Protein Binding Profiles Research Articles

Graphene Oxide Nanosheets Toxicity in Mice Is Dependent on Protein Corona Composition and Host Immunity.

Two-dimension graphene oxide (GO) nanosheets with high and low serum protein binding profiles (high/low hard-bound protein corona/HChigh/low) are used in this study as model materials and screening tools to investigate the underlying roles of the protein corona on nanomaterial toxicities in vivo. We proposed that the in vivo biocompatibility/nanotoxicity of GO is protein corona-dependent and host immunity-dependent. The hypothesis was tested by injecting HChigh/low GO nanosheets in immunocompetent ICR/CD1 and immunodeficient NOD-scid II2rγnull mice and performed histopathological and hematological evaluation studies on days 1 and 14 post-injection. HClow GO induced more severe acute lung injury compared to HChigh GO in both immunocompetent and immunodeficient mice, with the effect being particularly pronounced in immunocompetent animals. Additionally, HClow GO caused more significant liver injury in both types of mice, with immunodeficient mice being more susceptible to its hepatotoxic effects. Moreover, administration of HClow GO resulted in increased hematological toxicity and elevated levels of serum pro-inflammatory cytokines in immunocompromised and immunocompetent mice, respectively. Correlation studies were conducted to explore the impact of distinct protein corona compositions on resulting toxicities in both immunocompetent and immunodeficient mice. This facilitated the identification of consistent patterns, aligning with those observed in vitro, thus indicating a robust in vitro-in vivo correlation. This research will advance our comprehension of how hard corona proteins interact with immune cells, leading to toxicity, and will facilitate the development of improved immune-modulating nanomaterials for therapeutic purposes.

Abstract 1848: The Cellular Thermal Shift assay and its applications in Target ID, MoA determination and biomarker discovery

Abstract With the Cellular Thermal Shift Assay (CETSA) celebrating its first decade since the PoC publication, the method has gained much interest in the basic science field as well as become an industry standard in applied drug discovery setting. In the last few years it has become apparent that in addition to providing information about drug-protein interaction, CETSA based thermal profiling of cells can help describe additional aspects of cell biology as it gives insight into the activating and rewiring of protein-protein interaction networks inside the cell upon stimuli. In the work presented here, we have used CETSA coupled to high resolution mass spectrometric readout to profile the response patterns of about 8000 proteins to several hundred molecular probes and marketed drugs. The goal has been to provide a map of wanted and unwanted effects following treatment and to correlate it to alternative readouts, by for example cellular imaging and cell death assays. We have applied the CETSA protocol for close to five hundred compounds, many in several cell-lines (HepG2, U87MG and K562). The experimental setup includes the step of treating the cells with a certain compound at a fixed concentration. After incubation for an hour, we heated the cells to a range of temperatures, followed by a lysis protocol and pooling of the samples. After this, the samples have been centrifuged to separate the soluble from insoluble fraction. The soluble fraction has then been prepared for MS acquisition using TMT based multiplexing. Typically, this allowed us to monitor changes in thermal stability among more than 8,000 proteins, as a consequence of compound treatment. This data reveals specific compound “fingerprints” that can be used to decipher mode of action and identify biomarkers. Despite the short incubation time, proteins with compound-induced thermal stability shifts are not only targets/off-targets (direct binders), but also downstream, and sometimes upstream, pathway members and general cellular responses. All data combined makes the basis of the Target Engagement Atlas. Here we can see clusters of compounds, sharing similar protein binding profiles. Among the most prominent ones are mTOR inhibitors, NSAIDs, as well as tubulin binders. Alternatively, one can use this resource to reveal clusters of “pharmacologically” associated protein networks. We show that pharmacological perturbation allows identification of clusters representing tight protein complexes (for example ribosomes), known metabolic pathways (folate biosynthesis), molecular functions (kinases), as well as networks of proteins linked only by ligand binding specificity. CETSA MS allows for unbiased, cellular, and molecular profiling of compound effects. Together with data from clinical settings, as well as in vivo/in vitro end point assays it is possible to correlate the CETSA patterns to wanted but also unwanted responses. Citation Format: Tomas Friman, Alexey Chernobrovkin, Tuomas Tolvanen, Erin Gilson, Stina Lundgren, Victoria Brehmer, Daniel Martinez Molina. The Cellular Thermal Shift assay and its applications in Target ID, MoA determination and biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1848.

Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins.

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.

A simple and rapid HPLC-UV method for the determination of valproic acid in human plasma using microwave-assisted derivatization with phenylhydrazine hydrochloride

This study presents an efficient high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for monitoring valproic acid (VPA) level in human plasma. This method is distinguished by its simplicity, cost-effectiveness, and rapid execution, addressing the limitations associated with other advanced analytical techniques like liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and immunoassays, which are generally complex and costly for routine application. A challenge in analyzing VPA is its non-linear protein binding profile and the absence of a chromophore in its structure, making direct detection difficult. To overcome this, the study developed an efficient HPLC-UV for VPA determination in human plasma, utilizing a simplified and rapid microwave-assisted derivatization process. Due to the lack of a chromophore in VPA structure, this work developed a microwave-assisted derivatization of VPA using phenylhydrazine hydrochloride (PH HCl). The process optimization was achieved at 450 W for 50 s, facilitating effective HPLC-UV detection. The derivatized product was characterized using 1H nuclear magnetic resonance (NMR) and Fourier transform infrared spectrometer (FT-IR). The derivative, identified as (Z)-N-phenyl-2-propylpentanehydrazonic acid, demonstrated specificity in plasma analysis with no detectable interference. The method exhibited a linear response for VPA concentrations ranging from 30 to 150 μg/mL, with a correlation coefficient exceeding 0.99. Recovery varied between 86.7% and 107%, with a maximum coefficient of variation (CV) of 10.0%. The findings suggest that the microwave-assisted derivatization technique substantially improves the feasibility and cost-effectiveness of HPLC-UV for the analysis of VPA in plasma. This method provides a viable alternative to conventional HPLC methodologies, offering a balance of efficiency and economic practicality for VPA quantification.

Inferring Protein-DNA Binding Profiles at Interspersed Repeats Using HiChIP and PAtChER.

Alignment of short-read sequencing data to interspersed genomic repeats, such as transposable elements, can be problematic. This is especially true for evolutionarily young elements, which have not sufficiently diverged from each other to produce distinct and uniquely mappable reads. Mapping difficulties pose a challenge for studying the portfolio of epigenetic modifications and other chromatin regulators that bind to transposons and dictate their activity, which are typically studied using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Since ChIP-seq requires chromatin fragmentation to achieve appropriate resolution, longer reads do not appreciably improve mappability. Here, we present an experimental and computational protocol that couples ChIP-seq with 3D genome folding information to produce protein binding profiles with dramatically increased coverage at interspersed repeats.

Differential mitochondrial protein interaction profile between human translocator protein and its A147T polymorphism variant.

The translocator protein (TSPO) has been implicated in mitochondrial transmembrane cholesterol transport, brain inflammation, and other mitochondrial functions. It is upregulated in glial cells during neuroinflammation in Alzheimer's disease. High affinity TSPO imaging radioligands are utilized to visualize neuroinflammation. However, this is hampered by the common A147T polymorphism which compromises ligand binding. Furthermore, this polymorphism has been linked to increased risk of neuropsychiatric disorders, and possibly reduces TSPO protein stability. Here, we used immunoprecipitation coupled to mass-spectrometry (IP-MS) to establish a mitochondrial protein binding profile of wild-type (WT) TSPO and the A147T polymorphism variant. Using mitochondria from human glial cells expressing either WT or A147T TSPO, we identified 30 WT TSPO binding partners, yet only 23 for A147T TSPO. Confirming that A147T polymorphism of the TSPO might confer loss of function, we found that one of the identified interactors of WT TSPO, 14-3-3 theta (YWHAQ), a protein involved in regulating mitochondrial membrane proteins, interacts much less with A147T TSPO. Our data presents a network of mitochondrial interactions of TSPO and its A147T polymorphism variant in human glial cells and indicate functional relevance of A147T in mitochondrial protein networks.

