfaces. The focus is on hole-trapping at metal-oxygen bonds, denoted M-OH*, in the context of the oxygen evolution reaction (OER) from water. The potential energy surface for the hole-polaron defines the structural distortions from the periodic lattice and the resulting "active" site of catalysis. This perspective will highlight how current and future time-resolved, multi-modal probes can use spectroscopic signatures of M-OH* polarons to obtain kinetic and structural information on the individual reaction steps of OER. A particular motivation is to provide the background needed for eventually relating this information to relevant catalytic descriptors by free energies. Finally, the formation of the O-O chemical bond from the consumption of M-OH*, required to release O2 and store energy in H2, will be discussed as the next target for experimental investigations.

Ultrafast vibrational dynamics of the tyrosine ring mode and its application to enkephalin insertion into phospholipid membranes as probed by two-dimensional infrared spectroscopy.

Enkephalins are small opioid peptides whose binding conformations are catalyzed by phospholipid membranes. Binding to opioid receptors is determined by the orientation of tyrosine and phenylalanine side chains. In this work, we investigate the effects of different charged phospholipid headgroups on the insertion of the tyrosine side chain into a lipid bilayer using a combination of 2D IR spectroscopy, anharmonic DFT calculations, and third order response function modeling. The insertion is probed by using the ∼1515 cm-1 tyrosine ring breathing mode, which we found exhibits rich vibrational dynamics on the picosecond timescale. These dynamics include rapid intramolecular vibrational energy redistribution (IVR), where some of the energy ends up in a dark state that shows up as an anharmonically shifted combination band. The waiting-time dependent 2D IR spectra also show an unusual line shape distortion that affects the extraction of the frequency-frequency correlation function (FFCF), which is the dynamic observable of interest that reflects the tyrosine side chain's insertion into the lipid bilayer. We proposed three models to account for this distortion: a hot-state exchange model, a local environment dependent IVR model, and a coherence transfer model. A qualitative analysis of these models suggests that the local environment dependent IVR rate best explains the line shape distortion, while the coherence transfer model best reproduced the effects on the FFCF. Even with these complex dynamics, we found that the tyrosine ring mode's FFCF is qualitatively correlated with the degree of insertion expected from the different phospholipid headgroups.

DNA methylation markers panel can improve prediction of response to neoadjuvant chemotherapy in luminal B breast cancer.

Despite the advantages of neoadjuvant chemotherapy (NACT), associated toxicity is a serious complication that renders monitoring of the patients' response to NACT highly important. Thus, prediction of tumor response to treatment is imperative to avoid exposure of potential non-responders to deleterious complications. We have performed genome-wide analysis of DNA methylation by XmaI-RRBS and selected CpG dinucleotides differential methylation of which discriminates luminal B breast cancer samples with different sensitivity to NACT. With this data, we have developed multiplex methylation sensitive restriction enzyme PCR (MSRE-PCR) protocol for determining the methylation status of 10 genes (SLC9A3, C1QL2, DPYS, IRF4, ADCY8, KCNQ2, TERT, SYNDIG1, SKOR2 and GRIK1) that distinguish BC samples with different NACT response. Analysis of these 10 markers by MSRE-PCR in biopsy samples allowed us to reveal three top informative combinations of markers, (1) IRF4 and C1QL2; (2) IRF4, C1QL2, and ADCY8; (3) IRF4, C1QL2, and DPYS, with the areas under ROC curves (AUCs) of 0.75, 0.78 and 0.74, respectively. A classifier based on IRF4 and C1QL2 better meets the diagnostic panel simplicity requirements, as it consists of only two markers. Diagnostic accuracy of the panel of these two markers is 0.75, with the sensitivity of 75% and specificity of 75%.

Risk factors and urinary biomarkers of non-albuminuric and albuminuric chronic kidney disease in patients with type 2 diabetes.