Protein Binding and Population Pharmacokinetics of Dexmedetomidine after Prolonged Infusions in Adult Critically Ill Patients

PurposeDexmedetomidine (DEX) is a highly selective α2-adrenoceptor agonist with high protein binding of 94%. Critical illness may affect protein binding and the pharmacokinetic (PK) parameters of many drugs, including DEX. In critically ill patients receiving prolonged infusions of DEX, there is little information documenting the relationship between key pathophysiologic factors and DEX protein binding or PK parameters. The purpose of this study was to characterize the protein binding and PK profile of prolonged DEX infusion in critically ill patients. MethodsCritically ill, adult intensive care unit patients at a university hospital in Hong Kong were studied. The association between the pathophysiologic changes of critical illness and protein binding was evaluated using a generalized estimating equation. A population pharmacokinetic model to establish the PK profile of DEX was developed, and key pathophysiologic covariate effects of severity of illness, organ dysfunction measures, and altered protein binding on DEX PK parameters in this critically ill population were evaluated. FindingsA total of 22 critically ill patients and 1 healthy control were included. Mean protein binding of DEX in the critically ill patients was 90.4% (95% CI, 89.1–91.7), which was 4% lower than that in the healthy control. The PK data were adequately described by a 2-compartment model. The estimated population mean (relative standard error [RSE]) values of systemic clearance (CL), volume of distribution of the central compartment (V2), intercompartmental clearance (Q), and Vd in the peripheral compartment (V3) were 38.6 (11.7) L/h, 32.1 (46.1) L, 114.5 (58.3) L/h and 95.1 (30.6) L, respectively. The corresponding estimated interindividual variability expressed as CV% (RSE) was 52.4 (23.8) for CL, 172.9 (19.3) for V2, 123.7 (33.7) for Q, and 106 (39.9) for V3. No significant explanatory pathophysiologic covariates were identified. ImplicationsAlthough a marginally significant reduction of protein binding in critically ill patients was demonstrated, the magnitude of the difference was unlikely to be of clinical significance. Higher alanine aminotransferase concentration was associated with decreased protein binding. No significant pathophysiologic covariates were associated with the observed PK parameters. The high interindividual variability of PK parameters supports the current practice of dose titration to ensure the desired clinical effects of DEX infusion in the intensive care unit setting.

The m6A landscape of polyadenylated nuclear (PAN) RNA and its related methylome in the context of KSHV replication.

Polyadenylated nuclear (PAN) RNA is a long noncoding transcript involved in Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation and regulation of cellular and viral gene expression. We have previously shown that PAN RNA has dynamic secondary structure and protein binding profiles that can be influenced by epitranscriptomic modifications. N6-methyladenosine (m6A) is one of the most abundant chemical signatures found in viral RNA genomes and virus-encoded RNAs. Here, we combined antibody-independent next-generation mapping with direct RNA sequencing to address the epitranscriptomic status of PAN RNA in KSHV infected cells. We showed that PAN m6A status is dynamic, reaching the highest number of modifications at the late lytic stages of KSHV infection. Using a newly developed method, termed selenium-modified deoxythymidine triphosphate (SedTTP)-reverse transcription (RT) and ligation assisted PCR analysis of m6A (SLAP), we gained insight into the fraction of modification at identified sites. By applying comprehensive proteomic approaches, we identified writers and erasers that regulate the m6A status of PAN, and readers that can convey PAN m6A phenotypic effects. We verified the temporal and spatial subcellular availability of the methylome components for PAN modification by performing confocal microscopy analysis. Additionally, the RNA biochemical probing (SHAPE-MaP) outlined local and global structural alterations invoked by m6A in the context of full-length PAN RNA. This work represents the first comprehensive overview of the dynamic interplay that takes place between the cellular epitranscriptomic machinery and a specific viral RNA in the context of KSHV infected cells.

Unexpected Vancomycin Pharmacokinetic Profile Secondary to Macromolecular Complexing: A Case Series.

The optimal dosing and monitoring of vancomycin has been largely debated for decades, with key guideline changes for recommended monitoring in 2009 and 2020. Current and past practices for pharmacokinetic dose optimization use serum drug assays to guide dose adjustment to effectively balance efficacy and the risks of toxicity. These assays detect both bound and unbound serum concentrations. Vancomycin is believed to be 50%-55% protein bound in most cases; however, some variability in this parameter has been previously published. The authors report 2 cases of abnormal vancomycin pharmacokinetics discovered based on unexpected serum levels during routine clinical care. Unexpected vancomycin levels, observed during clinical care for 2 separate patients, were further evaluated to determine the source of the abnormal pharmacokinetics. In case 1, serial dilution was performed to assure that assay interference was not associated with the significant elevation (>100 mg/L). In both cases, samples were filtered using a Millipore Centrifree 30 KDa centrifugal filter to separate bound vancomycin, with a Protein G spin kit used to bind IgG and remove IgG complexes from the patient sample. In case 2, a polyethylene glycol precipitation was also performed to precipitate large-molecular-weight complexes. In both cases, laboratory analysis revealed abnormal vancomycin protein-binding profiles with macromolecular complex formation. Immunoglobulin G played a role in the macrocomplex in both patients. In cases of unusual or unexpected vancomycin pharmacokinetics in the absence of renal dysfunction, an abnormal protein-binding profile should be considered. Bound vancomycin may yield elevated serum levels, leading to poorly informed dose adjustments and risk for treatment failure. Given implications for therapeutic drug monitoring and unknown impacts on efficacy and toxicity, further investigations into population incidence and risk factors for abnormal protein binding of vancomycin are warranted.

Modeling protein target search in human chromosomes

Several processes in the cell, such as gene regulation, start when key proteins recognise and bind to short DNA sequences. However, as these sequences can be hundreds of million times shorter than the genome, they are hard to find by simple diffusion: diffusion-limited association rates may underestimate $in~vitro$ measurements up to several orders of magnitude. Moreover, the rates increase if the DNA is coiled rather than straight. Here we model how this works $in~vivo$ in mammalian cells. We use chromatin-chromatin contact data from state-of-the-art Hi-C experiments to map the protein target-search onto a network problem. The nodes represent a DNA segment and the weight of the links is proportional to measured contact probabilities. We then put forward a master equation for the density of searching protein that allows us to calculate the association rates across the genome analytically. For segments where the rates are high, we find that they are enriched with active genes and have high RNA expression levels. This paper suggests that the DNA's 3D conformation is important for protein search times $in~vivo$ and offers a method to interpret protein-binding profiles in eukaryotes that cannot be explained by the DNA sequence itself.

Cannabidiol (CBD) as a Promising Anti-Cancer Drug.

Simple SummaryThe use of cannabinoids containing plant extracts as herbal medicine can be traced back to as early as 500 BC. In recent years, the medical and health-related applications of one of the non-psychotic cannabinoids, cannabidiol or CBD, has garnered tremendous attention. In this review, we will discuss the most recent findings that strongly support the further development of CBD as a promising anti-cancer drug.Recently, cannabinoids, such as cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), have been the subject of intensive research and heavy scrutiny. Cannabinoids encompass a wide array of organic molecules, including those that are physiologically produced in humans, synthesized in laboratories, and extracted primarily from the Cannabis sativa plant. These organic molecules share similarities in their chemical structures as well as in their protein binding profiles. However, pronounced differences do exist in their mechanisms of action and clinical applications, which will be briefly compared and contrasted in this review. The mechanism of action of CBD and its potential applications in cancer therapy will be the major focus of this review article.

A review of the current understanding of nanoparticles protein corona composition.

Upon entering into the biological environments, the surface of the nanoparticles is immediately coated with proteins and form the so-called a protein corona due to which a nanoparticle changes its “synthetic” identity to a new “biological” identity. Different types of nanoparticles have different protein binding profiles, which is why they have different protein corona composition and therefore it cannot be said that there is a universal protein corona. The composition and amount of protein in the corona depends on the physical and chemical characteristics of the nanoparticles, the type of biological medium and the exposure time. Protein corona increases the diameter but also changes the composition of the surface of the nanoparticles and these changes affect biodistribution, efficacy, and toxicity of the nanoparticles.

Characterization of plasma protein binding in two mouse models of humanized liver, PXB mouse and humanized TK-NOG mouse

The unbound fractions in plasma (f up) in two mouse models of humanized liver mice, PXB and humanized TK-NOG mice, were compared with human f up values using equilibrium dialysis method. A good relationship between f up values obtained from PXB mice and humans was observed; the f up of 34/39 compounds (87.2%) in PXB mice were within 3-fold of human f up. In contrast, a weak correlation was observed between human and humanized TK-NOG mouse f up values; the f up of 15/24 compounds (62.5%) in humanized TK-NOG mice were within 3-fold of human f up. As different profiles of plasma protein binding (PPB) profiles were observed between PXB and humanized TK-NOG mice, f up evaluation is necessary in each mouse model to utilize these humanized liver mice for pharmacological, drug–drug interaction (DDI), and toxicity studies. The unbound fraction in the mixed plasma of human and SCID mouse plasma (85:15) was well correlated with f up in PXB mice (38/39 compounds within a 3-fold). Thus, this artificial PXB mouse plasma could be used to evaluate PPB.