BACKGROUND: A number of recent studies indicate a transformation in the natural course of chronic kidney disease (CKD) in type 2 diabetes (T2D) patients: an increasing prevalence of declined renal function without proceeding to the accompanying elevation of albuminuria. It has been suggested that albuminuric and non-albuminuric CKD patterns could be different in their phenotypes and pathogenic mechanisms. AIM: To identify the risk factors and biomarkers of albuminuric and non-albuminuric patterns of CKD in patients with T2D. METHODS: Three hundred sixty patients with T2D duration ≥ 10 years were included in this observational cross-sectional study. The associations of a panel of demographic and clinical characteristics, complications, comorbidities, and metabolic and hematology parameters with albuminuric and non-albuminuric CKD patterns were analyzed. The urinary excretion of nephrin and podocin, two podocyte-specific markers, and WAP-four-disulfide core domain protein 2 (WFDC-2), a marker of tubulointerstitial fibrosis, was determined by ELISA in comparison with healthy controls. RESULTS: Non-albuminuric CKD was associated with age ≥ 65 years (P = 0.0001), female sex (P = 0.04), diabetes duration ≥ 15 years (P = 0.0009), and the use of diuretics (P = 0.0005). Male sex (P = 0.01), smoking (P = 0.01), waist-to-hip ratio >1.0 (P = 0.01) and hemoglobin A1c (HbA1c) > 8.0% (P = 0.005) were risk factors for elevated albuminuria not accompanied by a decrease in estimated glomerular filtration rate (eGFR). Duration of diabetes ≥ 15 years and the use of calcium channel blockers were risk factors for albuminuria with decreased eGFR (both P = 0.01). In multivariate logistic regression analysis, age, HbA1c, female sex and diuretics were significant predictors for reduced eGFR, while waist-to-hip ratio, HbA1c and male sex were associated with elevated urinary albumin-to-creatinine ratio (UACR). Excretion of nephrin and podocin was increased in patients with albuminuria, regardless of decline in renal function (P < 0.001), correlating positively with UACR. The urinary excretion of WFDC-2 was markedly higher in men than in women (P < 0.000001). Men with T2D demonstrated increased WFDC-2 levels independently of the CKD pattern (all P < 0.05). In T2D women, WFDC-2 excretion was increased in those with reduced renal function (P ≤ 0.01), correlating negatively with eGFR. CONCLUSION: The data provide further evidence that albuminuric and non-albuminuric CKD phenotypes correspond to different pathways of diabetic kidney disease progression.

Optimized noise reduction scheme for heterodyne spectroscopy using array detectors.

In this work, we optimize and further advance a noise reduction scheme for heterodyne spectroscopy. This scheme linearly combines data from reference detectors to predict the noise statistics in the signal detector through an optimized coefficient matrix. We validate this scheme for visible white-light-continuum and 800-nm light sources using un-matched CMOS arrays and show that the signal-to-noise ratio can approach the noise floor of the signal detector while using only ~5% of the energy for reference detection. We also optimize the strategy for estimating the coefficient matrix in practical applications. When combined with elaborate algorithms to perform pixel data compression and expansion, our scheme is applicable in difficult situations, including when the sample position is rapidly scanned, when detectors exhibit nonlinear response, and/or when laser fluctuations are large. The scheme is generalized to scenarios with complex chopping or phase cycling patterns, and a simple approach is provided for the chopping case. Finally, a robust and computationally efficient method is devised to remove multiplicative noise.

General noise suppression scheme with reference detection in heterodyne nonlinear spectroscopy.

We devised a novel two-step reference scheme that can greatly suppress the additive and convolutional noises in heterodyne nonlinear spectroscopy. To optimally remove additive noise, we fully utilized the spectral correlation in multi-channel reference data through a linear combination and regression algorithm. Using our pump-probe 2D IR spectrometer, we demonstrated that our scheme can improve the signal-to-noise ratio by 10-30 times and reach the noise floor of the signal detector. The new algorithm is guaranteed to reduce noise, enables the use of unmatched reference detectors, and does not introduce baseline shift or signal distortion. This scheme is applicable to many heterodyne spectroscopic techniques.