In vivo pharmacokinetics, distribution, and excretion of an anticancer agent isolated from red ginseng, in rat

The compound 20(S),25-epoxydammarane-3β,12β,24α-triol (24-hydroxy-panaxadiol or 24-OH-PD), isolated from the red Panax ginseng CA Meyer possesses anticancer activity. Our aim was to study the pharmacokinetic characteristics of 24-OH-PD, which is essential for pre-clinical research during the development of new drugs. In this study, a simple and sensitive ultra-performance liquid chromatography-mass spectrometry (LC-MS/MS) method was established and used for studying the pharmacokinetics, in vitro protein binding, tissue distribution, and elimination profiles of 24-OH-PD in rats. 24-OH-PD was characterized by linear pharmacokinetics in the dose range of 2.5–10 mg/kg and had relatively longer half-life (4.82–5.45 h) than the other ginsenosides. It had a wide tissue distribution profile in rats and was primarily distributed in the lung. Within 96 h of intravenous administration, 13.84% of 24-OH-PD was excreted out via feces and 0.02% via urine in its unchanged form. In conclusion, a simple LC-MS/MS method with high sensitivity and selectivity was established for the quantification of 24-OH-PD.

Impact of Holder pasteurization on essential elements from human donor milk: Total contents and protein-binding profiles

Holder pasteurization is a thermal treatment commonly applied to milk in human milk banks. However, the application of heating processes can affect the actual composition of milk. In this work, the effect of Holder pasteurization on essential elements in donor human breast milk was studied, including total contents and protein-binding profile of Cu, Fe, Se and Zn. The total contents were determined by inductively coupled plasma mass spectrometry (ICP-MS) and chemical speciation studies were conducted by size exclusion chromatography (SEC) coupled to an ICP-MS and to an UV–vis detector. Total concentrations (μg/L) of Cu (0.19 ± 0.01), Fe (0.35 ± 0.01), Se (0.010 ± 0.003) and Zn (0.57 ± 0.01) were obtained before pasteurization and concentrations of Cu (0.19 ± 0.01), Fe (0.36 ± 0.02), Se (0.008 ± 0.002) and Zn (0.57 ± 0.01) were obtained after pasteurization. Their protein-binding profiles did not change significantly for the majority of the species, indicating a low degree of interconversion due to the elevation of the temperature. However, protein profiles changed after pasteurization, indicating that the losses occurred mainly in the apo form (metal-free form) of proteins rather than in their holo forms (metallo-species).

Discovery of a novel 3,4-dimethylcinnoline carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping.

This Letter details our efforts to replace the 2,4-dimethylquinoline carboxamide core of our previous M4 PAM series, which suffered from high predicted hepatic clearance and protein binding. A scaffold hopping exercise identified a novel 3,4-dimethylcinnoline carboxamide core that provided good M4 PAM activity and improved clearance and protein binding profiles.

Design, synthesis and identification of silicon-containing HCV NS5A inhibitors with pan-genotype activity

Modification of a HCV NS5A inhibitor, ombitasvir, led to the identification of 10d with improved pan-genotype NS5A inhibition and better pharmacokinetic properties. The key structural changes to ombitasvir include bioisosteric replacement of carbon with silicon atom. Compared with ombitasvir, the activity of anti-HCV genotypes (GT 1 to 6) of 10d is increased to some extent, especially the inhibitory activity against genotype 3a and 6a is increased by more than seven times, and the dog's in vivo pharmacokinetics properties were also superior to ombitasvir. Further drug evaluation showed that 10d was similar to ombitasvir on plasma protein binding and liver distribution profiles, with no cytotoxicity and no inhibitory effect on both CYP 450 and hERG ligand binding. However, permeability assay results indicated that 10d was not the substrate of P-gp or BCRP transporter, which is different from that of ombitasvir. The results of a 14-day repeat-dose toxicity study identified no toxicity with 10d. Our findings in preclinical tests suggest that the silicon-containing compound 10d could be worthy of continued study as a potential drug candidate.

A Review on Pharmacokinetics of Levetiracetam in Neonates.

Neonatal seizures are the most common clinical manifestation of Central Nervous System (CNS) dysfunction and are associated with various neurological sequelae. There are currently no evidence-based guidelines for the management of neonatal seizures and currently used drugs such as phenobarbital, and phenytoin have limited efficacy and potential toxicities. Newer second line anticonvulsant, levetiracetam, has been used in refractory neonatal seizures despite limited data and off-label use. In this review, we will discuss various pharmacological properties of levetiracetam when used in neonatal population. A PubMed search for MEDLINE was undertaken to look for studies using the terms "Levetiracetam", AND "Neonates" as key words from year 1995 to January 2017. Relevant articles were selected and information was extracted about pharmacokinetics, pharmacodynamics and clinical uses of levetiracetam in neonates. Levetiracetam is an active, water-soluble S-enantiomer of racemic pyrrolidine acetamide which exerts its antiepileptic action by binding to the synaptic vesicle protein within the brain. Metabolism of levetiracetam does not include the CYP P450 system and it is mainly eliminated through kidneys after rapid absorption. Also, no significant interactions with other drugs have been identified. Unlike other commonly used antiepileptic drugs, levetiracetam is not bound to plasma proteins, thereby, reducing the chances of toxicity and severe, life threatening side effects have not been reported. In fact, it has been shown to prevent neuro-degeneration after hypoxia/ischemia in rodent models of epilepsy. Levetiracetam has been emerging as a potential therapeutic option for refractory neonatal convulsions owing to its non-hepatic elimination, linear pharmacokinetics, low protein binding and better safety profile.

Predicting gene expression from genome wide protein binding profiles

Abstract High-throughput technologies such as chromatin immunoprecipitation (IP) followed by next generation sequencing (ChIP-seq) in combination with gene expression studies have enabled researchers to investigate relationships between the distribution of chromosome-associated proteins and the regulation of gene transcription on a genome-wide scale. Several attempts at integrative analyses have identified direct relationships between the two processes. However, a comprehensive understanding of the regulatory events remains elusive. This is in part due to the scarcity of robust analytical methods for the detection of binding regions from ChIP-seq data. In this paper, we have applied a recently proposed Markov random field model for the detection of enriched binding regions under different biological conditions and time points. The method accounts for spatial dependencies and IP efficiencies, which can vary significantly between different experiments. We further defined the enriched chromosomal binding regions as distinct genomic features, such as promoter, exon, intron, and distal intergenic, and then investigated how predictive each of these features are of gene expression activity using machine learning techniques, including neural networks, decision trees and random forest. The analysis of a ChIP-seq time-series dataset comprising six protein markers and associated microarray data, obtained from the same biological samples, shows promising results and identified biologically plausible relationships between the protein profiles and gene regulation.

Quantitative study of zinc and metallothioneins in the human retina and RPE cells by mass spectrometry-based methodologies

The retina contains the highest concentration of zinc in the human eye and it is primarily associated with the retinal pigment epithelium (RPE). Metallothioneins (MTs) are the main cytosolic zinc-ion-binding proteins, exerting a tight control in the number of atoms of Zn-bound to the MTs related with their antioxidant and neuroprotective functions. In order to study the Zn-MT system in retina and RPE, we have implemented mass spectrometry (MS)-based technologies: two complementary element detection methodologies (HPLC- and laser ablation (LA)-ICP-MS) have been successfully employed to study metal content in the human eye as well as to perform speciation studies of Zn-MTs. First, Zn-elemental distribution was studied on cryogenic ocular sections by LA-ICP-MS. Quantitative images of Zn along RPE cell layer and the retina were obtained with a laser beam diameter of 25µm, showing a preferential distribution in the RPE. We carried out then the quantitative speciation of Zn, Fe, and Cu in the water-soluble protein fractions of RPE and retina to study their protein binding profile using HPLC-ICP-MS, where Zn is mainly associated to low molecular mass proteins (i.e., MTs). Finally, the effect of addition of different inductors, such as metal (i.e., 68ZnSO4), dexamethasone (DEX) and erythropoietin, was investigated in an in vitro cellular model of human RPE cells (HRPEsv), again using HPLC-ICP-MS in combination with stable isotopes and mathematical calculations based on isotope dilution and isotope pattern deconvolution. Exogenous Zn and DEX were found to increase MT proteins synthesis and exerted a stoichiometric transition in MT proteins in HRPEsv cells.